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Pharmacokinetic-pharmacodynamic modeling of the analgesic effect of ibuprofen and codeine

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Pharmacokinetic-pharmacodynamic modeling of the analgesic effect of ibuprofen and codeine
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Kaltenbach, Matthieu L., 1962-
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English
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viii, 143 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Analgesics ( jstor )
Dosage ( jstor )
Evoked potentials ( jstor )
Morphine ( jstor )
Pain ( jstor )
Pharmacokinetics ( jstor )
Physiological stimulation ( jstor )
Plasmas ( jstor )
Tooth pulp ( jstor )
Writing tablets ( jstor )
Analgesics ( mesh )
Codeine -- pharmacokinetics ( mesh )
Codeine -- pharmacology ( mesh )
Department of Pharmaceutics thesis Ph.D ( mesh )
Dissertations, Academic -- College of Pharmacy -- Department of Pharmaceutics -- UF ( mesh )
Dose-Response Relationship, Drug ( mesh )
Ibuprofen -- pharmacokinetics ( mesh )
Ibuprofen -- pharmacology ( mesh )
Research ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1992.
Bibliography:
Bibliography: leaves 127-142.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Matthieu L. Kaltenbach.

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PHARMACOKINETIC-PHARMACODYNAMIC MODELING OF THE
ANALGESIC EFFECT OF IBUPROFEN AND CODEINE

















By

MATTHIEU L. KALTENBACH


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1992














Dedicated to my wife, Marie-Pierre,

and our children, Ccile, Pauline, and Emmanuelle













ACKNOW LEDGMENTS


I would like to offer my appreciation and grateful thanks to Dr. Hartmut

Derendorf for his friendship, guidance, and continuous encouragement during the

course of the work embodied in this dissertation. I would also like to thank the

members of my supervisory committee, Dr. Gunther Hochhaus, Dr. Hans Schreier, and

Dr. Charles Vierck, for their advice throughout my doctoral research.

My special thanks are extended to Dr. Betty Grundy for her time, fruitful

suggestions, and constant support with the evoked potentials experiments. Without

her, this research project would not have been possible.

I would like to acknowledge and thank Dr. John Perrin who supervised parts of

the drug analysis, and Mr. Fred Bauman and Dr. Andr6 Mauderli for their help in

setting up the tooth pulp experiments.

I take this opportunity to express my sincere appreciation to Martina Butschkau,

Maritza Cediel, Jan Mtller, Sumia Mohammed, Gotelind Mullersman, Pam Riley,

Shashank Rohatagi, Carolina Vivas, and especially Marjorie Rigby for their valuable

help with the drug analysis.

I would like to thank Dr. Daniel Robinson for introducing me to the wonderful

world of computers and their applications in pharmacy.

Last but not least, I would like to extend my sincere appreciation and warmest

thanks to my parents, parents in-law, and to my wife for believing in me, being there

whenever I needed them, and doing whatever they could to help me.














TABLE OF CONTENTS


page

A C KN O W LEDG M EN TS .............................................................................................. iii

A BSTRA CT ..................................................................................................................... vii

CHAPTERS

1 IN TRO D UCTIO N ...................................................... ........ ....... ................... 1

2 LITERA TURE REV IEW ................................................................................... 5
The C concept of Pain .................................................................. ...................... 5
Pain: A M ultidim ensional Experience ....................................................... 5
The Physiology of Pain............................................................................... 7
N ociceptors............................................................................................ 7
Central pain pathw ays........................................................................ 8
Pain m odulation.................................................................................... 11
Pain M easurem ent and A ssessm ent............................................................... 12
Unidim ensional Pain Rating Scales .......................................................... 13
Categorical rating scales...................................................................... 14
Visual analog scales............................................................................. 15
M ultidim ensional Pain Rating Scales...................................................... 17
Physiologic Correlates of Pain ................................................................... 18
Evoked Potentials in Pain Research .......................................... .......... ............. 19
Evoked Potentials....................................................................................... 19
N om enclature ....................................................................................... 21
C lassification of evoked potentials..................................................... 21
The Pain Stim ulus...................................................................................... 23
M echanical stim ulation ........................................................................ 24
Therm al stim ulation ............................................................................ 24
Electrical stim ulation ............................................................................ 24
Tooth Pulp Evoked Potentials.................................................................... 25
Evoked Potentials as Correlates of Pain ................................................... 26
Effect of A nalgesics on Evoked Potentials ............................................... 30
Centrally acting analgesics.................................................................. 31
Peripherally acting analgesics ............................................................. 32
Anesthetics ........................................................................................... 33
A advantages of Evoked Potential M ethods .............................................. 34
Ibuprofen............................................................................................................ 35
Physicochem ical Properties........................................................................ 35
Pharm acology............................................................................................. 36
Pharm acokinetics ........................................................................................ 38
A bsorption............................................................................................ 38
Distribution .......................................................................................... 38








M etabolism ........................................................................................... 39
Elim nation ........................................................................................... 41
Toxicity........................................................................................................ 42
Therapeutic Uses......................................................................................... 42
Dosage and Adm inistration...................................................................... 43
Codeine .............................................................................................................. 43
Physicochem ical Properties........................................................................ 43
Pharm acology............................................................................................. 44
Pharm acokinetics ....................................................................................... 46
Absorption............................................................................................ 46
Distribution .......................................................................................... 46
M etabolism ........................................................................................... 46
Elim nation ........................................................................................... 48
Toxicity........................................................................................................ 48
Therapeutic Uses......................................................................................... 49
D osage and A dm inistration...................................................................... 49

3 EXPERIM EN TA L............................................................................................. 50
Drug A says ..................................................................................................... 50
Codeine Analysis ....................................................................................... 50
Chem icals and reagents ....................................................................... 50
Instrum entation ................................................................................... 51
Extraction procedure ........................................................................... 51
Chrom atographic conditions.............................................................. 53
Ibuprofen A analysis .................................................................................... 53
Chem icals and reagents ....................................................................... 53
Instrum entation .................................................................................... 53
Extraction procedure ........................................................................... 54
Chrom atographic conditions............................................................... 54
Pharm acokinetic Study.................................................................................... 55
Experim ental Design .................................................................................. 55
Subjects........................................................................................................ 55
Drug A dm inistration .................................................................................. 56
Blood Sam pling........................................................................................... 56
Estim ation of Pharm acokinetic Param eters............................................. 56
Statistical A analysis ..................................................................................... 57
Pharm acodynam ic Study................................................................................. 57
Experim ental Design .................................................................................. 58
Subjects........................................................................................................ 58
Drug A dm inistration ................................................................................. 58
Blood Sam pling........................................................................................... 59
Dental Stim ulation ..................................................................................... 59
Determ nation of Pain Threshold ............................................................. 61
Evoked Potential Recording...................................................................... 61
Estim ation of Pharm acokinetic Param eters............................................. 65
Estim ation of Pharm acodynam ic Param eters.......................................... 66
Subjective pain ratings ........................................................................ 67
Tooth pulp evoked potentials............................................................. 68
Pharmacokinetic-Pharmacodynamic Modeling...................................... 69
Statistical A analysis ..................................................................................... 70

4 RESU LTS A N D DISCUSSIO N ........................................................................ 71
Analytical Procedures...................................................................................... 71
Ibuprofen Analysis .................................................................................... 71








Chrom atography .................................................................................. 71
Linearity.............................................................................................. 72
Reproducibility..................................................................................... 72
Codeine A analysis ....................................................................................... 72
Chrom atography .................................................................................. 73
Linearity................................................................................................ 74
Reproducibility..................................................................................... 74
Pharm acokinetic Study.................................................................................... 77
Ibuprofen Pharm acokinetics..................................................................... 78
Codeine Pharm acokinetics........................................................................ 85
Pharm acodynam ic Study................................................................................. 92
Dental Stim ulation ..................................................................................... 92
Evoked Potential Recording...................................................................... 94
Pharm acokinetic M easurem ents................................................................ 104
Pharm acodynam ic M easurem ents............................................................ 108
Pharmacokinetic-Pharmacodynamic Modeling....................................... 111

5 CO N C LUSIO N .................................................................................................. 118

APPENDIX

TPEP TO O LKIT ....................................................................................................... 120

BIBLIO G RA PH Y ............................................................................................................ 127

BIO G RA PH ICA L SKETC H ........................................................................................... 143














Abstract of Dissertation Presented to the Graduate School of the
University of Florida in Partial Fulfillment of the
Requirements for the Degree of
Doctor of Philosophy


PHARMACOKINETIC-PHARMACODYNAMIC MODELING OF THE
ANALGESIC EFFECT OF IBUPROFEN AND CODEINE


MATTHIEU L. KALTENBACH


December 1992


Chairman: Hartmut Derendorf, Ph.D.
Major Department: Pharmaceutics


Understanding the relationship between drug concentration and drug effect is

usually a prerequisite for designing an optimal drug therapy. In the case of analgesic

drugs, such pharmacokinetic-pharmacodynamic correlations have rarely been

described. Therefore, the aim of this study was to investigate the pharmacokinetics and

pharmacodynamics of ibuprofen and codeine when given alone or in combination,

using electrical tooth pulp stimulation as an experimental pain model.

The first part of this study investigated the pharmacokinetics of codeine and

ibuprofen when given alone or in combination. It consisted of a double-blind, cross-over

study, with twenty-four healthy subjects receiving oral doses of ibuprofen (400 mg),

codeine (60 mg), and four different codeine-ibuprofen combinations containing 400 mg

of ibuprofen with either 30 or 60 mg of codeine. Plasma concentrations were measured

by reversed-phase liquid chromatography with fluorescence and UV detection.

Pharmacokinetic parameters were estimated by non-compartmental data analysis. No








relevant change in pharmacokinetic parameters was observed following concomitant

administration of codeine and ibuprofen.

The second part of this study looked at the pharmacokinetics and

pharmacodynamics of codeine and ibuprofen when given alone or in combination. It

consisted of a double-blind, placebo controlled, cross-over study performed on seven

healthy volunteers. Four different treatments were investigated: placebo, ibuprofen

400 mg, codeine 60 mg, and an ibuprofen-codeine combination containing 400 mg of

ibuprofen and 30 mg of codeine. Pharmacokinetic parameters were estimated by

compartmental data analysis. Pharmacodynamic effects were monitored on visual

analog pain rating scales and on amplitudes of electrophysiologic cortical brain

potentials evoked by electrical tooth pulp stimulation.

Following oral administration, the pharmacokinetics of orally administered

ibuprofen and codeine were adequately described by an open one-compartment body

model with first-order input. Statistically significant differences between active drugs

and placebo were observed for all pharmacodynamic parameters. For these

pharmacodynamic effects, an integrated pharmacokinetic-pharmacodynamic model

was investigated to relate the plasma concentration-time profiles into effect-time

profiles. This model suggested a maximum pain relief effect of about 50% for each drug

and the combination. Due to the limited number of subjects and of effect measurements,

further pharmacokinetic-pharmacodynamic studies are needed to more fully define the

observed dose-effect relationship.














CHAPTER 1
INTRODUCTION


Analgesia is a major concern in clinical practice and one of the most problematic

areas in pain research. Despite many studies, it is evident that research on the

assessment of analgesic states has had little influence on medical care or guidelines for

clinical pain control. Many patients still experience inadequate pain relief (Bonica,

1990a; Donovan et al., 1987; Marks and Sachar, 1973; Sriwatanakul et al., 1983b). Pain is

recognized as the most frequent medical symptom by the National Institutes of Health.

Among pharmaceutical products used for pain relief, orally effective opioids

such as codeine are often prescribed in combination with peripherally acting analgesics

such as aspirin or acetaminophen. Analgesic mixtures containing codeine were among

the 10 most frequently dispensed drugs in the United States in 1991 (La Piana

Simonsen, 1992). The combination of an opioid with a non-opioid appears to be rational

because the mechanisms of action differ, and the analgesic effects of the individual

drugs are additive (Beaver, 1975, 1984). Since the non-opioids have a ceiling analgesic

effect and the dosage of opioids should be limited to prevent adverse effects,

combinations of this type may also provide greater pain relief with a minimum of

adverse effects in a convenient dosage form for the patient (Beaver, 1975, 1981, 1984).

However, there is still no consistent evidence from human clinical trials to substantiate

these rationales for any particular combination. Recently, a growing number of well-

controlled clinical trials have demonstrated that appropriately chosen combinations of

opioids with either aspirin or acetaminophen do in fact achieve these objectives (Bentley

and Head, 1987; Forbes et al. 1984a, 1984b, 1986).








Newer non-steroidal anti-inflammatory drugs constitute a potential source of

more effective peripherally acting components for opioid-containing analgesic

combinations. Among these, the combination of codeine with ibuprofen (a propionic

acid derivative with anti-inflammatory and analgesic activities) in a single oral dosage

form is currently investigated (Cater et al., 1985; Cooper et al., 1982; Frame et al., 1986;

Friedman et al., 1990; McQuay et al., 1989; Norman et al., 1985; Sunshine et al., 1987).

Codeine and ibuprofen have been studied extensively since their introduction.

Their pharmacology and pharmacokinetics are well defined in the literature (Gilman et

al., 1985). Yet, no study has clearly established the relationship between plasma levels of

these drugs and the degree of analgesia attained. The establishment of such correlations

is usually a prerequisite for optimal drug therapy and may lead to more effective use of

drugs. Ultimately, this will result in greater predictability of therapeutic response and

the possibility of reducing the incidence or severity of adverse effects (Colbumrn, 1987). In

the case of analgesic drugs, however, correlations between in vivo drug concentrations

and pharmacodynamic effects have seldom been described because of the inherent

difficulty of quantifying the degree of pain relief (Inturrisi and Colburn, 1986; Inturrisi

et al., 1987, 1990; Laska et al., 1986b). Studies employing electrical tooth pulp

stimulation have demonstrated that dental dolorimetry is a reliable way of producing a

relatively pure pain experience in human laboratory subjects (Azerad and Woda, 1977;

Mumford and Bowsher, 1976). Moreover, there is now considerable evidence that late

brain potentials evoked by tooth pulp stimulation reflect pharmacologically induced

pain relief in man, particularly when used in conjunction with other scaling procedures

(Bromm, 1985; Chapman and Jacobson, 1984; Chapman et al., 1979; Chen et al., 1979;

Hill and Chapman, 1989; Lekic and Cenic, 1992; Rohdewald et al., 1980,1982a, 1982b;

Rohdewald and Keuth, 1990). Using these methods, it is possible to measure the

analgesic effect as a function of time after drug administration.








Although much has been done during recent years to establish models that

describe relationships between drug concentrations in the body and pharmacological

effects (Colburn, 1981, 1987, 1988; Colbum and Brazzell, 1986; Holford and Sheiner,

1981, 1982), only limited information exists on such relationships for analgesics (Chay et

al., 1992; Inturrisi and Colburn, 1986; Laska et al., 1986b; Keuth and Rohdewald, 1989).

Several clinical trials have demonstrated that the mean analgesic responses of patients

with pain vary as a function of the logarithm of the dose (Inturrisi et al., 1987, 1990;

Inturrisi and Colbumrn, 1986; Velagapudi et al., 1990, 1991). To provide a basis for more

rational drug therapy, however, it is also necessary to consider the very important

relationship between the kinetics of the drug in the body and the time course of the

pharmacological effects produced.

The present study was designed to further evaluate the efficacy of ibuprofen and

codeine as analgesics and to investigate the relationship between drug concentrations

and effect as a function of time. The ability to concurrently measure drug concentrations

in biological fluids and pharmacodynamic effects provides the opportunity to explore

the relationship existing between the disposition of ibuprofen and codeine and their

analgesic effect. The objective of these studies is the development of a method for

defining this relationship in quantitative terms in order to allow predictions of

pharmacodynamic effects and development of optimal dosing regimens.

In addition, this study assesses the feasibility of an ibuprofen-codeine

combination. Despite the effectiveness of the new generation of peripherally acting

analgesics, these drugs do not provide the same qualitative effects as the narcotic

analgesics. Therefore, a combination could be useful in treating pain when a centrally

mediated analgesic effect is desirable.

The objectives of this study were

STo improve available methods for obtaining tooth pulp evoked potentials.





4


* To compare the pharmacokinetics of ibuprofen and codeine when given alone or in

combination.

* To compare the pharmacodynamics (analgesia) of ibuprofen and codeine when

given alone or in combination.

* To define the dose-effect relationship of ibuprofen and codeine when given alone or

in combination.

The ultimate objective of these studies is to better understand the dose/effect

relationship of analgesic drugs to provide patients with better pain relief.














CHAPTER 2
LITERATURE REVIEW


Pain, a major concern of humankind throughout history, has engendered

numerous efforts to understand its nature and to achieve its control. Pain is a common

and complex human experience, and the most frequent reason patients seek medical

advice. Despite its clinical importance, pain research was neglected until three decades

ago. Advances in diagnosis and therapy over the past twenty to thirty years hold the

promise of a better future.

This chapter reviews the concept of pain, its physiology, and the methods used

in its assessment. Special emphasis is placed on evoked potentials. Finally, current

knowledge on the model drugs used in this research, ibuprofen and codeine, is

presented.



The Concept of Pain


Pain: A Multidimensional Experience

Pain is one of the most personal yet universal human experiences. Originally,

pain was thought to be a punishment from the gods. In fact, the word pain is derived

from the Latin peone, meaning penalty or punishment (Stimmel, 1983). In the

seventeenth century, Descartes postulated that pain was a sensation in which the brain

played an important role. His theory, also known as the specificity theory, described the

pain system as a direct channel from the skin to the brain. This concept underwent little

change until the nineteenth century, when von Frey and Goldscheider introduced the

concepts of sensory input, central summation, neuroreceptors, and nociceptors. Their






6

theories proposed a specific pain system carrying messages from pain receptors in the

skin to a pain center in the brain (Melzack and Wall, 1983).

More recently, it became clear that pain is also influenced by a host of

psychological, social, and environmental factors in addition to stimulus intensity. It is

generally accepted that pain is a primary sensory modality, but it is also recognized that

pain is far more complex than simple sensation. Sensory mechanisms are necessary but

not sufficient for the definition of pain. Modem concepts of pain emphasize its

multidimensional nature (Beecher, 1957; Chapman and Jacobson, 1984; Melzack and

Wall, 1983; Syrjala and Chapman, 1984). In addition to nociception, pain involves an

intricate interaction of emotional, motivational, cognitive and environmental factors (see

Figure 2-1). Each pain has unique qualities, and the pain of a toothache is obviously

different from that of a pinprick. In contrast to von Frey's theory, pain is now

recognized to be one of the most complex human experiences.




Emotions
Motivations






/ Nociception

Socio-cultural J Cognition
learning No.- Discrimination



Figure 2-1. Multidimensional model of pain.
Adapted from Syrjala and Chapman (1984).




In light of these problems, the International Association for the Study of Pain

(1979) compiled a list of terms and definitions, and defined pain in scientific terms as






7

"an unpleasant sensory and emotional experience associated with actual or potential

tissue damage, or described in terms of such damage."


The Physiology of Pain

Within the last twenty years, remarkable progress has been made in

understanding the physiology of pain. The discovery of a highly organized central

nervous system network for the modulation of pain, and the isolation and description

of the endorphins greatly advanced our understanding of pain transmission.

Nevertheless, the physiology of pain is complex and many areas remain unknown.

The physiology of pain involves nociceptors, combined with an intricate system

of afferent and efferent neuronal connections.

Nociceptors

Most pain originates when specific nerve endings are stimulated and nerve

impulses are transmitted to the brain through the pain pathways. These receptors, also

known as nociceptors, can be classified into one of two types: mechanothermal

(unimodal) receptors and polymodal receptors (Bonica, 1990b; Nathan, 1976).

Mechanothermal nociceptors are mainly present in the skin and respond to

strong pressure applied to a wide area of skin and strong stimuli such as a pinprick or

sudden application of heat (greater than 45C). They warn of potential damage and are

the afferent part of the withdrawal reflexes. This type of receptor is mainly associated

with small myelinated primary afferent neurons designated A6 type that transmit

impulses rapidly. Stimulation of this type of receptor results in "first" or "rapid" pain

which occurs early after injury and is usually sharp, well-localized, and pricking

(Bonica, 1990b).

Polymodal receptors are the free nerve endings of unmyelinated primary

afferent neurons of the C type. They are widely distributed throughout most tissues

and respond to tissue damage. Their classification as polymodal indicates that they






8

respond to tissue damage caused by mechanical, thermal, or chemical stimuli. In

addition, they respond to chemical mediators formed or released as a result of tissue

damage. These impulses are transmitted more slowly than impulses from the

mechanothermal receptors and travel along unmyelinated C type nerve fibers. They are

responsible for "second" or "slow" pain after injury (slower in onset, prolonged, dull,

aching, and poorly localized).



Table 2-1. Properties of different types of afferent fibers involved in conducting
nociceptive impulses.

Fiber type
A6 C
Myelination yes no
Diameter 1-5 im 0.25-1.5 pm
Velocity 5-30 m/s 1.0-2.5 m/s
Threshold high high
Stimuli light pressure, heavy pressure light pressure, heavy pressure
heat (> 45C), chemicals, cold heat (> 45C), chemicals, warmth


While A6 nociceptors have a constant threshold below which stimuli are not

perceived, C fibers respond to a wide range of stimulus intensities, and both types of

fibers appear to have a role in coding pain intensity. However, it is the variation in the

tolerance of pain at the level of the central nervous system, not the threshold at the

periphery, which accounts for the wide variations in analgesic requirements among

patients (Bonica, 1990b).

Central pain pathways

Both A5 and C afferent fibers enter the spinal cord through the dorsal root. After

entering the spinal cord, the majority of A5 and C fibers terminate superficially in the

grey matter of the dorsal horn which is arranged in a series of laminae. It is in the spinal

cord that the first processing of painful stimuli occurs. The A6 fibers terminate in






9

lamina I and the C fibers in the substantial gelatinosa (lamina II). In addition, both A5

and C fibers synapse directly (or via intermediate neurons) in the deeper layers of the

dorsal horn with ascending fibers which cross the mid-line to join the spinothalamic

tract (Bonica, 1990b; Stimmel, 1983).

Pain transmission can be inhibited at the spinal cord level by inhibitory

interneurons or from descending inhibitory fibers. The best known theory describing

how painful stimuli may be altered at the spinal level is the "gate theory" put forward

by Melzack and Wall in 1965. They postulated that painful stimuli have to pass through

a gate in order to be relayed on to the central nervous system (see Figure 2- 2). This gate

can be closed by non-painful sensory input carried by large myelinated fibers from

mechanoreceptors responding to low-threshold stimuli (A3 fibers). In addition, the gate

may be closed as a result of descending inhibitory systems (Melzack and Wall, 1983).

These originate in the brain stem (including the periaqueductal grey matter) and some

of the descending tracts activate inhibitory interneurons of the enkephalinergic type in

the dorsal horn. Therefore, pain may be viewed as a complex summation of nociceptive

and non-nociceptive neuronal stimulation (Fields, 1984).

Ascending nociceptive impulses are transmitted mainly through the

spinothalamic tract. Fibers from the dorsal horn project to lateral and medial areas of

the thalamus, from where further transmission of impulses is to areas of the sensory

and motor cortex. This is the major route for the sensory-discriminatory aspects of pain.

Ascending impulses may also travel more slowly, via the spinoreticular tract,

terminating in the pontomedullary reticular formation. These slower impulses have a

major role in the arousal-motivational aspects of pain sensation, resulting from their

connections with the limbic system. They also reach the hypothalamus and are

responsible for the autonomic effects associated with pain (Melzack and Wall, 1983;

Stimmel 1983; Zimmermann, 1984).


















Large diameter


Small


Figure 2-2.


Schematic representation of the gate control theory.
This model includes excitatory (open circle) and inhibitory (closed circle)
links from the substantial gelatinosa (SG) to the transmission cell (T).
From Melzack and Wall (1983).


*Somalosensory
cortex


Descending---
Systems


--Spinotholamic troct


Figure 2-3. Afferent and efferent pathways involved with nociception and pain.
From Stimmel (1983).






11

Descending pathways are mainly inhibitory in nature. Stimuli are produced in

response to cortical and subcortical activation, responding to sustained peripheral pain

input. These descending impulses may be visualized as controls over the "gate",

thereby modulating pain input (Bonica, 1990b; Fields, 1984; Melzack and Wall, 1965,

1983; Nathan, 1976).

Pain modulation

Scientific evidence gathered over the last twenty years made it clear that many

different mechanisms modulate (i.e. facilitate or inhibit) the transmission of nociceptive

information at every level of the nervous system. The first evidence of the existence of a

pain modulation system was the observation that electrical stimulation of discrete areas

of the brain profoundly inhibited responses to painful stimuli (Fields, 1974; Basbaum

and Fields, 1978). At about the same time, the discovery of opiate receptors and of

enkephalins and endorphins provided insight into the manner in which narcotic

analgesics relieve pain. It is now generally accepted that pain modulation is achieved

through a series of complex interactions between inputs from the periphery,

interneurons in the spinal cord, and descending control systems from the brain (Bonica

et al., 1990; Fields, 1984; Zimmermann, 1984). These interactions involve a number of

endogenous biochemical agents called neurotransmitters or neuromodulators.

At the periphery, tissue damage caused by injury, disease, or inflammation

releases endogenous pain-producing substances into the extracellular fluid that

surrounds the nociceptors. These chemicals include H' and K' ions, serotonin,

histamine, prostaglandins, bradykinin, substance P, and others (Bonica et al., 1990;

Stimmel, 1983). In the dorsal horn of the spinal cord, a neurotransmitter involved in

pain transmission is the peptide substance P, which is released after stimulation of A5

and C fibers. Opioid receptors are also present in the dorsal horn, particularly in the

substantial gelatinosa, and they are thought to play an inhibitory role with enkephalins

acting as neurotransmitters in the inhibitory interneurons. Enkephalins are located in






12

the synapses between nerve fibers throughout the central and peripheral nervous

systems. They serve as neurotransmitters by binding to specific receptors and inhibiting

the release of substance P following nociceptive stimulation (Chapman and Bonica,

1983). A variety of other neurotransmitters and neuromodulators have been identified.

They include norepinephrine, serotonin, angiotensin II, somatostatin, cholecystokinin,

neurotensin, and dynorphin (Bonica et al., 1990; Stimmel, 1983).

In summary, although considerable progress has been achieved in the

understanding of the neurophysiology of pain, the modulation of nociceptive impulses

is complex, and the relationship between analgesic drugs and neurotransmitters in the

control of pain transmission is still under investigation.



Pain Measurement and Assessment

One of the most critical aspects in the investigation of pain is the question of its

assessment. Pain is a highly complex phenomenon that by its very nature renders

objective assessment difficult. Pain is a subjective experience which is difficult to

express and quantify, and is not directly measurable. Further difficulties occur since

individual patients react to similar painful stimuli in quite different ways. In addition,

many factors are known to influence pain response. They include race, sex, age, culture,

personality; the type, duration, and intensity of the pain; and psychological variables

such as fear, anxiety, bias, and distraction (Beecher, 1957). However, the measurement

of pain is essential for the study of its mechanisms and for the evaluation of pain

control methods.

Experimental pain differs fundamentally from clinical pain as seen by the

physician. Experimental pain can always be discontinued. The subjects are informed

about the experimental procedure and are often interested in the success of the

experiment. This leads to a predominant rational component of pain, so that what we

really measure is the sensory-discriminative component of pain. On the other hand,






13

clinical pain is essentially characterized by an aversive, emotional component which is

much more difficult to measure. In most cases, pain has a source somewhere in the

nociceptive pathways including central centers of pain processing, so that both

components of pain interact and influence each other (Bromm, 1984a).

Generally, responses to pain may be separated into three types: somatic reflex

reactions, autonomic nervous system reactions, and sensory reactions that depend upon

a high level of integrative central nervous system processing (Chapman and Syrjala,

1990). The first two types of reactions fall mainly into the category of involuntary

responses, whereas the last type tends to involve voluntary responses. Thus, obtaining

a meaningful, reliable, and objective measure of pain is difficult.

There have been two major approaches to quantifying pain. The first centers on

quantifying the subjective reports of patients. This can provide information on the

cognitive and affective, as well as sensory, dimensions of pain. The second approach

uses physiologic correlates of pain states. The most successful of these attempts has

been with evoked potentials. These methods all consist of noninvasive means of

observing the magnitude of pain from manipulation of external parameters (Syrjala and

Chapman, 1984).


Unidimensional Pain Rating Scales

Although the multidimensional nature of pain has been widely recognized, the

most common approach to pain assessment continues to be the unidimensional self-

report scale, on which patients provide a rating of pain intensity or pain relief.

Traditionally, studies of the human pain experience in the laboratory have relied on

categorical and visual analog scales (Bromm, 1984a; Buchsbaum et al., 1981a; Chapman

and Syrjala, 1990; Syrjala and Chapman, 1984).








Categorical rating scales

One of the simplest way of assessing the pain experience is the categorical rating

scale. This type of scale generally consists of a series of words (e.g. none, mild,

moderate, and severe) that are used to measure pain intensity or relief (see Figure 2-4).

The patient is then asked to estimate the intensity of his subjective pain experience by

selecting a category.



4-point Pain Intensity Category Scale
0 I 2 3
I i I
None Mild Moderate Severe



5-point Pain Relief Category Scale
0 I 2 3 4
II I I I
None Mild Moderate Lots Complete


Figure 2-4. Examples of categorical pain intensity and pain relief scales.



To assess the magnitude of the analgesic effect, several measures derived from

individual pain scores can be calculated. Most researchers assume equidistant

categories, and assign the numerical values of 0 for none, 1 for mild, 2 for moderate,

and 3 for severe. To account for differences in baseline pain intensity among patients,

pain intensity scores are first converted into "pain intensity difference" (PID) scores by

subtracting them from the pain score taken at baseline:

PID = P10o PI,

where PID is the pain intensity difference, PIt is the pain intensity at time t, and Pi1 the

pain intensity at baseline. Positive scores indicate reduction in pain, thus making the

pain intensity difference scores analogous to pain relief scores.






15

Pain intensity difference and pain relief scores are also commonly summed over

the observation period to provide estimates of the area under the effect-time curve.

These estimates are known as the summed pain intensity differences and the total pain

relief scores:

SPID= PIDt At

TOTPAR = Z PRt At

where SPID is the sum of the pain intensity differences, PIDt is the pain intensity

difference at time t, At is the time elapsed since the previous observation, TOTPAR is

the total pain relief, and PRt is the pain relief score at time t.

The efficacy of different analgesic treatments may then be evaluated by

comparing hourly scores, peak scores, SPID scores, and TOTPAR scores (Max and

Laska, 1991).

Due to their simplicity, categorical rating scales are among the most widely used

methods to assess the human pain experience. They are understood by most patients,

require little time to complete, and can be analyzed easily (Max and Laska, 1991).

However, these scales have been criticized on several counts. First, they often do not

have enough categories to allow patients to accurately describe their pain intensity,

resulting in a poor sensitivity (Chapman and Syrjala, 1990; Huskisson, 1974; Max and

Laska, 1991; Syrjala and Chapman, 1984). Similarly, words can have different nuances

of meaning, and ratings may vary from subject to subject (Wallenstein et al., 1980).

Another disadvantage of the categorical rating scales is the limited nature of the verbal

categories. Although categories are generally assigned equal step integer values, little is

known of the true size of the distances between pain scores (Huskisson, 1974; Laska et

al, 1986a; Max and Laska, 1991; Wallenstein et al., 1980).

Visual analog scales
Visual analog scales have been used in psychology since the beginning of the

century. It is only in recent years that they have been applied to the measurement of






16

pain (Huskisson, 1974). Visual analog scales consist of a line, usually 10 cm in length,

whose ends are labeled with descriptors of the pain experience (e.g. "no pain" and

"worst pain possible"). The patient is asked to mark the line at a point corresponding

to the severity of his pain. The distance (in millimeters) between the start of the line and

the mark is then taken as an indicator of the pain severity (Max and Laska, 1991; Scott

and Huskisson, 1976, 1979b).



o Pin Worst Pain
No Pain Possible


Figure 2-5. Example of visual analog scale for pain intensity.



Visual analog pain scales offer several advantages: simplicity, sensitivity, and

good reproducibility (Huskisson, 1983). Also, because visual analog scales are

continuous, they avoid the boundary error of category scales. They have generally been

found to be as or more sensitive than categorical scales and produce results that

correlate well with those of descriptive scales (Huskisson, 1974; Ohnhaus and Adler,

1975; Rohdewald and Keuth, 1990; Scott and Huskisson, 1976; Wallenstein et al., 1980).

Problems with visual analog scales include failure to understand the concept, and the

choice of appropriate statistics for data analysis (Chapman and Syrjala, 1990; Max and

Laska, 1991; Syrjala and Chapman, 1984). They are commonly used in clinical trials to

establish the value of analgesic treatments. Visual analog scales can also be used to

compare pain severity in the same patient at different times or in groups of patients

receiving different treatments (Scott & Huskisson, 1979a).

Visual analog scales have also been used to assess pain relief. In this case, the

line is labeled with descriptors such as "no pain relief" and "complete pain relief."

Patients can then evaluate their relief and mark the scale at any point along the line.






17

Measurements are made in terms of millimeters from the "no pain relief" boundary,

providing a 100-point relief scale (Wallenstein, 1991).

Like categorical relief scales, relief visual analog scales serve to expand the part

of the scale corresponding to slight relief, potentially increasing the sensitivity over that

offered by the visual analog scale for pain intensity (Max and Laska, 1991; Quiding and

Haggquist, 1983; Schachtel, 1991; Wallenstein, 1984, 1991; Wallenstein et al., 1980).

Wallenstein (1984, 1991) also found a linear relationship between categorical and visual

analog relief scales. Their interpretation is straightforward and provides a direct reading

of analgesia. It is the parameter of choice in pharmacokinetic studies correlating

analgesic effects with blood levels (Wallenstein, 1991).


Multidimensional Pain Rating Scales
Another approach to evaluate the pain experience in man relies on the use of

extensive questionnaires that assess the multiple dimensions of pain. Multidimensional

scales recognize the complex nature of the pain experience.

Among the different types of questionnaires, the most popular and most widely

used is the McGill Pain Questionnaire. It was first introduced by Melzack in 1975, and

has since been translated in many languages. This questionnaire was designed to

provide information on the sensory, affective, and evaluative dimensions of pain

(Chapman and Syrjala, 1990; Syrjala and Chapman, 1984). It consists of twenty sets of

words that describe pain. Patients are asked to select those sets that are relevant to their

pain, and to circle the word that best applies in each set. Pain scores may then be

summed for each dimension, with the total score being called the pain rating index

(Melzack, 1975).

Numerous studies have demonstrated the validity and reliability of the McGill

Pain Questionnaire (Gracely, 1990; Melzack, 1984). However, the complexity of its

vocabulary may limit its use with poorly educated patients. It remains primarily






18

suitable for clinical pain research (Chapman and Syrjala, 1990; Gracely, 1990; Syrjala

and Chapman, 1984).


Physiologic Correlates of Pain

Pain is a subjective experience and, on the ground of its large emotional

component, is quite dependent on the condition of the person being affected. But pain is

also a consequence of the activation of nociceptive afferents, and neural activity can be

measured with the methods commonly applied in sensory physiology. Consequently, a

number of attempts have been made to measure pain through physiologic indicators.

Many confounding factors are known to compromise subjective report measures. To

objectify these measures, several physiological parameters accompanying pain have

been investigated.

Over the past thirty years, many investigations have been undertaken to

correlate pain with muscle tension, the nociceptive flexion reflex (withdrawal reflex),

skin resistance reaction, certain autonomic parameters, and central nervous system

variables (Bromm, 1984a; Hill and Chapman, 1989; Syrjala and Chapman, 1984; Wilier,

1984; Wolff, 1979). Yet, no consistent relationship has been found between the

individual pain reports and these physiologic measurements (Bromm and Treede, 1980;

Bromm and Scharein, 1982b; Syrjala and Chapman, 1984). In addition, the usefulness of

these measurements is limited by their tendency to decrease over time, irrespective of

pain perception, and their sensitivity to stress (Bromm and Scharein, 1982b; Bromm and

Treede, 1980; Syrjala and Chapman, 1984). To date, the most stable and reliable

correlations with subjective pain reports have been obtained with cerebral evoked

potentials (Bromm, 1984a, 1985; Bromm and Scharein, 1982b; Buchsbaum, 1984;

Chapman and Jacobson, 1984; Chudler and Dong, 1983; Fernandes de Lima et al., 1982;

Hill and Chapman, 1989; Syrjala and Chapman, 1984).








Evoked Potentials in Pain Research


Evoked Potentials
Evoked potentials are event-related electrical signals of brain activity that can be

recorded from the scalp when precisely controlled stimuli are delivered. The

appearance of an evoked cerebral potential then gives evidence that the specific sensory

system of the brain does receive the activated peripheral impulse pattern elicited by the

applied stimulus (Chiappa, 1990; Spehlmann, 1985).

Evoked potentials consist of a series of peaks or waves, each characterized by

their polarity (positive or negative), latency (in milliseconds), and amplitude (in

microvolts). They are obtained by processing the electroencephalographic signals (EEG)

that occur following stimulation.

The basic problem in evoked potential recording is to distinguish the evoked

activity (signal) from the ongoing electrical activity of the brain (noise). This is done by

computer-assisted signal processing techniques, most often using methods that sum or

average successive responses to iterative simulation (see Figure 2-6). In this way,

potentials that are time-locked to the stimulus will be enhanced, while events not in

phase with the stimulus will be reduced (i.e. background EEG activity, artifacts,

instrumentation noise).

The number of EEG epochs to be averaged depends on the ratio of the

amplitude of the stimulus-locked components in the recording (signal) to the amplitude

of the unrelated components (noise). Because averaging procedures reduce the noise,

the signal to noise ratio improves with the number of responses averaged (see Figure

2-6). However, this relationship is not linear: the ratio of evoked potential signal to

on-going EEG signal ("noise") increases as the square root of the number of repetitions

(Chiappa, 1990).





















1 2 3 4 5 6

a
I sec
b C 1


3
4
5
6


125uV


25uV


300ms


Computer averaging of evoked potentials.
The top line (a) shows a continuous EEG recording; Six 300 ms EEG
epochs taken after stimulation are arranged vertically with the same time
scale (b) or with an expanded time scale (c); The samples are averaged in
(d) and (e) to enhance features common to the individual responses.
From Spehlmann (1981).


2W'

3


5
6


Sf300ms
__300ms


Figure 2-6.








Nomenclature

Evoked potentials are customarily displayed as a plot of voltage against time (see

Figure 2-7). Although a universal nomenclature for naming evoked potentials peaks

has not been established, waves are commonly labeled based on their polarity and

nominal latency. For example, a peak labeled as P250 indicates a positive wave

occurring 250 ms after stimulation.



40
P250
30
5, 2O
I20 P /o
1S 0- '80 \
S 07
E -I0 0
< \
-20- N 150
.30 ------. -----*-
0 100 200 300 400 500
Time [ms]


Figure 2-7. A typical evoked potential: Nomenclature of events.




Classification of evoked potentials

Many types of evoked potentials can be distinguished according to stimulation

and recording methods. For example, evoked potentials may be divided by stimulus

type into visual evoked potentials (VEP), auditory evoked potentials (AEP), and

somatosensory evoked potentials (SEP). Similarly, evoked potentials may be defined by

recording method as far-field recordings, early near-field recordings, or late near-field

recordings (Chapman et al., 1979; Chapman and Jacobson, 1984; Chiappa, 1990;

Grundy, 1983a, 1983b; Spehlmann, 1981, 1985).








Far-field evoked potentials are those recorded at a distance from the neural

generator. Near-field potentials are recorded from electrodes close to the neural

structures from which they arise. Short-latency potentials reflect activity in sensory

receptors and subcortical sensory afferent pathways. When recorded from the scalp,

these potentials are in the far field. Near-field evoked potentials, observed in scalp

recordings between 20 and 80 milliseconds after onset, are called intermediate-latency

potentials. They reflect the arrival of sensory information at primary sensory areas of

the cerebral cortex. Late cortical event-related potentials occur between 80 and

500 milliseconds after stimulus onset. It is generally assumed that they reflect the

processed event, i.e. appreciation, interpretation, and meaning associated with the

stimulus, rather than simple event detection. Late near-field recordings, sensitive to

attention and expectation, are of behavioral and perceptual interest (Cruccu et al., 1983;

Haider, 1967; Leandri et al., 1985; Rosenfeld et al., 1985). They depend on consciousness

and their recording can be seriously affected by synchrony in the EEG such as

prominent alpha rhythm. For example, there is an inverse relationship between the

amount of alpha activity present in the pre-stimulus EEG and the peak amplitude of the

late cortical event-related potentials (Bromm, 1984a; Bromm and Scharein, 1982a).

Contemporary work on pain and evoked potentials is predominantly concerned

with late cortical event-related responses latenciess between 80 and 500 ms). Verbal

reports of subjects rarely reflect pure sensations, but rather include a mixture of

sensation, emotional reaction and interpretation. Similarly, late cortical responses are

supposed to reflect these higher processes and the pain sensation as felt by the subject

(Chapman et al., 1979).

Late cortical evoked potentials are used in pain research. Correlations between

subjective pain report and various components of evoked potentials have been reported

(Bromm, 1984a, 1985; Chapman et al., 1979; Carmon et al., 1978; Chen et al., 1979;

Chudler and Dong, 1983; Fernandes de Lima et al., 1982; Harkins and Chapman, 1978;








Rohdewald and Keuth, 1990; Rohdewald et al., 1980). Evoked potentials have also been

used by pain researchers to evaluate the efficacy of different analgesics (Bromm et al.,

1983; Buchsbaum, 1984; Buchsbaum et al., 1981b; Butler et al., 1983; Chapman et al.,

1982; Chen and Chapman, 1980; Derendorf et al., 1982,1984; Rohdewald et al., 1982a,

1982b, 1983). The major use of evoked potentials in pain research has focused on

experimental pain.


The Pain Stimulus

Pain can be evoked by stimulation in many different sensory modalities. When

the stimulus intensity is great enough, pain can be evoked by tactile, acoustic, or visual

stimulation (Chudler and Dong, 1983). To be useful in pain research, however, an ideal

pain-producing stimulus should be

* exclusively painful, but barely noxious in order to avoid tissue damage,

* easy to apply and remove,

* quantifiable and repeatable.

Additionally, for obtaining evoked potentials, the stimulus must be of brief

duration so that the evoked potential can be time-locked to the stimulus and the

stimulus artifact does not obscure the potentials of interest. Another essential

requirement is the necessity to randomize inter-stimulus intervals and/or stimulus

intensities in order to keep the arousal state of the subject high and constant and, as a

consequence, to minimize the effects of habituation or sensitization (Bromm, 1985).

Although most researchers prefer short electrical or thermal pain stimuli, such

as electrical tooth pulp stimulation or laser-emitted short radiant heat pulses, three

main types of stimulation have been used in experimental pain research: mechanical,

thermal, and electrical stimulation.








Mechanical stimulation

Various mechanical stimulation methods have been used to generate pain

(Beecher, 1957). Although mechanical stimulation can generate painful sensations, the

stimulus may not be limited to excitation of nociceptive afferents alone. It is possible

that other afferent input related to touch and pressure may also be activated by skin

contact. Therefore, non-nociceptive input may contribute to some or all of the

components of the evoked potential generated by this method (Chudler and Dong,

1983).

Thermal stimulation

Thermal stimulation methods are among the oldest and most widely used

methods in the production of experimental pain. Cutaneous thermal stimulation is

precise, simple, and allows a fairly rapid collection of data. At moderate temperatures,

this method permits rapid repetition of the stimuli without injury (Hardy et al., 1940).

Like mechanical stimulation, thermal stimulation (i.e. laser-emitted radiant heat) has the

advantage of eliciting a sensation that is considered natural. But it also brings the risk of

contamination of the evoked potential by input related to sensory modalities other than

pain (Bromm and Treede, 1987; Carmon et al., 1976,1978,1980; Chudler and Dong,

1983; Morgan et al., 1984).

Electrical stimulation

Electrical stimulation is probably the most common method of generating pain

in evoked potential research because it is easy to apply, repeat and quantify

(Buchsbaum, 1984). Proponents of electrical stimulation argue that it has advantages

similar to that of thermal stimulation. Electrical stimulation induces a clearly detectable

pain sensation and the stimulus intensity can be exactly determined. In addition, a

number of parameters may be manipulated to modify pulse pattern and waveform

(Notermans 1966). Nevertheless, cutaneous electrical stimulation has many

disadvantages. First, electrical stimulation is an unnatural type of stimulation. Second,








it is evident that sensory systems other than pain are activated by cutaneous electrical

stimuli. Finally, low intensity electrical stimulation may recruit activity of non-

nociceptive AP nerve fibers. Therefore, evoked potentials elicited by cutaneous electrical

stimulation, like those produced by thermal and mechanical stimulation, are subject to

contamination by sensory modalities other than pain (Chudler and Dong, 1983).


Tooth Pulp Evoked Potentials

The tooth pulp has been considered by many to be the ideal site for the study of

pain. Its use in pain research is based upon the observation that pain is the only

sensation produced by stimulating intra-dental (pulpal) nerves.

Neuroanatomical evidence indicates that tooth pulp is innervated almost

exclusively by small myelinated A6 and unmyelinated C fibers originating in the

trigeminal ganglion (Matthews, 1979; Mumford and Bowsher, 1976; Sessle, 1979). The

unmyelinated fibers comprise 50 to 75% of the total sensory innervation. Hence, it is not

surprising that electrical tooth pulp stimulation elicits primarily, if not solely, painful

sensations (Azerad and Woda, 1977; Matthews and Searle, 1976). "Pre-pain" sensations

have also been reported following near-threshold electrical stimulation, indicating that

tooth pulp is innervated not only by A5 and C nociceptive afferents, but also by a small

number of large diameter Ap afferents. (Azerad and Woda, 1977)

Only a few experiments have described the quality of the sensation evoked by

electrical stimulation of the human tooth pulp (Azerad and Woda, 1977; Chatrian et al.,

1982). It has now become apparent, however, that low intensity electrical tooth pulp

stimulation results in a "pre-pain" sensation (sometimes described as a pure prick that

is not painful). Higher stimulus intensities cause sharp pain, and the highest stimulus

intensities cause an unpleasant dull ache (Azerad and Woda, 1977; Greenwood et al.,

1972; Matthews, 1979). Because single fiber studies in animals have shown that Ap

fibers are more sensitive than A5 fibers, and that C fibers are the least sensitive, it is








probable that "pre-pain", sharp pain, and dull ache correspond to AP, A6, and C fiber

activation (Burgess et al., 1990). For practical purposes, dental stimulation may be seen

as differing from cutaneous stimulation because of its quite small "pre-pain" range

(range between stimulus detection and pain threshold).

Chatrian (1975a, 1975b) first introduced tooth pulp evoked potentials in human

research as an objective monitor of pain. Soon after, Harkins and Chapman (1978) and

Chen et al. (1979) reported that the amplitude of late components of tooth pulp evoked

potentials could be considered a functional correlate of pain. Thereafter, numerous pain

modulating procedures (narcotic analgesics, non-steroidal anti-inflammatory drugs,

local anesthetics, acupuncture) have been evaluated by means of tooth pulp evoked

potentials. Now, it has been clearly established that tooth pulp evoked potentials are a

sensitive and reliable way to investigate human pain in the laboratory. Figure 2-7

shows a typical example of a tooth pulp evoked potential.


Evoked Potentials as Correlates of Pain

Pioneering work in the field of evoked potential correlates of human pain was

initiated by Chatrian and co-workers (Chatrian et al., 1975a, 1975b). These researchers

succeeded in demonstrating that there is a consistent evoked response associated with

pain. They also observed that the phenomenon was most apparent when sampled from

vertex with reference to inion (see Figure 2-8).

The important findings reported by these investigators were that

* Electrical dental stimulation elicits no sensations other than pain.

* Stimulation of a devitalized tooth does not produce evoked potentials or any

subjective sensations. Thus, the results cannot have been due to passive spread of

the stimulating current to sources of artifacts.






















R VU A-*. r -^






\ ^ "**.,......S..


\


Scalp distribution of tooth pulp evoked potentials.
Black triangles refer to standard electrode placements of the
international 10-20 system; white triangles represent supplementary
electrodes.
From Chatrian et al. (1975a).


V


6Ic)V N- 50
600msec


Figure 2-8.









* A patient congenitally insensitive to pain but with intact sensations in other sensory

modalities failed to show any cerebral responses or subjective responses to strong

electrical tooth pulp shocks.



These results assessing the validity of using evoked potentials in the study of

pain in humans have led many researchers to investigate the correlations existing

between evoked potentials and subjective reports of pain.

Evoked potentials bear striking similarities, whether they are elicited by painful

electrical or mechanical skin stimuli, or by electrical tooth pulp stimuli (see Figure 2-9).

Differences due to the different kinds of stimulation appear in the early and

intermediate latency components with peak latencies of less than 80 milliseconds, but

these early components have not been evaluated systemically in pain research.




PV EL )UV MEC
1 0 [ 10 n z 8
0 0


t ,,I t I I I I I I I I
0 200 400 0 zoo 400
tiMns) t(rns)

V DEN PV LAS
10 10n. n 11




I I I I
0 200 400 0 1000
t(Urns) t(ms)


Figure 2-9. Evoked potentials obtained with various pain stimuli.
EL: electrical skin stimulation; MEC: mechanical skin stimulation;
DEN: electrical tooth pulp stimulation; LAS: laser-emitted thermal
stimulation.
From Bromm (1984b).








The late potentials used in pain research can be described by a positive signal

occurring at approximately 80 ms after stimulus onset (P80), followed by a distinct

negativity between 120 and 200 ms (N150), and another positive signal (P250) between

200 and 400 ms (Chapman et al., 1979).

In their initial study, Harkins and Chapman (1978) delivered faint, mild and

strong painful stimuli to volunteers in order to determine whether a relationship

between stimulus intensity and the evoked potential response could be demonstrated.

Evoked potential peak-to-peak amplitude increased as stimulus intensity was

augmented, and there was a linear relationship between the logarithm of the stimulus

intensity and the amplitude of the various evoked potential components. In contrast,

peak latency was unaffected by changes in stimulus intensity. Partial correlation

analysis of the data revealed that amplitudes of the later components (N150 and P250

components) were significantly correlated with subjective pain ratings. The amplitude

of the P80 component showed no relationship to the subjective pain report. Rather, it

correlated with stimulus intensity (Chen et al., 1979).

These results were confirmed by Bromm (Bromm, 1985; Bromm and Scharein,

1982a) in studies using multivariate statistical data analysis methods (Bromm, 1984b;

Strenge and Gundel, 1983). This suggests that the late cortical evoked potential may be

useful as a physiological correlate of the psychophysiological processes underlying the

perception of pain. Thus, evoked potential amplitudes may track the intensity of the

pain experienced by subjects (see Figure 2-10).

It should be noted that the amplitude of the cerebral evoked potential may vary

as a function of the stimulus repetition rate: longer inter-stimulus intervals produce

larger evoked potentials than shorter inter-stimulus intervals (Chapman et al., 1981;

Jacobson et al., 1985). In studying differential effects of painful stimuli on averaged

evoked potentials, however, the particular inter-stimulus interval does not need to be

taken into account so long as it is consistent. Moreover, it has been demonstrated that









inter-stimulus intervals do not significantly affect latencies of the averaged evoked

potential components so long as the inter-stimulus intervals are greater than 2 seconds

(Jacobson et al., 1985).


MECHANICAL STIMULATION



5 r
53




53





I I I 1 I I
0 (ms 00


ELECTRICAL STIMULATION

EEG V5vll VV
n 40


K1

K2


K3


K4


0 I I I I
0 (msi 50


Figure 2-10. Evoked potentials obtained with mechanical and electrical stimulation
illustrating differences in cerebral responses to stimuli of increasing
intensity (K1 and K2 denote faint and mild tactile sensations, K3 and K4
faint and mild pain).
From Bromm and Scharein (1982b).






Effect of Analgesics on Evoked Potentials

The effects of a wide variety of analgesics on evoked potentials have been

studied by a number of investigators. Early studies suggested consistent and significant

changes in evoked potential waveforms when an analgesic treatment is administered. It

has been established that the dental P250 amplitude is proportional to pain intensity

and decreases when pain is attenuated by various therapeutic treatments. Waveform

comparisons and the consistency of findings suggest the reliability and consistency of

the evoked potential technique. Minor variations in site of stimulation and method of


Cz
29sns54
JS








peak measurement do not affect the general finding that analgesics induce a decrease in

evoked potential amplitude.

Centrally acting analgesics
Evoked potentials have been used to study opiate analgesics. Chapman et al.

first reported preliminary evidence that an opiate could affect the evoked potential

waveform and reduce the subjective pain report in subjects experiencing painful tooth

pulp stimulation (Chen et al., 1979; Harkins and Chapman, 1978). The effects observed

in the major negative and positive peaks were very similar to those reported by

Buchsbaum et al. (1981a; 1981b), who gave morphine to subjects experiencing painful

skin stimulation. Smaller amplitudes of the N120 component were found following

morphine than after placebo. This effect was more marked at higher stimulus

intensities. The morphine effect was opposite to that observed with naloxone:

amplitudes of most components of the evoked potential decreased with morphine and

increased with naloxone (Buchsbaum, 1984; Buchsbaum et al., 1977).

Later, Bromm et al. (1983) demonstrated the dose-dependent reversal of opioid

analgesia by increasing doses of naloxone, measuring pain ratings and late component

evoked potential amplitudes. Their results showed that, as compared to placebo, the

opiate agonist tilidine significantly reduces both the amplitude of the N150-P320

component and the subjective pain report. Naloxone and placebo alone had little or no

effect on either evoked potentials or subjective pain report.

To further pursue opiate analgesia and narcotic antagonism, Butler et al. (1983)

investigated the effect of fentanyl (0.1 mg IV) on evoked potentials (see Figure 2-11).

They found that the evoked potential amplitude was diminished by administration of

fentanyl and that dose-dependent reversal by naloxone could be demonstrated.

Additionally, other potent narcotics such as meperidine have also been shown to

remarkably influence the amplitudes of the N150 and P250 components of the evoked

cerebral potential (Grundy and Brown, 1980).









ODental
Stimulus N155
N55
Baseline N330
P90

P240
Fentanyl f-'-
0. mg.

Noloxone /\
0,4 mq


100pv
lOOmeec



Figure 2-11: Effect of fentanyl and its reversal after administration of naloxone on
tooth pulp evoked potentials.
From Butler et al. (1983).




Recently, Chapman and co-workers compared the analgesic effects of alfentanil,

fentanyl, and morphine on experimental pain (Chapman et al., 1988,1990; Hill and

Chapman, 1989; Hill et al., 1986). Their results showed a reduction in evoked potential

amplitudes following drug administration, with marked variations in analgesic profiles

between drugs. They attributed these variations to the different physicochemical

characteristics (lipophilicity, ionization) of each drug.

Peripherally acting analgesics

Because of their weak analgesic efficacy, peripheral analgesics are more difficult

to evaluate than the more potent central analgesics. Evoked potentials have been used

to study the analgesic effect of aspirin. In their initial study, Chen and Chapman (1980)

reported that the late components of the dental evoked potential were reduced by

acetylsalicylic acid (975 mg p.o.), as were pain reports during dental stimulation.

Derendorf et al. (1982,1984) and Rohdewald et al. (1980,1982a, 1982b, 1983)

confirmed these results in their studies on the effects of acetylsalicylic acid,

acetaminophen, salicylamide, and metamizole on tooth pulp evoked potentials.








Moreover, they also demonstrated that statistically significant differences in drug

dosage (500 mg versus 1000 mg of acetylsalicylic acid) and bioavailability could be

detected by evoked potential methods but not by subjective pain reports (Rohdewald,

1980). Thus, evoked potentials techniques appear to be more sensitive for quantitation

of pain attenuation than subjective methods.

Later, Buchsbaum (1984) reported that acetylsalicylic acid (1000 mg p.o.)

significantly decreased N120 amplitudes following cutaneous electrical stimulation

when compared to placebo. Similarly, Keuth and Rohdewald (1989) described the time

course of the analgesic effect of propyphenazone (1000 mg p.o.) in man using visual

analog pain rating scales and tooth pulp evoked potential amplitudes. Their results

differentiated active drug from placebo as soon as 20 to 40 minutes after drug

administration and for up to 2.5 hours.

More recently, Arendt-Nielsen et al. (1991) evaluated the analgesic efficacy of

single oral doses of acetaminophen (1000 mg) and of a codeine acetaminophen

combination (60 and 1000 mg respectively). They found that evoked potential

amplitudes were significantly diminished following administration of both active drugs

as compared to placebo, and that the combination acted faster and provided a longer

lasting analgesia than acetaminophen alone. On the other hand, Cox et al. (1987) could

not differentiate the analgesic effect of a 400 mg ibuprofen tablet from placebo by using

tooth pulp evoked potentials.

Anesthetics
The effects of local anesthetics on evoked potentials have been studied by

several researchers. Gehrig et al. (1981) reported that lidocaine infiltration at the apex of

a tooth obliterated the evoked potential and resulted in a subjective report of no pain,

whereas saline infiltration had no effect. Chatrian et al. (1975a) showed that after a

mandibular nerve block with mepivacaine, evoked potentials could not be elicited by

tooth pulp stimulation in the distribution of this nerve.








Nitrous oxide effects on tooth pulp evoked potential have been studied by

several researchers (Benedetti et al., 1982; Chapman and Benedetti, 1979; Estrin et al.,

1988). Chapman and Benedetti observed that 33% nitrous oxide in oxygen significantly

decreased the mean amplitudes of all late components and also decreased subjective

pain report (Chapman and Benedetti, 1979). Benedetti et al. (1982) subsequently

described a dose-response relationship between nitrous oxide concentration, evoked

potential amplitude and subjective pain report. Although the linearity of the

relationship was unclear, they hypothesized that evoked potential amplitudes were a

reliable correlate of nitrous oxide analgesia.


Advantages of Evoked Potentials Methods

The described studies using evoked potentials strongly suggest the usefulness of

this technique to measure analgesic activity. Analgesic effects are sensitively revealed

by evoked potential methods in healthy subjects, with little difference between evoked

potential results and clinical efficacy. Evoked potential techniques can be used

repetitively to follow the time course of drug action (Bromm and Scharein, 1982b;

Buchsbaum, 1984). Moreover, evoked potential methods can detect differences in pain

attenuation for different analgesic treatments, different bioavailabilities and even

different dosages of the same drug (i.e. 0.5 or 1.0 g of acetylsalicylic acid), whereas

subjective methods usually cannot do this in the same number of subjects (Derendorf et

al., 1982; Rohdewald et al., 1982a; 1982b).

In short, evoked potential methods are more sensitive for the quantitation of

pain perception than subjective pain reports, so that evoked potential studies can be

done in smaller groups of subjects than would be necessary with other commonly

employed techniques.







Ibuprofen

Ibuprofen is one in a series of 2-phenylpropionic acid derivatives discovered

over a fifteen year period at the Boots Pure Drug Company (Nottingham, England)

during the early 1960s. It is the parent compound of a class of non-steroidal anti-

inflammatory drugs: the propionic acid derivatives. It was introduced in the United

Kingdom in 1967 and became the first propionic acid derivative approved by the Food

and Drug Administration for use as an anti-inflammatory agent in the treatment of

rheumatic diseases in 1974. It was later approved as an analgesic for the treatment of

mild to moderate pain. On the basis of its good safety record, it became available as a

nonprescription analgesic in 1984 (Adams, 1992).


Physicochemical Properties
Ibuprofen, (R,S)-2-(4-isobutylphenyl)-propionic acid, occurs as a white to off-

white crystalline powder with a slight characteristic odor and taste. It has a molecular

weight of 206, and a pKa of 4.6 (Dietzel et al., 1990). Its melting point ranges between 74

and 77C. It is only slightly soluble in water (1 mg/mL), but readily soluble in alcohol

and most organic solvents (Baselt and Cravey, 1989; Davis, 1975; McEvoy, 1990).


Cl^ ff--" H CH3^ \ ---OH3
CH3\ CH3 CH CHCH2 H
CH-CH / / CCH CH -CH / /-- H
C3 COOH CH3 COOH


Figure 2-12. Chemical structure of ibuprofen.
Left: R(-)-isomer; Right: S(+)-isomer.



As with other propionic acid derivatives, ibuprofen possesses one chiral center

and thus exists as a racemic mixture of the R(-) and S(+)-isomers (see Figure 2-12).

However, its pharmacological activity is associated mainly, if not entirely, with the








S(+)-isomer (Adams et al., 1967,1976). The R(-)-isomer has very little, if any, anti-

inflammatory activity and serves no therapeutic purpose other than as a prodrug for

the active S(+) isomer.


Pharmacology

Ibuprofen has pharmacologic actions similar to those of other non-steroidal anti-

inflammatory drugs such as aspirin, phenylbutazone, and indomethacin. Ibuprofen has

shown anti-inflammatory, antipyretic, and analgesic activity in both animals and

humans (Flower et al., 1985). Higher doses are required for anti-inflammatory effects

than for analgesia.

The exact mechanism of action of ibuprofen is not known. The currently held

view is that the analgesic and anti-inflammatory effects of non-steroidal anti-

inflammatory drugs result primarily from their reversible inhibition of cyclooxygenase,

an enzyme responsible for the biosynthesis of prostaglandins (Benedetti and Butler,

1990; Flower et al., 1985; Kantor, 1979; McEvoy, 1990).




Phospholipids
Phospholipase
INon-steroidal
IArachidonmc Acid I
i----- ---i Anti-inflammatory drugs

Cyclooxygenase


Figure 2-13. Outline of arachidonic acid metabolism.








Prostaglandins play an important role in regulating a number of physiologic

processes, including inflammation and pain modulation. They are released from tissues

by a variety of stimuli such as chemical or mechanical stimulation. The observation that

aspirin and other non-steroidal anti-inflammatory drugs selectively inhibit

prostaglandin synthesis led Vane to propose that the therapeutic and toxic effects of

those drugs may be due to the inhibition of prostaglandin biosynthesis (Vane, 1971).

While prostaglandins possess no pain-producing properties themselves, prostaglandins

of the E type have been shown to sensitize afferent nerve endings to the effects of

bradykinin and histamine. Thus, the most likely explanation for the analgesic effect of

non-steroidal anti-inflammatory drugs is the prevention of sensitization of sensory

nerve endings by the inhibition of prostaglandin biosynthesis (Benedetti and Butler,

1990; Ferreira 1980; Higgs, 1980; Vane, 1971).

Recent findings by several laboratories suggested that other mechanisms may

account for some of the anti-inflammatory effects of non-steroidal anti-inflammatory

drugs. However, the importance of these non-prostaglandin-mediated effects such as

inhibition of the 15-lipooxygenase pathway and interference with leukocyte migration

and function remains unknown (Goodwin, 1984; Kantor, 1979,1984).

In addition to its analgesic and anti-inflammatory effect, ibuprofen has been

shown to reduce fever in animals and humans. This antipyretic effect is thought to be

caused by a central mechanism, possibly as a result of inhibition of prostaglandin

synthesis at the hypothalamus level (Benedetti and Butler, 1990).

Ibuprofen has also been shown to cause gastric irritation, tissue erosion, and

gastric bleeding. These effects are thought to result from the direct irritation of the

gastric mucosa, and from the indirect inhibition of prostaglandin synthesis in the

stomach (Benedetti and Butler, 1990; Kantor, 1979, 1984; McEvoy, 1990).

Ibuprofen, like other non-steroidal anti-inflammatory drugs, alters platelet

aggregation by inhibiting platelet cyclooxygenase, which in turn blocks thromboxane








A2 production. This effect is reversible and may result in a slight prolongation in

bleeding time. Prothrombin time remains unaffected (Kantor, 1979; McEvoy, 1990).


Pharmacokinetics

The basic pharmacokinetics of ibuprofen are well documented (Albert and

Gemrnaat, 1984; Busson, 1986; Geisslinger et al., 1989; Kantor, 1984). The discovery of an

in vivo inversion process led to a renewed academic interest targeted at elucidating the

pharmacokinetics of the individual enantiomers. Ibuprofen undergoes an in vivo

inversion of configuration in humans: R(-)-ibuprofen is stereoselectively inverted to

S(+)-ibuprofen (Adams et al., 1967,1976; Hutt and Caldwell, 1983; Mills et al., 1973).

Absorption

Following oral administration, ibuprofen is rapidly and almost completely

absorbed from the gastrointestinal tract. Its bioavailability after oral administration has

been estimated to be between 80 and 100% (Albert and Gemrnaat, 1984; Albert et al., 1984;

Lockwood et al., 1983a; Wagner et al., 1984). The administration of food and/or

antacids only alters minimally the rate and extent of ibuprofen absorption (Albert and

Gernaat, 1984; Albert et al., 1984; Szpunar et al., 1987). Peak plasma concentrations

(17-36 gg/mL after a 400 mg oral dose) are usually observed within 1.5 to 2 hours after

drug administration. Time to peak and peak plasma concentrations of both enantiomers

are similar, indicating a lack of stereoselective absorption for the enantiomers of

ibuprofen (Day et al., 1988; Lee et al., 1985).

Distribution

Ibuprofen, as well as other arylpropionic acid derivatives, is extensively bound

to plasma proteins. Approximately 90-99% of a dose is bound to plasma proteins

(Albert and Gernaat, 1984; Albert et al., 1984). It exhibits nonlinear plasma protein

binding at doses larger than 800 mg (Lockwood et al., 1983; Mills et al., 1973; Wagner et

al., 1984). Recent studies suggested that there might be an enantiomer-enantiomer








interaction with regard to protein binding: R(-)-ibuprofen may compete with

S(+)-ibuprofen for binding on the human serum albumin molecule (Evans et al., 1989;

Lee et al., 1985). However, the extent of such stereoselectivity remains to be determined.

Due to its high protein binding, ibuprofen is not extensively distributed into the

body and has an average volume of distribution of 10 L for both enantiomers (Albert

and Gemrnaat, 1984; Lee et al., 1985).

Both R(-) and S(+)-ibuprofen isomers diffuse slowly into synovial fluid.

Concentrations in synovial fluid fluctuate much less and remain higher than those in

plasma. The mean ratio of the total concentration in synovial fluid to that in plasma

seven hours after dosing is about 1.25 (Netter et al., 1989). Studies by Day et al. (1988)

on the stereoselective disposition of ibuprofen enantiomers in synovial fluid showed

that concentrations of the active S(+) enantiomer in synovial fluid were always larger

than those of the R(-) isomer.

Ibuprofen and its metabolites pass easily across the placenta and they appear in

the milk of lactating women at approximately 1% of the maternal plasma concentration

(Albert and Gemrnaat, 1984).

Metabolism
Ibuprofen is extensively metabolized in the liver by hydroxylation,

carboxylation and glucuronidation (see Figure 2-14). Major metabolites are generated

by addition of an hydroxy group at the 2 position to form 2-[4-(2-hydroxy-2-

methylpropyl)phenyl] propionic acid, or by oxidation of a methyl substituent to the

corresponding carboxylic acid to yield 2-[4-(2-carboxypropyl) phenyl] propionic acid

(Adams et al., 1967; Mills et al., 1973). Formation of ester glucuronides conjugates is

stereoselective and favors the S(+) enantiomer of ibuprofen (Lee et al., 1985).

Ibuprofen was the first 2-arylpropionic acid derivative shown to undergo a

metabolic chiral inversion of the inactive R(-)-isomer to its pharmacologically active

S(+)-antipode and, subsequently, has become the most studied compound of this








group. Mills et al. (1973) first examined the fate of the optical isomers of ibuprofen

given separately to man. They reported that ibuprofen undergoes an in vivo isomeric

R(-) to S(+) inversion in humans while there is no measurable inversion of the S(+)-

isomer to R(-)-ibuprofen. Conclusive evidence for the chiral inversion of R(-)-ibuprofen

was later obtained in studies by Wechter and co-workers (1974) using deuterium

labeled ibuprofen.




C'--&H-CH,-12 OH
o COOH

CH, CF3 C3 C CH3 CHI

OH COO OOH OH COOH

HO CH, =\
,C--C -- H H-


HO /^\CH3
cH
0 COOH



Figure 2-14. Metabolic pathways of ibuprofen in man.




The mechanism of the chiral inversion involves the stereoselective formation of a

coenzyme A thioester of R(-)-ibuprofen, subsequent inversion by a non-stereoselective

coenzyme A racemase, and release of the S(+) enantiomer by hydrolysis (Hutt and

Caldwell, 1983, Wechter et al., 1974). The S(+)-enantiomer of ibuprofen is not a substrate

for the coenzyme A synthetase and therefore is excluded from the reaction (Hutt and

Caldwell, 1983).

The mechanism of the chiral inversion of ibuprofen was later confirmed by

studies on the incorporation of ibuprofen into adipose tissue. Williams et al. (1986)








observed a stereoselective uptake of R(-)-ibuprofen into "hybrid triglycerides" in rats.

Since coenzyme A thioester of carboxylic acids can replace the natural fatty acids in

triacylglycerols to form toxic "hybrid triglycerides", these findings confirmed the

stereospecific role of coenzyme A thioester formation in the inversion of R(-)-ibuprofen

(Hutt and Caldwell, 1983). In contrast, S(+)-ibuprofen is not significantly incorporated

into adipose tissue following chronic treatment with this enantiomer.




R(-)-ibuprofen -CoASH--- R(-)-ibuprofen-S-CoA
Racemase

S(+)-ibuprofen 4 S(+)-ibuprofen-S-CoA
CoASH -



Figure 2-15. Mechanism of the chiral inversion of ibuprofen in man.



The enzyme system responsible for this inversion has not been clearly defined

yet. However, different investigators suggested either presystemic inversion in the

gastrointestinal tract and/or systemic inversion in the liver as possible sites for this

metabolism (Cox, 1988; Cox et al., 1985,1988; Jamali, 1988; Jamali et al., 1988; Knadler

and Hall, 1989; Mehvar and Jamali, 1988). The metabolic system responsible for this

inversion also seems to be present in the rectum as substantial differences have also

been noticed between the enantiomers plasma concentrations after administration of

ibuprofen suppositories (Jamali, 1988). An average of 63% of an administered dose of

R(-) ibuprofen is stereospecifically inverted to the S(+) enantiomer (Lee et al., 1985).

Elimination
Ibuprofen is eliminated primarily via the kidneys into the urine. More than 80%

of an ibuprofen dose is recovered in the urine after 24 hours, primarily as conjugated








hydroxy- and carboxy-metabolites. Only negligible amounts of ibuprofen (< 1%) are

excreted unchanged (Albert and Gernaat, 1984; Albert et al., 1984; Lee et al., 1985).

Biliary excretion may also occur, as found in animal studies, and account for as

much as 20% of the dose through the biliary tract and feces (Dietzel et al., 1990).

However, no study has investigated an enterohepatic recirculation of ibuprofen in

human subjects. Since 99% of ibuprofen excreted in urine in man is metabolized, it is

very unlikely that much unchanged drug gets into the bile or feces (Albert and Gernaat,

1984; Mills et al., 1973).

Both enantiomers follow parallel time courses and have similar elimination half-

lives of about 2 hours (Cox and Smith, 1987; Cox et al., 1988; Jamali et al., 1988; Lee et

al., 1985). This rapid elimination may explain, to some extent, the low toxicity of

ibuprofen when compared with other non-steroidal anti-inflammatory drugs.


Toxicity

Ibuprofen is generally well tolerated. The majority of adverse reactions reported

in clinical trials have been subjective and mild in nature. The most frequent type of

adverse reaction occurring with ibuprofen is gastrointestinal in nature. In controlled

clinical trials, the percentage of patients reporting one or more gastrointestinal

complaints ranged from 5 to 15%. Common reactions include epigastric pain, nausea,

heartburn, and sensation of "fullness" in the gastrointestinal tract. Occult blood loss is

uncommon. Other side effects of ibuprofen have been reported less frequently

(incidence < 3%). They include thrombocytopenia, skin rashes, headache, dizziness,

blurred vision, toxic amblyopia, fluid retention, and edema (Davis, 1975; Flower et al.,

1985; Kantor, 1979; Knodel, 1992; McEvoy, 1990).


Therapeutic Uses
The approved indications for the use of ibuprofen include the symptomatic

treatment of rheumatoid arthritis, osteoarthritis, and acute gouty arthritis. It is also used








as an analgesic for the symptomatic treatment of mild to moderate pain, especially pain

associated to soft tissues injuries, primary dysmenorrhea, post partum, and oral or

ophthalmic surgery (Flower et al., 1985; McEvoy, 1990).


Dosage and Administration
Ibuprofen is available as plain or film-coated tablets containing 200 to 800 mg

(Motrin, Nuprin, Rufen'?), and as a pediatric suspension containing 20 mg/mL

(PediaprofenR). Its usual adult dosage in the treatment of mild to moderate pain is 400

mg every four to six hours as necessary for relief of pain. The total daily dose should

not exceed 1200 mg. For the symptomatic treatment of acute and chronic rheumatoid

arthritis and osteoarthritis its usual adult dosage is 300-400 mg three or four times

daily. Dosage should be adjusted according to the response and tolerance of the patient

and should not exceed 3200 mg daily. Optimum therapeutic response usually occurs

within two weeks after beginning ibuprofen therapy. For antipyresis, the initial adult

dosage of ibuprofen is 200 mg every four to six hours. Dosage may be increased to 400

mg every four to six hours if fever is not adequately reduced but should not exceed

1200 mg daily (Flower et al., 1985; McEvoy, 1990).



Codeine

Codeine is a narcotic analgesic occurring naturally in opium. It was first isolated

by Robiquet in 1832. It is usually produced commercially by methylation of morphine

which is present in much higher concentrations in opium.


Physicochemical Properties
Codeine (3-methoxymorphine) is a phenanthrene derivative opiate agonist with

analgesic and antitussive properties. Like other morphine derivatives, codeine is a weak

base with a pKa value of 8.2 (Baselt and Cravey, 1989; Muhtadi and Hassan, 1981). It








occurs as a colorless or white crystalline powder with a molecular weight of 300

(anhydrous codeine base).



CH30



0

N-CH3

CH


Figure 2-16. Chemical structure of codeine.



Codeine is slightly soluble in water and freely soluble in alcohol. Phosphate and

sulfate salts of codeine are freely soluble in water (2000 mg/mL), and only slightly

soluble in alcohol (8 mg/mL). Codeine is levorotary in its natural form, and exhibits a

maximum in its UV spectrum in water at 284 nm (Grasselli and Ritchey, 1975).


Pharmacology
Codeine, like other opioids, has widespread pharmacologic effects on almost

every organ and function in the human body. The most important targets are the

central nervous system and the gastrointestinal system.

In man, codeine produces analgesia, drowsiness, change in mood, and mental

clouding. A significant feature of the analgesia is that it occurs without loss of

consciousness. Unlike the non-steroidal anti-inflammatory drugs which have a ceiling

effect for analgesia, opioids act in a dose-dependent manner and usually can control all

types of pain. The pain relief obtained by codeine is relatively selective in that other

sensory modalities are not modified. Patients frequently report that although their pain

is still present, it no longer bothers them.








Although the exact mechanism of action of codeine is still unknown, important

advances in the understanding of the mode of action of opioids have been made with

the discovery of opiate receptors and endogenous opioid peptides. Opioids agonists

produce analgesia and other effects by binding to specific receptors in the brain and

spinal cord. At the spinal cord level, opioids impair or inhibit the transmission of

nociceptive input from the periphery to the central nervous system in a dose-related

manner. At the level of the basal ganglia, opioids activate a descending inhibitory

system that modulates peripheral nociceptive input at the spinal cord level. Opioids

also alter the emotional response to pain by acting on the limbic system (Benedetti and

Butler, 1990; Jaffe and Martin, 1985; McEvoy, 1990). Recent evidence indicates that the

major mechanism of analgesia after oral, intramuscular, or intravenous administration

is the activation of the descending anti-nociceptive system. The activation of opioid

receptors in the substantial gelatinosa requires higher opioid concentrations, and may

only be attained after intraspinal administration (Benedetti and Butler, 1990).

Other central effects of codeine include nausea and vomiting by direct

stimulation of the chemoreceptor trigger zone. Codeine also depresses respiration by

decreasing the responsiveness of the medullary respiratory center to carbon dioxide

tension. Similarly, codeine suppresses the cough reflex by a direct effect on the cough

center in the medulla of the brain. It also appears to exert a drying effect on respiratory

tract mucosa, and to increase viscosity of bronchial secretions (Benedetti and Butler,

1990; Jaffe and Martin, 1985; McEvoy, 1990; Way and Adler, 1962).

At the gastrointestinal level, codeine increases smooth muscle tone and

decreases propulsive peristaltic contractions, thereby causing constipation. It also

causes a marked increase in pressure in the biliary tract, especially of the sphincter of

Oddi (Benedetti and Butler, 1990; Jaffe and Martin, 1985). Other effects of codeine

include miosis, urinary retention, and peripheral vasodilatation.








Pharmacokinetics

Codeine pharmacokinetics in man have been extensively investigated (Clouet,

1971; Findlay et al., 1977, 1978, 1986; Guay et al., 1987; Hull et al., 1982; Quiding et al.,

1986; Rogers et al., 1982; Shah and Williams, 1990; Way, 1968; Way and Adler, 1962,

1968). However, controversy still exists concerning the relative importance of some of

its active metabolites on its analgesic efficacy.

Absorption

Following oral administration, codeine is rapidly absorbed from the

gastrointestinal tract. Its bioavailability after oral administration was found to be

between 50 and 60% (Findlay et al., 1977; Spahn et al., 1983), and almost complete after

intramuscular administration (Brunson and Nash, 1975; Findlay et al., 1977).

Peak plasma concentrations (100 ng/mL after a 60 mg oral dose) are usually

observed within one to two hours after drug administration (Quiding et al., 1986; Shah

and Mason, 1990).

Distribution

Codeine is about 25% bound to plasma proteins (Judis, 1977; Findlay et al.,

1978). It has an approximate volume of distribution of 7.0 L/kg indicating extensive

distribution into the extravascular space (Findlay et al., 1977; Shah and Mason, 1990).

Codeine, like most basic amines, rapidly leaves the blood and concentrates in tissues,

particularly parenchymatous tissues such as the brain, liver, kidney, lung, and adrenal

glands (Way and Adler, 1962).

Metabolism
Codeine is primarily metabolized in the liver by conjugation with glucuronic

acid. Other less important metabolic pathways include 0-demethylation to morphine

and N-demethylation to norcodeine (Adler, 1952,1958; Adler et al., 1955; Muhtadi and

Hassan, 1981; Sindrup et al., 1991). These primary metabolites of codeine are also

metabolized further as outlined in figure 2-17.









-- 6-glucuronidation- Codeine-6-glucuronide 0-demethylation...

6-glucuronidation Morphine-6-glucuronide

Codeine O0-demethylation- l Morphine -3-glucuronidation- Morphine-3-glucuronide

N-demethylation-- Normorphine

N-demethylation- |Norcodeine 6-glucuronidation INorcodeine-6-glucuronide



Figure 2-17. Codeine metabolism in humans.
Adapted from Sindrup et al. (1991)



The hepatic biotransformation of codeine to morphine led many to believe that

codeine may exert its analgesic effect through partial conversion to morphine (Adler

and Latham, 1950; Findlay et al., 1978; Yue et al., 1). This assumption was supported by

the low affinity of codeine for the it opiate receptor and by the marked in vivo analgesic

efficacy of morphine, normorphine, morphine-6-glucuronide, and norcodeine (Lasagna

and Kornfeld, 1958; Osborne et al., 1988). However, studies by Quiding et al. (1986) and

Shah and Mason (1990) questioned the role of morphine on the analgesic effect of

codeine because of the very low plasma concentrations of morphine present after single

or repeated doses of codeine. In a more recent study, Chen et al. (1990) observed

morphine formation in the brain of rats and suggested that the analgesic effect of

codeine may stem from morphine formed in the brain. Yue et al. (1989,1991a, 1991b)

and Sindrup et al. (1991) later confirmed this hypothesis by investigating the analgesic

efficacy of codeine in extensive and poor metabolizers. As a result of a genetic

polymorphism of a particular hepatic cytochrome P450, 6% to 10% of the white

population are classified as poor metabolizers and are virtually unable to demethylate

codeine to morphine. Their results indicated that the analgesic efficacy of codeine in

poor metabolizers was either absent or weaker than in extensive metabolizers, thus








confirming the hypothesis that morphine formation is at least partly responsible for the

analgesic efficacy of codeine.

Elimination
Codeine is primarily excreted in urine as conjugated morphine and norcodeine

metabolites, with less than 10% of the administered dose excreted unchanged (Way and

Adler, 1968). While measurable amounts of unchanged morphine and norcodeine can

be recovered in urine after oral administration of large doses of codeine, only trace

amounts of these metabolites are excreted following therapeutic doses (Adler, 1958).

Renal elimination is rapid, with about two-third of the dose being recovered in the urine

six hours after drug administration (Adler et al., 1955). Negligible amounts of codeine

and its metabolites (< 1%) appear in the feces (Adler et al., 1955).

Codeine has an elimination half life of about two to three hours (Findlay et al.,

1977,1978; Quiding et al., 1986; Yue et al., 1991a, 1991b).


Toxicity

Codeine shares the toxic potentials of the opiate agonists. The most common

side effects observed after the administration of therapeutic doses of codeine include

lightheadedness, dizziness ,sedation, nausea, vomiting and sweating. Other adverse

reactions include euphoria, dysphoria, weakness, headache, insomnia, anorexia,

gastrointestinal distress, constipation, bradycardia, and urinary retention.

Severe overdosage with codeine is characterized by respiratory depression,

Cheyne-Stokes respiration, cyanosis, extreme somnolence progressing to stupor or

coma, and sometimes bradycardia or hypotension. In severe overdosage, apnea,

circulatory collapse, cardiac arrest and death may occur. Codeine toxicity can be

successfully treated by intravenous administration of the narcotic antagonist naloxone

(Jaffe and Martin, 1985; McEvoy, 1990).








As with other opiate agonists, codeine can produce drug dependence and

therefore has the potential for being abused. Psychological and physical dependence, as

well as tolerance, may develop upon repeated administration of codeine (Benedetti and

Butler, 1990; Cutting, 1972, Jaffe and Martin, 1985; McEvoy, 1990).


Therapeutic Uses

Codeine is a mild analgesic indicated for the symptomatic relief of mild to

moderate pain which is not relieved by non-opiate analgesics. Codeine is also used,

alone or in combination with other antitussives or expectorants, in the symptomatic

relief of nonproductive cough (Cutting, 1972, Jaffe and Martin, 1985; McEvoy, 1990).


Dosage and Administration

Codeine is usually given orally as a phosphate, sulfate, or hydrochloride salt for

the relief of cough and mild to moderate pain. The phosphate salt may also be given

parenterally for the relief of pain by intra-muscular or sub-cutaneous injection.

Dosages vary widely depending upon respective indications. As an analgesic,

the usual oral dosage of codeine is 30 to 60 mg every four to six hours, as needed for the

relief of pain. Against cough, the usual adult dose is 10 to 20 mg every four to six hours,

not to exceed 120 mg daily. As with other opiate agonists, the smallest effective dose

should be given in order to minimize the development of tolerance and physical

dependence (Cutting, 1972, Jaffe and Martin, 1985; McEvoy, 1990).














CHAPTER 3
EXPERIMENTAL



This research consisted of two separate studies. The first study centered on

determination of the pharmacokinetic parameters of codeine and ibuprofen in healthy

volunteers. The second study examined both pharmacokinetics and the pharmaco-

dynamic effect, analgesia, of codeine and ibuprofen.



Drug Assays



Determination of codeine and ibuprofen concentrations in biological fluids

required establishment of two high-performance liquid chromatographic assays.


Codeine Analysis

Plasma codeine concentrations were determined by the high-performance liquid

chromatographic method of Mohammed et al. (1992).

Chemicals and reagents

Codeine phosphate, USP reference standard, was provided by Boots

Pharmaceuticals (Shreveport, Louisiana, USA), and used as received. The internal

standard, N-isopropylcodeine, was synthesized by the Department of Medicinal

Chemistry at the University of Florida by alkylation of norcodeine. It was purified

before use.

HPLC-grade acetonitrile, hexane, and methylene chloride were purchased from

Fisher Scientific (Fair Lawn, New Jersey, USA). HPLC grade 1-octanesulfonic acid








(sodium salt) was obtained from Kodak (Rochester, New York, USA). All other reagents

were of analytical grade and were purchased from Fisher Scientific.

Instrumentation
The chromatographic system consisted of a Constametric IIIG high pressure

pump (LDC Milton-Roy, Riviera Beach, Florida, USA), an autosampler (model ISS-100,

Perkin Elmer Corporation, Norwalk, Connecticut, USA) fitted with a 50 pL injection

loop, a 15 cm x 4.6 mm I.D., 5 prm particle size Zorbax' cyanopropylsilane column

(DuPont Instruments, Wilmington, Delaware, USA), and a fluorescence detector with a

xenon lamp (model 650-10 S, Perkin Elmer, Norwalk, Connecticut, USA). A guard

column filled with Zorbax cyanopropylsilane packing material was placed before the

analytical column. Chromatograms were recorded on a Hewlett-Packard integrator

(model 3392A, Palo Alto, California, USA).

Extraction procedure

To 1.0 mL of plasma were added 1.0 mL of 0.05 M phosphate buffer adjusted to

pH 8, and an appropriate amount of internal standard (N-isopropylcodeine) to give

peak-height ratios between 0.2 and 5. The mixture was vortexed for 30 seconds. A

6.0 mL volume of hexane:methylene chloride (2:1, v:v) was added and the mixture

shaken for 5 minutes. The phases were separated by centrifugation for 10 minutes at

3000 revolutions per minute, and the organic phase was transferred into another tube.

The extraction was repeated with another 6.0 mL of fresh solvent. The combined

organic phases were extracted with 1 mL of 0.05 M acetate buffer adjusted to pH 3 and

discarded. The acidic aqueous phase was neutralized by addition of 1.0 mL of 0.1 M

sodium hydroxide and re-extracted with 6 mL of the hexane:methylene chloride

mixture. The organic phase was transferred to a clean tube and evaporated to dryness

under a nitrogen stream. The dried residue was dissolved in 100 PL of mobile phase

before injection into the chromatographic system.










1.0 mL Plasma pH 8
+ Internal Standard
I
Hexane/Methylene Chloride


Aqueous Phase Organic Phase
1
0.05 M Acetate Buffer pH 3


Aqueous Phase Organic Phase
I
0.1 N Sodium Hydroxide
Hexane/Methylene Chloride


Organic Phase Aqueous Phase



Evaporate
Nitrogen Stream



Reconstitute in Mobile Phase
Inject in HPLC System


Figure 3-1. Extraction procedure for codeine analysis.








Chromatographic conditions

Reversed phase chromatography was performed at room temperature. The

mobile phase consisted of a mixture of 0.05 M phosphate buffer and acetonitrile (83:17,

v:v). 1-octanesulfonic acid (0.005 M) was added as an ion-pairing reagent. The pH was

adjusted to 4.9 by addition of 85% phosphoric acid. The mobile phase was filtered

through a 0.2 gm filter and degassed by sonication before use. The flow rate was

1.2 mL/min. Fluorescence detection was performed with excitation and emission

wavelengths of 285 nm and 345 nm, respectively.


Ibuprofen Analysis

Ibuprofen was assayed in plasma by a reversed phase liquid chromatographic

assay procedure.

Chemicals and reagents

Ibuprofen and the internal standard, flurbiprofen, were provided by Boots

Pharmaceuticals (Shreveport, Louisiana, USA), and used as received.

HPLC-grade acetonitrile and all other reagents (analytical grade) were

purchased from Fisher Scientific (Fair Lawn, New Jersey, USA).

Instrumentation

The high-performance liquid chromatography system consisted of a

Constametric IIIG high pressure pump (LDC Milton-Roy, Riviera Beach, Florida, USA),

a manual injection valve with a 50 pL loop (Negretti and Zambra, Southampton, UK), a

15 cm x 4.6 mm I.D., 5 gm particle size, Zorbax octadecylsilane column (DuPont

Instruments, Wilmington, Delaware, USA), and a variable-wavelength UV detector

(Spectromonitor D, LDC Milton Roy). A guard column filled with Zorbax

octadecylsilane packing material was placed before the analytical column.

Chromatograms were recorded on a Hewlett-Packard integrator (model 3392A, Palo

Alto, California, USA).







Extraction procedure
To 1.0 mL of plasma were added 50 iL of an internal standard solution
(400 pg/mL flurbiprofen), 0.5 mL of 2.5 M sulfuric acid, and 12 mL of hexane. The tubes
were shaken for 3 minutes and centrifuged for 10 minutes at 2500 revolutions per

minute. The organic layer was transferred into a clean tube and evaporated to dryness
under a nitrogen steam in a water bath at 50C. The dried residue was then
reconstituted in 100 gL of mobile phase and injected into the chromatographic system.



1.0 mL Plasma
+ Internal Standard
1
2.5 M Sulfuric Acid
Hexane


Aqueous Phase Organic Phase



Evaporate
Nitrogen Stream



Reconstitute in Mobile Phase
Inject in HPLC System


Figure 3-2. Extraction procedure for ibuprofen analysis.



Chromatographic conditions
Reversed phase chromatography was performed at room temperature. The
mobile phase consisted of a mixture of 0.1 M phosphate buffer and acetonitrile (40:60,
v:v). The pH was adjusted to 3.70 by addition of 85% phosphoric acid. The mobile








phase was filtered through a 0.2 pm filter and degassed by sonication before use. The

flow rate was 1.10 mL/min. The UV absorbance was monitored at 254 nm.



Pharmacokminetic Study



The aim of this study was to compare the pharmacokinetics of single oral doses

of codeine and ibuprofen when given alone or in combination. The clinical part of this

study was performed by Harris Inc., Lincoln, Nebraska, USA. The analytical part was

performed in the Department of Pharmaceutics at the University of Florida.


Experimental Design

This study was a double-blind, six-way cross-over study comparing the

pharmacokinetics of ibuprofen and codeine when given alone or in combination. Upon

entering the study, subjects were randomly assigned to one of six dosing groups:

1. Ibuprofen 400 mg in one tablet

2. Codeine 60 mg in one tablet

3. Codeine 30 mg and ibuprofen 400 mg in two separate tablets

4. Codeine 60 mg and ibuprofen 400 mg in two separate tablets

5. Codeine 30 mg and ibuprofen 400 mg in one combination tablet

6. Codeine 60 mg and ibuprofen 400 mg in one combination tablet

After a two-day washout period, subjects were crossed over to receive the

remaining study drugs that they had not previously received.


Subjects

Twenty four healthy subjects between the ages of 18 and 50 years participated in

the study. Healthy was defined as the absence of any acute or chronic illness as

determined by medical history, physical examination, and laboratory testing. They were

fully informed about the investigational procedure and the intake of analgesic drugs.








They had no history of allergic reactions to codeine or ibuprofen. In compliance with

the Declaration of Helsinki specifying the rights of human subjects during clinical

studies, written informed consent was obtained from each participant.


Drug Administration

Subjects were fasted for at least eight hours overnight and could not drink water

two hours before dosing. Immediately after a standardized breakfast, a pre-dose blood

sample was drawn, and a single oral dose of drug was ingested with approximately

100 mL of water. Water was permitted two hours after dosing and subjects could

resume a normal living pattern no sooner than four hours following drug

administration. All doses were taken at approximately 8:00 AM. Subjects remained

seated during the first two hours following the dose of test medication.


Blood Sampling

Blood samples were collected immediately prior to dosing and at 0.5, 0.75, 1,

1.25, 1.5, 1.75, 2, 3, 4, 6, 8, and 14 hours after drug administration by means of an

indwelling catheter placed in a convenient vein in the arm of the subject.

Blood samples (7.0 mL) were harvested into Venojectf tubes containing 14 mg of

potassium oxalate and 17.5 mg of sodium fluoride (Terumo Medical, Elkton, Maryland,

USA). Samples were centrifuged, the plasma separated, frozen, and stored at -200C

until assayed for codeine and ibuprofen.


Estimation of Pharmacokinetic Parameters
The pharmacokinetic parameters of codeine and ibuprofen were determined for

each subject using standard non-compartmental data analysis techniques (Gibaldi and

Perrier, 1982). The maximum plasma concentration (C.ax) and the time to reach the

maximum plasma concentration (tmax) were obtained by direct interpretation of the

data. The overall elimination rate constant (ke) was obtained from the terminal slope of








a log-linear plot of the individual plasma concentrations versus time. The area under

the curve (AUCJ), the area under the first moment curve (AUMCJ), the mean residence

time (MRT), the half-life (tL,), and the apparent oral clearance (Cl0po) were calculated

according to the following equations:


AUC, = (ti-ti t, )Cn
,-i 2 ke


AUMC., 2i + '.(t ti ) +-- +I
2k, k;

MRT = AUMC,
AUC,

ln2
2 ke

C Dose
"- AUC,


Statistical Analysis
Statistical comparisons of each pharmacokinetic parameter were performed

using two-way analysis of variance (ANOVA) with subject, sequence, and treatment as

factors. In cases where treatment effects were significant (p < 0.05), pairwise

comparisons were evaluated with Tukey's multiple range test (Bolton, 1984).


Pharmacodynamic Study


The aim of this study was to compare the pharmacodynamic effect (i.e.

analgesia) of single oral doses of codeine and ibuprofen when given alone or in

combination.








Experimental Design

This study was a double-blind, placebo controlled, four-way cross-over study

comparing the pharmacodynamic effect (i.e. analgesia) of codeine and ibuprofen when

given alone or in combination. Upon entering the study, subjects were randomly

assigned to one of four dosing groups:

1. Ibuprofen 400 mg

2. Codeine 60 mg

3. Codeine 30 mg and ibuprofen 400 mg

4. Placebo

Each dose consisted of two tablets and one capsule that were matched for color

and appearance. After a two-day washout period, subjects were crossed over to receive

the remaining study drugs that they had not previously received.


Subjects

Eight healthy male students (ages 23-32) participated in this series of

experiments as paid volunteers. They were free of neurological disease and were fully

informed about the investigational procedure and the intake of analgesic drugs. They

were screened for medical contraindications and for analgesic use and abuse.

Before testing, all subjects participated in a training session involving

familiarization with the stimulation and recording procedures. No placebo or drug was

given. Subjects were informed of the general objective of the study and signed written

informed consents. All procedures were subjected to review and approval by the

University of Florida Health Center Institutional Review Board, and by the Veterans

Administration Subcommittee for Clinical Investigation.


Drug Administration

Subjects did not eat for at least two hours before dosing, but liquids were

allowed. Immediately after threshold determination, a single oral dose of the test drug








was ingested with approximately 100 mL of water. Food was withheld for two hours

following drug administration, and caffeine intake was limited to each subject's regular

coffee consumption. All doses were taken at approximately 12:00 PM. Subjects

remained seated during the first two hours following the dose of test medication.


Blood Sampling

To avoid repeated venipuncture, blood samples were collected via an indwelling

catheter (Quick-Cath 16G 2 in., Baxter Healthcare Corporation, Deerfield, Illinois, USA)

placed in a convenient vein in the arm of the subject. The catheter was flushed at thirty

minute intervals with a 0.9% sodium chloride solution U.S.P. to maintain patency of the

vein (Epperson, 1984).

Venous blood samples (10 mL) were collected prior to, and at 0.5, 1, 1.5, 2, 2.5, 3,

4, 5, and 6 hours after drug administration in plain (silicone coated) Vacutainet tubes

(Becton Dickinson and Company, Rutherford, New Jersey ,USA). After coagulation, the

serum was separated, transferred into a clean tube, and kept at -20C until assayed for

codeine and ibuprofen.


Dental Stimulation

Pain was induced by electrically stimulating a healthy upper front tooth. Bipolar

electrical stimuli were applied to one upper incisor using 4 mm diameter electrodes

mounted in an individual impression of the subject's upper front teeth.

The individual dental impression was made with orthodontic acrylic resin

(L.D. Caulk Company, Milford, Delaware, USA) from an exact stone replica of the

subject's upper dentition. All electrical connections resided within the resin to minimize

the risk of current spread. Stainless steel springs (Hi-T rectangular wire, 0.019" x 0.025",

Unitek/3M, Monrovia, California, USA) were used to push electrodes against the

enamel of the stimulated tooth and maintain a good contact (see Figure 3-3). The








resistance of the contact between electrodes and tooth was minimized by coating the tip

of the electrode with a drop of electrolyte jelly (Matthews and Searle, 1974, 1976).







Acrylic resin

.... -Electrode

Spring

To stimulator






Figure 3-3. Dental form used for tooth pulp stimulation.



The electrical stimuli consisted of 0.5 millisecond square wave pulses applied

semi-randomly with an interstimulus interval in the range of 2 to 4 seconds (mean

3 seconds). They were generated by a Grass 548 Stimulator (Grass Instrument

Company, Quincy, Massachusetts, USA) with stimulus isolation (model SIU5, Grass

Instrument Company) and constant current units (Model CCU1A, Grass Instrument

Company).

The intensity of the current needed to evoke pain was based on the value of the

measured threshold of sensation (lowest current necessary to evoke a sensation). The

threshold of sensation was determined for each subject before drug intake and the

current intensity noted. This current intensity was then doubled and the resulting

painful stimulus was applied and kept constant throughout the course of the

experiment. The stimulated tooth was kept dry by wiping it with cotton followed by








insertion of dental cotton rolls (Healthco International, Boston, Massachusetts, USA)

between the upper lip and gum.





Stimulator Stimulus Constant
Trigger-- Isolation Unit -- Current Unit Subject
Grass S48 Grass SIU5 Grass CCUIA




Figure 3-4. Block diagram of the stimulation system.





Determination of Pain Threshold

On each study day, threshold of sensation was defined as the lowest current

intensity necessary to evoke a sensation. It was determined by each subject by

manipulating a dial on the constant current unit until the first sensation was felt at the

tooth. The current intensity was then read on a digital multimeter (model DM 7143,

Goldstar Precision, Cerritos, California, USA) by the investigator. After turning the

knob for producing higher and slightly painful stimulation intensities, the subject was

asked to turn the knob back to lower intensities and relocate the position of the

threshold of sensation. The current intensity was then read and the mean of the

corresponding two current intensities was recorded as threshold by the investigator.

This individual threshold measurement was used as the basis for painful tooth pulp

stimulation, with all subsequent stimuli being delivered at twice the threshold current.


Evoked Potential Recording

Spontaneous electroencephalographic activity (EEG) was recorded from leads

placed over midline regions of the brain. All electrodes were placed according to the

International Ten-Twenty System of electrode placement (see Figure 3-5). This system








is based on measurements of head circumference, the distance from nasion to inion, and

the distance from ear to ear. Even numbers are to the right of the midline, and odd

numbers are to the left (Jasper, 1958).



Fpl I ~Fp2
0
F7F F3 Fz F4 F

T3 C3 Cz C4 T4
Al 0 0 0 0 0 A2
TS P3 Pz P4 T6
01 02
00


Figure 3-5 Electrode names and locations in the International Ten-Twenty System.



Samples of electrophysiologic brain activity were obtained from Fz, Cz, and Pz

with reference to Al, with an electrode affixed to Fpz as ground. Electrodes were

maintained on the scalp between effect measurements. The positioning of the electrodes

was chosen to maximize the signal-to-noise ratio.

The skin at each electrode site was prepared by cleaning with isopropyl alcohol,

then rubbing with a cotton swab dipped in a conductive abrasive cream (Omni-Prep,

D.O. Weaver and Company, Aurora, Colorado, USA). Electrodes (10 mm gold plated

disk electrodes, type E5GH, Grass Instrument Company, Quincy, Massachusetts) were

then held in place on the skin while collodion (Mavidon Corporation, Palm City,

Florida, USA) was applied around the rim and dried rapidly with compressed air.

Electrodes were filled with conductive jelly (Mavidon Corporation).

Inter-electrode impedances were measured prior to initial recording and at

hourly intervals during the experiment on an analog electrode impedance meter (Model

EZM1E, Grass Instrument Company). They were matched and maintained below 3 kQ








during the course of an experiment, with readings generally in the 1-2 kQ range.

Physical disturbance of electrodes and wires was minimized by twisting and taping the

electrodes cables together.














Figure 3-6 EEG recording electrodes (cup shaped, gold-plated, 10 mm diameter,
2 mm hole).




Evoked potential measurements were carried out under uniform conditions

(minimum noise environment, dimmed light, closed eyes, room temperature).

Measurements for a certain volunteer were always performed at the same time of the

day to avoid the circadian rhythm in pain perception (time of day around 12:00AM).

After a preliminary training session, in which the subject was familiarized with

the experimental procedure, the four experimental sessions were separated by intervals

of at least two days. During experiments, the subjects were seated in a reclining chair.

They were instructed to be alert but relaxed, and to minimize eye movements or blinks.

They were also closely observed to guard against possible artifacts. Each experimental

session lasted four hours.

The EEG potentials were amplified, filtered (low frequency filter: 0.5 Hz; high

frequency filter: 70 Hz), and recorded on paper (electroencephalograph model 1A97,

Nicolet Biomedical Instruments, Madison, Wisconsin, USA). These recordings provided

control of the quality of the signal stored as well as an assessment of the subject's level








of alertness. The signal was then fed into a digital computer (Brain Atlas III, model 175,

version 2.308, Bio-logic Systems Corporation, Mundelein, Illinois, USA) and processed

by an electrophysiological data analysis system for on-line averaging. The block

diagram below illustrates the interaction of the various system components and the

flow of information through the system.


Digital Computer _____
Bio-logic Brain Atlas


Trigger F
EEG
SNicolet IA97 I
1 ^L
A


Stimulator
Grass $48i Subject )
IGrass S48 /


Figure 3-7. Block diagram of the evoked potential recording system.







Table 3-1. System components and associated functions.


Component
Brain Atlas

Electroencephalograph
Stimulator
Stimulus isolation unit
Constant current unit


Function
Triggering of stimulator and EEG
Signal digitizing and averaging
Signal amplification and filtering
Dental stimulation
Subject isolation
Current intensity regulation








Processing of evoked potential data involved sampling and digitization of an

epoch of the analog EEG signal followed by on-line averaging (see Figure 3-8). The

sweep time was 512 ms with a sampling rate of 512 samples per second (2 ms inter-

sample interval). Automatic artifact rejection was on during the data collection process,

and 16 EEG epochs were collected for each individual waveform. The averaged evoked

potentials were graphed and stored on disk.


Figure 3-8. Block diagram illustrating the processing of evoked potential data.




Estimation of Pharmacokinetic Parameters

The pharmacokinetic parameters of codeine and ibuprofen were determined by

standard compartmental data analysis techniques (Gibaldi and Perrier, 1982). The

plasma concentration time profiles of codeine and ibuprofen were fitted to a

bi-exponential equation corresponding to a one-compartment model with first-order

input, using the equation:

Cp = A e-Aet -A-e e-kt








where Cp is the drug concentration at time t, k, is the absorption rate constant, ke is the

elimination rate constant, and t is the time after drug administration.

The equation was fitted to the experimental data by use of the poly-exponential

curve stripping and fitting program Rstrip (release 5.0, Micromath Scientific, Salt Lake

City, Utah). From these results, the time to reach the maximum plasma concentration

(tmax), the area under the curve (AUC,), the area under the first moment curve

(AUMCJ), the mean residence time (MRT), the elimination half life (t,), and the

apparent oral clearance (Clpo) were calculated according to the following equations:

SIn k, in k,
tfl k-



AUMC, -. )




MRT= AUMC,
AUC,

ln2
t./I = k---


CI D
CP" A AUC,--'-

The maximum plasma concentration (Cmax) was the fitted plasma concentration

at the time of the maximum plasma concentration (tmax).


Estimation of Pharmacodynamic Parameters
Three different indicators of analgesic action were used to correlate drug effects.

Analgesia was recorded before drug intake, and at 0.5, 1, 1.5, 2, 3, and 4 hours after
drug administration








Subjective pain ratings

Subjective pain ratings were derived from two different visual analog scales

(pain intensity and pain relief scales) presented to the subjects immediately after each

dental stimulation. To improve accuracy of subjective measurements, subjects were

shown their previous scores when making an assessment of pain severity (Scott and

Huskisson, 1979). Both scales consisted of a 10 cm line bounded by markers of least to

greatest value along which subjects made pencil marks (see figure 3-9).



No Pain I I Severe Pain

No Complete
Pain Relief Pain Relief


Figure 3-9. Visual analog scales.
Top: Pain intensity scale; Bottom: Pain relief scale.




Pain intensity and pain relief scores were determined by measuring, in

millimeters, the distance between the beginning of the scale and the subject's mark.

To assess the magnitude of the analgesic effect, several measures derived from

individual pain scores were calculated. To account for differences in baseline pain

intensity among patients, pain intensity difference efficacy values (PID) were calculated

as percentages of the value determined before drug intake:

PIDt =100. P pi
PIo

where PIDt is the percent change in pain intensity at time t, PI, the pain intensity at time

t, and PO the pain intensity before drug administration. Positive values indicate

reduction in pain, thus making the pain intensity difference scores analogous to pain

relief scores.








Pain relief scores were determined by measuring the distance (in millimeters)

between the endpoint labeled "no pain relief" and the mark made by the subject.

Pain intensity difference and pain relief scores were also summed over the

observation period to provide estimates of the area under the effect-time curve:

A UCpID = Z PIDt" At

AUC =PR Yp -At

where A UCPID is the sum of the pain intensity differences, PIDt is the pain intensity

difference at time t, At is the time elapsed since the previous observation, AUCPR is the

total pain relief, and PRt is the pain relief score at time t.

The efficacy of different analgesic treatments was then evaluated by comparing

hourly scores, AUCpID scores, and AUCPR scores.

Tooth pulp evoked potentials
Tooth pulp evoked potentials were recorded before drug intake and at 0.5, 1, 1.5,

2, 3, and 4 hours after drug administration. A computer program was developed to

retrieve evoked potential amplitudes at Fz, Cz, and Pz from the Brain Atlas computer

data files (see Appendix). Waveforms were then plotted and smoothed by application

of a moving average technique. For quantitative evaluation of the evoked potential, a

perpendicular was drawn through the height of the P250 peak crossing the line between

the N150 and N380 peaks (see Figure 3-10). This amplitude value (P250 peak to

extrapolated baseline) was measured and its percentage of change after drug intake was

taken as an indicator for the pharmacodynamic activity. This amplitude measurement

technique was chosen since it has proved to have the smallest variability in comparative

measurements (Rohdewald and Keuth, 1990; Rohdewald et al., 1980).

To account for large inter-individual differences in tooth pulp evoked potential

amplitudes, all amplitude values were expressed as a percentage of the amplitude

before drug intake in that individual:









EPt -- 100. PH, PHt
PHo

where EPt is the percent change in evoked potential amplitude at time t, PHt the

amplitude of the P250 peak at time t, and PH() the amplitude of the P250 peak before

drug administration.

Evoked potential scores were also summed over the observation period to

provide estimates of the area under the effect-time curve:

AUCEp = EP-At

where AUCEp is the sum of the changes in evoked potential amplitude and At is the

time elapsed since the previous observation.



40 ,---
30- P250

5:720-
'i10-
0-
= 0

E -10- N380
-20- N 150
-30'
0 100 200 300 400 500
Time [ms]


Figure 3-10. Determination of evoked potential amplitude.





Pharmacokinetic-Pharmacodynamic Modeling

The time course of drug effect was investigated by plotting the observed

analgesic effect as a function of time and as a function of measured plasma

concentration. In addition, the average effect data was fitted to an Ema model to

investigate the relationship existing between drug concentration and drug effect.








This model was selected for its simplicity and by the fact that analgesic effects

are mediated by specific drug-receptor interactions. This model predicts a maximal

effect when drug concentration is above a certain value, as well as no effect when no

drug is present:

E= E,, -Cp
EC50 + Cp

where E is the observed effect at time t, Ema. is the maximum drug effect, EC50 is the

concentration at which 50% of the maximum effect is reached, and Cp is the plasma

concentration at time t.

The experimental data was fitted to the equation by a nonlinear least-squares

regression program (Minsq release 4.02, Micromath Scientific, Salt Lake City, Utah,

USA).


Statistical Analysis

Statistical comparisons of pharmacokinetic and pharmacodynamic parameters

were performed using one-way and two-way analysis of variance (ANOVA) with

subject, sequence, and treatment as factors. In cases where treatment effects were

significant (p < 0.05), pairwise comparisons were evaluated with Tukey's multiple

range test (Bolton, 1984).














CHAPTER 4
RESULTS AND DISCUSSION





Analytical Procedures

Our objective was to develop simple, rapid, specific, and sensitive

chromatographic assay methods for the determination of codeine and ibuprofen in

biological fluids.


Ibuprofen Analysis

Many different chromatographic methods have been published in the literature

for the determination of ibuprofen in biological fluids. They include thin-layer

chromatography (Mills et al., 1973), gas chromatography (Mills et al., 1973), and

high-performance liquid chromatography (Cox et al., 1988; Ginman et al., 1985;

Lockwood and Wagner, 1982; Mehvar et al., 1988).

Our objective was to develop a simple, rapid, and specific high-performance

liquid chromatographic assay for the determination of ibuprofen in plasma. We used a

modification of the method of Ginman et al. (1985).

Chromatography

The analysis was performed using reversed-phase liquid chromatography with

UV detection. Separation of ibuprofen from endogenous plasma components was

achieved with an octadecylsilane column. The mobile phase consisted of a mixture of

0.1 M phosphate buffer and acetonitrile. Detection and quantitation of ibuprofen was

performed by monitoring the UV absorbance of the eluent at 254 nm.








Under the conditions described, ibuprofen and flurbiprofen were well separated

from each other and from endogenous plasma components. Their retention times were

4.2 and 3.2 minutes, respectively. Typical chromatograms of blank plasma and plasma

spiked with ibuprofen obtained after extraction are shown in Figure 4-1.

Linearity
Standard curves were established by spiking drug-free plasma with ibuprofen

and the internal standard (flurbiprofen). They were analyzed by linear regression. They

were linear over the concentration range expected in plasma (0 to 100 Pg/mL) with

correlation coefficients larger than 0.990.

Reproducibility

The accuracy and reproducibility of the method was investigated by spiking

drug-free plasma with different concentrations of ibuprofen. Five replicates of each

concentration were assayed. Table 4-1 shows that the reproducibility in plasma is good

with coefficients of variation of less than 10% in the low concentration range (10 ug/mL

and below), and of about 5% in the higher concentration range (above 10 ug/mL).

The recovery for ibuprofen after extraction from plasma using this method was

determined to be about 40%.


Codeine Analysis

Various analytical methods have been reported for the determination of codeine

in biological fluids. Among these, gas-liquid chromatographic methods have often been

used to quantify codeine in various media (Cone et al., 1983; Edlund, 1981; Zweidinger

et al., 1976), sometimes in combination with mass-spectrometry (Quiding et al., 1986).

Several high-performance liquid chromatographic methods have also been described

using either reversed-phase (Nitsche and Mascher, 1984; Tsina et al., 1982; Visser et al.,

1983) or ion-pairing techniques (Ginman et al., 1985; Kubiak and Munson, 1980).








Finally, radio immunoassay procedures have been reported in the study of the

pharmacokinetics of codeine in man (Findlay et al., 1977, 1978; Rogers et al., 1982).

These procedures are, however, not always sufficiently sensitive or selective for

the measurement of codeine at therapeutic concentrations. Some also involve tedious

sample preparation procedures, or require sophisticated equipment which is not always

available in clinical laboratories. The establishment of the disposition parameters of

codeine for pharmacokinetic investigations requires monitoring of low concentrations of

drug in biological fluids. Therefore, the sensitive, rapid, and selective high-performance

liquid chromatographic method of Mohammed et al., (1992) was selected to measure

codeine concentrations.

Chromatography

The analysis was performed using reversed-phase liquid chromatography with

fluorescence detection. Codeine was separated from endogenous plasma components

with a cyanopropylsilane column. The mobile phase consisted of a mixture of

acetonitrile and 0.05 M phosphate buffer. Octanesulfonic acid (0.005 M) was added to

the mobile phase to decrease the polarity of the eluate, leading to reasonable retention

times and improved peak shapes. N-isopropylnorcodeine was selected as internal

standard due to its structural similarity to codeine and its natural fluorescing

properties. Detection and quantitation was done by monitoring the fluorescence of the

eluent. Excitation (285 nm) and emission (345 nm) wavelengths were chosen from the

maxima of the excitation and emission spectra of a codeine solution.

Under the described conditions, codeine and the internal standard were well

separated from each other and from endogenous plasma components or metabolites.

The retention times for codeine and the internal standard were 4.5 and 6.8 minutes,

respectively. Typical chromatograms obtained from the analysis of blank plasma and

plasma spiked with 10 ng/mL of codeine are shown in Figure 4-2.








Linearity

Calibration curves, obtained by analyzing plasma samples spiked with codeine

in the concentration range of 10 to 400 ng/mL, were analyzed by linear regression. They

were linear in the investigated range with correlation coefficients larger than 0.995.

Reproducibility

The accuracy and reproducibility of the method was investigated by spiking

drug-free plasma with different concentrations of codeine. Five replicates of each

concentration were assayed. Table 4-2 shows that the reproducibility in plasma is good

with coefficients of variation of about 5% for concentrations larger than 50 ng/mL, and

of less than 20% for a concentration of 10 ng/mL.

The recovery for codeine after extraction from plasma using this method was

determined to be 85%. Using this procedure, plasma concentrations as low as 5 ng/mL

can be analyzed.



In summary, the described procedures represent simple, rapid, yet selective and

sensitive assay methods for the analysis of codeine and ibuprofen in biological samples.

Short analysis times (less than 10 minutes per sample) allowed the use of an

auto-injector for semi-automated processing of blood samples. They were successfully

applied to the determination of codeine and ibuprofen levels in all studies presented in

this dissertation.




























Chromatograms of ibuprofen (I) after extraction from plasma, using
flurbiprofen (F) as the internal standard. The two chromatograms
represent concentrations of 0 (left) and 22.1 pg/mL (right).


Table 4-1. Accuracy and reproducibility of ibuprofen assay.


Concentration

pg/mL

5

10

20

30

50


1

4.7

10.8

19.7

31.7

48.9


Assay, jg/mL

2 3

4.7 5.7

10.3 9.3

19.1 18.0

30.0 30.2

48.7 50.4


Figure 4-1.


4

5.5

9.3

18.8

28.2

55.0


5

4.9

10.0

19.3

29.2

50.3


Mean

pg/mL

5.1

9.9

19.0

30.5

50.7


Cv

%

9.20

6.54

3.36

4.34

5.03









[PrC


B-.,l^B


1IPrC.







C







S 5 I -
~J 4VJ.b



n-l-mb


Chromatograms of codeine (C) after extraction from plasma, using
N-isopropylnorcodemine (IPrC) as the internal standard. The two
chromatograms represent concentrations of 0 (left) and 10 ng/mL (right).


Table 4-2. Accuracy and reproducibility of codeine assay.


Concentration Assay, ng/mL Mean CV

ng/mL 1 2 3 4 5 ng/mL %

10 10.5 12.2 15.5 11.1 9.4 11.7 17.8

50 49.8 50.8 46.4 53.6 54.0 50.9 5.4

100 105.0 108.4 107.5 96.2 95.6 102.5 5.4

200 204.5 193.4 195.5 213.0 218.6 205.0 4.7

400 396.5 401.0 400.7 394.0 391.3 396.7 0.9


Figure 4-2.








Pharmacokinetic Study

The combination of ibuprofen and codeine in a single oral dosage form is

currently contemplated. Their pharmacokinetics in healthy subjects have been

extensively investigated. Yet, their pharmacokinetics when given together have not

been established, and the potential for a pharmacokinetic interaction exists. Ibuprofen is

highly bound to plasma proteins, and a change in its pharmacokinetic parameters due

to the presence of an interacting drug may produce undesirable pharmacological

effects. Its is then important to investigate the pharmacokinetics of codeine and

ibuprofen when given alone or in combination before any such combination can be

developed further.

The aim of this study was to compare the pharmacokinetics of single oral doses

of codeine and ibuprofen when given alone or in combination. Six different oral

formulations were examined:

1. Ibuprofen 400 mg in one tablet

2. Codeine 60 mg in one tablet

3. Codeine 30 mg and ibuprofen 400 mg in two separate tablets

4. Codeine 30 mg and ibuprofen 400 mg in one combination tablet

5. Codeine 60 mg and ibuprofen 400 mg in two separate tablets

6. Codeine 60 mg and ibuprofen 400 mg in one combination tablet

Twenty four subjects, matched for age and gender, were enrolled and completed

this study. They received on six different occasions an oral dose of the test drug. Plasma

samples were then harvested and analyzed to determine ibuprofen and codeine

concentrations. The pharmacokinetic parameters of codeine and ibuprofen were

determined in each subject by standard non-compartmental data analysis techniques.








Ibuprofen Pharmacokinetics

The pharmacokinetic parameters of ibuprofen are summarized in Tables 4-3

through 4-5 and representative plasma profiles are shown in Figures 4-3 through 4-7.

Figures 4-4 to 4-7 compare the mean ibuprofen plasma levels for each formulation to

the 400 mg oral dose of ibuprofen.

Following oral administration of a single 400 mg dose of ibuprofen, absorption

was rapid, with average peak plasma concentrations of 30.6 pg/mL appearing at

1.81 hours after dosing. The elimination of ibuprofen was rapid with an average

elimination half-life of 2.13 hours and mean residence time of 4.23 hours. These values

were in agreement with previously published data (Albert and Gemrnaat, 1984; Gillespie

et al., 1982; Lockwood et al., 1983a; Wagner et al., 1984).

The pharmacokinetic parameters of ibuprofen obtained after administration of

the four different ibuprofen-codeine combinations were not significantly different from

those obtained after sole administration of ibuprofen. As can be seen on Figures 4-4

through 4-7, the plots show close agreement between the disposition profile of the

different ibuprofen-codeine combinations and the reference (ibuprofen 400 mg).

However, the maximum concentration obtained after administration of ibuprofen 400

mg and codeine 60 mg in one combination tablet (34.9 tg/mL) was significantly higher

(p < 0.05) than the one obtained after ibuprofen 400 mg and codeine 30 mg in two

separate tablets (26.4 jtg/mL). Since the areas under the concentration-time curve, as

well as the other parameters of these two formulations were not significantly different,

it may well reflect a difference in absorption rate but not in extent. This may be

explained by differences in the manufacturing process of the combination tablet.
























Table 4-3. Pharmacokinetic parameters for ibuprofen given alone
(mean standard deviation).


Ibuprofen 400 mg p.o.


Camax (Lg/mL)

tmax (h)

AUC. ([g.h/mL)

AUMC. (pg.h2/mL)

MRT (h)

ke (h-1)

t, (h)

Clo (L/h)


30.6 10.2

1.81 0.82

102.1 28.8

440.7 231.4

4.23 1.44

0.350 0.094

2.13 0.62

4.27 1.39






















Table 4-4. Pharmacokinetic parameters for ibuprofen given in combination with
codeine (mean standard deviation).


Cmax (ig/mL)

tmax (h)

AUC, (pg.h/mL)

AUMC, (pg.h2/mL)

MRT (h)

ke (h-1)

t, (h)

Clo (L/h)


Ibuprofen 400 mg and codeine

30 mg as two tablets

26.4 11.3

1.89 1.22

105.1 44.4

471.1 242.4

4.76 1.76

0.312 0.122

2.37 0.80

4.21 1.12


Ibuprofen 400 mg and codeine

30 mg as one tablet

33.2 9.2

1.38 0.60

100.2 25.6

354.6 111.7

3.55 0.74

0.335 0.079

2.16 0.43

4.26 1.17


I























Table 4-5. Pharmacokinetic parameters for ibuprofen given in combination with
codeine (mean standard deviation).


Cmax (pg/mL)

tmax (h)

AUC, (pg.h/mL)

AUMC, (pg.h2/mL)

MRT (h)

ke (h-1)

t (h)

Clio (L/h)


Ibuprofen 400 mg and codeine

60 mg as two tablets

29.8 12.4

1.89 1.42

98.0 32.3

429.3 217.3

4.32 1.60

0.347 0.101

2.19 0.72

4.59 1.87


Ibuprofen 400 mg and codeine

60 mg as one tablet

34.9 8.9

1.09 0.55

104.2 29.8

356.2 129.1

3.39 0.61

0.342 0.078

2.12 0.44

4.14 1.12


f


































1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Time (h)


Plasma concentrations of ibuprofen (mean standard deviation).
E] Ibuprofen 400 mg in one tablet
A Codeine 30 mg and ibuprofen 400 mg in two separate tablets
A Codeine 30 mg and ibuprofen 400 mg in one combination tablet
0 Codeine 60 mg and ibuprofen 400 mg in two separate tablets
* Codeine 60 mg and ibuprofen 400 mg in one combination tablet


j-
%


100 4-
0.0


Figure 4-3.


























Time [h]


Figure 4-4.


Plasma concentrations of ibuprofen (mean standard deviation).
o Ibuprofen 400 mg in one tablet
* Codeine 30 mg and ibuprofen 400 mg in one combination tablet


100 -
0.0


1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0


Time [hi


Figure 4-5. Plasma concentrations of ibuprofen (mean standard deviation).
0 Ibuprofen 400 mg in one tablet
Codeine 30 mg and ibuprofen 400 mg in two separate tablets
























100 -4-
0.0


1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Time [hi


Plasma concentrations of ibuprofen (mean standard deviation).
0 Ibuprofen 400 mg in one tablet
* Codeine 60 mg and ibuprofen 400 mg in one combination tablet


100 o-
0.0


1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Time [h]


Plasma concentrations of ibuprofen (mean standard deviation).
o Ibuprofen 400 mg in one tablet
* Codeine 60 mg and ibuprofen 400 mg in two separate tablets


Figure 4-6.


Figure 4-7.








Codeine Pharmacokinetics
The pharmacokinetic parameters of codeine are summarized in Tables 4-6

through 4-8 and representative concentration-vs-time curves are shown in Figures 4-8

through 4-12. Figures 4-9 to 4-12 compare the mean plasma levels for each formulation

to the 400 mg oral dose of codeine.

A single 60 mg oral dose of codeine resulted in an average peak plasma

concentration of 122.8 ng/mL at 1.31 hours after dosing. The levels declined rapidly

with an average elimination half-life of 2.5 hours and mean residence time of 4.25 hours.

These parameters were in good agreement with literature values (Findlay et al., 1977,

1978; Yue et al., 1991a, 1991b; Quiding et al., 1986; Shah and Mason, 1990).

As can be seen on Figures 4-8 through 4-12, the plots show good agreement

between the disposition profile of the different ibuprofen-codeine combinations and the

reference (codeine 60 mg). Except for dose effects (i.e. 30 mg versus 60 mg), the

pharmacokinetic parameters of codeine obtained after administration of the four

different ibuprofen-codeine combinations were not significantly different from those

obtained after sole administration of codeine. In the case of the area under the curve

(AUC) and area under the first moment curve (AUMC), the dose-corrected parameters

were not significantly different.

As was seen with the ibuprofen data, however, the time of the maximum

concentration obtained after administration of ibuprofen 400 mg and codeine 60 mg in

one combination tablet (0.94 hour) was significantly longer (p < 0.05) than the one

observed after codeine 60 mg (1.31 hour). Since the other pharmacokinetic parameters

of these two formulations, and in particular the areas under the concentration-time

curve, were not significantly different, this may indicate a difference in absorption rate

but not in extent. This formulation was already shown to have a significantly reduced

the peak ibuprofen concentration when compared to ibuprofen 400 mg alone.






















Table 4-6. Pharmacokminetic parameters for codeine given alone
(mean standard deviation).


codeine 60 mg p.o.

122.8 48.5

1.31 0.57


Cmax (ng/mL)

tmax (h)

AUC. (ng.h/mL)

AUMC. (ng.h2/mL)

MRT (h)

ke (h-1)


464.4 113.5

2023.5 763.9

4.25 1.00

0.293 0.070

2.50 0.61

137.1 33.9


t, 2 (h)

CL (L/h)


L





















Table 4-7. Pharmacokinetic parameters for codeine given in combination with
ibuprofen (mean standard deviation).


Cma, (ng/mL)

tmax (h)

AUC. (ng-h/mL)

AUMC, (ng.h2/mL)

MRT (h)

ke (h-1)

t (h)

C1,o (L/h)


Ibuprofen 400 mg and codeine

30 mg as two tablets

63.5 17.5

1.19 0.44

242.1 54.6

1002.2 323.9

4.10 0.68

0.294 0.051

2.43 0.45

139.2 39.6


Ibuprofen 400 mg and codeine

30 mg as one tablet

69.0 22.5

1.02 0.35

241.9 60.8

992.5 349.5

4.09 0.81

0.292 0.055

2.46 0.50

137.1 43.0


I





















Table 4-8. Pharmacokinetic parameters for codeine given in combination with
ibuprofen (mean standard deviation).


Cmax (ng/mL)

tmax (h)

AUC. (ng.h/mL)

AUMC. (ng.h2/mL)

MRT (h)

ke (h-1)

t/,/ (h)

CLo (L/h)


Ibuprofen 400 mg and codeine

60 mg as two tablets

133.3 42.9

1.15 0.57

496.4 100.1

2071.9 642.6

4.11 0.79

0.292 0.053

2.45 0.49

123.3 25.5


Ibuprofen 400 mg and codeine

60 mg as one tablet

144.0 45.5

0.94 0.36

516.2 123.5

2054.3 599.4

3.97 0.66

0.292 0.051

2.45 0.47

123.8 30.3


I I





































1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

Time (h)


Plasma concentrations of codeine (mean standard deviation).
D Codeine 60 mg in one tablet
A Codeine 30 mg and ibuprofen 400 mg in two separate tablets
A Codeine 30 mg and ibuprofen 400 mg in one combination tablet
0 Codeine 60 mg and ibuprofen 400 mg in two separate tablets
* Codeine 60 mg and ibuprofen 400 mg in one combination tablet


_J
E

0)


L

ci
C-
O

1E
0


0
U


102





101


100 -
0.0


Figure 4-8.


























Time [h]


Plasma concentrations of codeine (mean standard deviation).
o Codeine 60 mg in one tablet
* Codeine 30 mg and ibuprofen 400 mg in one combination tablet


3.0 4.0
Time [h]


Figure 4-10.


Plasma concentrations of codeine (mean standard deviation).
0 Codeine 60 mg in one tablet
* Codeine 30 mg and ibuprofen 400 mg in two separate tablets


Figure 4-9.


10 -
0.0


























Time [hi


Figure 4-11.


Plasma concentrations of codeine (mean standard deviation).
0 Codeine 60 mg in one tablet
* Codeine 60 mg and ibuprofen 400 mg in one combination tablet


100 +-
0.0


Time [h]


Figure 4-12.


Plasma concentrations of codeine (mean standard deviation).
o Codeine 60 mg in one tablet
* Codeine 60 mg and ibuprofen 400 mg in two separate tablets








In summary, all the pharmacokinetic parameters studied were equivalent with

respect to the different formulations as compared to the control drug. Although one

formulation (ibuprofen 400 mg and codeine 60 mg in one combination tablet) displayed

a significantly faster absorption rate than some other dosage forms, the difference is

probably not clinically significant. No relevant effect of codeine on the disposition of

ibuprofen, and of ibuprofen and the disposition of codeine could be found.



Pharmacodynamic Study

The objective of this double-blind, placebo controlled study was to compare the

pharmacodynamic effect (i.e. analgesia) of single oral doses of codeine and ibuprofen

when given alone or in combination. Eight healthy male students (ages 23-32, mean

weight 72.1 kg 7.2, mean height 1.78 m 0.09) participated in this series of

experiments. One subject was eliminated after initial screening according to study

criteria and another subject's data was not included in the analysis due to difficulties

encountered in resolving the evoked signal from the background EEG.

Four treatments were investigated: Ibuprofen 400 mg, codeine 60 mg, an

ibuprofen-codeine combination (400 mg and 30 mg respectively), and placebo.


Dental Stimulation

The design of a reliable dental stimulation device for use in human pain research

presents special problems. Intact enamel has a very high resistivity and its thickness

may be as much as 1.5 mm in the stimulation area (Matthews and Searle, 1976).

Consequently, a number of dental stimulators have been described in the literature

(Martin and Chapman, 1979; Matthews and Searle, 1976; Rohdewald and Keuth, 1988).

Each design met with varying degrees of success, with contact degradation being one of

the most common problems encountered. Therefore, several prototypes were developed

to improve the quality of the contact between tooth enamel and electrodes. Many




Full Text
33
Moreover, they also demonstrated that statistically significant differences in drug
dosage (500 mg versus 1000 mg of acetylsalicylic acid) and bioavailability could be
detected by evoked potential methods but not by subjective pain reports (Rohdewald,
1980). Thus, evoked potentials techniques appear to be more sensitive for quantitation
of pain attenuation than subjective methods.
Later, Buchsbaum (1984) reported that acetylsalicylic acid (1000 mg p.o.)
significantly decreased N120 amplitudes following cutaneous electrical stimulation
when compared to placebo. Similarly, Keuth and Rohdewald (1989) described the time
course of the analgesic effect of propyphenazone (1000 mg p.o.) in man using visual
analog pain rating scales and tooth pulp evoked potential amplitudes. Their results
differentiated active drug from placebo as soon as 20 to 40 minutes after drug
administration and for up to 2.5 hours.
More recently, Arendt-Nielsen et al. (1991) evaluated the analgesic efficacy of
single oral doses of acetaminophen (1000 mg) and of a codeine acetaminophen
combination (60 and 1000 mg respectively). They found that evoked potential
amplitudes were significantly diminished following administration of both active drugs
as compared to placebo, and that the combination acted faster and provided a longer
lasting analgesia than acetaminophen alone. On the other hand, Cox et al. (1987) could
not differentiate the analgesic effect of a 400 mg ibuprofen tablet from placebo by using
tooth pulp evoked potentials.
Anesthetics
The effects of local anesthetics on evoked potentials have been studied by
several researchers. Gehrig et al. (1981) reported that lidocaine infiltration at the apex of
a tooth obliterated the evoked potential and resulted in a subjective report of no pain,
whereas saline infiltration had no effect. Chatrian et al. (1975a) showed that after a
mandibular nerve block with mepivacaine, evoked potentials could not be elicited by
tooth pulp stimulation in the distribution of this nerve.


138
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Mohammed SS, Butschkau M, and Derendorf H (1992). A reversed phase liquid
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applications. Biomed. Chromatogr. (in press).
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112
Finally, to define the contribution of pharmacokinetic factors to the analgesic
effect of codeine and ibuprofen, an attempt was made to link the observed
pharmacokinetics and pharmacodynamics. To accomplish this objective, the data was
analyzed with a direct-link Emax pharmacokinetic-pharmacodynamic model. This model
was selected for its simplicity and wide applicability, and because analgesic effects are
mediated by specific drug-receptor interactions. Also, this model predicts a maximal
effect when drug concentration is above a certain value, as well as no effect when no
drug is present. Using a nonlinear least-squares regression program, the experimental
data equation was fitted to the equation:
ax'Q7
EC50 + Cp
where £ is the observed effect at time t, Emax is the maximum drug effect, EC50 is the
concentration at which 50% of the maximum effect is reached, and Cp is the plasma
concentration at time t.
The pharmacodynamic estimates provided by this model included Emax, the
maximum attainable effect, and EC50 which reflects the sensitivity of the patient to the
drug effect. From these parameters, a complete concentration-effect curve can be
generated from experimental data.
The maximum effect, as estimated by Emax, was determined for each treatment.
However, due to large inter-subject variability in the effect data and the lack of data
points in the submaximum range, it was not possible to get a good estimate of EC50.
The utility and validity of pharmacokinetic and pharmacodynamic parameters
obtained from a pharmacokinetic-pharmacodynamic model require that the methods
used for both the pharmacokinetic and the pharmacodynamic measures be specific and
sensitive. This is accomplished more easily with the analytical methods used in
pharmacokinetics than for the methods used to obtain pharmacodynamic information


31
peak measurement do not affect the general finding that analgesics induce a decrease in
evoked potential amplitude.
Centrally acting analgesics
Evoked potentials have been used to study opiate analgesics. Chapman et al.
first reported preliminary evidence that an opiate could affect the evoked potential
waveform and reduce the subjective pain report in subjects experiencing painful tooth
pulp stimulation (Chen et al., 1979; Harkins and Chapman, 1978). The effects observed
in the major negative and positive peaks were very similar to those reported by
Buchsbaum et al. (1981a; 1981b), who gave morphine to subjects experiencing painful
skin stimulation. Smaller amplitudes of the N120 component were found following
morphine than after placebo. This effect was more marked at higher stimulus
intensities. The morphine effect was opposite to that observed with naloxone:
amplitudes of most components of the evoked potential decreased with morphine and
increased with naloxone (Buchsbaum, 1984; Buchsbaum et al., 1977).
Later, Bromm et al. (1983) demonstrated the dose-dependent reversal of opioid
analgesia by increasing doses of naloxone, measuring pain ratings and late component
evoked potential amplitudes. Their results showed that, as compared to placebo, the
opiate agonist tilidine significantly reduces both the amplitude of the N150-P320
component and the subjective pain report. Naloxone and placebo alone had little or no
effect on either evoked potentials or subjective pain report.
To further pursue opiate analgesia and narcotic antagonism, Butler et al. (1983)
investigated the effect of fentanyl (0.1 mg IV) on evoked potentials (see Figure 2-11).
They found that the evoked potential amplitude was diminished by administration of
fentanyl and that dose-dependent reversal by naloxone could be demonstrated.
Additionally, other potent narcotics such as meperidine have also been shown to
remarkably influence the amplitudes of the N150 and P250 components of the evoked
cerebral potential (Grundy and Brown, 1980).


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relevant change in pharmacokinetic parameters was observed following concomitant
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38
A2 production. This effect is reversible and may result in a slight prolongation in
bleeding time. Prothrombin time remains unaffected (Kantor, 1979; McEvoy, 1990).
Pharmacokinetics
The basic pharmacokinetics of ibuprofen are well documented (Albert and
Gemaat, 1984; Busson, 1986; Geisslinger et al., 1989; Kantor, 1984). The discovery of an
in vivo inversion process led to a renewed academic interest targeted at elucidating the
pharmacokinetics of the individual enantiomers. Ibuprofen undergoes an in vivo
inversion of configuration in humans: R(-)-ibuprofen is stereoselectively inverted to
S(+)-ibuprofen (Adams et al., 1967, 1976; Hutt and Caldwell, 1983; Mills et al., 1973).
Absorption
Following oral administration, ibuprofen is rapidly and almost completely
absorbed from the gastrointestinal tract. Its bioavailability after oral administration has
been estimated to be between 80 and 100% (Albert and Gemaat, 1984; Albert et al., 1984;
Lockwood et al., 1983a; Wagner et al., 1984). The administration of food and/or
antacids only alters minimally the rate and extent of ibuprofen absorption (Albert and
Gemaat, 1984; Albert et al., 1984; Szpunar et al., 1987). Peak plasma concentrations
(17-36 pg/mL after a 400 mg oral dose) are usually observed within 1.5 to 2 hours after
drug administration. Time to peak and peak plasma concentrations of both enantiomers
are similar, indicating a lack of stereoselective absorption for the enantiomers of
ibuprofen (Day et al., 1988; Lee et al., 1985).
Distribution
Ibuprofen, as well as other arylpropionic acid derivatives, is extensively bound
to plasma proteins. Approximately 90-99% of a dose is bound to plasma proteins
(Albert and Gemaat, 1984; Albert et al., 1984). It exhibits nonlinear plasma protein
binding at doses larger than 800 mg (Lockwood et al., 1983; Mills et al., 1973; Wagner et
al., 1984). Recent studies suggested that there might be an enantiomer-enantiomer


74
Linearity
Calibration curves, obtained by analyzing plasma samples spiked with codeine
in the concentration range of 10 to 400 ng/mL, were analyzed by linear regression. They
were linear in the mvestigated range with correlation coefficients larger than 0.995.
Reproducibility
The accuracy and reproducibility of the method was investigated by spiking
drug-free plasma with different concentrations of codeine. Five replicates of each
concentration were assayed. Table 4-2 shows that the reproducibility in plasma is good
with coefficients of variation of about 5% for concentrations larger than 50 ng/ mL, and
of less than 20% for a concentration of 10 ng/mL.
The recovery for codeine after extraction from plasma using this method was
determined to be 85%. Using this procedure, plasma concentrations as low as 5 ng/mL
can be analyzed.
In summary, the described procedures represent simple, rapid, yet selective and
sensitive assay methods for the analysis of codeine and ibuprofen in biological samples.
Short analysis times (less than 10 minutes per sample) allowed the use of an
auto-injector for semi-automated processing of blood samples. They were successfully
applied to the determination of codeine and ibuprofen levels in all studies presented in
this dissertation.


BIOGRAPHICAL SKETCH
Matthieu Kaltenbach was born on October 23,1962, to Jean and Colette
Kaltenbach. He graduated from "l'Ecole Secondaire et Technique de lTmmacule
Conception" in Saint Dizier, France, in 1980. He entered Pharmacy School at the
University of Reims Champagne Ardenne, France, and graduated with a Pharm.D.
degree in May 1986. He came to the United States in August 1986 and entered graduate
school in the College of Pharmacy at the University of Florida. He worked under the
guidance of Dr. Hartmut Derendorf.
The author received his Doctor of Philosophy degree in the pharmaceutical
sciences in 1992. He accepted a position as an associate faculty member in the
Department of Pharmacology and Pharmacokinetics at the College of Pharmacy of the
University of Reims Champagne Ardenne, France.
143


CHAPTER 1
INTRODUCTION
Analgesia is a major concern in clinical practice and one of the most problematic
areas in pain research. Despite many studies, it is evident that research on the
assessment of analgesic states has had little influence on medical care or guidelines for
clinical pain control. Many patients still experience inadequate pain relief (Bonica,
1990a; Donovan et al., 1987; Marks and Sachar, 1973; Sriwatanakul et al., 1983b). Pain is
recognized as the most frequent medical symptom by the National Institutes of Health.
Among pharmaceutical products used for pain relief, orally effective opioids
such as codeine are often prescribed in combination with peripherally acting analgesics
such as aspirin or acetaminophen. Analgesic mixtures containing codeine were among
the 10 most frequently dispensed drugs in the United States in 1991 (La Piaa
Simonsen, 1992). The combination of an opioid with a non-opioid appears to be rational
because the mechanisms of action differ, and the analgesic effects of the individual
drugs are additive (Beaver, 1975,1984). Since the non-opioids have a ceiling analgesic
effect and the dosage of opioids should be limited to prevent adverse effects,
combinations of this type may also provide greater pain relief with a minimum of
adverse effects in a convenient dosage form for the patient (Beaver, 1975, 1981, 1984).
However, there is still no consistent evidence from human clinical trials to substantiate
these rationales for any particular combination. Recently, a growing number of well-
controlled clinical trials have demonstrated that appropriately chosen combinations of
opioids with either aspirin or acetaminophen do in fact achieve these objectives (Bentley
and Head, 1987; Forbes et al. 1984a, 1984b, 1986).
1


79
Table 4-3. Pharmacokinetic parameters for ibuprofen given alone
(mean standard deviation).
Ibuprofen 400 mg p.o.
Cmax (Hg/mL)
30.6 10.2
^max 0"0
1.81 0.82
AUCm (pg-h/mL)
102.1 28.8
AUMC^ (pg-h2/ mL)
440.7 231.4
MRT (h)
4.23 1.44
ke (h-1)
0.350 0.094
(h)
2.13 0.62
Clpo (L/h)
4.27 1.39


136
Jamali F (1988). Pharmacokinetics of enantiomers of chiral non-steroidal anti
inflammatory drugs. Eur. ]. Drug Mctab. Pharmacokinet., 13:1-9.
Jamali F, Singh N, Pasutto FM, Russell AS, and Coutts RT (1988). Pharmacokinetics of
ibuprofen enantiomers in humans following oral administration of tablets with
different absorption rates. Pharm. Res., 5:40-43.
Jasper HH (1958). The ten twenty electrode system of the International Federation.
Electroencephalogr. Clin. Neurophysiol., 10:371-375.
Judis J (1977). Binding of codeine, morphine and methadone to human serum proteins.
/. Pharm. Sci.r 66:802-806.
Kantor TG (1979). Ibuprofen. Ann. Int. Med., 91:877-882.
Kantor TG (1984). Summary: Ibuprofen Past, present, and future. Am. J. Med.,
77:121-125.
Keuth V and Rohdewald P (1989). Propyphenazone: Pharmacokinetics and
pharmacodynamics. Pharm. Ztg. Wiss., 2:21-26.
Knodel LC (1992). NSAID adverse effects and interactions: Who is at risk. Am. Pharm.,
32:39-47.
Kubiak EJ, and Munson JW (1980). High-performance liquid chromatographic analysis
of codeine in syrups using ion-pair formation. J. Pharm. Sci., 69:152-156.
Lahoda R, Stacher G, and Bauer P (1977). Experimentally induced pain: Measurement of
pain threshold and pain tolerance using a new apparatus for electrical stimulation of
the skin. Int. J. Clin. Pharmacol., 15:51-56.
La Piaa Simonsen L (1992). Top 200 drugs of 1991: What are pharmacists dispensing
most often. Pharmacy Times, 4:47-66.
Lasagna L and Komfeld TJ (1958). Analgesic potency of normorphine in patients with
postoperative pain. ]. Pharmacol., 124:260-263.
Laska EM, Meisner M, Takeuchi K, Wanderling JA, Siegel C, and Sunshine A (1986a).
An analytic approach to quantifying pain scores. Pharmacotherapy, 6:276-282.
Laska EM, Sunshine A, Marrero I, Olson N, Siegel C, and McCormick N (1986). The
correlation between blood levels of ibuprofen and clinical analgesic response. Clin.
Pharmacol. Th.er., 40:1-7.
Leandri M, Campbell JA, and Lahuerta J (1985). Effect of attention on tooth-pulp evoked
potentials. In Advances in pain research and therapy, volume 9. Edited by HL Fields,
R Dubner, and F Cervero. Raven Press, New York, pp. 331-336.
Lee EJD, Williams K, Graham G, and Champion D (1985). Stereoselective disposition of
ibuprofen enantiomers in man. Br. J. Clin. Pharmacol., 19:669-674.
Lekic D and Cenic D (1992). Pain and tooth pulp evoked potentials. Clin.
Electroencephalogr., 23:37-46.


73
Finally, radio immunoassay procedures have been reported in the study of the
pharmacokinetics of codeine in man (Findlay et al., 1977, 1978; Rogers et al., 1982).
These procedures are, however, not always sufficiently sensitive or selective for
the measurement of codeine at therapeutic concentrations. Some also involve tedious
sample preparation procedures, or require sophisticated equipment which is not always
available in clinical laboratories. The establishment of the disposition parameters of
codeine for pharmacokinetic investigations requires monitoring of low concentrations of
drug in biological fluids. Therefore, the sensitive, rapid, and selective high-performance
liquid chromatographic method of Mohammed et al., (1992) was selected to measure
codeine concentrations.
Chromatography
The analysis was performed using reversed-phase liquid chromatography with
fluorescence detection. Codeine was separated from endogenous plasma components
with a cyanopropylsilane column. The mobile phase consisted of a mixture of
acetonitrile and 0.05 M phosphate buffer. Octanesulfonic acid (0.005 M) was added to
the mobile phase to decrease the polarity of the eluate, leading to reasonable retention
times and improved peak shapes. N-isopropylnorcodeine was selected as internal
standard due to its structural similarity to codeine and its natural fluorescing
properties. Detection and quantitation was done by monitoring the fluorescence of the
eluent. Excitation (285 nm) and emission (345 nm) wavelengths were chosen from the
maxima of the excitation and emission spectra of a codeine solution.
Under the described conditions, codeine and the internal standard were well
separated from each other and from endogenous plasma components or metabolites.
The retention times for codeine and the internal standard were 4.5 and 6.8 minutes,
respectively. Typical chromatograms obtained from the analysis of blank plasma and
plasma spiked with 10 ng/ mL of codeine are shown in Figure 4-2.


55
phase was filtered through a 0.2 pm filter and degassed by sonication before use. The
flow rate was 1.10 mL/min. The UV absorbance was monitored at 254 nm.
Pharmacokinetic Study
The aim of this study was to compare the pharmacokinetics of single oral doses
of codeine and ibuprofen when given alone or in combination. The clinical part of this
study was performed by Harris Inc., Lincoln, Nebraska, USA. The analytical part was
performed in the Department of Pharmaceutics at the University of Florida.
Experimental Design
This study was a double-blind, six-way cross-over study comparing the
pharmacokinetics of ibuprofen and codeine when given alone or in combination. Upon
entering the study, subjects were randomly assigned to one of six dosing groups:
1. Ibuprofen 400 mg in one tablet
2. Codeine 60 mg in one tablet
3. Codeine 30 mg and ibuprofen 400 mg in two separate tablets
4. Codeine 60 mg and ibuprofen 400 mg in two separate tablets
5. Codeine 30 mg and ibuprofen 400 mg in one combination tablet
6. Codeine 60 mg and ibuprofen 400 mg in one combination tablet
After a two-day washout period, subjects were crossed over to receive the
remaining study drugs that they had not previously received.
Subjects
Twenty four healthy subjects between the ages of 18 and 50 years participated in
the study. Healthy was defined as the absence of any acute or chronic illness as
determined by medical history, physical examination, and laboratory testing. They were
fully informed about the investigational procedure and the intake of analgesic drugs.


18
suitable for clinical pain research (Chapman and Syrjala, 1990; Gracely, 1990; Syrjala
and Chapman, 1984).
Physiologic Correlates of Pain
Pain is a subjective experience and, on the ground of its large emotional
component, is quite dependent on the condition of the person being affected. But pain is
also a consequence of the activation of nociceptive afferents, and neural activity can be
measured with the methods commonly applied in sensory physiology. Consequently, a
number of attempts have been made to measure pain through physiologic indicators.
Many confounding factors are known to compromise subjective report measures. To
objectify these measures, several physiological parameters accompanying pain have
been investigated.
Over the past thirty years, many investigations have been undertaken to
correlate pain with muscle tension, the nociceptive flexion reflex (withdrawal reflex),
skin resistance reaction, certain autonomic parameters, and central nervous system
variables (Bromm, 1984a; Hill and Chapman, 1989; Syqala and Chapman, 1984; Wilier,
1984; Wolff, 1979). Yet, no consistent relationship has been found between the
individual pain reports and these physiologic measurements (Bromm and Treede, 1980;
Bromm and Scharein, 1982b; Syrjala and Chapman, 1984). In addition, the usefulness of
these measurements is limited by their tendency to decrease over time, irrespective of
pain perception, and their sensitivity to stress (Bromm and Scharein, 1982b; Bromm and
Treede, 1980; Syrjala and Chapman, 1984). To date, the most stable and reliable
correlations with subjective pain reports have been obtained with cerebral evoked
potentials (Bromm, 1984a, 1985; Bromm and Scharein, 1982b; Buchsbaum, 1984;
Chapman and Jacobson, 1984; Chudler and Dong, 1983; Fernandes de Lima et al., 1982;
Hill and Chapman, 1989; Syrjala and Chapman, 1984).


CHAPTER 5
CONCLUSION
Among analgesic mixtures, the combination of codeine, an opioid, with
lbuprofen, a non-opioid, appears to be rational because their mechanisms of action
differ. Since the non-opioids have a ceiling analgesic effect and the dosage of opioids
should be limited to prevent adverse effects, combinations of this type may also provide
greater pain relief with a minimum of adverse effects in a convenient dosage form for
the patient.
Consequently, the present study was designed to evaluate the pharmacokinetics
and efficacy of ibuprofen and codeine as analgesics, and to investigate the relationship
existing between their drug concentrations and the analgesic effect as a function of time.
To accomplish these goals, two high-performance liquid chromatographic assay
procedures were developed to determine ibuprofen and codeine levels in biological
fluids. The use of either fluorescence (for codeine) or UV detection (for ibuprofen)
allowed the detection and quantification of therapeutic concentrations of both drugs in
biological fluids in a reproducible manner.
These assay procedures enabled us to investigate the pharmacokinetics of
codeine and ibuprofen when given alone or in combination. Pharmacokinetic
parameters of both codeine and ibuprofen obtained in this study were comparable to
previously published values. In addition, this pharmacokinetic study established for the
first time the disposition characteristics of both ibuprofen and codeine when given in a
single combination dosage form. Results showed that codeine and ibuprofen do not
interact with each other and thus can be given safely together in a single oral dosage
form.
118


ACKNOWLEDGMENTS
I would like to offer my appreciation and grateful thanks to Dr. Hartmut
Derendorf for his friendship, guidance, and continuous encouragement during the
course of the work embodied in this dissertation. 1 would also like to thank the
members of my supervisory committee, Dr. Giinther Hochhaus, Dr. Hans Schreier, and
Dr. Charles Vierck, for their advice throughout my doctoral research.
My special thanks are extended to Dr. Betty Grundy for her time, fruitful
suggestions, and constant support with the evoked potentials experiments. Without
her, this research project would not have been possible.
I would like to acknowledge and thank Dr. John Perrin who supervised parts of
the drug analysis, and Mr. Fred Bauman and Dr. Andr Mauderli for their help in
setting up the tooth pulp experiments.
I take this opportunity to express my sincere appreciation to Martina Butschkau,
Maritza Cediel, jan Mller, Sumia Mohammed, Gotelind Mullersman, Pam Riley,
Shashank Rohatagi, Carolina Vivas, and especially Marjorie Rigby for their valuable
help with the drug analysis.
I would like to thank Dr. Daniel Robinson for introducing me to the wonderful
world of computers and their applications in pharmacy.
Last but not least, I would like to extend my sincere appreciation and warmest
thanks to my parents, parents in-law, and to my wife for believing in me, being there
whenever I needed them, and doing whatever they could to help me.


69
EP, = 100
PH0
where £P( is the percent change in evoked potential amplitude at time t, PHt the
amplitude of the P250 peak at time t, and PH0 the amplitude of the P250 peak before
drug administration.
Evoked potential scores were also summed over the observation period to
provide estimates of the area under the effect-time curve:
AUCep ^EPt At
where AUCEP is the sum of the changes in evoked potential amplitude and At is the
time elapsed since the previous observation.
Figure 3-10. Determination of evoked potential amplitude.
Pharmacokinetic-Pharmacodynamic Modeling
The time course of drug effect was investigated by plotting the observed
analgesic effect as a function of time and as a function of measured plasma
concentration. In addition, the average effect data was fitted to an Emax model to
investigate the relationship existing between drug concentration and drug effect.


80
Table 4-4. Pharmacokinetic parameters for ibuprofen given in combination with
codeine (mean standard deviation).
Ibuprofen 400 mg and codeine
30 mg as two tablets
Ibuprofen 400 mg and codeine
30 mg as one tablet
Cmax (Hg/mL)
26.4 11.3
33.2 9.2
tmax (h)
1.89 1.22
1.38 0.60
AUCk (pg-h/ mL)
105.1 44.4
100.2 25.6
AUMC* (pg-h2/ mL)
471.1 242.4
354.6 111.7
MRT (h)
4.76 1.76
3.55 0.74
ke^'1)
0.312 0.122
0.335 0.079
t./2(h)
2.37 0.80
2.16 0.43
Cipo (L/h)
4.21 1.12
4.26 1.17


139
Owen JA and Sitar DS (1983). Morphine analysis by high-performance liquid
chromatography. J. Chromatogr., 276:202-207.
Park GR and Fulton B (1991). The management of acute pain. Oxford University Press,
Oxford, England.
o
Quiding H, Anderson P, Bondeson U, Borus LO, and Hynning PA (1986). Plasma
concentrations of codeine and its metabolite, morphine, after single and repeated
oral administration. Eur. /. Clin. Pharmacol., 30:673-677.
Quiding H and Haggquist SO (1983). Visual analogue scale and the analysis of
analgesic action. Eur. J. Clin. Pharmacol., 24:475-478.
Rogers JF, Findlay JWA, Hull JH, Butz RF, Jones EC, Bustrack JA, and Welch RM (1982).
Codeine disposition in smokers and non-smokers. Clin. Pharmacol. Ther., 32:218-227.
Rohdewald P, Derendorf H, Drehsen G, Eiger CE, and Knoll O (1982a). Changes in
cortical evoked potentials as correlates of the efficacy of weak analgesics. Pain,
12:329-341.
Rohdewald P, Derendorf H, Eiger CE, and Knoll O (1980). Quantification of the
attenuation of pain by means of evoked potentials after application of weak
analgesics. Z. EEG-EMG, 11:199-204.
Rohdewald P, Derendorf H, Eiger CE, and Knoll O (1982b). Evoked potentials as
objective parameters for the analgesic response of weak analgesics. Adv. Neurol.,
32:375-381.
Rohdewald P, Drehsen G, Milsmann E, and Derendorf H (1983). Relationship between
saliva levels of metamizole metabolites, bioavailability and analgesic efficacy.
Drug Res., 33:985-988.
Rohdewald P and Keuth V (1988). Algesimetry: one way to differentiate analgesics.
Pharm. Ztg., 133:152-160.
Rohdewald P and Keuth V (1990). Evaluation of algesimetric parameters on the basis of
tooth pulp stimulation in humans. Anesth. Prog., 37:4-10.
Rosenfeld JP, Silvia R, Weitkunat R, and Dowman R (1985). Operant control of human
somatosensory evoked potentials alters experimental pain perception. In Advances
in pain research and therapy, volume 9. Edited by HL Fields, R Dubner, and F Cervero.
Raven Press, New York, pp. 113-116.
Schachtel BP (1991). Pain measurement scales in single-dose comparisons. In Advances
in pain research and therapy, volume 18. Edited by MB Max, RK Portenoy, and EM
Laska. Raven Press, New York, pp. 113-116.
Scott J and Huskisson EC (1976). Graphic representation of pain. Pain, 2:175-184.
Scott J and Huskisson EC (1979a). Accuracy of subjective measurements made with or
without previous scores: an important source of error in serial measurements of
subjective states. Ann. Rheum. Dis., 38:558-559.


44
occurs as a colorless or white crystalline powder with a molecular weight of 300
(anhydrous codeine base).
O
CH
Figure 2-16. Chemical structure of codeine.
Codeine is slightly soluble in water and freely soluble in alcohol. Phosphate and
sulfate salts of codeine are freely soluble in water (2000 mg/mL), and only slightly
soluble in alcohol (8 mg/mL). Codeine is levorotary in its natural form, and exhibits a
maximum in its UV spectrum in water at 284 nm (Grasselli and Ritchey, 1975).
Pharmacology
Codeine, like other opioids, has widespread pharmacologic effects on almost
every organ and function in the human body. The most important targets are the
central nervous system and the gastrointestinal system.
In man, codeine produces analgesia, drowsiness, change in mood, and mental
clouding. A significant feature of the analgesia is that it occurs without loss of
consciousness. Unlike the non-steroidal anti-inflammatory drugs which have a ceiling
effect for analgesia, opioids act in a dose-dependent manner and usually can control all
types of pain. The pain relief obtained by codeine is relatively selective in that other
sensory modalities are not modified. Patients frequently report that although their pain
is still present, it no longer bothers them.


.y
Figure 2-8. Scalp distribution of tooth pulp evoked potentials.
Black triangles refer to standard electrode placements of the
international 10-20 system; white triangles represent supplementary
electrodes.
From Chatrian et al. (1975a).


CHAPTER 2
LITERATURE REVIEW
Pain, a major concern of humankind throughout history, has engendered
numerous efforts to understand its nature and to achieve its control. Pain is a common
and complex human experience, and the most frequent reason patients seek medical
advice. Despite its clinical importance, pain research was neglected until three decades
ago. Advances in diagnosis and therapy over the past twenty to thirty years hold the
promise of a better future.
This chapter reviews the concept of pain, its physiology, and the methods used
in its assessment. Special emphasis is placed on evoked potentials. Finally, current
knowledge on the model drugs used in this research, ibuprofen and codeine, is
presented.
The Concept of Pain
Pain: A Multidimensional Experience
Pain is one of the most personal yet universal human experiences. Originally,
pain was thought to be a punishment from the gods. In fact, the word pain is derived
from the Latin peone, meaning penalty or punishment (Stimmel, 1983). In the
seventeenth century, Descartes postulated that pain was a sensation in which the brain
played an important role. His theory, also known as the specificity theory, described the
pain system as a direct channel from the skin to the brain. This concept underwent little
change until the nineteenth century, when von Frey and Goldscheider introduced the
concepts of sensory input, central summation, neuroreceptors, and nociceptors. Their
5


26
probable that "pre-pain", sharp pain, and dull ache correspond to Ap, A5, and C fiber
activation (Burgess et al., 1990). For practical purposes, dental stimulation may be seen
as differing from cutaneous stimulation because of its quite small "pre-pain" range
(range between stimulus detection and pain threshold).
Chatrian (1975a, 1975b) first introduced tooth pulp evoked potentials in human
research as an objective monitor of pain. Soon after, Harkins and Chapman (1978) and
Chen et al. (1979) reported that the amplitude of late components of tooth pulp evoked
potentials could be considered a functional correlate of pain. Thereafter, numerous pain
modulating procedures (narcotic analgesics, non-steroidal anti-inflammatory drugs,
local anesthetics, acupuncture) have been evaluated by means of tooth pulp evoked
potentials. Now, it has been clearly established that tooth pulp evoked potentials are a
sensitive and reliable way to investigate human pain in the laboratory. Figure 2-7
shows a typical example of a tooth pulp evoked potential.
Evoked Potentials as Correlates of Pain
Pioneering work in the field of evoked potential correlates of human pain was
initiated by Chatrian and co-workers (Chatrian et al., 1975a, 1975b). These researchers
succeeded in demonstrating that there is a consistent evoked response associated with
pain. They also observed that the phenomenon was most apparent when sampled from
vertex with reference to inion (see Figure 2-8).
The important findings reported by these investigators were that
Electrical dental stimulation elicits no sensations other than pain.
Stimulation of a devitalized tooth does not produce evoked potentials or any
subjective sensations. Thus, the results cannot have been due to passive spread of
the stimulating current to sources of artifacts.


76
IPrC IPrC:
C
iij rr-'f it 1ri r
oaoo oa Ffca&a deajgfl Tlo
Figure 4-2. Chromatograms of codeine (C) after extraction from plasma, using
N-isopropylnorcodeine (IPrC) as the internal standard. The two
chromatograms represent concentrations of 0 (left) and 10 ng/mL (right).
Table 4-2. Accuracy and reproducibility of codeine assay.
Concentration
ng/mL
1
Assay, ng/mL
2 3 4
5
Mean
ng/mL
CV
%
10
10.5
12.2
15.5
11.1
9.4
11.7
17.8
50
49.8
50.8
46.4
53.6
54.0
50.9
5.4
100
105.0
108.4
107.5
96.2
95.6
102.5
5.4
200
204.5
193.4
195.5
213.0
218.6
205.0
4.7
400
396.5
401.0
400.7
394.0
391.3
396.7
0.9


TABLE OF CONTENTS
page
ACKNOWLEDGMENTS i
ABSTRACT vii
CHAPTERS
1 INTRODUCTION.... 1
2 LITERATURE REVIEW 5
The Concept of Pain 5
Pain: A Multidimensional Experience 5
The Physiology of Pain 7
Nociceptors... 7
Central pain pathways 8
Pain modulation 11
Pain Measurement and Assessment 12
Unidimensional Pain Rating Scales 13
Categorical rating scales 14
Visual analog scales 15
Multidimensional Pain Rating Scales 17
Physiologic Correlates of Pain 18
Evoked Potentials in Pain Research 19
Evoked Potentials 19
Nomenclature 21
Classification of evoked potentials 21
The Pain Stimulus 23
Mechanical stimulation 24
Thermal stimulation 24
Electrical stimulation 24
Tooth Pulp Evoked Potentials 25
Evoked Potentials as Correlates of Pain 26
Effect of Analgesics on Evoked Potentials 30
Centrally acting analgesics 31
Peripherally acting analgesics 32
Anesthetics 33
Advantages of Evoked Potential Methods 34
Ibuprofen 35
Physicochemical Properties 35
Pharmacology 36
Pharmacokinetics 38
Absorption 38
Distribution 38
iv


39
interaction with regard to protein binding: R(-)-ibuprofen may compete with
S(+)-ibuprofen for binding on the human serum albumin molecule (Evans et al., 1989;
Lee et al., 1985). However, the extent of such stereoselectivity remains to be determined.
Due to its high protein binding, ibuprofen is not extensively distributed into the
body and has an average volume of distribution of 10 L for both enantiomers (Albert
and Gemaat, 1984; Lee et al., 1985).
Both R(-) and S(+)-ibuprofen isomers diffuse slowly into synovial fluid.
Concentrations in synovial fluid fluctuate much less and remain higher than those in
plasma. The mean ratio of the total concentration in synovial fluid to that in plasma
seven hours after dosing is about 1.25 (Netter et al., 1989). Studies by Day et al. (1988)
on the stereoselective disposition of ibuprofen enantiomers in synovial fluid showed
that concentrations of the active S(+) enantiomer in synovial fluid were always larger
than those of the R(-) isomer.
Ibuprofen and its metabolites pass easily across the placenta and they appear in
the milk of lactating women at approximately 1% of the maternal plasma concentration
(Albert and Gernaat, 1984).
Metabolism
Ibuprofen is extensively metabolized in the liver by hydroxylation,
carboxylation and glucuronidation (see Figure 2-14). Major metabolites are generated
by addition of an hydroxy group at the 2 position to form 2-[4-(2-hydroxy-2-
methylpropyl)phenyl] propionic acid, or by oxidation of a methyl substituent to the
corresponding carboxylic acid to yield 2-[4-(2-carboxypropyl) phenyl] propionic acid
(Adams et al., 1967; Mills et al., 1973). Formation of ester glucuronides conjugates is
stereoselective and favors the S(+) enantiomer of ibuprofen (Lee et al., 1985).
Ibuprofen was the first 2-arylpropionic acid derivative shown to undergo a
metabolic chiral inversion of the inactive R(-)-isomer to its pharmacologically active
S(+)-antipode and, subsequently, has become the most studied compound of this


53
Chromatographic conditions
Reversed phase chromatography was performed at room temperature. The
mobile phase consisted of a mixture of 0.05 M phosphate buffer and acetonitrile (83:17,
v:v). 1-octanesulfonic acid (0.005 M) was added as an ion-pairing reagent. The pH was
adjusted to 4.9 by addition of 85?f> phosphoric acid. The mobile phase was filtered
through a 0.2 pm filter and degassed by sonication before use. The flow rate was
1.2 mL/min. Fluorescence detection was performed with excitation and emission
wavelengths of 285 nm and 345 nm, respectively.
Ibuprofen Analysis
Ibuprofen was assayed in plasma by a reversed phase liquid chromatographic
assay procedure.
Chemicals and reagents
Ibuprofen and the internal standard, flurbiprofen, were provided by Boots
Pharmaceuticals (Shreveport, Louisiana, USA), and used as received.
HPLC-grade acetonitrile and all other reagents (analytical grade) were
purchased from Fisher Scientific (Fair Lawn, New Jersey, USA).
Instrumentation
The high-performance liquid chromatography system consisted of a
Constametric 1I1G high pressure pump (LDC Milton-Roy, Riviera Beach, Florida, USA),
a manual injection valve with a 50 pL loop (Negretti and Zambra, Southampton, UK), a
15 cm x 4.6 mm I.D., 5 pm particle size, Zorbax octadecylsilane column (DuPont
Instruments, Wilmington, Delaware, USA), and a variable-wavelength UV detector
(Spectromonitor D, LDC Milton Roy). A guard column filled with Zorbax
octadecylsilane packing material was placed before the analytical column.
Chromatograms were recorded on a Hewlett-Packard integrator (model 3392A, Palo
Alto, California, USA).


APPENDIX
TPEP TOOLKIT PROGRAM
TPEP Toolkit is a computer program designed to process evoked potential data
collected by the Bio-logic Brain Atlas System. TPEP Toolkit reads and processes ASCII
files generated by the Bio-logic Brain Atlas System, and creates new ASCII files (with a
CSV extension) containing 2 to 10 columns. Brain Atlas ASCII files are created from
Biologic Brain Atlas binary files (DAT extension) through bank mathematics (bank math
menu item in the map analysis program).
Figure A-l. TPEP Toolkit: User interface.
120


119
The second part of this research dealt with the evaluation of the analgesic
efficacy of codeine and ibuprofen when given alone or in a combination. Three different
pharmacodynamic parameters were used to compare the analgesic efficacy of the
different analgesics: a pain intensity analog scale, a pam relief analog scale, and tooth
pulp evoked potentials.
First, a method was developed to obtain and record tooth pulp evoked
potentials in humans in the laboratory. A safe and reliable dental stimulator was
designed to deliver painful electrical stimuli to the tooth pulp. By using an individual
impression of one's upper front teeth, we improved on currently available methods for
obtaining tooth pulp evoked potentials. The main advantage of this technique over
existing methods lies in the small number of EEG epochs necessary to obtain a
consistent waveform, thus minimizing the effects of habituation. In addition, the
stimulus can be repetitively administered over extended periods of time, without
significant deterioration in the evoked potential. This method was used successfully to
evaluate the analgesic efficacy of codeine and ibuprofen when given alone or in a
combination.
Results of the pharmacodynamic study indicated that the analgesic effect (i.e.
area under the effect-time curve) of ibuprofen and/or codeine was significantly larger
than placebo. Active drugs, however could not be distinguished from one another. The
analgesia produced by weak analgesics has always been difficult to detect in small
numbers of subjects. Nevertheless, evoked potential methods, and in particular tooth
pulp evoked potentials, are extremely sensitive and may allow the use of smaller
groups of subjects than are needed with other pain assessment methods.
The dose-effect relationship of ibuprofen and codeine when given alone or in
combination could not be determined. Further studies with smaller doses or a larger
number of effect measurements are needed to better characterize these relationships.


8
respond to tissue damage caused by mechanical, thermal, or chemical stimuli. In
addition, they respond to chemical mediators formed or released as a result of tissue
damage. These impulses are transmitted more slowly than impulses from the
mechanothermal receptors and travel along unmyelinated C type nerve fibers. They are
responsible for "second" or "slow" pain after injury (slower in onset, prolonged, dull,
aching, and poorly localized).
Table 2-1. Properties of different types of afferent fibers involved in conducting
nociceptive impulses.
AS
Fiber type
C
Myelination
yes
no
Diameter
1-5 pm
0.25-1.5 pm
Velocity
5-30 m/s
1.0-2.5 m/s
Threshold
high
high
Stimuli
light pressure, heavy pressure
heat (> 45C), chemicals, cold
light pressure, heavy pressure
heat (> 45C), chemicals, warmth
While A5 nociceptors have a constant threshold below which stimuli are not
perceived, C fibers respond to a wide range of stimulus intensities, and both types of
fibers appear to have a role in coding pain intensity. However, it is the variation in the
tolerance of pain at the level of the central nervous system, not the threshold at the
periphery, which accounts for the wide variations in analgesic requirements among
patients (Bonica, 1990b).
Central pain pathways
Both AS and C afferent fibers enter the spinal cord through the dorsal root. After
entering the spinal cord, the majority of A5 and C fibers terminate superficially in the
grey matter of the dorsal horn which is arranged in a series of laminae. It is in the spinal
cord that the first processing of painful stimuli occurs. The AS fibers terminate in


104
Pharmacokinetic Measurements
To ensure reliable determination of drug levels, the pharmacokinetic parameters
of codeine and ibuprofen were also estimated in the subjects participating in the
pharmacodynamic study. They were determined by compartmental analysis after
fitting the concentration data to a bi-exponential equation corresponding to a one
compartment model with first-order input. They were compared against values
determined in the preceding pharmacokinetic study.
Tables 4-9 and 4-10 summarize the pharmacokinetic parameters obtained after
curve fitting. Figures 4-22 and 4-23 present the fitted log plasma concentration-time
curves for ibuprofen following administration of ibuprofen alone and of the ibuprofen
codeine combination. Figures 4-24 and 4-25 present the fitted log plasma concentration
time curves for codeine following administration of codeine alone and of the ibuprofen
codeine combination.
From the figures it can be observed that the disposition profiles of ibuprofen and
codeine were not significantly different from the ones observed previously. Absorption
and distribution were complete with maximum plasma concentrations observed at one
to two hours after dosing in all the subjects, thus indicating a fairly rapid absorption
from the gastrointestinal tract. Elimination half lives were similar to the ones
determined in the pharmacokinetic study.
Statistical analysis of the data revealed no significant differences between the
pharmacokinetic parameters of codeine and ibuprofen obtained by model-dependent
and model-independent data analysis techniques.


47
6-glucuronidation Codeine-6-glucuronide O-demethylation
-6-glucuronidation N Morphine-6-glucuronide
Codeine ^O-demethylation--] Morphine i3-glucuronidation] Morphine-3-glucuronide
N-demethylation
Normorphine
N-demethylationNNorcodeinei6-glucuromdation-HNorcodeine-6-glucuronide
Figure 2-17. Codeine metabolism in humans.
Adapted from Sindrup et al. (1991)
The hepatic biotransformation of codeine to morphine led many to believe that
codeine may exert its analgesic effect through partial conversion to morphine (Adler
and Latham, 1950; Findlay et al., 1978; Yue et al., 1). This assumption was supported by
the low affinity of codeine for the p opiate receptor and by the marked in vivo analgesic
efficacy of morphine, normorphine, morphine-6-glucuronide, and norcodeine (Lasagna
and Komfeld, 1958; Osborne et al., 1988). However, studies by Quiding et al. (1986) and
Shah and Mason (1990) questioned the role of morphine on the analgesic effect of
codeine because of the very low plasma concentrations of morphine present after single
or repeated doses of codeine. In a more recent study, Chen et al. (1990) observed
morphine formation in the brain of rats and suggested that the analgesic effect of
codeine may stem from morphine formed in the brain. Yue et al. (1989, 1991a, 1991b)
and Sindrup et al. (1991) later confirmed this hypothesis by investigating the analgesic
efficacy of codeine in extensive and poor metabolizers. As a result of a genetic
polymorphism of a particular hepatic cytochrome P450, 6% to 10% of the white
population are classified as poor metabolizers and are virtually unable to demethylate
codeine to morphine. Their results indicated that the analgesic efficacy of codeine in
poor metabolizers was either absent or weaker than in extensive metabolizers, thus


81
Table 4-5. Pharmacokinetic parameters for ibuprofen given in combination with
codeine (mean standard deviation).
Ibuprofen 400 mg and codeine
60 mg as two tablets
Ibuprofen 400 mg and codeine
60 mg as one tablet
Cmax (Hg/mL)
29.8 12.4
34.9 1 8.9
W (h)
1.8911.42
1.09 1 0.55
AUC. (pg-h/mL)
98.0 32.3
104.21 29.8
AUMQ (pg-h2/mL)
429.3 217.3
356.21129.1
MRT (h)
4.32 1.60
3.39 10.61
ke (h-i)
0.347 + 0.101
0.342 1 0.078
V2 (h)
2.19 0.72
2.121 0.44
CU (L/h)
4.5911.87
4.1411.12


87
Table 4-7. Pharmacokinetic parameters for codeine given in combination with
ibuprofen (mean standard deviation).
Ibuprofen 400 mg and codeine
30 mg as two tablets
Ibuprofen 400 mg and codeine
30 mg as one tablet
Cmax (ng/mL)
63.5 17.5
69.0 22.5
tmax (h)
1.1910.44
1.0210.35
AUCm (ng-h/ml)
242.1 54.6
241.9 1 60.8
AUMC^ (ng h2/mL)
1002.2 323.9
992.5 1 349.5
MRT (h)
4.10 0.68
4.09 10.81
K (h-1)
0.294 0.051
0.2921 0.055
k2 (h)
2.43 0.45
2.46 10.50
Clpo (L/h)
139.2 1 39.6
137.1143.0


30
inter-stimulus intervals do not significantly affect latencies of the averaged evoked
potential components so long as the inter-stimulus intervals are greater than 2 seconds
(Jacobson et al., 1985).
MECHANICAL SIIMUIAIION
ELECTRICAL STIMULATION
l/uV)
51" -/vn/vA/AaT'~/'
EEG
n -40
Figure 2-10. Evoked potentials obtained with mechanical and electrical stimulation
illustrating differences in cerebral responses to stimuli of increasing
intensity (K1 and K2 denote faint and mild tactile sensations, K3 and K4
faint and mild pain).
From Bromm and Scharein (1982b).
Effect of Analgesics on Evoked Potentials
The effects of a wide variety of analgesics on evoked potentials have been
studied by a number of investigators. Early studies suggested consistent and significant
changes in evoked potential waveforms when an analgesic treatment is administered. It
has been established that the dental P250 amplitude is proportional to pain intensity
and decreases when pain is attenuated by various therapeutic treatments. Waveform
comparisons and the consistency of findings suggest the reliability and consistency of
the evoked potential technique. Minor variations in site of stimulation and method of


20
Figure 2-6. Computer averaging of evoked potentials.
The top line (a) shows a continuous EEG recording; Six 300 ms EEG
epochs taken after stimulation are arranged vertically with the same time
scale (b) or with an expanded time scale (c); The samples are averaged in
(d) and (e) to enhance features common to the individual responses.
From Spehlmann (1981).


133
Fessler RG (1988). Physiology, anatomy, and pharmacology of pain perception. In
Assessing chronic pain: A multidisciplinary clinic handbook. Edited by PM Camic
and FD Brown. Springer-Verlag, New York, pp. 5-19.
Fields HL (1984). Neurophysiology of pain and pain modulation. Am. J. Med., 77:2-8.
Findlay jWA, Butz RF, and Welch RM (1977). Codeine kinetics as determined by
radioimmunoassay. Clin. Pharmacol. Ther., 22:439-446.
Findlay JWA, Jones EC, Butz RF, and Welch RM (1978). Plasma codeine and morphine
concentrations after therapeutic oral doses of codeine-containing analgesics. Clin.
Pharmacol. Ther., 24:60-68.
Findlay JWA, Fowle ASE, Butz RF, Jones EC, Weatherley BC, Welch RM, and Posner J
(1986). Comparative disposition of codeine and pholcodine in man after single oral
doses. Br. J. Clin. Pharmacol., 22:61-65.
Flower RJ, Moneada S, and Vane JR (1985). Analgesic-antipyretics and anti
inflammatory agents; Drugs employed in the treatment of gout. In The
pharmacological basis of therapeutics. Edited by AG Gilman, LS Goodman, TW Rail,
and F Murad. Seventh edition. Macmillan Publishing, New York, pp. 674-715.
Forbes JA, Barkaszi AB, Ragland RN, and Hankie JJ (1984a). Analgesic effect of
acetaminophen, phenyltoxolamine, and their combination in postoperative oral
surgery pain. Pharmacotherapy, 4:221-226.
Forbes JA, Keller CK, Smith JW, Zeleznock JR, Sevelius H, and Beaver WT (1986).
Analgesic effect of naproxen sodium, codeine, a naproxen-codeine combination, and
aspirin on the postoperative pain of oral surgery. Pharmacotherapy, 6:211-218.
Forbes JA, Kolodny AL, Chachich BM, and Beaver WT (1984b). Nalbuphine,
acetaminophen, and their combination in postoperative pain. Clin. Pharmacol. Ther.,
35:843-851.
Frame JW, Fisher SE, Pickvance NJ, and Skene AM (1986). A double-blind placebo-
controlled comparison of three ibuprofen/codeine combinations and aspirin. Br. /.
Oral Maxillofac. Surg., 24:122-129.
Friedman H, Seckman C, Stubbs C, Oster H and Royer G (1990). Multiple-dose safety
study of ibuprofen/codeine and aspirin/codeine combinations. /. Clin. Pharmacol.,
30:64-69.
Garrett ER and Gurkan T (1978). Pharmacokinetics of morphine and its surrogates I:
comparison of sensitive assays of morphine in biological fluids and application to
morphine pharmacokinetics in the dog. /. Pharm. Sci., 67:1512-1517.
Gehrig JD, Colpitts YH, and Chapman CR (1981). Effects of local anesthetic infiltration
on brain potentials evoked by painful dental stimulation. Anesth. Analg., 60:779-782.
Geisslinger G., Stock KP, Bach GL, Loew D, and Brue K (1989). The pharmacokinetics
of ibuprofen in man. Eur. j. Clin. Pharmacol., 36[Suppl]:A181.
Gibaldi M and Perrier D (1982). Pharmacokinetics. Marcel Dekker, New York.


113
for which indirect or subjective indexes of effect are often employed. Measurement of
subjective responses such as pain intensity or pain relief are subject to many variables.


41
observed a stereoselective uptake of R(-)-ibuprofen into "hybrid triglycerides" in rats.
Since coenzyme A thioester of carboxylic acids can replace the natural fatty acids in
triacylglycerols to form toxic "hybrid triglycerides", these findings confirmed the
stereospecific role of coenzyme A thioester formation in the inversion of R(-)-ibuprofen
(Hutt and Caldwell, 1983). In contrast, S(+)-ibuprofen is not significantly incorporated
into adipose tissue following chronic treatment with this enantiomer.
R(-)-ibuprofen -CoASH R(-)-ibuprofen-S-CoA
Racemase
I
V
S(+)-ibuprofen 4 ¡ S(+)-ibuprofen-S-CoA
CoASH 41
Figure 2-15. Mechanism of the chiral inversion of ibuprofen in man.
The enzyme system responsible for this inversion has not been clearly defined
yet. However, different investigators suggested either presystemic inversion in the
gastrointestinal tract and/or systemic inversion in the liver as possible sites for this
metabolism (Cox, 1988; Cox et al., 1985,1988; Jamali, 1988; Jamali et al., 1988; Knadler
and Hall, 1989; Mehvar and Jamali, 1988). The metabolic system responsible for this
inversion also seems to be present in the rectum as substantial differences have also
been noticed between the enantiomers plasma concentrations after administration of
ibuprofen suppositories (Jamali, 1988). An average of 63% of an administered dose of
R(-) ibuprofen is stereospecifically inverted to the S(+) enantiomer (Lee et al., 1985).
Elimination
Ibuprofen is eliminated primarily via the kidneys into the urine. More than 80%
of an ibuprofen dose is recovered in the urine after 24 hours, primarily as conjugated


58
Experimental Design
This study was a double-blind, placebo controlled, four-way cross-over study
comparing the pharmacodynamic effect (i.e. analgesia) of codeine and ibuprofen when
given alone or in combination. Upon entermg the study, subjects were randomly
assigned to one of four dosing groups:
1. Ibuprofen 400 mg
2. Codeine 60 mg
3. Codeine 30 mg and ibuprofen 400 mg
4. Placebo
Each dose consisted of two tablets and one capsule that were matched for color
and appearance. After a two-day washout period, subjects were crossed over to receive
the remaining study drugs that they had not previously received.
Subjects
Eight healthy male students (ages 23-32) participated in this series of
experiments as paid volunteers. They were free of neurological disease and were fully
informed about the investigational procedure and the intake of analgesic drugs. They
were screened for medical contraindications and for analgesic use and abuse.
Before testing, all subjects participated in a training session involving
familiarization with the stimulation and recording procedures. No placebo or drug was
given. Subjects were informed of the general objective of the study and signed written
informed consents. All procedures were subjected to review and approval by the
University of Florida Health Center Institutional Review Board, and by the Veterans
Administration Subcommittee for Clinical Investigation.
Drug Administration
Subjects did not eat for at least two hours before dosing, but liquids were
allowed. Immediately after threshold determination, a single oral dose of the test drug


72
Under the conditions described, ibuprofen and flurbiprofen were well separated
from each other and from endogenous plasma components. Their retention times were
4.2 and 3.2 minutes, respectively. Typical chromatograms of blank plasma and plasma
spiked with ibuprofen obtained after extraction are shown in Figure 4-1.
Linearity
Standard curves were established by spiking drug-free plasma with ibuprofen
and the internal standard (flurbiprofen). They were analyzed by linear regression. They
were linear over the concentration range expected in plasma (0 to 100 pg/mL) with
correlation coefficients larger than 0.990.
Reproducibility
The accuracy and reproducibility of the method was investigated by spiking
drug-free plasma with different concentrations of ibuprofen. Five replicates of each
concentration were assayed. Table 4-1 shows that the reproducibility in plasma is good
with coefficients of variation of less than 10% in the low concentration range (10 pg/mL
and below), and of about 5% in the higher concentration range (above 10 pg/mL).
The recovery for ibuprofen after extraction from plasma using this method was
determined to be about 40%.
Codeine Analysis
Various analytical methods have been reported for the determination of codeine
in biological fluids. Among these, gas-liquid chromatographic methods have often been
used to quantify codeine in various media (Cone et al., 1983; Edlund, 1981; Zweidinger
et al., 1976), sometimes in combination with mass-spectrometry (Quiding et al., 1986).
Several high-performance liquid chromatographic methods have also been described
using either reversed-phase (Nitsche and Mascher, 1984; Tsina et al., 1982; Visser et al.,
1983) or ion-pairing techniques (Ginman et al., 1985; Kubiak and Munson, 1980).


Concentration (ng/mL)
89
Figure 4-8. Plasma concentrations of codeine (mean standard deviation).
Codeine 60 mg in one tablet
A Codeine 30 mg and ibuprofen 400 mg in two separate tablets
A Codeine 30 mg and ibuprofen 400 mg in one combination tablet
O Codeine 60 mg and ibuprofen 400 mg in two separate tablets
Codeine 60 mg and ibuprofen 400 mg in one combination tablet


4
To compare the pharmacokinetics of ibuprofen and codeine when given alone or in
combination.
To compare the pharmacodynamics (analgesia) of ibuprofen and codeine when
given alone or in combination.
To define the dose-effect relationship of ibuprofen and codeine when given alone or
in combination.
The ultimate objective of these studies is to better understand the dose/effect
relationship of analgesic drugs to provide patients with better pain relief.


19
Evoked Potentials in Pain Research
Evoked Potentials
Evoked potentials are event-related electrical signals of brain activity that can be
recorded from the scalp when precisely controlled stimuli are delivered. The
appearance of an evoked cerebral potential then gives evidence that the specific sensory
system of the brain does receive the activated peripheral impulse pattern elicited by the
applied stimulus (Chiappa, 1990; Spehlmann, 1985).
Evoked potentials consist of a series of peaks or waves, each characterized by
their polarity (positive or negative), latency (in milliseconds), and amplitude (in
microvolts). They are obtained by processing the electroencephalographic signals (EEG)
that occur following stimulation.
The basic problem in evoked potential recording is to distinguish the evoked
activity (signal) from the ongoing electrical activity of the brain (noise). This is done by
computer-assisted signal processing techniques, most often using methods that sum or
average successive responses to iterative simulation (see Figure 2-6). In this way,
potentials that are time-locked to the stimulus will be enhanced, while events not in
phase with the stimulus will be reduced (i.e. background EEG activity, artifacts,
instrumentation noise).
The number of EEG epochs to be averaged depends on the ratio of the
amplitude of the stimulus-locked components in the recording (signal) to the amplitude
of the unrelated components (noise). Because averaging procedures reduce the noise,
the signal to noise ratio improves with the number of responses averaged (see Figure
2-6). However, this relationship is not linear: the ratio of evoked potential signal to
on-going EEG signal ("noise") increases as the square root of the number of repetitions
(Chiappa, 1990).


92
In summary, all the pharmacokinetic parameters studied were equivalent with
respect to the different formulations as compared to the control drug. Although one
formulation (ibuprofen 400 mg and codeine 60 mg in one combination tablet) displayed
a significantly faster absorption rate than some other dosage forms, the difference is
probably not clinically significant. No relevant effect of codeine on the disposition of
ibuprofen, and of ibuprofen and the disposition of codeine could be found.
Pharmacodynamic Study
The objective of this double-blind, placebo controlled study was to compare the
pharmacodynamic effect (i.e. analgesia) of single oral doses of codeine and ibuprofen
when given alone or in combination. Eight healthy male students (ages 23-32, mean
weight 72.1 kg 7.2, mean height 1.78 m 0.09) participated in this series of
experiments. One subject was eliminated after initial screening according to study
criteria and another subject's data was not included m the analysis due to difficulties
encountered in resolving the evoked signal from the background EEG.
Four treatments were investigated: Ibuprofen 400 mg, codeine 60 mg an
ibuprofen-codeine combination (400 mg and 30 mg respectively), and placebo.
Dental Stimulation
The design of a rehable dental stimulation device for use in human pain research
presents special problems. Intact enamel has a very high resistivity and its thickness
may be as much as 1.5 mm in the stimulation area (Matthews and Searle, 1976).
Consequently, a number of dental stimulators have been described in the literature
(Martin and Chapman, 1979; Matthews and Searle, 1976; Rohdewald and Keuth, 1988).
Each design met with varying degrees of success, with contact degradation being one of
the most common problems encountered. Therefore, several prototypes were developed
to improve the quality of the contact between tooth enamel and electrodes. Many


25
it is evident that sensory systems other than pain are activated by cutaneous electrical
stimuli. Finally, low intensity electrical stimulation may recruit activity of non
nociceptive Ap nerve fibers. Therefore, evoked potentials elicited by cutaneous electrical
stimulation, like those produced by thermal and mechanical stimulation, are subject to
contammation by sensory modalities other than pain (Chudler and Dong, 1983).
Tooth Pulp Evoked Potentials
The tooth pulp has been considered by many to be the ideal site for the study of
pain. Its use in pain research is based upon the observation that pain is the only
sensation produced by stimulating intra-dental (pulpal) nerves.
Neuroanatomical evidence indicates that tooth pulp is innervated almost
exclusively by small myelinated A8 and unmyelinated C fibers originating in the
trigeminal ganglion (Matthews, 1979; Mumford and Bowsher, 1976; Sessle, 1979). The
unmyelinated fibers comprise 50 to 75% of the total sensory innervation. Hence, it is not
surprising that electrical tooth pulp stimulation elicits primarily, if not solely, painful
sensations (Azerad and Woda, 1977; Matthews and Searle, 1976). "Pre-pain" sensations
have also been reported following near-threshold electrical stimulation, indicating that
tooth pulp is innervated not only by A5 and C nociceptive afferents, but also by a small
number of large diameter Ap afferents. (Azerad and Woda, 1977)
Only a few experiments have described the quality of the sensation evoked by
electrical stimulation of the human tooth pulp (Azerad and Woda, 1977; Chatrian et al.,
1982). It has now become apparent, however, that low intensity electrical tooth pulp
stimulation results in a "pre-pain" sensation (sometimes described as a pure prick that
is not painful). Higher stimulus intensities cause sharp pain, and the highest stimulus
intensities cause an unpleasant dull ache (Azerad and Woda, 1977; Greenwood et al.,
1972; Matthews, 1979). Because single fiber studies in animals have shown that Ap
fibers are more sensitive than A8 fibers, and that C fibers are the least sensitive, it is


3
Although much has been done during recent years to establish models that
describe relationships between drug concentrations in the body and pharmacological
effects (Colburn, 1981,1987, 1988; Colburn and Brazzell, 1986; Holford and Sheiner,
1981,1982), only limited information exists on such relationships for analgesics (Chay et
al, 1992; Inturrisi and Colburn, 1986; Laska et al., 1986b; Keuth and Rohdewald, 1989).
Several clinical trials have demonstrated that the mean analgesic responses of patients
with pain vary as a function of the logarithm of the dose (Inturrisi et al., 1987,1990;
Inturrisi and Colburn, 1986; Velagapudi et al., 1990,1991). To provide a basis for more
rational drug therapy, however, it is also necessary to consider the very important
relationship between the kinetics of the drug in the body and the time course of the
pharmacological effects produced.
The present study was designed to further evaluate the efficacy of ibuprofen and
codeine as analgesics and to investigate the relationship between drug concentrations
and effect as a function of time. The ability to concurrently measure drug concentrations
in biological fluids and pharmacodynamic effects provides the opportunity to explore
the relationship existing between the disposition of ibuprofen and codeine and their
analgesic effect. The objective of these studies is the development of a method for
defining this relationship in quantitative terms in order to allow predictions of
pharmacodynamic effects and development of optimal dosing regimens.
In addition, this study assesses the feasibility of an lbuprofen-codeine
combination. Despite the effectiveness of the new generation of peripherally acting
analgesics, these drugs do not provide the same qualitative effects as the narcotic
analgesics. Therefore, a combination could be useful in treating pain when a centrally
mediated analgesic effect is desirable.
The objectives of this study were
To improve available methods for obtaining tooth pulp evoked potentials.


45
Although the exact mechanism of action of codeine is still unknown, important
advances in the understanding of the mode of action of opioids have been made with
the discovery of opiate receptors and endogenous opioid peptides. Opioids agonists
produce analgesia and other effects by binding to specific receptors in the brain and
spinal cord. At the spinal cord level, opioids impair or inhibit the transmission of
nociceptive input from the periphery to the central nervous system in a dose-related
manner. At the level of the basal ganglia, opioids activate a descending inhibitory
system that modulates peripheral nociceptive input at the spinal cord level. Opioids
also alter the emotional response to pain by acting on the limbic system (Benedetti and
Butler, 1990; Jaffe and Martin, 1985; McEvoy, 1990). Redent evidence indicates that the
major mechanism of analgesia after oral, mtramuscular, or intravenous administration
is the activation of the descending anti-nociceptive system. The activation of opioid
receptors in the substantia gelatinosa requires higher opioid concentrations, and may
only be attained after intraspinal administration (Benedetti and Butler, 1990).
Other central effects of codeine include nausea and vomiting by direct
stimulation of the chemoreceptor trigger zone. Codeine also depresses respiration by
decreasing the responsiveness of the medullary respiratory center to carbon dioxide
tension. Similarly, codeine suppresses the cough reflex by a direct effect on the cough
center in the medulla of the brain. It also appears to exert a drying effect on respiratory
tract mucosa, and to increase viscosity of bronchial secretions (Benedetti and Butler,
1990; Jaffe and Martin, 1985; McEvoy, 1990; Way and Adler, 1962).
At the gastrointestinal level, codeine increases smooth muscle tone and
decreases propulsive peristaltic contractions, thereby causing constipation. It also
causes a marked increase in pressure in the biliary tract, especially of the sphincter of
Oddi (Benedetti and Butler, 1990; Jaffe and Martin, 1985). Other effects of codeine
include miosis, urinary retention, and peripheral vasodilatation.



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S(+)-isomer (Adams et al., 1967, 1976). The R(-)-isomer has very little, if any, anti
inflammatory activity and serves no therapeutic purpose other than as a prodrug for
the active S(+) isomer.
Pharmacology
Ibuprofen has pharmacologic actions similar to those of other non-steroidal anti
inflammatory drugs such as aspirin, phenylbutazone, and indomethacin. Ibuprofen has
shown anti-inflammatory, antipyretic, and analgesic activity in both animals and
humans (Flower et al., 1985). Higher doses are required for anti-inflammatory effects
than for analgesia.
The exact mechanism of action of ibuprofen is not known. The currently held
view is that the analgesic and anti-inflammatory effects of non-steroidal anti
inflammatory drugs result primarily from their reversible inhibition of cyclooxygenase,
an enzyme responsible for the biosynthesis of prostaglandins (Benedetti and Butler,
1990; Flower et al., 1985; Kantor, 1979; McEvoy, 1990).
Figure 2-13. Outline of arachidonic acid metabolism.


14
Categorical rating scales
One of the simplest way of assessing the pain experience is the categorical rating
scale. This type of scale generally consists of a series of words (e.g. none, mild,
moderate, and severe) that are used to measure pain intensity or relief (see Figure 2-4).
The patient is then asked to estimate the intensity of his subjective pain experience by
selecting a category.
4-point Pain Intensity Category Scale
0
1
1
1
2
I
3
1
1
None
1
Mild
1
Moderate
1
Severe
5-point Pain Relief Category Scale
0
I
1
i
2
I
3
I
4
I
1
None
1
Mild
1
Moderate
1
Lots
1
Complete
Figure 2-4. Examples of categorical pain intensity and pain relief scales.
To assess the magnitude of the analgesic effect, several measures derived from
individual pain scores can be calculated. Most researchers assume equidistant
categories, and assign the numerical values of 0 for none, 1 for mild, 2 for moderate,
and 3 for severe. To account for differences in baseline pain intensity among patients,
pain intensity scores are first converted into "pain intensity difference" (PID) scores by
subtracting them from the pain score taken at baseline:
PID = PI0 PIt
where PID is the pain intensity difference, PIt is the pain intensity at time t, and PI0 the
pain intensity at baseline. Positive scores indicate reduction in pain, thus making the
pain intensity difference scores analogous to pain relief scores.


13
clinical pain is essentially characterized by an aversive, emotional component which is
much more difficult to measure. In most cases, pain has a source somewhere in the
nociceptive pathways including central centers of pain processing, so that both
components of pain interact and influence each other (Bromm, 1984a).
Generally, responses to pain may be separated into three types: somatic reflex
reactions, autonomic nervous system reactions, and sensory reactions that depend upon
a high level of integrative central nervous system processing (Chapman and Syrjala,
1990). The first two types of reactions fall mainly into the category of involuntary
responses, whereas the last type tends to involve voluntary responses. Thus, obtaining
a meaningful, reliable, and objective measure of pain is difficult.
There have been two major approaches to quantifying pain. The first centers on
quantifying the subjective reports of patients. This can provide information on the
cognitive and affective, as well as sensory, dimensions of pain. The second approach
uses physiologic correlates of pain states. The most successful of these attempts has
been with evoked potentials. These methods all consist of noninvasive means of
observing the magnitude of pain from manipulation of external parameters (Syrjala and
Chapman, 1984).
Unidimensional Pain Rating Scales
Although the multidimensional nature of pain has been widely recognized, the
most common approach to pain assessment continues to be the unidimensional self-
report scale, on which patients provide a rating of pain intensity or pain relief.
Traditionally, studies of the human pain experience in the laboratory have relied on
categorical and visual analog scales (Bromm, 1984a; Buchsbaum et al, 1981a; Chapman
and Syrjala, 1990; Syrjala and Chapman, 1984).


57
a log-linear plot of the individual plasma concentrations versus time. The area under
the curve (AUC.J, the area under the first moment curve (AUMQJ, the mean residence
time (MRT), the half-life (tV2), and the apparent oral clearance (Cl ) were calculated
according to the following equations:
In 2
Statistical Analysis
Statistical comparisons of each pharmacokinetic parameter were performed
using two-way analysis of variance (ANOVA) with subject, sequence, and treatment as
factors. In cases where treatment effects were significant (p < 0.05), pairwise
comparisons were evaluated with Tukey's multiple range test (Bolton, 1984).
Pharmacodynamic Study
The aim of this study was to compare the pharmacodynamic effect (i.e.
analgesia) of single oral doses of codeine and ibuprofen when given alone or in
combination.


22
Far-field evoked potentials are those recorded at a distance from the neural
generator. Near-field potentials are recorded from electrodes close to the neural
structures from which they arise. Short-latency potentials reflect activity in sensory
receptors and subcortical sensory afferent pathways. When recorded from the scalp,
these potentials are in the far field. Near-field evoked potentials, observed in scalp
recordings between 20 and 80 milliseconds after onset, are called intermediate-latency
potentials. They reflect the arrival of sensory information at primary sensory areas of
the cerebral cortex. Late cortical event-related potentials occur between 80 and
500 milliseconds after stimulus onset. It is generally assumed that they reflect the
processed event, i.e. appreciation, interpretation, and meaning associated with the
stimulus, rather than simple event detection. Late near-field recordings, sensitive to
attention and expectation, are of behavioral and perceptual interest (Cruccu et al., 1983;
Haider, 1967; Leandri et al., 1985; Rosenfeld et al., 1985). They depend on consciousness
and their recording can be seriously affected by synchrony in the EEG such as
prominent alpha rhythm. For example, there is an inverse relationship between the
amount of alpha activity present in the pre-stimulus EEG and the peak amplitude of the
late cortical event-related potentials (Bromm, 1984a; Bromm and Scharein, 1982a).
Contemporary work on pain and evoked potentials is predominantly concerned
with late cortical event-related responses (latencies between 80 and 500 ms). Verbal
reports of subjects rarely reflect pure sensations, but rather include a mixture of
sensation, emotional reaction and interpretation. Similarly, late cortical responses are
supposed to reflect these higher processes and the pain sensation as felt by the subject
(Chapman et al., 1979).
Late cortical evoked potentials are used in pain research. Correlations between
subjective pain report and various components of evoked potentials have been reported
(Bromm, 1984a, 1985; Chapman et al., 1979; Carmon et al., 1978; Chen et al., 1979;
Chudler and Dong, 1983; Fernandes de Lima et al., 1982; Harkins and Chapman, 1978;


95
+ T
10.0 i^V
1.0 s
Figure 4-13. Resting electroencephalogram of subjects 1 (top) and 2 (bottom); Subjects
were alert and had eyes closed during the recording.


60
resistance of the contact between electrodes and tooth was minimized by coating the tip
of the electrode with a drop of electrolyte jelly (Matthews and Searle, 1974, 1976).
Figure 3-3. Dental form used for tooth pulp stimulation.
The electrical stimuli consisted of 0.5 millisecond square wave pulses applied
semi-randomly with an interstimulus interval in the range of 2 to 4 seconds (mean
3 seconds). They were generated by a Grass S48 Stimulator (Grass Instrument
Company, Quincy, Massachusetts, USA) with stimulus isolation (model SIU5, Grass
Instrument Company) and constant current units (Model CCU1A, Grass Instrument
Company).
The intensity of the current needed to evoke pain was based on the value of the
measured threshold of sensation (lowest current necessary to evoke a sensation). The
threshold of sensation was determined for each subject before drug intake and the
current intensity noted. This current intensity was then doubled and the resulting
painful stimulus was applied and kept constant throughout the course of the
experiment. The stimulated tooth was kept dry by wiping it with cotton followed by


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
li'. Qkj^ctQjJL
Hartmut Derendorf, Chair
Associate Professor of Pharmaceutics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Associate Professor of Pharmaceutics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Ikw Vi u ^
Hans Schreier
Associate Professor of Pharmaceutics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
This dissertation was submitted to the Graduate Faculty of the College of
Pharmacy and to the Graduate School and was accepted as partial fulfillment of the
requirements for the degree of Doctor of Philosophy.
December, 1992
Â¥
Dean, College of Pharmacy
Dean, Graduate School


123
Case 2: Channel = PZ
Case 3: Channel = ALL
End Select
End Sub
Sub BTN_Exit_Click ()
Temp% = WinHelp(hWnd, HelpFile, HELP_QUIT, 0)
End
End Sub
Sub BTN_Help_Click ()
Temp% = WinHelp(hWnd, HelpFile, HELP_CONTENTS, 0)
End Sub
Sub BTN_Rename_Click ()
' Exit if no file selected; Else rename Bio-logic file and update file list box
If Filel.Filename = "" Then
Beep: MsgBox "Cannot rename file: No file selected..."
Exit Sub
Else
RenameFile: Filel.Refresh
End If
End Sub
Sub BTN_RenameAll_Click ()
If Filel.ListCount > 0 Then
' Select and rename each file starting with a T
For LoopIndex% = 0 To Filel.ListCount -1
Filel.Listlndex = Looplndex%: RenameFile
Next Looplndex%
Filel.Refresh: Filel.Listlndex = 0
End If
End Sub
Sub BTN_Extract_Click ()
If Filel.Filename = "" Then
Beep: MsgBox "Cannot extract EEG channel(s): No file selected..."
Exit Sub
End If
Screen. Mousepoin ter = HOURGLASS
' Get Bio-logic ASCII filename
SourcePath = Dirl.Path
If Right$(SourcePath, 1) <> "\" Then SourcePath = SourcePath + "\"
BLFilename = SourcePath + Filel.Filename
' Read Fz, Cz, and Pz amplitudes from Bio-logic ASCII file
Readout.Caption = "Reading + UCase$(BLFilename) + "..."
Readout.Refresh: Gauge.FloodShowpct = TRUE
Open BLFilename For Input As #1


124
ReDim EPData(0 To 3, 0 To 255)
i% =0
Do While Not EOF(l)
EPData(0, i%) = i% 2 1 Epoch is 512 ms
Input #1, Vari, Var2, Var3, Var4, Var5, EPData(l, i%), Var
Input #1, Vari, Var2, Var3, EPData(2, i%), Var4, Var5, Var
Input #1, Vari, EPData(3, i%), Var2, Var3, Var4, Var5, Var
If i% Mod 8 = 0 Then
Gauge.FloodPercent = (i% 100 \ 255): Gauge.Refresh
End If
i% = i% + 1
Loop
Close #1
Gauge.FloodPercent = 0: Gauge.FloodShowpct = FALSE
' Create new filename by appending suffix at the end of existing filename
Select Case Channel
Case FZ
NewFilename = Mid$(BLFilename, 1, Len(BLFilename) -4) + "FZ.CSV"
Case CZ
NewFilename = Mid$(BLFilename, 1, Len(BLFilename) 4) + "CZ.CSV"
Case PZ
NewFilename = Mid$(BLFilename, 1, Len(BLFilename) 4) + "PZ.CSV"
Case ALL
NewFilename = Mid$(BLFilename, 1, Len(BLFilename) -4) + ".CSV"
End Select
' Write Fz, Cz, and/or Pz amplitudes to new file
Readout.Caption = "Writing + UCase$(NewFilename) + "..."
Read out. Refresh
Gauge. FloodShowpct = TRUE
Open NewFilename For Output As #2
If CHK_Labels. Value = TRUE Then 1 Write labels on first line of file
Select Case Channel
Case FZ
If CHK_Time.Value Then Write #2, "Time (ms)", "Fz (pV)"
Else Write #2, "Fz (pV)"
Case CZ
If CHK_Time.Value Then Write #2, "Time (ms)", "Cz (pV)"
Else Write #2, "Cz (pV)"
Case PZ
If CHK_Time.Value Then Write #2, "Time (ms)", "Pz (pV)"
Else Write #2, "Pz (pV)"
Case ALL
If CHK_Time.Value Then Write #2, "Time (ms)", "Fz(pV)", "Cz(pV)", "Pz(pV)"
Else Write #2, "Fz (pV)", "Cz (pV)", "Pz (pV)"
End Select
End If
For i% = 0 To 255 Write time, amplitude on other lines of file
Select Case Channel


102
Figure 4-20. Effect of treatment on tooth pulp evoked potentials before (left) and
90 minutes after drug administration (right).
Subject 5.
From top to bottom: placebo, ibuprofen, codeine, and ibuprofen-codeine.


56
They had no history of allergic reactions to codeine or ibuprofen. In compliance with
the Declaration of Helsinki specifying the rights of human subjects during clinical
studies, written informed consent was obtained from each participant.
Drug Administration
Subjects were fasted for at least eight hours overnight and could not drink water
two hours before dosing. Immediately after a standardized breakfast, a pre-dose blood
sample was drawn, and a single oral dose of drug was ingested with approximately
100 mL of water. Water was permitted two hours after dosing and subjects could
resume a normal living pattern no sooner than four hours following drug
administration. All doses were taken at approximately 8:00 AM. Subjects remained
seated during the first two hours following the dose of test medication.
Blood Sampling
Blood samples were collected immediately prior to dosing and at 0.5, 0.75,1,
1.25, 1.5, 1.75, 2, 3, 4, 6, 8, and 14 hours after drug administration by means of an
indwelling catheter placed in a convenient vein in the arm of the subject.
Blood samples (7.0 mL) were harvested into Venoject tubes containing 14 mg of
potassium oxalate and 17.5 mg of sodium fluoride (Terumo Medical, Elkton, Maryland,
USA). Samples were centrifuged, the plasma separated, frozen, and stored at -20C
until assayed for codeine and ibuprofen.
Estimation of Pharmacokinetic Parameters
The pharmacokinetic parameters of codeine and ibuprofen were determined for
each subject using standard non-compartmental data analysis techniques (Gibaldi and
Perrier, 1982). The maximum plasma concentration (Cmax) and the time to reach the
maximum plasma concentration (tmax) were obtained by direct interpretation of the
data. The overall elimination rate constant (ke) was obtained from the terminal slope of


32
Dental
Baseline
Fentanyl
0.1 mg.
Figure 2-11: Effect of fentanyl and its reversal after administration of naloxone on
tooth pulp evoked potentials.
From Butler et al. (1983).
Recently, Chapman and co-workers compared the analgesic effects of alfentanil,
fentanyl, and morphine on experimental pain (Chapman et al., 1988, 1990; Hill and
Chapman, 1989; Hill et al., 1986). Their results showed a reduction in evoked potential
amplitudes following drug administration, with marked variations in analgesic profiles
between drugs. They attributed these variations to the different physicochemical
characteristics (lipophilicity, ionization) of each drug.
Peripherally acting analgesics
Because of their weak analgesic efficacy, peripheral analgesics are more difficult
to evaluate than the more potent central analgesics. Evoked potentials have been used
to study the analgesic effect of aspirin. In their initial study, Chen and Chapman (1980)
reported that the late components of the dental evoked potential were reduced by
acetylsalicyiic acid (975 mg p.o.), as were pain reports during dental stimulation.
Derendorf et al. (1982, 1984) and Rohdewald et al. (1980, 1982a, 1982b, 1983)
confirmed these results in their studies on the effects of acetylsalicyiic acid,
acetaminophen, salicylamide, and metamizole on tooth pulp evoked potentials.


CHAPTER 4
RESULTS AND DISCUSSION
Analytical Procedures
Our objective was to develop simple, rapid, specific, and sensitive
chromatographic assay methods for the determmation of codeine and ibuprofen in
biological fluids.
Ibuprofen Analysis
Many different chromatographic methods have been published in the literature
for the determmation of ibuprofen in biological fluids. They include thin-layer
chromatography (Mills et al., 1973), gas chromatography (Mills et al., 1973), and
high-performance liquid chromatography (Cox et al., 1988; Ginman et al., 1985;
Lockwood and Wagner, 1982; Mehvar et al., 1988).
Our objective was to develop a simple, rapid, and specific high-performance
liquid chromatographic assay for the determmation of ibuprofen in plasma. We used a
modification of the method of Ginman et al. (1985).
Chromatography
The analysis was performed using reversed-phase liquid chromatography with
UV detection. Separation of ibuprofen from endogenous plasma components was
achieved with an octadecylsilane column. The mobile phase consisted of a mixture of
0.1 M phosphate buffer and acetonitrile. Detection and quantitation of ibuprofen was
performed by monitoring the UV absorbance of the eluent at 254 nm.
71


48
confirming the hypothesis that morphine formation is at least partly responsible for the
analgesic efficacy of codeine.
Elimination
Codeine is primarily excreted in urine as conjugated morphine and norcodeine
metabolites, with less than 10% of the administered dose excreted unchanged (Way and
Adler, 1968). While measurable amounts of unchanged morphine and norcodeine can
be recovered in urine after oral administration of large doses of codeine, only trace
amounts of these metabolites are excreted following therapeutic doses (Adler, 1958).
Renal elimination is rapid, with about two-third of the dose being recovered in the urine
six hours after drug administration (Adler et al., 1955). Negligible amounts of codeine
and its metabolites (< 1%) appear in the feces (Adler et al., 1955).
Codeine has an elimination half life of about two to three hours (Findlay et al.,
1977,1978; Quiding et al., 1986; Yue et al., 1991a, 1991b).
Toxicity
Codeine shares the toxic potentials of the opiate agonists. The most common
side effects observed after the administration of therapeutic doses of codeine include
lightheadedness, dizziness ,sedation, nausea, vomiting and sweating. Other adverse
reactions include euphoria, dysphoria, weakness, headache, insomnia, anorexia,
gastrointestinal distress, constipation, bradycardia, and urinary retention.
Severe overdosage with codeine is characterized by respiratory depression,
Chevne-Stokes respiration, cyanosis, extreme somnolence progressing to stupor or
coma, and sometimes bradycardia or hypotension. In severe overdosage, apnea,
circulatory collapse, cardiac arrest and death may occur. Codeine toxicity can be
successfully treated by intravenous administration of the narcotic antagonist naloxone
(Jaffe and Martin, 1985; McEvoy, 1990).


Abstract of Dissertation Presented to the Graduate School of the
University of Florida in Partial Fulfillment of the
Requirements for the Degree of
Doctor of Philosophy
PHARMACOKINETIC-PHARMACODYNAMIC MODELING OF THE
ANALGESIC EFFECT OF IBUPROFEN AND CODEINE
MATTHIEU L. KALTENBACH
December 1992
Chairman: Hartmut Derendorf, Ph.D.
Major Department: Pharmaceutics
Understanding the relationship between drug concentration and drug effect is
usually a prerequisite for designing an optimal drug therapy. In the case of analgesic
drugs, such pharmacokinetic-pharmacodynamic correlations have rarely been
described. Therefore, the aim of this study was to investigate the pharmacokinetics and
pharmacodynamics of ibuprofen and codeine when given alone or in combination,
using electrical tooth pulp stimulation as an experimental pain model.
The first part of this study investigated the pharmacokinetics of codeine and
ibuprofen when given alone or in combination. It consisted of a double-blind, cross-over
study, with twenty-four healthy subjects receiving oral doses of ibuprofen (400 mg),
codeine (60 mg), and four different codeine-ibuprofen combinations containing 400 mg
of ibuprofen with either 30 or 60 mg of codeine. Plasma concentrations were measured
by reversed-phase liquid chromatography with fluorescence and UV detection.
Pharmacokinetic parameters were estimated by non-compartmental data analysis. No


86
Table 4-6. Pharmacokinetic parameters for codeine given alone
(mean standard deviation).
codeine 60 mg p.o.
Cmax (ng/mL)
122.8 48.5
W (h)
1.31 0.57
AUC^, (ng-h/mL)
464.4 113.5
AUMCa (ng-h2/ mL)
2023.5 763.9
MRT (h)
4.25 1.00
ke (A1)
0.293 0.070
ti/2 (h)
2.50 0.61
Clpo (L/h)
137.1 33.9


134
Gilman AG, Goodman LS, Rail TW, and Murad F (1985). The Pharmacological basis of
therapeutics. Seventh edition. Macmillan Publishing, New York.
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ibuprofen in plasma by high-performance liquid chromatography. /. Pharm. Biomed
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Gracely RH (1990). Measuring pain in the clinic. Anesth. Prog., 37:88-92.
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Arch. Oral Biol, 17:701-709.
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anesthesiology, volume 12. Lippincott, New York, pp. 111-125.
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Anesthesiology, 58:72-87.
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potentials. Electroenccphalogr. Clin. Neurophysiol., 50:177P.
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analgesia. In Advances in pain research and therapy, volume 12. Edited by JD Loeser.
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21
Nomenclature
Evoked potentials are customarily displayed as a plot of voltage against time (see
Figure 2-7). Although a universal nomenclature for naming evoked potentials peaks
has not been established, waves are commonly labeled based on their polarity and
nominal latency. For example, a peak labeled as P250 indicates a positive wave
occurring 250 ms after stimulation.
Figure 2-7. A typical evoked potential: Nomenclature of events.
Classification of evoked potentials
Many types of evoked potentials can be distinguished according to stimulation
and recording methods. For example, evoked potentials may be divided by stimulus
type into visual evoked potentials (VEP), auditory evoked potentials (AEP), and
somatosensory evoked potentials (SEP). Similarly, evoked potentials may be defined by
recording method as far-field recordings, early near-field recordings, or late near-field
recordings (Chapman et al., 1979; Chapman and Jacobson, 1984; Chiappa, 1990;
Grundy, 1983a, 1983b; Spehlmann, 1981, 1985).


116
Concentration (ng/ml)
Figure 4-30. Codeine effect-concentration curves (mean SEM).
(Top: PID; Center: PR; bottom: EP).
O Codeine 60 mg
Ibuprofen 400 mg and codeine 30 mg


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94
Evoked Potential Recording
Samples of electrophysiologic brain activity were recorded from Fz, Cz, and Pz
with reference to Al (see Figures 4-13 to 4-15 for baseline EEG tracings recorded before
drug intake). Processing of evoked potential data involved sampling and digitization of
512 ms epochs of the analog EEG signal followed by on-line averagmg. Only sixteen
EEG epochs were necessary to obtain a consistent waveform. This represents an
improvement over existing methods which often require 40 to 120 repetitions to obtain
an evoked potential. The small number of repetitions also minimized the effect of
habituation on the evoked potential and allowed repeated measurements to be taken
over time.
Evoked potentials obtained after painful stimulation of the tooth pulp were
characterized by a negative peak at 150 milliseconds post stimulation, followed by a
positive peak at about 250 milliseconds and another negative peak at about 380
milliseconds (see Figures 4-16 to 4-21). These waveforms were similar in shape and in
latencies to waveforms obtained by other investigators (Chapman et al., 1979;
Derendorf et al., 1982; Rohdewald and Keuth, 1990).


115
Concentration (ng/ml)
Figure 4-29. Codeine effect-concentration curves (mean SEM).
(Top: PID; Center: PR; bottom: EP).
Codeine 60 mg.


43
as an analgesic for the symptomatic treatment of mild to moderate pain, especially pain
associated to soft tissues injuries, primary dysmenorrhea, post partum, and oral or
ophthalmic surgery (Flower et al., 1985; McEvoy, 1990).
Dosage and Administration
Ibuprofen is available as plain or film-coated tablets containing 200 to 800 mg
(Motrin, Nuprin, Rufen), and as a pediatric suspension containing 20 mg/mL
(Pediaprofen" ). Its usual adult dosage in the treatment of mild to moderate pain is 400
mg every four to six hours as necessary for relief of pain. The total daily dose should
not exceed 1200 mg. For the symptomatic treatment of acute and chronic rheumatoid
arthritis and osteoarthritis its usual adult dosage is 300-400 mg three or four times
daily. Dosage should be adjusted according to the response and tolerance of the patient
and should not exceed 3200 mg daily. Optimum therapeutic response usually occurs
within two weeks after beginning ibuprofen therapy. For antipyresis, the initial adult
dosage of ibuprofen is 200 mg every four to six hours. Dosage may be increased to 400
mg every four to six hours if fever is not adequately reduced but should not exceed
1200 mg daily (Flower et al., 1985; McEvoy, 1990).
Codeine
Codeine is a narcotic analgesic occurring naturally in opium. It was first isolated
by Robiquet in 1832. It is usually produced commercially by methylation of morphine
which is present in much higher concentrations in opium.
Physicochemical Properties
Codeine (3-methoxymorphine) is a phenanthrene derivative opiate agonist with
analgesic and antitussive properties. Like other morphine derivatives, codeine is a weak
base with a pKa value of 8.2 (Baselt and Cravey, 1989; Muhtadi and Hassan, 1981). It


67
Subjective pain ratings
Subjective pain ratings were derived from two different visual analog scales
(pain intensity and pain relief scales) presented to the subjects immediately after each
dental stimulation. To improve accuracy of subjective measurements, subjects were
shown their previous scores when making an assessment of pain severity (Scott and
Huskisson, 1979). Both scales consisted of a 10 cm line bounded by markers of least to
greatest value along which subjects made pencil marks (see figure 3-9).
No Pain 1
Severe Pain
No
Pain Relief
Complete
Pain Relief
Figure 3-9. Visual analog scales.
Top: Pain intensity scale; Bottom: Pain relief scale.
Pain intensity and pain relief scores were determined by measuring, in
millimeters, the distance between the beginning of the scale and the subject's mark.
To assess the magnitude of the analgesic effect, several measures derived from
mdividual pain scores were calculated. To account for differences in baseline pain
intensity among patients, pain intensity difference efficacy values (PID) were calculated
as percentages of the value determined before drug intake:
PJ PI
PID, = 100 -
where PID, is the percent change in pain intensity at time t, PI, the pain intensity at time
t, and PI0 the pain intensity before drug administration. Positive values indicate
reduction in pain, thus making the pain intensity difference scores analogous to pain
relief scores.


93
factors were considered in the overall design of the stimulator, and variables such as the
internal diameter of the electrodes, hardness of the polymer used to build the form, use
of conductive rubber, and electrode positioning on tooth surface were investigated.
These designs were all based on an individual stone cast of the subject's upper
dentition.
The use of an individual dental impression allowed repetitive positioning of the
stimulating electrodes on the exact same area of the tooth. This impression was made in
orthodontic acrylic resin from an exact stone replica of the subject's upper dentition.
Springs were used to maintain electrodes firmly in place against the tooth surface, and
all electrical connections resided within the resin to minimize the risk of an electrical
shock to the subject.
Bipolar electrical stimuli were used to minimize the risk of current spread to the
periodontal tissues. In addition, a constant current stimulator was used to minimize the
effect of variations in enamel thickness on the current passed. To insure a good contact
and minimize the resistance between electrode and stimulated tooth, the tooth was kept
dry by wiping it with cotton followed by insertion of dental cotton rolls between the
upper lip and gum. The contact was further improved by application of a small drop of
dental conducting jelly on the tip of each electrode before each experimental session.
The described dental stimulation device was found safe and reliable. It was used
throughout the pharmacodynamic experiments. Electrical stimuli consisted of
0.5 millisecond square wave pulses applied semi-randomly with an interstimulus
interval in the range of 2 to 4 seconds (mean of 3 seconds). The intensity of the current
needed to evoke pain was calculated as twice the value of the measured threshold of
sensation (lowest current necessary to evoke a sensation).


121
Release Notes
TPEP Toolkit is designed to run under the Microsoft Windows graphical
environment (Microsoft Corporation, Redmond, WA). It was written and compiled with
Microsoft Visual Basic version 1.0 (Microsoft Corporation). It has been tested and used
successfully with release 2.308 (model 175) of the Brain Atlas software (Bio-logic
Systems Corporation).
Features
TPEP Toolkit allows you to specify which EEG channel to extract and what the
output should contain. TPEP Toolkit can extract amplitude values from Fz, Cz, and/or
Pz channels. In addition, the output can contain a time column and column labels (e.g.
time ms, amplitude fiV).
The Rename and Rename all commands remove the initial letter T from
selected Bio-logic ASCII files. The Extract and Extract all commands extract Fz, Cz,
and/or Pz amplitude data from selected Bio-logic ASCII files. New ASCII files with a
CSV extension (comma separated values) are created for import into other programs.
The Process experiment command extracts Cz amplitude data from 1 to 10 separate
Bio-logic ASCII files in the selected directory. A new ASCII file with a CSV extension is
created for import into other programs.
Source Code
GLOBAL.BAS
' Brain Atlas EP ASCII files contain 3 lines per time point and 256 time points
1 First line channels: Fpl, Fpz, Fp2, F7, F3, Fz, F4
' Second line channels: F8, T3, C3, Cz, C4, T4, T5
' Third line channels: P3, Pz, P4, T6, Ol, Oz, 02
' Variables holding file paths and names
Global SourcePath As String
Global BLFilename As String
Global NewFilename As String
Global EPdataQ As Single 1 Array holding time and evoked potential amplitude data


78
Ibuprofen Pharmacokinetics
The pharmacokinetic parameters of ibuprofen are summarized in Tables 4-3
through 4-5 and representative plasma profiles are shown in Figures 4-3 through 4-7.
Figures 4-4 to 4-7 compare the mean ibuprofen plasma levels for each formulation to
the 400 mg oral dose of ibuprofen.
Following oral administration of a single 400 mg dose of ibuprofen, absorption
was rapid, with average peak plasma concentrations of 30.6 pg/mL appearing at
1.81 hours after dosing. The elimination of ibuprofen was rapid with an average
elimination half-life of 2.13 hours and mean residence time of 4.23 hours. These values
were in agreement with previously published data (Albert and Gemaat, 1984; Gillespie
et al., 1982; Lockwood et aL, 1983a; Wagner et al., 1984).
The pharmacokinetic parameters of ibuprofen obtained after administration of
the four different ibuprofen-codeine combinations were not significantly different from
those obtained after sole administration of ibuprofen. As can be seen on Figures 4-4
through 4-7, the plots show close agreement between the disposition profile of the
different ibuprofen-codeine combinations and the reference (ibuprofen 400 mg).
However, the maximum concentration obtained after administration of ibuprofen 400
mg and codeine 60 mg in one combination tablet (34.9 pg/mL) was significantly higher
(p < 0.05) than the one obtained after ibuprofen 400 mg and codeine 30 mg in two
separate tablets (26.4 pg/mL). Since the areas under the concentration-time curve, as
well as the other parameters of these two formulations were not significantly different,
it may well reflect a difference in absorption rate but not in extent. This may be
explained by differences in the manufacturing process of the combination tablet.


108
Pharmacodynamic Measurements
The analgesic efficacy of codeine and ibuprofen was assessed by three
independent parameters: pain intensity difference (PID), pain relief (PR), and evoked
potential amplitude (EP). These parameters were also summed over the observation
period to provide estimates of the area under the effect-time curve (AUCPID, AUCPR,
and AUCep).
Effect-time relationships were investigated by plotting measured effect as a
function of time. Figure 4-26 presents PID, PR, and EP effect-time curves for the four
different formulations, it shows that all active drugs (ibuprofen, codeine, and their
combination) produced a stronger analgesic effect than placebo, as indicated by higher
hourly pain intensity difference (PID), pain relief (PR), and evoked potential scores (EP).
No reduction in analgesic effect was observed at the end of the data collection period
(4.0 hours). All three measures of analgesic effect were closely correlated to each other,
which is in agreement with previously published data from other authors (Bromm,
1984a; Rohdewald and Keuth, 1990).
The analgesic efficacy of codeine and ibuprofen was also assessed by cumulative
measures that provide estimates of the area under the effect-time curve. Figure 4-27
depicts those cumulative values (AUCP1D, AUCPR, and AUCEP) for each of the four
treatments investigated. All active drugs produced a stronger analgesic effect than
placebo and statistical analysis of the AUCPID and AUCPR scores revealed significant
differences between active drugs and placebo (p < 0.05). It was not possible, however, to
differentiate codeine from ibuprofen and from the combination.


126
End If
ReDim EPData(0 To 10, 0 To 255)
For i% 0 To Filel.ListCount -1
Filel.Listlndex = i%
ColNum% = ColNum% + 1
1 Get Bio-logic ASCII filename
BLFilename = SourcePath + Filel.Filename
Readout.Caption = "Reading Cz amplitude from + UCase$(BLFilename) +
Readout.Refresh: Gauge. FloodShowpct = TRUE
' Read amplitude values
Open BLFilename For Input As #1
j% = 0
Do While Not EOF(l)
EPData(0, j%) = j% 2 Epoch is 512 ms
Input #1, Vari, Var2, Var3, Var4, Var5, Var, Var7
Input #1, Vari, Var2, Var3, EPData(ColNum%, j%), Var4, Var5, Var
Input #1, Vari, Var2, Var3, Var4, Var5, Var, Var7
If j% Mod 8 = 0 Then
Gauge.FloodPercent = (j% 100 \ 256): Gauge.Refresh
End If
j% = j% + 1
Loop
Close #1
Gauge.FloodPercent = 0: Gauge.FloodShowpct = FALSE
Next i%
1 Create new filename by appending suffix at the end of existing filename
NewFilename = Mid$(BLFilename, 1, Len(BLFilename) 5) + "XL.CSV"
Open NewFilename For Output As #2
Readout.Caption = "Writing + UCase$(NewFilename) + Readout.Refresh
Gauge.FloodShowpct = TRUE
Write #2, "Amplitude at Cz (pV)" 1 Write labels on first line of file
Write #2, "Time (ms)", "Record 1", "Record 2", "Record 3", "Record 4", "Record 5",
"Record 6", "Record 7", "Record 8", "Record 9", "Record 10"
' Write time, amplitude on other lines of file
For i% = 0 To 255
Write #2, EPData(0, i%), EPDatafl, i%), EPData(2, i%), EPData(3, i%),
EPData(4, i%), EPData(5, i%), EPData(6, i%), EPData(7, i%),
EPData(8, i%), EPData(9, i%), EPData(10, i%)
If i% Mod 8 = 0 Then
Gauge.FloodPercent = (i% 100 \ 256): Gauge.Refresh
End If
Next i%
Close #2
Gauge.FloodPercent = 0: Gauge.FloodShowpct = FALSE
Filel.Pattern = "*.ASC": Filel.Refresh
Readout.Caption = "Select Biologic ASCII file to process..."
Screen.Mousepointer = DEFAULT
End Sub


Concentration (/xg/mL)
82
Figure 4-3. Plasma concentrations of ibuprofen (mean standard deviation).
Ibuprofen 400 mg in one tablet
A Codeine 30 mg and ibuprofen 400 mg in two separate tablets
ik Codeine 30 mg and ibuprofen 400 mg in one combination tablet
O Codeine 60 mg and ibuprofen 400 mg in two separate tablets
O Codeine 60 mg and ibuprofen 400 mg in one combination tablet


68
Pain relief scores were determined by measuring the distance (in millimeters)
between the endpoint labeled "no pain relief" and the mark made by the subject.
Pain intensity difference and pain relief scores were also summed over the
observation period to provide estimates of the area under the effect-time curve:
AUCPID =^PIDf At
AUCpr = ^ PR, At
where A UCpiD is the sum of the pain intensity differences, P/D, is the pain intensity
difference at time t, Af is the time elapsed since the previous observation, AUCPR is the
total pain relief, and PR, is the pain relief score at time t.
The efficacy of different analgesic treatments was then evaluated by comparing
hourly scores, AU.Cp¡D scores, and AUCPR scores.
Tooth pulp evoked potentials
Tooth pulp evoked potentials were recorded before drug intake and at 0.5,1,1.5,
2, 3, and 4 hours after drug administration. A computer program was developed to
retrieve evoked potential amplitudes at Fz, Cz, and Pz from the Brain Atlas computer
data files (see Appendix). Waveforms were then plotted and smoothed by application
of a moving average technique. For quantitative evaluation of the evoked potential, a
perpendicular was drawn through the height of the P250 peak crossing the line between
the N150 and N380 peaks (see Figure 3-10). This amplitude value (P250 peak to
extrapolated baseline) was measured and its percentage of change after drug intake was
taken as an indicator for the pharmacodynamic activity. This amplitude measurement
technique was chosen since it has proved to have the smallest variability in comparative
measurements (Rohdewald and Keuth, 1990; Rohdewald et al., 1980).
To account for large inter-individual differences in tooth pulp evoked potential
amplitudes, all amplitude values were expressed as a percentage of the amplitude
before drug intake in that individual:


63
during the course of an experiment, with readings generally in the 1-2 kQ range.
Physical disturbance of electrodes and wires was minimized by twisting and taping the
electrodes cables together.
Figure 3-6 EEG recording electrodes (cup shaped, gold-plated, 10 mm diameter,
2 mm hole).
Evoked potential measurements were carried out under uniform conditions
(minimum noise environment, dimmed light, closed eyes, room temperature).
Measurements for a certam volunteer were always performed at the same time of the
day to avoid the circadian rhythm in pain perception (time of day around 12:00AM).
After a preliminary training session, in which the subject was familiarized with
the experimental procedure, the four experimental sessions were separated by intervals
of at least two days. During experiments, the subjects were seated in a reclining chair.
They were instructed to be alert but relaxed, and to minimize eye movements or blinks.
They were also closely observed to guard against possible artifacts. Each experimental
session lasted four hours.
The EEG potentials were amplified, filtered (low frequency filter: 0.5 Hz; high
frequency filter: 70 Hz), and recorded on paper (electroencephalograph model 1A97,
Nicolet Biomedical Instruments, Madison, Wisconsin, USA). These recordings provided
control of the quality of the signal stored as well as an assessment of the subject's level


59
was ingested with approximately 100 mL of water. Food was withheld for two hours
following drug administration, and caffeine intake was limited to each subject's regular
coffee consumption. All doses were taken at approximately 12:00 PM. Subjects
remained seated during the first two hours following the dose of test medication.
Blood Sampling
To avoid repeated venipuncture, blood samples were collected via an indwelling
catheter (Quick-Cath 16G 2 in., Baxter Healthcare Corporation, Deerfield, Illinois, USA)
placed in a convenient vein in the arm of the subject. The catheter was flushed at thirty
minute intervals with a 0.9% sodium chloride solution U.S.P. to maintain patency of the
vein (Epperson, 1984).
Venous blood samples (10 mL) were collected prior to, and at 0.5, 1,1.5, 2, 2.5, 3,
4, 5, and 6 hours after drug administration in plain (silicone coated) Vacutainer tubes
(Becton Dickinson and Company, Rutherford, New jersey ,USA). After coagulation, the
serum was separated, transferred into a clean tube, and kept at -20C until assayed for
codeine and ibuprofen.
Dental Stimulation
Pain was induced by electrically stimulating a healthy upper front tooth. Bipolar
electrical stimuli were applied to one upper incisor using 4 mm diameter electrodes
mounted in an individual impression of the subject's upper front teeth.
The individual dental impression was made with orthodontic acrylic resin
(L.D. Caulk Company, Milford, Delaware, USA) from an exact stone replica of the
subject's upper dentition. All electrical connections resided within the resin to minimize
the risk of current spread. Stainless steel springs (Hi-T rectangular wire, 0.019" x 0.025",
Unitek/3M, Monrovia, California, USA) were used to push electrodes against the
enamel of the stimulated tooth and maintain a good contact (see Figure 3-3). The


98
O 128 256 384 512 O 128 256 384 512
O 126 256 384 512 O 128 256 384 512
Figure 4-16. Effect of treatment on tooth pulp evoked potentials before (left) and
90 minutes after drug administration (right).
Subject 1.
From top to bottom: placebo, ibuprofen, codeine, and ibuprofen-codeine.


10
Figure 2-2. Schematic representation of the gate control theory.
This model includes excitatory (open circle) and inhibitory (closed circle)
links from the substantia gelatinosa (SG) to the transmission cell (T).
From MeLzack and Wall (1983).
Figure 2-3. Afferent and efferent pathways involved with nociception and pain.
From Stimmel (1983).


54
Extraction procedure
To 1.0 mL of plasma were added 50 pL of an internal standard solution
(400 pg/mL flurbiprofen), 0.5 mL of 2.5 M sulfuric acid, and 12 mL of hexane. The tubes
were shaken for 3 minutes and centrifuged for 10 mmutes at 2500 revolutions per
minute. The organic layer was transferred into a clean tube and evaporated to dryness
under a nitrogen steam in a water bath at 50C. The dried residue was then
reconstituted in 100 pL of mobile phase and injected into the chromatographic system.
1.0 mL Plasma
+ Internal Standard
l
2.5 M Sulfuric Acid
Hexane
Evaporate
Nitrogen Stream
Reconstitute in Mobile Phase
Inject in HPLC System
Figure 3-2. Extraction procedure for ibuprofen analysis.
Chromatographic conditions
Reversed phase chromatography was performed at room temperature. The
mobile phase consisted of a mixture of 0.1 M phosphate buffer and acetonitrile (40:60,
v:v). The pH was adjusted to 3.70 by addition of 85% phosphoric acid. The mobile


140
Scott J and Huskisson EC (1979b). Vertical or horizontal visual analogue scales. Ann.
Rheum. Dis., 38:560.
Scott JC, Ponganis KV, and Stanski DR (1985). EEG quantitation of narcotic effect: the
comparative pharmacodynamics of fentanyl and alfentanil. Anesthesiology,
62:234-241.
Sessle BJ (1979). Is the tooth pulp a pure source of noxious input ? In Advances in pain
research and therapy, volume 3. Edited by JJ Bonica, JC Liebeskind, and DG Albe-
Fessard. Raven Press, New York, pp. 245-260.
Shafer SL and Varvel JR (1991). Pharmacokinetics, pharmacodynamics, and rational
opioid selection. Anesthesiology, 74:53-63.
Shah JC and Mason WD (1990). Plasma codeine and morphine concentrations after a
single oral dose of codeine phosphate. /. Clin. Pharmacol., 30:764-766.
Shah JC and Mason WD (1991). A dose-ranging study of the pharmacokinetics of
codeine phosphate following intravenous administration to rats. J. Pharm. Sci.,
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potentials in clinical diagnosis. Butterworth Publishers, Boston, Massachusetts.
Sriwatanakul K, Kelvie W, Lasagna L, Calimlim J, Weis OF, and Mehta G (1983a).
Studies with different types of visual analog scales for measurement of pain. Clin.
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16
pain (Huskisson, 1974). Visual analog scales consist of a line, usually 10 cm in length,
whose ends are labeled with descriptors of the pain experience (e.g. "no pain" and
"worst pain possible") The patient is asked to mark the line at a point corresponding
to the severity of his pain. The distance (in millimeters) between the start of the line and
the mark is then taken as an indicator of the pain severity (Max and Laska, 1991; Scott
and Huskisson, 1976, 1979b).
No Pain F
Figure 2-5. Example of visual analog scale for pain intensity.
Worst Pain
Possible
Visual analog pain scales offer several advantages: simplicity, sensitivity, and
good reproducibility (Huskisson, 1983). Also, because visual analog scales are
continuous, they avoid the boundary error of category scales. They have generally been
found to be as or more sensitive than categorical scales and produce results that
correlate well with those of descriptive scales (Huskisson, 1974; Ohnhaus and Adler,
1975; Rohdewald and Keuth, 1990; Scott and Huskisson, 1976; Wallenstein et al., 1980).
Problems with visual analog scales include failure to understand the concept, and the
choice of appropriate statistics for data analysis (Chapman and Syrjala, 1990; Max and
Laska, 1991; Syrjala and Chapman, 1984). They are commonly used in clinical trials to
establish the value of analgesic treatments. Visual analog scales can also be used to
compare pain severity in the same patient at different times or in groups of patients
receiving different treatments (Scott & Huskisson, 1979a).
Visual analog scales have also been used to assess pain relief. In this case, the
line is labeled with descriptors such as "no pain relief" and "complete pain relief."
Patients can then evaluate their relief and mark the scale at any point along the line.


122
Global Channel As Integer 1 Variable holding channel to be extracted
Global Const FZ = 0
Global Const CZ = 1
Global Const PZ = 2
Global Const ALL = 3
Global Const TRUE = -1
Global Const FALSE = 0
1 MousePointer
Global Const DEFAULT = 0
Global Const HOURGLASS = 11
' Help engine declarations.
Declare Function WinHelp Lib "USER" (ByVal hWnd As Integer, ByVal lpHelpFile As
String, ByVal wCommand As Integer, dwData As Any) As Integer
Global Const HelpFile = "WINTPEP.HLP"
Global Const HELP_QUIT = &H2 1 Terminate help
Global Const HELP_CONTENTS = &H3 1 Display index
WINTPEP.FRM
Sub Form_Load ()
Channel = CZ
End Sub
Sub RenameFile ()
1 Get Bio-logic ASCII filename
SourcePath = Dir 1. Path
If Right$(SourcePath, 1) <> "\" Then SourcePath = SourcePath + "\"
BLFilename = SourcePath + Filel. Filename
1 Rename Bio-logic file by removing first letter of original filename
If UCase$(Left$(Filel.Filename, 1)) = "T" Then
NewFilename = SourcePath + Right$(Filel.Filename, Len(Filel.Filename) 1)
Name BLFilename As NewFilename
End If
End Sub
Sub Drivel_Change ()
Dirl.Path = Drivel. Drive
End Sub
Sub Dirl_Change ()
Filel.Path = Dirl.Path
End Sub
Sub OPT_Channel_Click (Index As Integer, Value As Integer)
' display selected channels in picture
Head.Picture = PictureLib(Index).Picture
Select Case Index
Case 0: Channel = FZ
Case 1: Channel = CZ


141
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Neurophysiological correlates of pain. Edited by B Bromm. Elsevier, Amsterdam,
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24
Mechanical stimulation
Various mechanical stimulation methods have been used to generate pain
(Beecher, 1957). Although mechanical stimulation can generate painful sensations, the
stimulus may not be limited to excitation of nociceptive afferents alone. It is possible
that other afferent input related to touch and pressure may also be activated by skin
contact. Therefore, non-nociceptive input may contribute to some or all of the
components of the evoked potential generated by this method (Chudler and Dong,
1983).
Thermal stimulation
Thermal stimulation methods are among the oldest and most widely used
methods in the production of experimental pain. Cutaneous thermal stimulation is
precise, simple, and allows a fairly rapid collection of data. At moderate temperatures,
this method permits rapid repetition of the stimuli without injury (Hardy et al., 1940).
Like mechanical stimulation, thermal stimulation (i.e. laser-emitted radiant heat) has the
advantage of eliciting a sensation that is considered natural. But it also brings the risk of
contamination of the evoked potential by input related to sensory modalities other than
pain (Bromm and Treede, 1987; Carmon et al., 1976, 1978, 1980; Chudler and Dong,
1983; Morgan et al., 1984).
Electrical stimulation
Electrical stimulation is probably the most common method of generating pain
in evoked potential research because it is easy to apply, repeat and quantify
(Buchsbaum, 1984). Proponents of electrical stimulation argue that it has advantages
similar to that of thermal stimulation. Electrical stimulation induces a clearly detectable
pain sensation and the stimulus intensity can be exactly determined. In addition, a
number of parameters may be manipulated to modify pulse pattern and waveform
(Notermans 1966). Nevertheless, cutaneous electrical stimulation has many
disadvantages. First, electrical stimulation is an unnatural type of stimulation. Second,


100
Figure 4-18. Effect of treatment on tooth pulp evoked potentials before (left) and
90 minutes after drug administration (right).
Subject 3.
From top to bottom: placebo, ibuprofen, codeine, and ibuprofen-codeine.


9
lamina 1 and the C fibers in the substantia gelatinosa (lamina II). In addition, both A8
and C fibers synapse directly (or via intermediate neurons) in the deeper layers of the
dorsal horn with ascending fibers which cross the mid-line to join the spinothalamic
tract (Bonica, 1990b; Stimmel, 1983).
Pain transmission can be inhibited at the spinal cord level by inhibitory
interneurons or from descending inhibitory fibers. The best known theory describing
how painful stimuli may be altered at the spinal level is the "gate theory" put forward
by Melzack and Wall in 1965. They postulated that painful stimuli have to pass through
a gate in order to be relayed on to the central nervous system (see Figure 2-2). This gate
can be closed by non-painful sensory input carried by large myelinated fibers from
mechanoreceptors responding to low-threshold stimuli (Ap fibers). In addition, the gate
may be closed as a result of descending inhibitory systems (Melzack and Wall, 1983).
These originate in the brain stem (including the periaqueductal grey matter) and some
of the descending tracts activate inhibitory intemeurons of the enkephalinergic type in
the dorsal horn. Therefore, pain may be viewed as a complex summation of nociceptive
and non-nociceptive neuronal stimulation (Fields, 1984).
Ascending nociceptive impulses are transmitted mainly through the
spinothalamic tract. Fibers from the dorsal horn project to lateral and medial areas of
the thalamus, from where further transmission of impulses is to areas of the sensory
and motor cortex. This is the major route for the sensory-discriminatory aspects of pain.
Ascending impulses may also travel more slowly, via the spinoreticular tract,
terminating in the pontomedullary reticular formation. These slower impulses have a
major role in the arousal-motivational aspects of pain sensation, resulting from their
connections with the limbic system. They also reach the hypothalamus and are
responsible for the autonomic effects associated with pain (Melzack and Wall, 1983;
Stimmel 1983; Zimmermann, 1984).


107
Figure 4-24. Plasma concentrations of codeine following oral administration of
codeine 60 mg (mean standard deviation).
Time [hi
Figure 4-25. Plasma concentrations of codeine following oral administration of
codeine 30 mg and ibuprofen 400 mg (mean standard deviation).


40
group. Mills et al. (1973) first examined the fate of the optical isomers of ibuprofen
given separately to man. They reported that ibuprofen undergoes an in vivo isomeric
R(-) to S(+) inversion in humans while there is no measurable inversion of the S(+)-
isomer to R(-)-ibuprofen. Conclusive evidence for the chiral inversion of R(-)-ibuprofen
was later obtained in studies by Wechter and co-workers (1974) using deuterium
labeled ibuprofen.
1
Figure 2-14. Metabolic pathways of ibuprofen in man.
The mechanism of the chiral inversion involves the stereoselective formation of a
coenzyme A thioester of R(-)-ibuprofen, subsequent inversion by a non-stereoselective
coenzyme A racemase, and release of the S(+) enantiomer by hydrolysis (Hutt and
Caldwell, 1983, Wechter et al., 1974). The S(+)-enantiomer of ibuprofen is not a substrate
for the coenzyme A synthetase and therefore is excluded from the reaction (Hutt and
Caldwell, 1983).
The mechanism of the chiral inversion of ibuprofen was later confirmed by
studies on the incorporation of ibuprofen into adipose tissue. Williams et al. (1986)


114
Concentration (/g/ml)
Figure 4-28. Ibuprofen effect-concentration curves (mean SEM).
(Top: PID; Center: PR; bottom: EP).
Ibuprofen 400 mg


110
Figure 4-27. Cumulative efficacy measures.
Top: AUCP1D; Center: AUCpR; Bottom: AUCEP.


88
Table 4-8. Pharmacokinetic parameters for codeine given in combination with
ibuprofen (mean 1 standard deviation).
Ibuprofen 400 mg and codeine
60 mg as two tablets
Ibuprofen 400 mg and codeine
60 mg as one tablet
cmax (ng/rnL)
133.3 42.9
144.0 45.5
tmax (h)
1.1510.57
0.94 0.36
AUC^ (ng-h/mL)
496.4 100.1
516.21123.5
AUMC (ng-h2/ mL)
2071.9 1 642.6
2054.3 1 599.4
MRT (h)
4.1110.79
3.9710.66
ke (h-i)
0.292 0.053
0.2921 0.051
Vz (h)
2.45 0.49
2.45 1 0.47
OpoiL/h)
123.3 25.5
123.8 130.3


90
Figure 4-9. Plasma concentrations of codeine (mean standard deviation).
O Codeine 60 mg in one tablet
Codeine 30 mg and ibuprofen 400 mg in one combination tablet
Time [hi
Figure 4-10. Plasma concentrations of codeine (mean standard deviation).
O Codeine 60 mg in one tablet
Codeine 30 mg and ibuprofen 400 mg in two separate tablets


65
Processing of evoked potential data involved sampling and digitization of an
epoch of the analog EEG signal followed by on-line averaging (see Figure 3-8). The
sweep time was 512 ms with a sampling rate of 512 samples per second (2 ms inter
sample interval). Automatic artifact rejection was on during the data collection process,
and 16 EEG epochs were collected for each individual waveform. The averaged evoked
potentials were graphed and stored on disk.
Figure 3-8. Block diagram illustrating the processing of evoked potential data.
Estimation of Pharmacokinetic Parameters
The pharmacokinetic parameters of codeine and ibuprofen were determined by
standard compartmental data analysis techniques (Gibaldi and Perrier, 1982). The
plasma concentration time profiles of codeine and ibuprofen were fitted to a
bi-exponential equation corresponding to a one-compartment model with first-order
input, using the equation:
Cp = A-e~k't -A-e-k'*


Concentration [fig/mU m Concentration
106
Time [h]
Plasma concentrations of ibuprofen following oral administration of
ibuprofen 400 mg (mean standard deviation).
Figure 4-23. Plasma concentrations of ibuprofen following oral administration of
codeine 30 mg and ibuprofen 400 mg (mean standard deviation).


64
of alertness. The signal was then fed into a digital computer (Brain Atlas III, model 175,
version 2.308, Bio-logic Systems Corporation, Mundelein, Illinois, USA) and processed
by an electrophysiological data analysis system for on-line averaging. The block
diagram below illustrates the interaction of the various system components and the
flow of information through the system.
Figure 3-7. Block diagram of the evoked potential recording system.
Table 3-1. System components and associated functions.
Component
Function
Brain Atlas
Triggering of stimulator and EEG
Signal digitizing and averaging
Electroencephalograph
Signal amplification and filtering
Stimulator
Dental stimulation
Stimulus isolation unit
Subject isolation
Constant current unit
Current intensity regulation


49
As with other opiate agonists, codeine can produce drug dependence and
therefore has the potential for being abused. Psychological and physical dependence, as
well as tolerance, may develop upon repeated administration of codeine (Benedetti and
Butler, 1990; Cutting, 1972, Jaffe and Martin, 1985; McEvoy, 1990).
Therapeutic Uses
Codeine is a mild analgesic indicated for the symptomatic relief of mild to
moderate pain which is not relieved by non-opiate analgesics. Codeine is also used,
alone or in combination with other antitussives or expectorants, in the symptomatic
relief of nonproductive cough (Cutting, 1972, Jaffe and Martin, 1985; McEvoy, 1990).
Dosage and Administration
Codeine is usually given orally as a phosphate, sulfate, or hydrochloride salt for
the relief of cough and mild to moderate pain. The phosphate salt may also be given
parenterally for the relief of pain by intra-muscular or sub-cutaneous injection.
Dosages vary widely depending upon respective indications. As an analgesic,
the usual oral dosage of codeine is 30 to 60 mg every four to six hours, as needed for the
relief of pain. Against cough, the usual adult dose is 10 to 20 mg every four to six hours,
not to exceed 120 mg daily. As with other opiate agonists, the smallest effective dose
should be given in order to minimize the development of tolerance and physical
dependence (Cutting, 1972, Jaffe and Martin, 1985; McEvoy, 1990).


75
Figure 4-1. Chromatograms of ibuprofen (I) after extraction from plasma, using
flurbiprofen (F) as the internal standard. The two chromatograms
represent concentrations of 0 (left) and 22.1 pg/mL (right).
Table 4-1. Accuracy and reproducibility of ibuprofen assay.
Concentration
iag/mL
1
Assay, pg/ mL
2 3 4
5
Mean
pg/mL
CV
%
5
4.7
4.7
5.7
5.5
4.9
5.1
9.20
10
10.8
10.3
9.3
9.3
10.0
9.9
6.54
20
19.7
19.1
18.0
18.8
19.3
19.0
3.36
30
31.7
30.0
30.2
28.2
29.2
30.5
4.34
50
48.9
48.7
50.4
55.0
50.3
50.7
5.03


23
Rohdewald and Keuth, 1990; Rohdewald et al., 1980). Evoked potentials have also been
used by pain researchers to evaluate the efficacy of different analgesics (Bromm et al.,
1983; Buchsbaum, 1984; Buchsbaum et al., 1981b; Butler et al., 1983; Chapman et al.,
1982; Chen and Chapman, 1980; Derendorf et al., 1982,1984; Rohdewald et al., 1982a,
1982b, 1983). The major use of evoked potentials in pain research has focused on
experimental pain.
The Pain Stimulus
Pain can be evoked by stimulation in many different sensory modalities. When
the stimulus intensity is great enough, pain can be evoked by tactile, acoustic, or visual
stimulation (Chudler and Dong, 1983). To be useful in pain research, however, an ideal
pain-producing stimulus should be
exclusively painful, but barely noxious in order to avoid tissue damage,
easy to apply and remove,
quantifiable and repeatable.
Additionally, for obtaining evoked potentials, the stimulus must be of brief
duration so that the evoked potential can be time-locked to the stimulus and the
stimulus artifact does not obscure the potentials of interest. Another essential
requirement is the necessity to randomize inter-stimulus intervals and/or stimulus
intensities in order to keep the arousal state of the subject high and constant and, as a
consequence, to minimize the effects of habituation or sensitization (Bromm, 1985).
Although most researchers prefer short electrical or thermal pain stimuli, such
as electrical tooth pulp stimulation or laser-emitted short radiant heat pulses, three
main types of stimulation have been used in experimental pain research: mechanical,
thermal, and electrical stimulation.


135
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Analgetics. Edited by G de Stevens. Academic Press, New York, pp. 75-122.
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evaluation of the effect of smoking on codeine pharmacokinetics and bioavailability
in normal human volunteers. Drug Intell. Clin. Pharm., 16:849-850.
Huskisson EC (1974). Measurement of pain. Lancet, 2:1127-1131.
Huskisson EC (1983). Visual analogue scales. In: Pain measurement and assessment.
Edited by R Melzack. Raven Press, New York, pp. 33-37.
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novel route with pharmacological consequences. /. Pharm. Pharmacol., 35:693-704.
Insel PA (1990). Analgesic-antipyretics and anti-inflammatory agents; drugs employed
in the treatment of rheumatoid arthritis and gout. In Tire pharmacological basis of
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and notes on usage. Pain, 6:249-252.
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Pharmacokinetic-pharmacodynamic relationships of methadone infusions in
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Jacobson RC, Chapman CR, and Gerlach R (1985). Stimulus intensity and inter-stimulus
interval effects on pain-related cerebral potentials. Electroencephalogr. Clin.
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Figure 4
Figure 4-
84
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Time [h]
6. Plasma concentrations of buprofen (mean standard deviation).
O Ibuprofen 400 mg in one tablet
# Codeine 60 mg and ibuprofen 400 mg in one combination tablet
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Time [hi
7. Plasma concentrations of ibuprofen (mean standard deviation).
O Ibuprofen 400 mg in one tablet
Codeine 60 mg and ibuprofen 400 mg in two separate tablets


70
This model was selected for its simplicity and by the fact that analgesic effects
are mediated by specific drug-receptor interactions. This model predicts a maximal
effect when drug concentration is above a certain value, as well as no effect when no
drug is present:
E_ £nux-CP
£Qo+ Cp
where E is the observed effect at time t, Emax is the maximum drug effect, £C50 is the
concentration at which 50% of the maximum effect is reached, and Cp is the plasma
concentration at time t.
The experimental data was fitted to the equation by a nonlinear least-squares
regression program (Minsq release 4.02, Micromath Scientific, Salt Lake City, Utah,
USA).
Statistical Analysis
Statistical comparisons of pharmacokinetic and pharmacodynamic parameters
were performed using one-way and two-way analysis of variance (ANOVA) with
subject, sequence, and treatment as factors. In cases where treatment effects were
significant (p < 0.05), pairwise comparisons were evaluated with Tukey's multiple
range test (Bolton, 1984).


97
vWV*-vw'"'sNW'*VV'*'V9W/v'^
+
10.0 ;;V
1.0 s
VM-v/vs>vv|/vn**''VVbYtyV^^
Figure 4-15. Resting electroencephalogram of subjects 5 (top) and 6 (bottom); Subjects
were alert and had eyes closed during the recording.


42
hydroxy- and carboxy-metabolites. Only negligible amounts of ibuprofen (< 1%) are
excreted unchanged (Albert and Gemaat, 1984; Albert et al., 1984; Lee et al., 1985).
Biliary excretion may also occur, as found in animal studies, and account for as
much as 20% of the dose through the biliary tract and feces (Dietzel et al., 1990).
However, no study has investigated an enterohepatic recirculation of ibuprofen in
human subjects. Since 99% of ibuprofen excreted in urine in man is metabolized, it is
very unlikely that much unchanged drug gets into the bile or feces (Albert and Gemaat,
1984; Mills et al., 1973).
Both enantiomers follow parallel time courses and have similar elimination half-
lives of about 2 hours (Cox and Smith, 1987; Cox et al., 1988; Jamali et al., 1988; Lee et
al., 1985). This rapid elimination may explain, to some extent, the low toxicity of
ibuprofen when compared with other non-steroidal anti-inflammatory drugs.
Toxicity
Ibuprofen is generally well tolerated. The majority of adverse reactions reported
in clinical trials have been subjective and mild in nature. The most frequent type of
adverse reaction occurring with ibuprofen is gastrointestinal in nature. In controlled
clinical trials, the percentage of patients reporting one or more gastrointestinal
complaints ranged from 5 to 15%. Common reactions include epigastric pain, nausea,
heartburn, and sensation of "fullness" in the gastrointestinal tract. Occult blood loss is
uncommon. Other side effects of ibuprofen have been reported less frequently
(incidence < 3%). They include thrombocytopenia, skin rashes, headache, dizziness,
blurred vision, toxic amblyopia, fluid retention, and edema (Davis, 1975; Flower et al.,
1985; Kantor, 1979; Knodel, 1992; McEvoy, 1990).
Therapeutic Uses
The approved indications for the use of ibuprofen include the symptomatic
treatment of rheumatoid arthritis, osteoarthritis, and acute gouty arthritis. It is also used


132
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51
(sodium salt) was obtamed from Kodak (Rochester, New York, USA). All other reagents
were of analytical grade and were purchased from Fisher Scientific.
Instrumentation
The chromatographic system consisted of a Constametric IIIG high pressure
pump (LDC Milton-Roy, Riviera Beach, Florida, USA), an autosampler (model ISS-100,
Perkin Elmer Corporation, Norwalk, Connecticut, USA) fitted with a 50 pL injection
loop, a 15 cm x 4.6 mm I.D., 5 pm particle size Zorbax cyanopropylsilane column
(DuPont Instruments, Wilmington, Delaware, USA), and a fluorescence detector with a
xenon lamp (model 650-10 S, Perkin Elmer, Norwalk, Connecticut, USA). A guard
column filled with Zorbax cyanopropylsilane packing material was placed before the
analytical column. Chromatograms were recorded on a Hewlett-Packard integrator
(model 3392A, Palo Alto, California, USA).
Extraction procedure
To 1.0 mL of plasma were added 1.0 mL of 0.05 M phosphate buffer adjusted to
pH 8, and an appropriate amount of internal standard (N-isopropylcodeine) to give
peak-height ratios between 0.2 and 5. The mixture was vortexed for 30 seconds. A
6.0 mL volume of hexane:methylene chloride (2:1, v:v) was added and the mixture
shaken for 5 minutes. The phases were separated by centrifugation for 10 minutes at
3000 revolutions per minute, and the organic phase was transferred into another tube.
The extraction was repeated with another 6.0 mL of fresh solvent. The combined
organic phases were extracted with 1 mL of 0.05 M acetate buffer adjusted to pH 3 and
discarded. The acidic aqueous phase was neutralized by addition of 1.0 mL of 0.1 M
sodium hydroxide and re-extracted with 6 mL of the hexane: methylene chloride
mixture. The organic phase was transferred to a clean tube and evaporated to dryness
under a nitrogen stream. The dried residue was dissolved in 100 pL of mobile phase
before injection into the chromatographic system.


91
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Time [h]
Figure 4-11. Plasma concentrations of codeine (mean standard deviation).
O Codeine 60 mg in one tablet
Codeine 60 mg and ibuprofen 400 mg in one combination tablet
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Time [h]
Figure 4-12. Plasma concentrations of codeine (mean standard deviation).
O Codeine 60 mg in one tablet
Codeine 60 mg and ibuprofen 400 mg in two separate tablets


131
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clearance and distribution in normal and fatty in situ perfused rat liver. J. Pharmaol.
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Clin. Pharmacol. Ther., 43:146.
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Comparative human study of ibuprofen enantiomer plasma concentrations
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9:539-549.
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ibuprofen plasma concentration and a neuroelectric correlate of analgesia. Pharm.
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administration. Clin. Pharmacol. Ther., 41:200.
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evoked potentials for pain quantitation. Physiol. Bchav., 31:339-342.


83
Figure 4-4. Plasma concentrations of ibuprofen (mean standard deviation).
O Ibuprofen 400 mg in one tablet
Codeine 30 mg and ibuprofen 400 mg in one combination tablet
Figure 4-5. Plasma concentrations of ibuprofen (mean standard deviation).
O Ibuprofen 400 mg in one tablet
Codeine 30 mg and ibuprofen 400 mg in two separate tablets


125
Case FZ
If CHKJTime.Value Then Write #2, EPData(0, i%), EPData(l, i%)
Else Write #2, EPData(l, i%)
Case CZ
If CHK_Time. Value Then Write #2, EPData(0, i%), EPData(2, i%)
Else Write #2, EPData(2, i%)
Case PZ
If CHK_Time.Value Then Write #2, EPData(0, i%), EPData(3, i%)
Else Write #2, EPData(3, i%)
Case ALL
If CHK_Time. Value Then Write #2, EPData(0, i%), EPData(l, i%),
EPData(2, i%), EPData(3, i%)
Else Write #2, EPData(l, i%)7 EPData(2, i%), EPData(3, i%)
End Select
If i% Mod 8 = 0 Then
Gauge.FloodPercent = (i% 100 \ 255): Gauge.Refresh
End If
Next i%
Close #2
Gauge.FloodPercent = 0: Gauge.FloodShowpct = FALSE
Readout.Caption = "Select Biologic ASCII file to process..."
Screen. Mousepom ter = DEFAULT
End Sub
Sub BTN_ExtractAll_Click ()
If Filel.ListCount > 0 Then
1 Select and extract data from each Bio-logic file individually
For LoopIndex% = 0 To Filel.ListCount -1
Filel.Listlndex = LoopIndex%: BTN_Extract_Click
Next LoopIndex%
Filel.Listlndex = 0
End If
End Sub
Sub BTN_ProcessExperiment_Click ()
If Filel.Filename = "" Then
Beep: MsgBox "Cannot process experiment: No file selected..."
Exit Sub
End If
Screen. Mousepoin ter = HOURGLASS
' Get Bio-logic filename prefix (first 4 characters)
SourcePath = Dirl.Path
If Right$(SourcePath, 1) <> "\" Then SourcePath = SourcePath + "\"
' Reset hie list box to only display files with the 4 letter prefix
Filel.Pattern = Left$(Filel.Filename, 4) + "*.ASC": Filel.Refresh
If Filel.ListCount > 10 Then
Beep: MsgBox "Cannot process experiment: more than 10 files selected..."
Exit Sub


130
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potentials in man: Possible central action in man. Exp. Brain Res., 39:359-364.
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46:1067-1074.


Metabolism 39
Elimination 41
Toxicity 42
Therapeutic Uses 42
Dosage and Administration 43
Codeine 43
Physicochemical Properties 43
Pharmacology 44
Pharmacokinetics 46
Absorption 46
Distribution 46
Metabolism 46
Elimination 48
Toxicity 48
Therapeutic Uses 49
Dosage and Administration 49
3 EXPERIMENTAL 50
Drug Assays 50
Codeine Analysis 50
Chemicals and reagents 50
Instrumentation 51
Extraction procedure 51
Chromatographic conditions 53
Ibuprofen Analysis 53
Chemicals and reagents 53
Instrumentation 53
Extraction procedure 54
Chromatographic conditions 54
Pharmacokinetic Study 55
Experimental Design 55
Subjects 55
Drug Administration 56
Blood Sampling 56
Estimation of Pharmacokinetic Parameters 56
Statistical Analysis 57
Pharmacodynamic Study 57
Experimental Design 58
Subjects 58
Drug Administration 58
Blood Sampling 59
Dental Stimulation 59
Determination of Pain Threshold 61
Evoked Potential Recording 61
Estimation of Pharmacokinetic Parameters 65
Estimation of Pharmacodynamic Parameters 66
Subjective pain ratings 67
Tooth pulp evoked potentials 68
Pharmacokinetic-Pharmacodynamic Modeling 69
Statistical Analysis 70
4 RESULTS AND DISCUSSION 71
Analytical Procedures 71
Ibuprofen Analysis 71
v


12
the synapses between nerve fibers throughout the central and peripheral nervous
systems. They serve as neurotransmitters by binding to specific receptors and inhibiting
the release of substance P following nociceptive stimulation (Chapman and Bonica,
1983). A variety of other neurotransmitters and neuromodulators have been identified.
They include norepinephrine, serotonin, angiotensin II, somatostatin, cholecystokinin,
neurotensin, and dynorphin (Bonica et al., 1990; Stimmel, 1983).
In summary, although considerable progress has been achieved in the
understanding of the neurophysiology of pain, the modulation of nociceptive impulses
is complex, and the relationship between analgesic drugs and neurotransmitters in the
control of pain transmission is still under investigation.
Pain Measurement and Assessment
One of the most critical aspects in the investigation of pain is the question of its
assessment. Pain is a highly complex phenomenon that by its very nature renders
objective assessment difficult. Pain is a subjective experience which is difficult to
express and quantify, and is not directly measurable. Further difficulties occur since
individual patients react to similar painful stimuli in quite different ways. In addition,
many factors are known to influence pain response. They include race, sex, age, culture,
personality; the type, duration, and intensity of the pain; and psychological variables
such as fear, anxiety, bias, and distraction (Beecher, 1957). However, the measurement
of pain is essential for the study of its mechanisms and for the evaluation of pain
control methods.
Experimental pain differs fundamentally from clinical pain as seen by the
physician. Experimental pain can always be discontinued. The subjects are informed
about the experimental procedure and are often interested in the success of the
experiment. This leads to a predominant rational component of pain, so that what we
really measure is the sensory-discriminative component of pain. On the other hand,


11
Descending pathways are mainly inhibitory in nature. Stimuli are produced in
response to cortical and subcortical activation, responding to sustained peripheral pain
input. These descending impulses may be visualized as controls over the "gate",
thereby modulating pain input (Bonica, 1990b; Fields, 1984; Melzack and Wall, 1965,
1983; Nathan, 1976).
Pain modulation
Scientific evidence gathered over the last twenty years made it clear that many
different mechanisms modulate (i.e. facilitate or inhibit) the transmission of nociceptive
information at every level of the nervous system. The first evidence of the existence of a
pain modulation system was the observation that electrical stimulation of discrete areas
of the brain profoundly inhibited responses to painful stimuli (Fields, 1974; Basbaum
and Fields, 1978). At about the same time, the discovery of opiate receptors and of
enkephalins and endorphins provided insight into the manner in which narcotic
analgesics relieve pain. It is now generally accepted that pain modulation is achieved
through a series of complex interactions between inputs from the periphery,
intemeurons in the spinal cord, and descending control systems from the brain (Bonica
et al., 1990; Fields, 1984; Zimmermann, 1984). These interactions involve a number of
endogenous biochemical agents called neurotransmitters or neuromodulators.
At the periphery, tissue damage caused by injury, disease, or inflammation
releases endogenous pain-producing substances into the extracellular fluid that
surrounds the nociceptors. These chemicals include H+ and K+ ions, serotonin,
histamine, prostaglandins, bradykinin, substance P, and others (Bonica et al., 1990;
Stimmel, 1983). In the dorsal horn of the spinal cord, a neurotransmitter involved in
pain transmission is the peptide substance P, which is released after stimulation of A5
and C fibers. Opioid receptors are also present in the dorsal horn, particularly in the
substantia gelatinosa, and they are thought to play an inhibitory role with enkephalins
acting as neurotransmitters in the inhibitory intemeurons. Enkephalins are located in


101
0 128 256 384 512 0 128 256 384 512
Figure 4-19. Effect of treatment on tooth pulp evoked potentials before (left) and
90 minutes after drug administration (right).
Subject 4.
From top to bottom: placebo, ibuprofen, codeine, and ibuprofen-codeine.


6
theories proposed a specific pain system carrying messages from pain receptors in the
skin to a pain center in the brain (Melzack and Wall, 1983).
More recently, it became clear that pain is also influenced by a host of
psychological, social, and environmental factors in addition to stimulus intensity. It is
generally accepted that pain is a primary sensory modality, but it is also recognized that
pain is far more complex than simple sensation. Sensory mechanisms are necessary but
not sufficient for the definition of pain. Modern concepts of pain emphasize its
multidimensional nature (Beecher, 1957; Chapman and Jacobson, 1984; Melzack and
Wall, 1983; Syrjala and Chapman, 1984). In addition to nociception, pain involves an
intricate interaction of emotional, motivational, cognitive and environmental factors (see
Figure 2-1). Each pain has unique qualities, and the pain of a toothache is obviously
different from that of a pinprick. In contrast to von Frey's theory, pain is now
recognized to be one of the most complex human experiences.
Emotions
Motivations
Figure 2-1. Multidimensional model of pain.
Adapted from Syrjala and Chapman (1984).
In light of these problems, the International Association for the Study of Pain
(1979) compiled a list of terms and definitions, and defined pain in scientific terms as


66
where Cp is the drug concentration at time t, kn is the absorption rate constant, ke is the
elimination rate constant, and t is the time after drug administration.
The equation was fitted to the experimental data by use of the poly-exponential
curve stripping and fitting program Rstrip (release 5.0, Micromath Scientific, Salt Lake
City, Utah). From these results, the time to reach the maximum plasma concentration
(tmax), the area under the curve (AUCJ, the area under the first moment curve
(AUMCJ, the mean residence time (MRT), the elimination half life (tV2), and the
apparent oral clearance (Clpo) were calculated according to the following equations:
t
max
I n kn In ke
K-K
AUC
CO
AUMC = A-
MRT =
AUMC
AUCrr
Cl =
po A L/C.
The maximum plasma concentration (Cmax) was the fitted plasma concentration
at the time of the maximum plasma concentration (tmax).
Estimation of Pharmacodynamic Parameters
Three different indicators of analgesic action were used to correlate drug effects.
Analgesia was recorded before drug intake, and at 0.5, 1,1.5, 2, 3, and 4 hours after
drug administration


117
Time (h)
Figure 4-31. Ibuprofen effect-time curves (mean SEM).
(Top: PID; Center: PR; bottom: EP).
O Ibuprofen 400 mg
Ibuprofen 400 mg and codeine 30 mg


Ill
Pharmacokinetic-Pharmacodynamic Modeling
The concentration-effect relationships of ibuprofen and codeine were
investigated by plotting the observed analgesic effect as a function of measured plasma
concentration. Figures 4-28 and 4-29 present the concentration-effect curves obtained
after administration of ibuprofen and codeine.
Examination of Figures 4-28 and 4-29 reveals that in both cases, concentration-
effect plots present a maximum analgesic effect of about 50 to 60%. However, a lack of
data points in the sub-maximal range prevented any kind of modeling to be done.
An interesting feature of the codeine concentration-effect curve is the presence of
a counter-clockwise hysteresis loop, thus indicating the presence of a lag-time between
the time-course of effect and plasma concentration. This delay could be explained by the
formation of numerous active metabolites.
The establishment of the contribution of codeine to the analgesic efficacy of the
combination was investigated by comparing the effect-time curves of ibuprofen and of
the combination. Since the ibuprofen doses were similar (400 mg ), direct comparison of
the two curves is possible. As can be seen on Figure 4-31, the time course of the
analgesic effect of ibuprofen given alone is almost superimposable to the one of the
combination. This indicates that the contribution of codeine to the analgesic effect of the
combination is minimal. The addition of codeine probably does not add much to the
analgesic effect of ibuprofen.
Due to different codeine doses, the evaluation of the contribution of ibuprofen to
the analgesic efficacy of the combination cannot be done with simple effect-time
profiles. Instead, a plot of the analgesic effect of codeine as a function of codeine
concentration may be used. As shown on Figure 4-30, slight differences in the slopes of
the individual effect-time curves may indicate that ibuprofen adds some effect to the
effect of codeine in the combination.


96
+
10.0 pV
1.0 s
H
Figure 4-14. Resting electroencephalogram of subjects 3 (top) and 4 (bottom); Subjects
were alert and had eyes closed during the recording.


7
"an unpleasant sensory and emotional experience associated with actual or potential
tissue damage, or described in terms of such damage."
The Physiology of Pain
Within the last twenty years, remarkable progress has been made in
understanding the physiology of pain. The discovery of a highly organized central
nervous system network for the modulation of pain, and the isolation and description
of the endorphins greatly advanced our understanding of pain transmission.
Nevertheless, the physiology of pain is complex and many areas remain unknown.
The physiology of pain involves nociceptors, combined with an intricate system
of afferent and efferent neuronal connections.
Nociceptors
Most pain originates when specific nerve endings are stimulated and nerve
impulses are transmitted to the brain through the pain pathways. These receptors, also
known as nociceptors, can be classified into one of two types: mechanothermal
(ummodal) receptors and polymodal receptors (Bonica, 1990b; Nathan, 1976).
Mechanothermal nociceptors are mainly present in the skin and respond to
strong pressure applied to a wide area of skin and strong stimuli such as a pinprick or
sudden application of heat (greater than 45C). They warn of potential damage and are
the afferent part of the withdrawal reflexes. This type of receptor is mainly associated
with small myelinated primary afferent neurons designated A6 type that transmit
impulses rapidly. Stimulation of this type of receptor results in "first" or "rapid" pain
which occurs early after injury and is usually sharp, well-localized, and pricking
(Bonica, 1990b).
Polymodal receptors are the free nerve endings of unmyelinated primary
afferent neurons of the C type. They are widely distributed throughout most tissues
and respond to tissue damage. Their classification as polymodal indicates that they


85
Codeine Pharmacokinetics
The pharmacokinetic parameters of codeine are summarized in Tables 4-6
through 4-8 and representative concentration-vs-time curves are shown in Figures 4-8
through 4-12. Figures 4-9 to 4-12 compare the mean plasma levels for each formulation
to the 400 mg oral dose of codeine.
A single 60 mg oral dose of codeine resulted in an average peak plasma
concentration of 122.8 ng/mL at 1.31 hours after dosing. The levels declined rapidly
with an average elimination half-life of 2.5 hours and mean residence time of 4.25 hours.
These parameters were in good agreement with literature values (Findlay et al., 1977,
1978; Yue et al., 1991a, 1991b; Quiding et al., 1986; Shah and Mason, 1990).
As can be seen on Figures 4-8 through 4-12, the plots show good agreement
between the disposition profile of the different ibuprofen-codeine combinations and the
reference (codeine 60 mg). Except for dose effects (i.e. 30 mg versus 60 mg), the
pharmacokinetic parameters of codeine obtained after administration of the four
different ibuprofen-codeine combinations were not significantly different from those
obtained after sole administration of codeine. In the case of the area under the curve
(AUC) and area under the first moment curve (AUMC), the dose-corrected parameters
were not significantly different.
As was seen with the ibuprofen data, however, the time of the maximum
concentration obtained after administration of ibuprofen 400 mg and codeine 60 mg in
one combination tablet (0.94 hour) was significantly longer (p < 0.05) than the one
observed after codeine 60 mg (1.31 hour). Since the other pharmacokinetic parameters
of these two formulations, and m particular the areas under the concentration-time
curve, were not significantly different, this may indicate a difference in absorption rate
but not in extent. This formulation was already shown to have a significantly reduced
the peak ibuprofen concentration when compared to ibuprofen 400 mg alone.


35
Ibuprofen
Ibuprofen is one in a series of 2-phenylpropionic acid derivatives discovered
over a fifteen year period at the Boots Pure Drug Company (Nottingham, England)
during the early 1960s. It is the parent compound of a class of non-steroidal anti
inflammatory drugs: the propionic acid derivatives. It was introduced in the United
Kingdom in 1967 and became the first propionic acid derivative approved by the Food
and Drug Administration for use as an anti-inflammatory agent in the treatment of
rheumatic diseases in 1974. It was later approved as an analgesic for the treatment of
mild to moderate pain. On the basis of its good safety record, it became available as a
nonprescription analgesic in 1984 (Adams, 1992).
Physicochemical Properties
Ibuprofen, (R,S)-2-(4-isobutylphenyl)-propionic acid, occurs as a white to off-
white crystalline powder with a slight characteristic odor and taste. It has a molecular
weight of 206, and a pKa of 4.6 (Dietzel et al., 1990). Its melting point ranges between 74
and 77C. It is only slightly soluble in water (1 mg/mL), but readily soluble in alcohol
and most organic solvents (Baselt and Cravey, 1989; Davis, 1975; McEvoy, 1990).
Figure 2-12. Chemical structure of ibuprofen.
Left: R(-)-isomer; Right: S(+)-isomer.
As with other propionic acid derivatives, ibuprofen possesses one chiral center
and thus exists as a racemic mixture of the R(-) and S(+)-isomers (see Figure 2-12).
However, its pharmacological activity is associated mainly, if not entirely, with the


34
Nitrous oxide effects on tooth pulp evoked potential have been studied by
several researchers (Benedetti et al., 1982; Chapman and Benedetti, 1979; Estrin et al.,
1988). Chapman and Benedetti observed that 33% nitrous oxide in oxygen significantly
decreased the mean amplitudes of all late components and also decreased subjective
pain report (Chapman and Benedetti, 1979). Benedetti et al. (1982) subsequently
described a dose-response relationship between nitrous oxide concentration, evoked
potential amplitude and subjective pain report. Although the linearity of the
relationship was unclear, they hypothesized that evoked potential amplitudes were a
reliable correlate of nitrous oxide analgesia.
Advantages of Evoked Potentials Methods
The described studies using evoked potentials strongly suggest the usefulness of
this technique to measure analgesic activity. Analgesic effects are sensitively revealed
by evoked potential methods in healthy subjects, with little difference between evoked
potential results and clinical efficacy. Evoked potential techniques can be used
repetitively to follow the time course of drug action (Bromm and Scharein, 1982b;
Buchsbaum, 1984). Moreover, evoked potential methods can detect differences in pain
attenuation for different analgesic treatments, different bioavailabilities and even
different dosages of the same drug (i.e. 0.5 or 1.0 g of acetylsalicylic acid), whereas
subjective methods usually cannot do this in the same number of subjects (Derendorf et
al., 1982; Rohdewald et al., 1982a; 1982b).
In short, evoked potential methods are more sensitive for the quantitation of
pain perception than subjective pain reports, so that evoked potential studies can be
done in smaller groups of subjects than would be necessary with other commonly
employed techniques.


29
The late potentials used in pain research can be described by a positive signal
occurring at approximately 80 ms after stimulus onset (P80), followed by a distinct
negativity between 120 and 200 ms (N150), and another positive signal (P250) between
200 and 400 ms (Chapman et al., 1979).
In their initial study, Harkins and Chapman (1978) delivered faint, mild and
strong painful stimuli to volunteers in order to determine whether a relationship
between stimulus intensity and the evoked potential response could be demonstrated.
Evoked potential peak-to-peak amplitude increased as stimulus intensity was
augmented, and there was a linear relationship between the logarithm of the stimulus
intensity and the amplitude of the various evoked potential components. In contrast,
peak latency was unaffected by changes in stimulus intensity. Partial correlation
analysis of the data revealed that amplitudes of the later components (N150 and P250
components) were significantly correlated with subjective pain ratings. The amplitude
of the P80 component showed no relationship to the subjective pain report. Rather, it
correlated with stimulus intensity (Chen et al., 1979).
These results were confirmed by Bromm (Bromm, 1985; Bromm and Scharein,
1982a) in studies using multivariate statistical data analysis methods (Bromm, 1984b;
Strenge and Gundel, 1983). This suggests that the late cortical evoked potential may be
useful as a physiological correlate of the psychophysiological processes underlying the
perception of pain. Thus, evoked potential amplitudes may track the intensity of the
pain experienced by subjects (see Figure 2-10).
It should be noted that the amplitude of the cerebral evoked potential may vary
as a function of the stimulus repetition rate: longer inter-stimulus intervals produce
larger evoked potentials than shorter inter-stimulus intervals (Chapman et al., 1981;
Jacobson et al., 1985). In studying differential effects of painful stimuli on averaged
evoked potentials, however, the particular inter-stimulus interval does not need to be
taken into account so long as it is consistent. Moreover, it has been demonstrated that


37
Prostaglandins play an important role in regulating a number of physiologic
processes, including inflammation and pain modulation. They are released from tissues
by a variety of stimuli such as chemical or mechanical stimulation. The observation that
aspirin and other non-steroidal anti-inflammatory drugs selectively inhibit
prostaglandin synthesis led Vane to propose that the therapeutic and toxic effects of
those drugs may be due to the inhibition of prostaglandin biosynthesis (Vane, 1971).
While prostaglandins possess no pain-producing properties themselves, prostaglandins
of the E type have been shown to sensitize afferent nerve endings to the effects of
bradykinin and histamine. Thus, the most likely explanation for the analgesic effect of
non-steroidal anti-inflammatory drugs is the prevention of sensitization of sensory
nerve endings by the inhibition of prostaglandin biosynthesis (Benedetti and Butler,
1990; Ferreira 1980; Higgs, 1980; Vane, 1971).
Recent findings by several laboratories suggested that other mechanisms may
account for some of the anti-inflammatory effects of non-steroidal anti-inflammatory
drugs. However, the importance of these non-prostaglandin-mediated effects such as
inhibition of the 15-lipooxygenase pathway and interference with leukocyte migration
and function remains unknown (Goodwin, 1984; Kantor, 1979, 1984).
In addition to its analgesic and anti-inflammatory effect, lbuprofen has been
shown to reduce fever in animals and humans. This antipyretic effect is thought to be
caused by a central mechanism, possibly as a result of inhibition of prostaglandin
synthesis at the hypothalamus level (Benedetti and Butler, 1990).
lbuprofen has also been shown to cause gastric irritation, tissue erosion, and
gastric bleeding. These effects are thought to result from the direct irritation of the
gastric mucosa, and from the indirect inhibition of prostaglandin synthesis in the
stomach (Benedetti and Butler, 1990; Kantor, 1979, 1984; McEvoy, 1990).
lbuprofen, like other non-steroidal anti-inflammatory drugs, alters platelet
aggregation by inhibiting platelet cyclooxygenase, which in turn blocks thromboxane


CHAPTER 3
EXPERIMENTAL
This research consisted of two separate studies. The first study centered on
determination of the pharmacokinetic parameters of codeine and ibuprofen in healthy
volunteers. The second study examined both pharmacokinetics and the pharmaco
dynamic effect, analgesia, of codeine and ibuprofen.
Drug Assays
Determination of codeine and ibuprofen concentrations in biological fluids
required establishment of two high-performance liquid chromatographic assays.
Codeine Analysis
Plasma codeine concentrations were determined by the high-performance liquid
chromatographic method of Mohammed et al. (1992).
Chemicals and reagents
Codeine phosphate, USP reference standard, was provided by Boots
Pharmaceuticals (Shreveport, Louisiana, USA), and used as received. The internal
standard, N-isopropylcodeine, was synthesized by the Department of Medicinal
Chemistry at the University of Florida by alkylation of norcodeine. It was purified
before use.
HPLC-grade acetonitrile, hexane, and methylene chloride were purchased from
Fisher Scientific (Fair Lawn, New Jersey, USA). HPLC grade 1-octanesulfonic acid
50


105
Table 4-9. Pharmacokinetic parameters of ibuprofen (mean standard deviation).
Ibuprofen 400 mg
Ibuprofen 400 mg
and codeine 30 mg
Cmax (Pg/mL)
25.2
25.4
tmax (h)
1.16
1.29
AUQ (pg-h/mL)
104.6
103.0
AUMC^ (pg-h2/ mL)
344.6
321.3
MRT (h)
3.30
3.12
ka (h->)
1.669 0.478
1.265 1.929
ke (h-)
0.371 0.080
0.429 0.311
t,2 (h)
1.87
1.62
CU (L/h)
3.82
3.88
Table 4-10. Pharmacokinetic parameters of codeine (mean standard deviation).
Codeine 60 mg
Codeine 30 mg
and ibuprofen 400 mg
Cmax (ng/mL)
122.2
75.7
tmax (h)
1.33
1.56
AUCk (ng-h/mL)
556.6
340.0
AUMC^ (ng-h2/mL)
1994.0
1145.5
MRT (h)
3.58
3.37
ka (h-1)
1.376 1.476
0.887 0.704
ke (h-i)
0.350 0.154
0.446 0.322
(h)
1.98
1.55
Opn (L/h)
107.8
88.2


28
A patient congenitally insensitive to pain but with intact sensations in other sensory
modalities failed to show any cerebral responses or subjective responses to strong
electrical tooth pulp shocks.
These results assessing the validity of using evoked potentials in the study of
pain in humans have led many researchers to investigate the correlations existing
between evoked potentials and subjective reports of pain.
Evoked potentials bear striking similarities, whether they are elicited by painful
electrical or mechanical skin stimuli, or by electrical tooth pulp stimuli (see Figure 2-9).
Differences due to the different kinds of stimulation appear in the early and
intermediate latency components with peak latencies of less than 80 milliseconds, but
these early components have not been evaluated systemically in pain research.
|UV DEN yuV LAS
Figure 2-9. Evoked potentials obtained with various pain stimuli.
EL: electrical skin stimulation; MEC: mechanical skin stimulation;
DEN: electrical tooth pulp stimulation; LAS: laser-emitted thermal
stimulation.
From Bromm (1984b).


Chromatography 71
Linearity 72
Reproducibility 72
Codeine Analysis 72
Chromatography 73
Linearity 74
Reproducibility 74
Pharmacokinetic Study 77
Ibuprofen Pharmacokinetics 78
Codeine Pharmacokinetics 85
Pharmacodynamic Study 92
Dental Stimulation 92
Evoked Potential Recording 94
Pharmacokinetic Measurements 104
Pharmacodynamic Measurements 108
Pharmacokinetic-Pharmacodynamic Modeling Ill
5 CONCLUSION 118
APPENDIX
TPEP TOOLKIT 120
BIBLIOGRAPHY 127
BIOGRAPHICAL SKETCH 143
vi


103
Figure 4-21. Effect of treatment on tooth pulp evoked potentials before (left) and
90 minutes after drug administration (right).
Subject 6.
From top to bottom: placebo, ibuprofen, codeine, and ibuprofen-codeine.


61
insertion of dental cotton rolls (Healthco International, Boston, Massachusetts, USA)
between the upper lip and gum.
Stimulator
Grass S48
Stimulus
Constant
Trigger
Isolation Unit
Current Unit
Grass SIUS
Grass CCUIA
Subject
Figure 3-4. Block diagram of the stimulation system.
Determination of Pain Threshold
On each study day, threshold of sensation was defined as the lowest current
intensity necessary to evoke a sensation. It was determined by each subject by
manipulating a dial on the constant current unit until the first sensation was felt at the
tooth. The current intensity was then read on a digital multimeter (model DM 7143,
Goldstar Precision, Cerritos, California, USA) by the investigator. After turning the
knob for producing higher and slightly painful stimulation intensities, the subject was
asked to turn the knob back to lower intensities and relocate the position of the
threshold of sensation. The current intensity was then read and the mean of the
corresponding two current intensities was recorded as threshold by the investigator.
This individual threshold measurement was used as the basis for painful tooth pulp
stimulation, with all subsequent stimuli being delivered at twice the threshold current.
Evoked Potential Recording
Spontaneous electroencephalographic activity (EEG) was recorded from leads
placed over midline regions of the brain. All electrodes were placed according to the
International Ten-Twenty System of electrode placement (see Figure 3-5). This system


62
is based on measurements of head circumference, the distance from nasion to inion, and
the distance from ear to ear. Even numbers are to the right of the midline, and odd
numbers are to the left (Jasper, 1958).
Figure 3-5 Electrode names and locations in the International Ten-Twenty System.
Samples of electrophysiologic brain activity were obtained from Fz, Cz, and Pz
with reference to Al, with an electrode affixed to Fpz as ground. Electrodes were
maintained on the scalp between effect measurements. The positioning of the electrodes
was chosen to maximize the signal-to-noise ratio.
The skin at each electrode site was prepared by cleaning with isopropyl alcohol,
then rubbing with a cotton swab dipped in a conductive abrasive cream (Omni-Prep,
D.O. Weaver and Company, Aurora, Colorado, USA). Electrodes (10 mm gold plated
disk electrodes, type E5GH, Grass Instrument Company, Quincy, Massachusetts) were
then held in place on the skin while collodion (Mavidon Corporation, Palm City,
Florida, USA) was applied around the rim and dried rapidly with compressed air.
Electrodes were filled with conductive jelly (Mavidon Corporation).
Inter-electrode impedances were measured prior to initial recording and at
hourly intervals during the experiment on an analog electrode impedance meter (Model
EZM1E, Grass Instrument Company). They were matched and maintained below 3 k£2


77
Pharmacokinetic Study
The combination of ibuprofen and codeine in a single oral dosage form is
currently contemplated. Their pharmacokinetics in healthy subjects have been
extensively investigated. Yet, their pharmacokinetics when given together have not
been established, and the potential for a pharmacokinetic interaction exists, ibuprofen is
highly bound to plasma proteins, and a change in its pharmacokinetic parameters due
to the presence of an interacting drug may produce undesirable pharmacological
effects. Its is then important to investigate the pharmacokinetics of codeine and
ibuprofen when given alone or in combination before any such combination can be
developed further.
The aim of this study was to compare the pharmacokinetics of single oral doses
of codeine and ibuprofen when given alone or in combination. Six different oral
formulations were examined:
1. Ibuprofen 400 mg in one tablet
2. Codeine 60 mg in one tablet
3. Codeine 30 mg and ibuprofen 400 mg in two separate tablets
4. Codeine 30 mg and ibuprofen 400 mg in one combination tablet
5. Codeine 60 mg and ibuprofen 400 mg in two separate tablets
6. Codeine 60 mg and ibuprofen 400 mg in one combination tablet
Twenty four subjects, matched for age and gender, were enrolled and completed
this study. They received on six different occasions an oral dose of the test drug. Plasma
samples were then harvested and analyzed to determine ibuprofen and codeine
concentrations. The pharmacokinetic parameters of codeine and ibuprofen were
determined in each subject by standard non-compartmental data analysis techniques.


PHARMACOKINETIC-PHARMACODYNAMIC MODELING OF THE
ANALGESIC EFFECT OF IBUPROFEN AND CODEINE
By
MATTHIEU L. KALTENBACH
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1992


2
Newer non-steroidal anti-inflammatory drugs constitute a potential source of
more effective peripherally acting components for opioid-containing analgesic
combinations. Among these, the combination of codeine with ibuprofen (a propionic
acid derivative with anti-inflammatory and analgesic activities) in a single oral dosage
form is currently investigated (Cater et al., 1985; Cooper et al., 1982; Frame et al., 1986;
Friedman et al., 1990; McQuay et al., 1989; Norman et al., 1985; Sunshine et al., 1987).
Codeine and ibuprofen have been studied extensively since their introduction.
Their pharmacology and pharmacokinetics are well defined in the literature (Gilman et
al., 1985). Yet, no study has clearly established the relationship between plasma levels of
these drugs and the degree of analgesia attained. The establishment of such correlations
is usually a prerequisite for optimal drug therapy and may lead to more effective use of
drugs. Ultimately, this will result in greater predictability of therapeutic response and
the possibility of reducing the incidence or severity of adverse effects (Colburn, 1987). In
the case of analgesic drugs, however, correlations between in vivo drug concentrations
and pharmacodynamic effects have seldom been described because of the inherent
difficulty of quantifying the degree of pain relief (Inturrisi and Colburn, 1986; Inturrisi
et al., 1987,1990; Laska et al., 1986b). Studies employing electrical tooth pulp
stimulation have demonstrated that dental dolorimetry is a reliable way of producing a
relatively pure pain experience in human laboratory subjects (Azerad and Woda, 1977;
Mumford and Bowsher, 1976). Moreover, there is now considerable evidence that late
brain potentials evoked by tooth pulp stimulation reflect pharmacologically induced
pain relief in man, particularly when used in conjunction with other scaling procedures
(Bromm, 1985; Chapman and Jacobson, 1984; Chapman et al., 1979; Chen et al., 1979;
Hill and Chapman, 1989; Lekic and Cenic, 1992; Rohdewald et al., 1980,1982a, 1982b;
Rohdewald and Keuth, 1990). Using these methods, it is possible to measure the
analgesic effect as a function of time after drug administration.


46
Pharmacokinetics
Codeine pharmacokinetics in man have been extensively investigated (Clouet,
1971; Findlay et al., 1977, 1978, 1986; Guay et al., 1987; Hull et al., 1982; Quiding et al,
1986; Rogers et al., 1982; Shah and Williams, 1990; Way, 1968; Way and Adler, 1962,
1968). However, controversy still exists concerning the relative importance of some of
its active metabolites on its analgesic efficacy.
Absorption
Following oral administration, codeine is rapidly absorbed from the
gastrointestinal tract. Its bioavailability after oral administration was found to be
between 50 and 60% (Findlay et al., 1977; Spahn et al., 1983), and almost complete after
intramuscular administration (Brunson and Nash, 1975; Findlay et al, 1977).
Peak plasma concentrations (100 ng/mL after a 60 mg oral dose) are usually
observed within one to two hours after drug administration (Quiding et al., 1986; Shah
and Mason, 1990).
Distribution
Codeine is about 25% bound to plasma proteins (Judis, 1977; Findlay et al.,
1978). It has an approximate volume of distribution of 7.0 L/kg indicating extensive
distribution into the extravascular space (Findlay et al., 1977; Shah and Mason, 1990).
Codeine, like most basic amines, rapidly leaves the blood and concentrates in tissues,
particularly parenchymatous tissues such as the brain, liver, kidney, lung, and adrenal
glands (Way and Adler, 1962).
Metabolism
Codeine is primarily metabolized in the liver by conjugation with glucuronic
acid. Other less important metabolic pathways include O-demethylation to morphine
and N-demethylation to norcodeine (Adler, 1952, 1958; Adler et al., 1955; Muhtadi and
Hassan, 1981; Sindrup et al., 1991). These primary metabolites of codeine are also
metabolized further as outlined in figure 2-17.


Dedicated to my wife, Marie-Pierre,
and our children, Ccile, Pauline, and Emmanuelle


15
Pain intensity difference and pain relief scores are also commonly summed over
the observation period to provide estimates of the area under the effect-time curve.
These estimates are known as the summed pain intensity differences and the total pain
relief scores:
SPlD = ^PIDrAt
TOTPAR = ^ PR, At
where SPID is the sum of the pain intensity differences, PIDt is the pain intensity
difference at time t, At is the time elapsed since the previous observation, TOTPAR is
the total pain relief, and PRt is the pain relief score at time t.
The efficacy of different analgesic treatments may then be evaluated by
comparing hourly scores, peak scores, SPID scores, and TOTPAR scores (Max and
Laska, 1991).
Due to their simplicity, categorical rating scales are among the most widely used
methods to assess the human pain experience. They are understood by most patients,
require little time to complete, and can be analyzed easily (Max and Laska, 1991).
However, these scales have been criticized on several counts. First, they often do not
have enough categories to allow patients to accurately describe their pain intensity,
resulting in a poor sensitivity (Chapman and Syrjala, 1990; Huskisson, 1974; Max and
Laska, 1991; Syrjala and Chapman, 1984). Similarly, words can have different nuances
of meaning, and ratings may vary from subject to subject (Wallenstein et al., 1980).
Another disadvantage of the categorical rating scales is the limited nature of the verbal
categories. Although categories are generally assigned equal step integer values, little is
known of the true size of the distances between pain scores (Huskisson, 1974; Laska et
al, 1986a; Max and Laska, 1991; Wallenstein et al., 1980).
Visual analog scales
Visual analog scales have been used in psychology since the beginning of the
century. It is only in recent years that they have been applied to the measurement of


17
Measurements are made in terms of millimeters from the "no pain relief" boundary,
providing a 100-point relief scale (Wallenstein, 1991).
Like categorical relief scales, relief visual analog scales serve to expand the part
of the scale corresponding to slight relief, potentially increasing the sensitivity over that
offered by the visual analog scale for pain intensity (Max and Laska, 1991; Quiding and
Hggquist, 1983; Schachtel, 1991; Wallenstein, 1984, 1991; Wallenstein et al., 1980).
Wallenstein (1984, 1991) also found a linear relationship between categorical and visual
analog relief scales. Their interpretation is straightforward and provides a direct reading
of analgesia. It is the parameter of choice in pharmacokinetic studies correlating
analgesic effects with blood levels (Wallenstein, 1991).
Multidimensional Pain Rating Scales
Another approach to evaluate the pain experience in man relies on the use of
extensive questionnaires that assess the multiple dimensions of pain. Multidimensional
scales recognize the complex nature of the pain experience.
Among the different types of questionnaires, the most popular and most widely
used is the McGill Pain Questionnaire, it was first introduced by Melzack in 1975, and
has since been translated in many languages. This questionnaire was designed to
provide information on the sensory, affective, and evaluative dimensions of pain
(Chapman and Syrjala, 1990; Syrjala and Chapman, 1984). It consists of twenty sets of
words that describe pain. Patients are asked to select those sets that are relevant to then-
pain, and to circle the word that best applies in each set. Pain scores may then be
summed for each dimension, with the total score being called the pain rating index
(Melzack, 1975).
Numerous studies have demonstrated the validity and reliability of the McGill
Pain Questionnaire (Gracely, 1990; Melzack, 1984). However, the complexity of its
vocabulary may limit its use with poorly educated patients. It remains primarily


109
Time (h)
Figure 4-26. PID (top), PR (center), and EP (bottom) effect-time curves.
Placebo
Ibuprofen 400 mg
O Codeine 60 mg
Ibuprofen 400 mg and codeine 30 mg


99
O 128 256 384 512 O 128 256 384 512
Figure 4-17. Effect of treatment on tooth pulp evoked potentials before (left) and
90 minutes after drug administration (right).
Subject 2.
From top to bottom: placebo, ibuprofen, codeine, and ibuprofen-codeine.


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52
1.0 ml Plasma pH 8
+ Internal Standard
Hexane/Methylene Chloride
Aqueous Phase Organic Phase
0.05 M Acetate Buffer pH 3
Aqueous Phase Organic Phase
0.1 N Sodium Hydroxide
Hexane/Methylene Chloride
Organic Phase Aqueous Phase
1
Evaporate
Nitrogen Stream

Reconstitute in Mobile Phase
Inject in HPLC System
Figure 3-1. Extraction procedure for codeine analysis.