Pharmacokinetics of buprenorphine in dogs

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Pharmacokinetics of buprenorphine in dogs
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Chandran, V. Ravi, 1955-
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Buprenorphine -- metabolism   ( mesh )
Buprenorphine -- administration & dosage   ( mesh )
Dogs   ( mesh )
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theses   ( marcgt )
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Thesis:
Thesis (Ph. D.)--University of Florida, 1986.
Bibliography:
Includes bibliographical references (leaves 348-351).
Statement of Responsibility:
by V. Ravi Chandran.
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Typescript.
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Vita.

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University of Florida
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PHARMACOKINEICS OF BUPRENORPHINE IN DOGS


By

V. RAVI CHANDRAN




















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FIDRIDA IN
PARTIAL FULF~IIDGET OF THE RIEQUIREMENTS
FOR THE DEGREEE OF DOCTOR OF PHIID~SOPHY



UNIVERSITY OF~ FIDRIDA


1986














ACKNIOWLEDGEMENT


I gratefully acknowledge Dr. Edward R. G~arrett for his numerous and

varied contributions. I also thank him for his guidance, training,

support and facilities during my graduate career, which made this

dissertation possible.

My special thanks are extended to Dr. Jurgen Venitz for his time,

fruitful discussions and helpful suggestions, and to Dr. Larry J. Peters

and ~Dr. August H. Battles for animal preparations.

I wish to thank the current and past members of the "Beehive" for

their help, support, advice and friendship.

I acknowledge the members of my supervisory committee,

Dr. John H. Perrin, Dr. Hartmut C. Derendorf, Dr. James W. Simp~kins,

Dr. Michael J. Katovich, Dr. C. Lindsay Devane, Dr. John A. Zoltewicz

for their contributions.

I take this opportunity to express my sincere accpreciation to

Dr. Bernard Desoize, Dr. Peter Langguth, Mrs. Marjorie Rigby, Mrs. Kathy

Eberst, Mr. George Perry and Mr. Thomas Miller for their valuable help.

















TABLE OF CONTENTS



ACKNOWLEDGEMIENTS ... .. .. .. .. .,. ... .. ii

ABSTRACT .. .. ... .. .. .. ... .Iv

INTRODUCTION...............,..... ........ 1

EXPERIMENTAL .... .................... 20

IV BOLUS STUDIES. ................... ...... 30

URINARY EXCRETION OF BUPRENORPHONE .. ... .. .. .. 86

TV INFUSION STUDIES ... ... ... .. .. .. .. 131

PHARMAOOKINETICS OF THE IV ADMINISTERED MEABLTE.. .. ... 214

SUMIA~RY AND CONCLUSIONS. .. .. .. .. .. ... .. .. ..241

APP~ENDIX I -PROGRAM "MULTI"................... 247

APPENDIX II FITTING OF DATA TO EQUATIONS .. .. .... 251

APPENDIX III KRUSKA;L-WLIS TEST. .. .. ... ... .. 258

APPENDIX TV TABLES OF RAW DATA. .. .. .. ... .. .. .. 261

GLO>SSARY OF TERMS. .. .. .. .. .. .. .. .. .. 346




BIOGRAPHICAL SKETCH. .. ... .. .. .. .. ... .. 52












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




PHARMACOKINETICS OF BUPRENORPHINE IN DOGS




By

V. Ravi Chandran


December 1986



Chairman: Dr. Edward R. Garrett
Cochairman: Dr. John H. Perrin
Major Department: Pharmaceutical Sciences

Specific and sensitive reverse-phase HPI assays of buprenorphine

and its metabolite in biological fluids wee developed with

sensitivities of 2-6 ng/ml using fluorimetric detection. Upon acute

bolus administration of buprenorphine in six dogs within the 0.7-2.6

mg/kg dose range, accurate estimation of the terminal rate constant and

the derived total body clearance were not feasible due to the lack of

sufficient quantifiable terminal plasma points at less than 5 ng/ml

sensitivity. The terminal plasma concentrations could not be increased

by increasing the bolus dose since such high doses would have

significant toxicity. This toxicity was circumvented and the terminal

plasma concentrations were increased by infusing 3.7-4.8 mg/kg doses of

buprenorphine over 3 h in six studies in six dogs. The tl-erminal rate









constants of the IV infusion studies averaged 34 + 3.7 h with an

averaged total body clearance of 212 + 35 ml/min. The apparent volumes of

distribution of buprenorphine referenced to the total plasma

concentration were 35 L (V central compartment volume) and 617 L

(Vd, total body volume) indicative of a highly bound, sequestered or
lipophilic drug.

Unchanged buprenorphine is insignificantly really (<0.5% of the

dose) and biliary (<0.5%) excreted. The major route of buprenorphine

disposition is by hepatic conjugation to glucuronide which is eliminated

into the bile (about 95%) with only small amounts appearing in urine

(<1% as metabolite) Minor metabolites excreted in the bile accounted

for about 3% of the administered dose.

Direct IV administration of the metabolite gave a terminal

balf-life of 6 h. Unlike intravenously administered morphine glucuronide

which was not excreted in the bile, more than 90% of the systemically

circulating metabolite was excreted in bile and only 10% in urine.

The oral bioavailability estimated from the areas under the

buprenorphine plasma concentration-time curve following IV and oral

administration of buprenorphine in the dogs was 3-6%. In a bile

cannulated dog, intraduodenally administered metabolite demonstrated 6%

enterohepatic recirculation of the conjugate.

There were no apparent correlations of the buprenorphine time

course with cardiovascular parameters such as heart rate, ECG and blood

pressure. Miotic effect was significant. Respiratory depression was

observed during the first 4 h after IV bolus injection, but not during

the infusion studies.














INTRODUCTION

Buprenorphine (1) is a derivative of the morphine alkaloid

thebaine. It is a strong analgesic with marked narcotic activity. Since

the mid-sixties, its therapeutic potential as a morphine-type analgesic

at low doses and antagonistic activity at high doses, has been well

documented.1 Buprenorphine has been claimed to have an advantage over

morphine in that the dose does not need to be increased during several
weeks of chronic administration.2

Pharmacodynamic and Therapeutic Studies

Bup~renorphine has displayed narcotic agonist and antagonist

properties in animals and man. Agonistic effects often exhibited a bell

shaped dose-response curve, as occurs with pentazocine,3 and subjective

opiate-like effects reached a maximum at a dose of about 0.2 to 0.8 mg

subcutaneously in man.4 The onset of agonistic effects (peak effects

about 6 h after subcutaneous or IM injection) in man was slower than

with morphine but the duration of such effects was longer (about 72

hours) than with morphine.5 Also, the analgesic potency of

buprenorphine was about 25 times that of morphine (on a per unrit weight

basis) .6

Therapeutic Trials

In a comparative study of the treatment of chronic pain of

malignant origin by intramuscularly administered buprenorphine and

morphine, 27 patients received buprenorphine (0.3 mg) and morphine (10

mg) in a double-blind, single-dose within-patient study.? There were n~o









significant differences ~in the intensity of analgesic effect or the time
to reach it.7 Howe~ver, buprenorphine had a significantly longer

duration of action than morphine. Sedation was the most frequent side

effect but dizziness, nausea and vomiting wEere also seen.7] Compared to

morphine, buprenorphine showed significantly higher incidences of side
effects, greater severity and earlier onset, and longer duration.7

Following both treatments there were small but significant decreases in

pulse rate, blood pressure and respiratory rate.7
Antinociceptive actions (blockade of impulses at the peripheral

pain sensitive nerves) of buprenorphine and morphine given intrathecally
in conscious rats wee compared.8 After intrathecal injection the peak

(30 min) antinociceptive potencies of buprenorphine or morphine were
similar.8 The analgesic profiles of buprenorphine and morphine (0.3 mg

and 10 mg respectively) were compared in a double-blind non-crossover

multiple dose study (IM administration) in Imn.9 When the patient

complained of moderate to severe post-operative pain after upper
abdominal surgery, the first test dose of either drug was given. The

drugs gave an equal decrease in pain intensity, suggesting a relative

potency of 33:1.9 An average of 0.51 mg of buprenorphine or 16 mg of
morphine had to be administered for satisfactory initial analgesia. A
faster decrease in the rate of respiration was observed after

buprenorphine than after morphine, but ultimately both the drugs gave
the same minimum rate of respiration.9 These results were comparable to

those reported elsewhiere.7

An oral combination of buprenorphine and paracetamol was compared

to paracetamo1 in a single-dose double-blind study in m~an for the
initial acute treatment of post-operative pain.10 One hundred and









twenty patients undergoing orthopedic operations wee divided into four

groups of 30 patients each. The four treatments were 1, 1.5 or 2 mg~ of

buprenorphine combined with paracetamol 1000 mg or paracetamol (1000 mg))

alone. There were no significant differences among the groups in

analgesia measured by the observer and by the pain intensity scoring by

the patients over the first six hour. The oral combSinations of

buprenorphine and paracetamo1 produced a significant increase in

duration of analgesia beyond 6 hours over that of paracetalmo1 alone at

all three dose concentrations. A significant increase in side effects

was seen only at the highest dose of buprenorphine-paracetamol

combination compared with paracetamrol alone.10

In a study designed to assess the development of drug dependence,

rats were chronically treated subcutaneously for 4 days with

buprenorphine.11 These rats showed only weak signs of withdrawal upon

cessation of a treatment or upon challenge with naloxone.11 More

intense withdrawal syrp~toms were induced when morphine was substituted

for buprenorphine. Even one injection of morphine, given 12 h after the

last buprenorphine treatment, led to withdrawal symptoms with naloxone.

Naloxone did not cause withdrawal in naive rats treated with this dose

of morphine. Thus, according to these authors,11 and contrary to a few

claims in the literature, buprenorphine induced dependence like other

opiates. The authors argue that the intensity of withdrawal is less

severe due to slow dissociation of the drug from the receptors.11

The neurochemical effects of buprenorphine were compared to those

of morphine and haloperidol in rats.13 The effect of a wide range of

doses of buprenorphine (0.001 10 mg/kg, subcutaneous administration)

was studied a) with normal concentrations of dopamine, noradrenaline,









5-hydroxytryptamine etc. and b) with lower concentrations of dopamine

and noradrenaline in rat brain following the treatment with alpha-methyl

para-tyrosine (alpha-MpTr) which is a inhibitor of catecholamine

synthesis. Morphine and haloperidol were used as reference agents.

Buprenorphine increased the alpha-M~pT induced rate of dopamine depletion

but did not deplete norepinephrine. Similar results were obtained with

a higher dose (30 mg/kg) of morphine but it increased the alphaT

induced depletion of norepinephrine. Apparently similar effects of

buprenorphine and haloperidol on dopaminergic neurotransmission wee

distinguished by pretreating the rats with naloxone (which antagonized

the effect of buprenorphine, and prevented dopamine depletion) These

neurochemical results were claimed to support the view that one site of

action of buprenorphine is on opiate receptors located on the

dopaminergic neurons.13

In a double-blind comparison between fentanyl and buprenorphine in

supplemented nitrous oxide analgesia, buprenorphine or fentanyl (0.3 and

0.125 mg respectively administered IV) were used as supplements in 40

patients undergoing major abdominal surgery.14 Initially both narcotics

appeared to suppress tachycardia and increase arterial pressure in

response to surgery but 80% of the patients who received fentanyl

eventually required a further supplement of halothane (0.5%) but no

patient who received buprenorphine required halothane. Recovery from

analgesia was similar in both groups, but the duration of analgesia

after the operation was significantly greater for buprenorphine (12 h)

than fentanyl (3 h) .14

In a double-blind randomized non-crossover trial, 47 patients

received either morphine (10 mg) or buprenorphine (0.3 mg) by regular IM









injection for 24 h after abdominal surgery.15 In this study, the tw~o

drugs werie equally effective at the dose ratio 1:33, buprenorphine to

morphine which is comparable to the results reported elsewhere.7,9

O~ne hundred twenty-six patients undergoing upper and low~ner

abdominal surgery were studied post-operatively to compare the analgesic

effect of IM morphine, sublingual buprenorphine and self administered IV

pethidinre.16 There were no significant differences among analgesic

regimens in respect to subjective pain scores or static and dynamic lung

volumes assessed at 24 h, 48 h and 5 days after operation. Sublingual

buprenorphine produced more nausea and sedation than the other regimens,

but the results wee not clinically important. These authorsl6 report

that buprenorphine offered considerable advantages in terms of ease of

administration .

When diamorphine and buprenorphine were compared in the relief of

chest pain in man, sublingual administration appeared to be as effective

as the IV route, but the onset of action was slow.17 There were no

significant changes in the systemic or pulmonary arterial blood pressure

or heart rate after IV buprenorphine.17 A randomized double-blind

controlled trial of equivalent doses of buprenorphine and diamorphine

showed no significant differences between the drugs in terms of pain

relief and duration of action.17 The occurence of nausea, vomiting and

other side effects was similar in the two groups. The onset of action of

buprenorhine was slightly but significantly slower than that of

diamorphine.17

Buprenorphine and pethidine were compared in a double-blind study

of on-demand IV analgesia. Buprenorphine was about 600 times as potent

as pethidine.18 The incidence of side effects was similar with both









drugs. The quality of analgesia subjectively assessed was the same with

both drugs using this method of administration.1 These authors claim

that buprenorphine is a powerful analgesic agent that may be given

intravenously provided that its low potential for abuse is

substantiated .

In a smaller number of patients with chronic pain, usually due to

cancer, sublingually given buprenorphine (up to 0.8 mg 4 hourly)

provided adequate pain relief for periods up to several months, but side

effects (usually nausea and vomiting) required discontinuation of

treatment in about 1/3 to 1/2 of the ambulatory patients.19

Following anesthesia with fentanyl in 180 patients, buprenorphine

(usually 0.4 to 0.8 mg~ IV) reversed some of the anesthetic effects while

producing continued analgesia that lasted about 8-12 h after a

single-dose.20 The antagonistic activity however, was frequently

short-lived, declining rapidly after 90 to 120 min and a second dose of

buprenorphine was of ten required to prevent the re-emergence of

anesthetic effects.20

The efficacy of buprenorphine has been compared to lofentanil and

to saline placebo by extradural administration in the management of

post-operative pain in sixty patients.21 In a double-blind study, these

orthopedic patients wFere randomly assigned to three equal groups to

determine the analgesic effects, duration of action and side effects of

the extradural administration of lofentanil (5 ug) buprenorphine (0.3

mrg) or physiological saline.21 No systemic analgesics were given

before, during or after surgery, and all the patients had operations on

the lowe extremities under extradural analgesia (lign~caine or

bupivacaine) Upon administration of the test drug as soon as pain









occurred in the post-operative period, a long duration of action and a

marked analgesic effect was observed with lofentanil. A shorter duration

of action and less pain suppression occured with buprenorphine and a

rather marked placebo effect was seen with saline. The only side effect

noticed was drowcsiness in 3 patients in the lofentanil group and in 2

patients in the buprenorphine group. 21
In a randomized double-blind trial22 comparing analgesia produced

by combinations of droperidol with either buprenorphine or morphine,

bupre~norphine wa~s claimed to be as satisfactory as morphine to produce

analgesia during maFjor surgery in 60 patients, with no difference in the
incidence of uxn~toward side effects.

Epidural buprenorphine was investigated as a post-operative

analgesic in a randomized double-blind study of 158 patients given
intraoperative epidural analgesia with 2% mepivacaine or 0.5%

bupivacaine for orthopedic surgery of the lowerI extremity.23 At the end

of the surgery, the patients were given epidurally in 15 ml saline,

either 0.15 mg of buprenorphine (n=38) or 0.3 mg (n=37) A control group

received nlo epidural injection (n=47) The above 3 groups received 2%

mepivacaine as intraoperative anesthetic. A fourth group (n=36) received

0.3 mg buprenorphine in 15 ml saline, after intraoperative use of 0.5%

bulpivacaine. The patients rated post-operative pain. Analgesia after
0.15 mg of buprenorphine was superior to that after saline injection,

and 0.3 mg buprenorphine was superior to both saline injection and to

0.15 mg of buprenorphine until 12th hour.23 Analgesia after bupivacaine

followed by 0.3 mgl of buprenorphine was not significantly different than

analgesia seen after mepivacaine followed by 0.3 mg of buprenorphine.
These results are comparable to those reported elsewhere.21









Respiratory effects. The respiratory depressant activity (such as
decreased respiratory rate, increased arterial Pa 23 and decreased

arterial PaO2) of single equianalgesic doses of buprenorphine and

morphine appear to be similar in rats and rabbits.1,2,2 The extent of

buprenorphine-induced respiratory depression against dose plateaued in

animals, whereas such an effect was not clearly demonstrated in man,26

which showed dose-related respiratory depression within the therapeutic

dose range (0.3 mrg to 0.6 mg) The time to reach peak respiratory

depression in man was slower after intramuscular buprenorphine than

after morphine (3 h vs 1 h) and the duration of such an effect waRs

longer.26 There appears to be no completely reliable specific

antagonist for buprenorphine-induced respiratory depression since even

high doses of naloxone produced only partial reversal.26 However, the

respiratory stimulant drug doxapram has reversed respiratory depression

due to buprenorphine in a few healthy volunteers and in a few

patients. 26

Cardiovascular effects. Hemodynamic changes in healthy volunteers

after IM (0.15 to 0.6 mg~) sublingual (0.4 to 0.8 mg) or oral (1 to 4

mgI) doses of buprenorphine include dose related reductions in heart rate

(up to 25%) and small decreases in systolic blood pressure (about

10%) .26 These results are comparable to the cardiovascular effects of

morphine.26 Similar effects occurred in anesthetized patients undergoing

surgery and in a few patients with myocardial infarctions. However, in
the latter group the heart rate was found to be relatively

unperturbed. 27

Addiction potential of buprenorphine. Buprenorphine appeared to

have a lower addiction potential than the opioid agonist pentazocine in









animals. However the extent to which such results can be extrapolated to

man was uncertain., 2 In a single-dose addiction study in 5

volunteers, high (8 mg daily) intramuscular doses of buprenorphine,

administered up to 1 to 2 months produced a slowly emerging withdrawal

syndrome on abstinence from the drug.5,6 Though the results wnere

indicative of lesser addiction potential compared to morphine,

definitive statements about addiction cannot be made until it has been

more widely used in patients with chronic pain with repeated doses over

an extended period of time.1

Receptor binding studies. Receptor binding studies wee undertaken
to elucidate the opioid binding characteristics of fentanyl and

buprenorphine,~b~ and to investigate differences between thmem.29

Buprenorphine showed slow receptor equilibration (30 min) but with high

affinity to multiple sites. The dissociation was claimed to be slow

(half-life = 166 min) and incomplete (50% binding after 1 h) This

contrasted with the receptor binding of fentanyl, which achieved rapid

equilibrium (within 10 min) and dissociated equally rapidly (half-life=

6.8 min) and completely (100% by 1 h) Using competitive displacement

studies, it was claimed that buprenorphine displacement of fentanyl was

concentration and time dependent over the ranges (equimolar

buprenorphine and fentanyl concentrations, 2 nmol/1iter) encountered in

clinical use. Howe~nver, buprenorphine binding was displaced with only

high concentrations of other opicids.29

Binding of buprenorphine to the rat forebrain (telencephelon,

diencephelon and mesencephelon) was claimed to be stereospecific,

saturable and had high affinity.3 Maximum binding (B a) was reached

by 30 min and dissociation from the receptor was slorw. The regional









distribution of buprenorphine binding sites in the rat brain was claimed

to be qualitatively similar to the distribution of naloxone and

dihydromorphine binding sites. The Bm for this receptor binding of

buprenorphdne was about 2 times that for the mu-opiate receptor drugs

and three times the Bm for the delta-opiate receptor ligands (such as

enkephalins) Buprenorphine was also found to be very potent in

displacing naloxone, dihydromorphine and met-enkephalin. Since mu-

receptors bind with exogenous opioids (such as morphine) and delta-

receptors bind with endogenous opioids (such as enkephalins) the above

findings suggest that buprenorphine binds to both mu- and

delta-receptors. 30

Side effects.

Moderate to marked drow~siness has been reported in about 40-50% of

the patients (up to 75% in some studies) but all such patients wlere

found to be easily arousable upon stim~ulation.1,13 Nausea and/or

vomiting occurred in 15% of the patients. Other minor side effects (e.g.

dizziness, sweating, headache or confusion) typical of strong

analgesics, have been reported with a widely varying incidence.

Respiratory depression, as determined by laboratory measurements of

respiratory functions, does occur with buprenorphine. Th~e extent of such

depression was similar to other opioid drugs administered in usual

clinical doses.1 Howevr, this was not a problem in clinical studies

which wciere usually conducted in fit patients.7- The effect of

buprenorphine on respiration in "poor risk" patients, such as those with

respiratory diseases or congestive heart failure, has not been

determined. However, it appears that buprenorphine would have the same

potential problems as morphine in this patient group.1








Dosage and Administration

Buprenorphine is presently available in Europe for parenteral
use.1 The recommended dose is 0.3 to 0.6 mg by IM or slow IV injection,

repeated every 6-8 h as needed. Administration of buprenorphiine to

patients already receiving large doses of narcotic drugs should be
undertaken with caution until the response is established, since its

antagonistic activity could conceivably cause withdrawal symptoms.1
Pharmacokinetic Studies

There is limited information available on the pharmacokinetic

properties of buprenorphine in Imn.1 It wnas stated without
documentation or citation of references that rapid absorption and peak

plasma concentrations were seen in rats on oral and IM dosings where the
oral dose was 4 times the IM dose.1 It was claimed that in primates and

in human volunteers, peak plasma concentrations were reached more slowly
after oral administration (2 h) than by IM injection (7 min). .1 u

concentrations wiere stated to be detectable in blood for longer times

after oral (24 h) than IM (7 h) administration of equivalent doses. In

man buprenorphine was claimed to be excreted unchanged in the feces, and

as glucuronide and N-dealkylated bupr~enorphine in urine.1 References of
studies supporting these data wiere not given.1

In a 3 h study, peak plasma buprenorphine concentration did not yet

occur in some patients after sublingual administration.33 In a

subsequent 10 h study with 15 post-operative patients, 5 patients

received a sublingual dose 0.4 mg of buprenorphine, five 0.8 mg and 5

received placebo at 3 h after a 0.3 mg IV dose of buprenorphi~ne. The

plasma buprenorphine concentration was measured by a specific
radioirmnunossay.34 The plasma concentration reached a peak level in an









average time of about 200 min in both the 0.4 mg and 0.8 mg groups

(range 90-360 min after the initial 3 h period) .34 The plasma drug

concentration in the 0.8 mg group were approximately twice that in the

0.4 mg group. The absolute bioavailability was estimated to be about 55%

of the IV route for both groups by the ratio of the area under the

plasma concentration versus time (AUC) for sublingual and IV

administration. Uptake of buprenorphine from the sublingual site was

claimed to be complete by 5 h after the dose was given.34 In this

study, cross-reactivity between buprenorphine and its metabolites was

not ruled out. ITwo modes of administration (IV followed by subcutaneous)

er~e carried out in each study which complicated the pharmacokinetic

analysis.,

Buprenorphine kinetics were studied in surgical patients using

radlioimmuanoassay.35 Buprenorp~hine wals measured in the plasma of 21

patients who received 0.3 mg IV. After 3 h, ten of these patients

received further dose of 0.3 mg IV, and 11 patients were given 0.3 mg

IM. Plasmra drug concentrations were measured up to 3 h after the second

dosing. Comparison of the pharmacokinetics in the same patient, awake

and anesthetized by general anesthesia, showed that the clearance was

significantly lower (900 ml/min) in the anesthetized state compared to

the unanesthetized state (1225 ml/min) Bioavailability was claimed to

be the same for both IV and IM administered drug. The peak plasma levels

were seen at 2-5 min and in 10 min respectively for IV and IM dosing

after the second dosing.35 Cross reactivity among buprenorphine and its

metabolites was not ruled out in this study. The sensitivity and limits

of detection for buprenorphine wee not given. Thus the terminal plasma

buprenorphine concentrations at less than 1 ng/ml are questionable.









Procedures for obtaining various pharmacokinetic parameters were not

given .

Plasma concentrations were correlated with clinical effects after a

single IV dose of buprenorphine (0.3 or 0.6 mg) in patients recovering

from surgery.36 Analgesia was greater at the high dose without any

apparent parallel increase in respiratory depression. Better analgesia

was reported if the first required post-operative dose of 0.3 mg~ has

been preceded by a similar loading dose or by the use of a larger dose

during surgery.36 This study was largely descriptive without rigorouxs

pharmacokinetjic analysis. The plasma concentrations obtained from a

number of patients wFere averaged to obtain a mean concentration. This is

not a valid pharmacokinetic technique.

Metabolism and Excretion

Higher amounts of polar metabolites were seen in plasma after oral

administration than after IM in rats.1 The premise of drug conjugation

in the gut wall was supported by studies with rat gut preparations.373

Buprenorphine has been found conjugated or N-dealkylated in bile or

tissues of animals, but unchanged in the brain. This is possibly

indicative of the fact that buprenorphine and not a derivative is

responsible for the narcotic activity.1

In a study of pharmacokinetics of buprenorphine after IM

administration to rats, dogs, rhesus monkeys and one human volunteer,

most of the dosed radioactive drug was excreted in the feces, indicating

biliary excretion with possible enterohepatic recirculation.39Ate I

administration of the tritiated buprenorphine (100 p g/kg) to the bile

duct cannulated rats, over 90% of the administered drug was excreted in

the bile within 48 h after dosing. The major metabolite in the bile was









buprenorphine glucuronide. N-dealkylated buprenorphine was also present.

Intraduodenal infusion of rats with bile obtained from other rats dosed

with radiolabelled drug produced a slow but extensive excretion drug

related metabolites in the bile of the recipient animal.39 The plasma

concentrations were not measured in this study. The assay techniques

were not specific for the parent drug or its metabolites. Dose

dependency was not studied.

In a chronically cannulated cow, 40 it was shwnm that the hepatic

extraction ratio for IV boluses of morphine, diamorphine, fentanyl,

methadone and buprenorphine increased towards a plateau value as the

portal vein drug concentration increased. The extraction ratio was

claimed to be independent of hepatic blood florw, but dependent on

concentration .

Disposition of radiolabelled buprenorphine in the rat after a

single 0.2 mgl/kg IV bolus dose and continuous administration via a

subcutaneous delivery system wee carried out.41 After IV injection,

tri-exp~onential decay of the drug from brain was seen with half-lives of

0.6, 2.3 and 7.2 h, respectively. Plasma half-lives were 0.5 and 1.4 h

(the third phase was not estimated) Decay half-life of the drug from

its high affinity binding sites in brain we~re 1.1 and 68.7 h

respectively. Fat and lung had higher concentrations than other tissues

or plasma. No metbolites of the drug were detected in brain.41.

Unrmetabolized drug excreted in the urine and feces one we~tek after IV

injection were 1.9 and 22.4% of the dose, respectively, and 92% of the

dose was accounted for in 1 week. Urinary metabolites (%) wer

conjugated buprenorphine 0.9; norbuprenorphine (free 9.4, conjugated









5.2); tentative 6-Cdesmethyl norbuprenorphine (free 5.4, conjugated

15.9) .41

Peak plasma concentration of buprenorphine occurred in 4 weeks after

s.c. implantation of a long-acting radiolabelled buprenorphine (10 mg)

pellet. The apparent dissociation half-lives of the drug from the Ilow-

and high-affinity binding sites in the brain were 4.6 and 6.8 weeks,

respectively. Fat, spleen and skeletal muscle had higher radioactivity

than other tissues and plasma. These authors41l state that high-affinity

binding of buprenorphine in brain and subsequent slow dissociation are

the factors responsible for its prolonged agonist and antagonist effects

and higher potency than other narcotic agonists.

Absorption and bioavailability. There a~Lre no published studies on

the oral absorption of this drug in man. It was claimed on the basis of

unpublished data that the peak plasma concentration of orally
administered radiolabelled buprenorphine in the rat was reached in 10

min with another peak in plasma appearing in about 5-8 h.1 This delayed

peak could be due to the late appearance of radiolabelled metabolites

(e.g., N-dealkylated buprenorphine and conjugates) in plasma.1 A

intramuscular dose of 20 pl g/kg gave blood peak concentration similar to

that of a 100 p g/kg oral dose. In monkeys, unpublished data were cited

to support the statements that peak blood concentrations of

radiolabelled drug were reached at 2 min, 2 h and between 2-4 h

respectively for IM, oral and sublingual admninistration.1 Also, it wasl

stated that in ~two healthy volunteers, peak blood concentrations were

reached rapidly after IM dosing (2 F g/kg) of radiolabelled

buprenorphine followed by a rapid decline. Peak concentrations wer

reached slowly at 2 h after the oral administration of 15 v g/kg of the









drug, followed by a biexponential decline of concentration.

Concentrations as low as 0.5 to 3.5 ng/ml were claimed to be detected by

a specific radioimmunoassay technique after IV aLnd IM administrations

(0.3 mg).1 In human volunteers a dose of 0.4 mg produced peak

concentrations of 1-2 ng/ml at about 2 h after oral administration.

References of studies supporting these data wee not cited.1

Systemic bioavailability of buprenorphine was studied in female

rats following single-doses (200 aug/kg) administered by six different

routes.4 Relative to the 100% bioavailability from the intra-arterial

route, the mean bioavailabilities were, IV 98%, intrarectal 54%,

intrahepatoportal 49%, sublingual 13% and intraduodenal 9.7%. AUC

analysis of buprenorphine concentrations in blood showed the relative

fractions of the drug excreted (first pass) by gut, liver and lung to be

0.8, 0.5 and 0.02 respectively. In vitro absorption studies showed that

poor bioavailability of intraduodenally administered buprenorphine was

not due to slow or incomplete absorption, but due to first-pass

me~tabolism. In this study, the authors42? computed AUC only up to 4 h

for the plasma data. The data showed ~two compartment model type

disposition for the drug in plasma. AUC would be different if calculated

up to time infinity.

In the above pharmacokinetic studies, low doses and subsequent low

terminal plasma concentrations have essentially limited the estimates of

terminal rate constants and half-lives of elimination. Sensitive and

selective assay techniques for buprenorphine and its metabolites in

biological fluids, and administration of large doses to a higher animal

such as dog could give acceptable pharmacokinetic parameters.









Protein binding. It was reported without documentation1 that

buprenorphine was highly bound (96%) to alpha- and beta-globulin

fractions of human plasma proteins in the concentration range 0-9 ng/ml.

The fraction of drug bound to dog plasma protein was determined by

measuring the drug concentration in plasma water after

ultracentrifugation.43 The fraction bound was estimated to be 0.945.

Binrding of buprenorphine to dog plasma proteins was also determined by

partitioning the drug into red blood cells. The estimation is based upon

the presumpgtion of established equilibria between drug in plasma water,

red blood cells and plasma proteins.44 By this technique, the fraction

of buprenorhine bound to plasma proteins was estimated as 0.983.43 This

relatively high plasma protein binding for the lipophilic buprenorphine

contrasts to the 26-36% plasma protein binding of morphine,45 naloxone,

and naltrexone.4

RBC Partition. Partition studies have shown that red blood

cell-plasma water partition coefficient of buprenorphine was 6-11.43

This is in contrast to 1.11 for morphine,45 1.83 for naltrexone, and

1.49 for naloxone.4

Physical properties. Fluorescence (excitation 285 nm, emission 350
nm) of buprenorphine provided excellent detection for HPLC assay in

biological fluids with a 5 ng/ml sensitivity.43 Buprenorphine

solvolysis was specific-acid and specific-base catalysed. It yielded a

stoichiometric final acid degradation product (3) a fluorescent

detectable, rearranged demethoxy analogue of buprenorphine. Alkaline

hydrolysis produced no fluorescence products.43 Acid hydrolysis also

produced a fluorescent-detectable transient dehydro intermediate (2)

that was also completely transformed into the demethoxy analogue (Scheme


























,CH2 ~


,CH,-


OH Cnz
- C -C -CHz
tsH 5


CHz
C C -CH,
CH, CH,


H
--+


+H


Mclhyl migrofion


(N


O e--c*
CzCHS
HO O OH


Cyclization


CH, -a


CHz
O as,__CH
C)~z
HO/ 0 \O' CH,


C -CHs

OCH, CH,


Scheme I


Ac~id Hydrolysis of Buprenorphine









I) Compound 2 was an excellent bioassay internal standard. Buprenorhine

was show~n to be highly stable at neutral pH values, even at elevated

temperatures.43 Estimrated buprenorphine p~Ga' values were 8.24 and 10
for the amnonium and phenolic groups respectively. The intrinsic aqueous

solubility of buprenorhine was 12.7 + 1.2 pg/ml at 23C.43

Assay methods. Few assay methods of buprenorhine in biological
fluids have been reported in the literature. A radioirmmunoassay47a

been used to determine plasma levels of parenteally administered

buprenorphine in dogs and humans.3,3 A selective ion monitoring

method (SIM) of the silylated buprenorphine in GC-M~S has been used to

determine the plasma levels of buprenorphine over a 20-3000 nlg/ml

concentration range.48 A GC assay with flame-ionization detection of

sily1 derivatives of buprenorhine was used in stability studies at 5-10

pg/ml of aqueous solutions.49 An HPIC assay with fluorescence detection
of buprenorhine in biological fluids has been reported and its

modification and imrprovement is presented in this dissertation.














EXPERIME~NTAL,

Materials. Analytical grade solvents and reagents were used.

Buprenorphine hydrochloride, 21-cyclopropyl-7-alpha- [ (s) -1-hydroxy-1, 2,

2-trimethylpropyl]-6,14 endo ethanotetrahydro-oripavine 1, (National

Institute for Drug Abuse, Rockville, MD)48 and the demethoxy analog, 3,

of buprenorphine (Addiction Research Center, lIexington, KY) 4 were used

as received. A standard sample of 21-cyclopropyl-7-alpha- [2- (3,

3-dimethyl-1-butenyl )] 6,14 enado ethanotetrahydro-oripavine 2, was

obtained from Dr.G. Lloyd Jones of Rickett & Colman, Pharmaceutical

Division, Kingston-upon-Hull, England.

Apparatus. An HPIC: (model M6000A pump, Waters Associates, Milford,

MA) equipped with a variable-wavelength fluorescence detector (model

600S Fluorescence Detector, Perkin-Elmer, Norwalk, CT) was used.

Injections were carried out with an auto samupler (WISP Autosampler,

Waters Associates) and the data were analysed by a microcomputer (Sigma

15, Data Station, Perkin Elmer) A separate HPIC pump (series 3B, Perkin

Elmer) equipped with a variable wavelength UV detector (model IC: 75,

Perkin Elmer) was used in some studies. A laboratory centrifuge wras used

in the separation of organic extract from biological fluids (Lab

Centrifuge, International Centrifuge Equipment Co., Needham Heights,

MA) .50

Liquid Chromatographic Procedures. Aliquots (50-100 yL) of the

solutions to be analyzed w~ere injected into the HPIC system equipped

with a packed [packing material was C18 5- pm Bondapak-reversed phase









(ODS-Hypersil), Shannon Southern Products Ltd., Cheshire, U.K.] 120 mn

i.d. stainless steel column [Knauer HPICI analytical column (unpacked) ,

Knauer A.G. Berlin, F.R.G.] which was maintained at 40 C. The usual

mobile phase flow rate was 1.5 mL/min of a 40:60 acetonitrile :acetate

buffer (pH 3.75, 0.05M) containing 0.0004M tetrabutylarmonium phosphate.

Fluorescence was effected at 285 nm excitation (slit 20 nm) and 350 nm

emission (slit 15 mn) and was used unless stated otherwise.43

Calibration Curves in Biological Fluids

Buprenorphine. Aliquots (1 mL) of plasma, urine or bile in each of

ten 15-mL, centrifuge tubes were spiked with 100 pL of 100-1000 ng/mil of

buprenorphine (1) Each solution contained 50 ng/mL; of the acid

degradation intermediate of buprenorphine, compound 2, as the internal

standard. The final sample contained no drug. Sodium borate-boric acid

buffer (1 mL at pH 9.1, 1 M) and 4.2 mTL of benzene er~e added to each

tube. The tubes were shaken for 20 min, centrifuged at 3000 rpm for 10

min, and 4 mtLT of each benzene extract was transferred to another set of

ten 15-mL centrifuge tubes. Hydrochloric acid (1 mL, 1 M) was added to

each tube and the tubes were shaken for 10 min and then centrifuged at

3000 rpm for 10 min. After removal of benzene layer by aspiration, 1 mTL

of both 1 M NaOH and pH 9.1 borate buffer (1 M) wee added to each of

the remaining aqueous phases. The pH values were confirmed or adjusted

to be between 9.05 to 9.15. Benzene (4.5 mLS) was added to each tube

which was shaken for 10 m~in and centrifuged at 3000 rpm for 10 min. The

benzene extract (4.00 mL) was transferred to a 5-mL vial (Reacti-v~ial,

Supelco, Inc~. Bellefonte, Pa.) and the benzene was evaporated under a

stream of nitrogen at 55 *C. Sodium acetate-acetic acid buffer (pH 3.75,

0.05 M, 100 y L) wa~s added to each of the Reacti-Vials and they were










vortexed for 30 s, and then 75 1IL of the solution was analyzed by HPIC.

Buprenorphine conjugates. Aliquots (1 mLJ) of plamsa, urine or bile

in each of ten 15-mL; centrifuge tubes were spiked with 100 l1L of

100-1000 ng/iL; of buprenorphine. The first sample contained no drug. To

each centrifuge tube, 1 mL of 6 N HC1 was added, and autoclaved at 15

lbs/sq.in pressure for 10 min. The tubes were allowd to equilibrate to

room temperature. To each tube containing the acid-transformed demethapry

buprenorphine, 50 FpL of unconverted buprenorphine (1 Ir g/mL;) was added

as internal standard. Ex~cess acid was neutralized with soduim carbon~ate.

The pH was adjusted to 9.1 with sodium borate-boric acid buffer (1 ah, 1

M) and 4.2 mL~ of benzene was added to each tube. The tubes wee shaken

for 20 min, centrifuged at 3000 rpm for 10 min, and 4 mL- of each benzene

extract was transferred to fresh 15-mL centrifuge tubes. Hydrochloric

acid (1 mTL, 1 M) was added to each tube and the tubes were shaken for 10

min and centrifuged at 3000 rpm for 10 min. After removal of the benzene

by aspiration, 1 mL of both 1.00 M NaCH and pH 9.1 borate buffer (1.00

M) were added to each reamining aqueous phases. The pH values were

confirmed or adjusted to be between 9.05 to 9.15. Benzene (4.5 mrL) ass

added to each tube which wras shaken for 10 min and centrifuged for 10

min at 3000 rpm. The benzene extract, (4.00 mL) was transferred to a 5

miL vial (Reacti-Vial) and the benzene was evaporated under a stream of

nitrogen at 55' C. Sodium acetate-acetic acid buffer (100 9 L, pH 3.75,

0.05 M) was added to each vial (Reacti-Vial) vortexed for 30 s, and 75

uL of the solution was analyzed by HPL~C.

Pharmacokinetic studies in dogs. Healthy mongrel male dogs (8) were

used for the pharmacokinetic investigations. Their blood analysis shoWed

no pathogenic abnormality or presence of microfilaria. The dogs w~ere









fasted for at least 17-24 h before each study and were given water ad

libitum. The animals were supported by a dog sling in a frame placed on

a laboratory table. The dogs were infused with intravenous saline (35

drops per min) for at least 3 h until drug administration, when the

intravenous drip was reduced to 20 drops per min. The animals were

catheterized 3 h before the study with a 30.5-cm standard catheter

(Intracath, 16 GA size, Deseret Medical Inc., Sandy, Utah) in the

jugular vein after local anesthesia with mepivacaine hydrochloride

(Carbocaine hydrochloride; Winthrop Laboratories, New York, NY) Second

catheter was also implanted in a foreleg vein (vena brachialis) in most

IV infusion studies. The drug was injected directly into the jugular

catheter, followed by flushing of the catheter with 25 mL of normal

saline. The catheter was connected via a three-way stopcock (Pharmacea,

Toa Alta, PR) to the saline infusion bottle (Mc~aw laboratories, Irvine,

CA) Blood samples (1-6 mrL) were collected in hteparinized Vacutainer

tubes (Becton Dickinson Vacutainers, Rutherford, NJJ) after the dead

volume of the catheter was filled with 5 mL of blood by aspiration with

an extra syringe. These aspirations were carefully aseptically

reinjected into the jugular vein. The heparinized blood samples were

immediately centrifuged at 3000 rpm for 10 min. The plasmas were removed

with sterile glass pipets and were frozen until analysed.

Urine was collected from the dogs through a urinary catheter

(Polyurethane whistle tip units, 6 FR size, McGaw Laboratories) at

intervals of 15-60 min for up to 24 h and at longer intervals for up to

1 week. Withdrawal times, volumes and urinary pH values were recorded

and portions of each sample were frozen until analyzed.

Infusion studies were carried out using Harvard Infusion pumpg










(Harvard Apparatus Co., Dover, MA) Buprenorphine-HC1 was dissolved in

normal saline (150 mL, concentration=0. 73-78 mg/mL), ultrasonicated for

30 min and infused into the jugular vein at the rate of 0.7026 mrL/min

for 162-175 min (studies 7-11) During infusion, blood samples were

collected from the brachialis vein. Post-infusion blood samples Je~re

collected from both jugular and brachialis veins. In dog study 12, the

drug solution was infused into the brachialis vein (using the same drug

concentration and flow rate as above) .

Dogs E, F and G underwent surgery.51 A 2% solution of thymalol

sodium was administered IV (6 mL/kg) to each dog and anesthesia was

maintained by halothane. After removal of the gallbladder, a screw-cap

was placed on the opposite side of the sphincter of Oddi and the

intestine was sewn to the abdominal wall (Fig 1) At least 1 month was

allowed for recovery from the surgery before the pharmacokinetic study

of buprenorphine in the bile-cannulated dogs. These dogs could be

repetitively used for bile cannulation studies by opening the screw-cap

and inserting a catheter (Fast Right Heart Cardiovascular catheter, 5 F

size, C.R. Bard Inc., Billerica, MA) into the bile duct. The balloon at

the tip of the above catheter was inflated with 0.8-1.0 m~L of air,

pulled back until the catheter was securely positioned at the inside

wall of the spincter of Oddi. Complete bile collection was effected in

such studies at intervals of 15-120 min for up to 26 h.

Isolation of buprenorphine conjugate from bile. ITwot liquid

chromatographic glass columns (40 X 2.5 cm) were packed with nonionic

Amberlite XAD-4 beads (Sigma Chemical Co., St. Louis, Mo.) by passing a

slurry of the packing material in distilled water through the column.

The perforated disk at the bottom end of the column retained the













H *H





a -


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AmbSerlite beads. Each column was washed with 500 mL of water followed by

250 mL~ of methanol. The columns w~ere closed at the bottom and soaked

with distilled water overnight. Pooled bile samples (150 mLt) collected

from dog studies 9 and 10 were diluted to 500 m~L with distilled water.

Aliquots (250 mLi~) wee passed through each column and ~wshed with 300 mL

of water until the eluent was colorless. Then, 300 mL of methanol Fwas

passed through each column. These methanolic eluates were combined and

completely evaporated to dryness under reduced pressure. The residue was

dissolved in sterile normal saline (120 mL) and the solution was

filtered through a 0.22 clm Millipore filter aided under reduced

pressure and strictly aseptic conditions. The final sterile solution was

infused into dog F (Study #13) at the rate of 14 mL~/min for 8.5 min. The

pooled bile samples collected from dog study #11 were similarly except

that chromatographic separation was achieved with only one Amiberlite

XAD-4 column. The final sterile solution was infused into dog G (Study

#14) at the rate of 14 ml/min for 7 min.

Analysis of the conjugate by enzymatic hydrolysis. The enzyme

8-g~lucuronidase ( B -glucuronide glucuronosohydrolase 0.76 mgT = 660,000

Fishrmn Units, Lo~t. #51F-9013, Sigma Chemical Co.) was dissolved (50 mg~)

in 20 mL acetate buffer (pH 3.8, 0.05 M) Aliquots (500 v L) of the

enzyme preparation and the internal standard (100 pCL of compound j2 1

ug/miL) were added to 500 9 L of diluted bile sample (1:10,000 dilution

made with distilled water) and the total volume was adjusted to 1.6 mLt

with pH 3.8 acetate buffer. The samples wFere incubated at 37'C for 24 h.

Diluted bile (1:10,000 dilution) samples containing no drug were spiked

with buprenorphine and treated in the same manner to establish an

appropriate calibration curve. The generated aglycone buprenorphine was









assayed by HPIC: separation and fluorimetric detection.

Catheter binding of buprenorphine. Buprenorphine-HC1 was dissolved

in normal saline (150 mL, concentration = 0.7576 mg/mL of base) The

solution was passed through the plastic catheter (Intracath) without

back pressure at the rate of 0.7026 mL/min for 1 h (1 study) and 3 h (2

studies) The internal surface of the catheter was washed with 25 mL of

normal saline and dried under a stream of air. Then benzene (250 ml) was

pumped through the plastic catheter (Intracath) at the rate of 14

ml/min, evaporated in a collecting flask at 70'C under reduced pressure.

The residue was reconstituted in acetate buffer (pH 3.75, 0.05 M) and

aliquots were assayed by HPIC separation and fluorimetric detection.

Buprenorphine-HC1 was dissolved in normal saline (0.7567 mg/mL- of

base) and passed through the plastic catheter (Intracath) at the rate of

0.7026 mL/min for 3 h. The interior surface of the catheter was washed

with 25 mL of normal saline. The saline was allowed to flow from an

infusion bag under the gravitational force at the rate of 45 drops/min

for 2 h and was collected (300 mL) The pH was adjusted to 9.1 and the

saline solution was extracted twice with 250 mrL portions of benzene. The

combined benzene layer was evaporated under reduced pressure at 70* C.

The residue was reconsituted in acetate buffer (pH 3.75, 0.05 M) and

aliquots were assayed by HPIC: separation and fluorimetric detection.

Buprenorphine (0.7567 mg/miL) in normal saline was passed through

the plastic catheter (Intracath) for 1 h at the flow rate of 0.7026

mL/min. At the end of 1 h, the catheter was washed with 25 mLt of normal

saline, and saline drip (45 drops/min) was continued. Fresh blank dog

blood (1-3 mL) was drawn through the catheter at 1, 2, 5, 10, 15, 20,

30, 45, 60, 120 min. The blood samples w~ere centrifuged at 3000 rpm for










10 min and the supernatent plasma was analysed for buprenorphine by HPICL

separation and fluorimetric detection. The experiment wa~s repeated

following the pumpqing of buprenorphine solution (using the same

concentration and flow rate as above) through the catheter for 3 h.

In-vivo study. In dog study #12, buprenorphine was infused (0.5236

mgJ/min for 177 min) into the left brachialis vein through the indwelling

plastic catheter (Intracath) Blood samples during infusion wer

collected from the jugular vein and the contralateral brachialis vein.

Upon cessation of infusion, the catheter through which the drug was

infused into the left brachialis vein was washed with 25 m~TL of lnormal

saline. Post-infusion blood samples were collected from the left

brachialis vein (site of infusion) through the plastic catheter, as well

as from the jugular and contralateral brachialis veins.















IV BOLUS STUDIES

Chromatographic assays of Buprenorphine and its conjugate. The HP~LC

assay methods developed for buprenorphine have been published.43

Chromatograms of the HPLC-assayed buprenorphine are given in Fig. 2. The

acid hydrolyzable conjugate (M) assay (Fig. 3) by fluorimetric detection

(285 nm excitation, slit width 15 nm and 350 nm emission, slit width 10

nm) was equally sensitive, Table 1 showcs the relevant statistics of

calibration curves. The standard errors of estimates of the

concentration about its regression on peak height ratio ranged from +

0.5 to + 3 ng/ml.

Some additional linear regressions of concentrations (C, ng/ml) of

buprenorphine in plasma with their standard errors of the parameter

estimates in accordance with


C + SER = ( m + sm ) PHR + b + sb Eq. 1


in the range 5-50 ng/ml were, C + 1.52 ng/ml = (61.19 + 1.84) PHIR 1.11

+ 0.808, r = 0.9968; C + 1.52 ng/ml = (76.64 + 2.518) PHR 3.04 +

1.068, r = 0.9962; C + 1.3 ng/ml = (54.34 + 2.6) PHR 5.01 + 1.42, r =

0.9943; C + 0.38 ng/ml = (72.21 + 1.8) PHR 12.3 + 0.98, r = 0.9991. In

the buprenorphine concentration range of 50-100 ng/ml, C + 0.97 ng/ml =

(75.49 + 1.76) PHR 13.45 + 2.1, r = 0.999; C + 2.42 ng/ml = (75.48 +

2.43) PHR 21.6 + 2.71, r = 0.9959; C + 1.78 ng/ml = (67.96 + 2.9) PB

+ 6.02 + 3.026, r = 0.9964; C + 0.64 ng/ml = (27.94 + 0.4) PHR + 17.1 +

0.85, r = 0.9995.
































Blank



I I 1 I I I I lr i


Plasma


Urine


1.).1111


Blank



24 6 810 12


2468
Minutes


10 12


Figure 2. Representative chromatograms after assay of buprenorphine (1,
60 ng/ml) with internal standard (2, 100 ng/ml) from plasma (a) and
urine (b) (The blank plasma and urine chromatograms without drug are
given underneath) Chromatogram of mixture of 25 .;g/ml of
buprenorphine, 1, with its products 2 and3 3 after acid degradation in 1
M HC1 for 3 min (c) (See also reference 43) .














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In urine, examples of regression equations for buprenorphine in the

range 5-100 ng/ml were, C + 1.59 ng/ml = (68.9 +- 1.11) PHR 13.93

1.12, r = 0.9991; C +- 0.89 ng/ml = (113 + 1.89) PHR 6.8 + 0.851, r =

0.9993; C j- 2.02 ng/ml = (66.5 +- 1.26) PHR 7.31 j- 1.17, r = 0.9977.

Exarrples of regression equations for buprenorphine conjugate (M) in

plasma in the range 10-50 ng/ml were, C 3- 2 ng/ml = (57.02 +- 2.13) PHR +

4.9 1.211, r = 0.9958; range 60-100 ng/ml; C + 1.12 ng/ml = (39.57 +;

1.247) PHR + 22.2 + 1.83, r = 0.9985; range 5-100 ng/ml, C + 1.4 ng/ml =

(73.8 + 0.99) PRR 2.46 0.8, r = 0.9991; range 10-90 ng/ml; C + 1.3

ng/ml = (50.14 + 0.8) PHR 1.51 + 0.92, r = 0.9991.

Examples of regression equations for buprenorphine conjugate (M) in

urine in the range 10-200 ng/ml were, C j- 1.8 ng/ml = (82 + 0.621) PHR +

0.72 + 0.82, r = 0.9997; range 10-120 ng/ml, C 4- 2.6 ng/ml = (92.99 1-

2.4) PHR 27 + 2.5, r = 0.998; C +- 1.81 ng/ml = (162 j- 2.9) PAR 13.19

+ 1.5, r = 0.9991, where PAR = peak area ratio; range 10-100 ng/ml, C +

2 ng/ml = (66.5 +- 1.26) PHR 7.31 j- 1.17, r = 0.9977. An example of

regression equation for buprenorphine conjugate (M) in bile in the range

10-200 ng/ml was: C + 6 ng/ml = (102 2.7) PKR 0.53 j- 2.63, r=

0.9956.

Twice the standard error of estimate of buprenorphine and the

retabolite concentrations (ng/ml) on peak height ratio ranged from 1-5

ng/ml (Taole 1), indicative of the sensitivity of the fluorimetric assay

of buprenorphine and its metabolite in biological fluids.

Plasma Pharmacokinetics and Volumes of Distribution. The plasma

concentration-time profile of buprenorphine could be fitted to a sum of

three exponentials (EIq. 2, Figs. 4-9). There may not be an unique linear

sum of three exponentials Cpcalc (estimated plasma concentration) that















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best fits the actual data Cp ; instead there may be several
exp
solutions with similar minimum sum of squares. A unique solution can be

obtained only if the drug transferred into other compartments or

transformed into metabolites were analysed in their respective

compartments.5j2 The plasma data of buprenorphine administered

intravenously to 6 dogs were fitted by nonlinear regression to a sum of

three exponentials (In study #1, Dog A, the data were also fitted to a

4l-compartme~nt model; Fig. 4, top inset) .53 The fitting was effected

with the computer program of Yamaoka et al.5 (See also appendix I) ,

where 1/Cp, the inverse of the plasma concentration was the weighting

factor (See appendix II for a discussion of different weighting

techniques) The validity of the triexponential equation,


Cpcl = Pe +"t Aea + Be Eq. 2

was confirmed by demonstration that regressions of the various studies

of the we~ighted residuals


E5 = ( Cp Cp ) / Cp Eq. 3
exp calc calc

against log Cpcalc gave mean residuals e slopes and intercepts,
which were not significantly different from zero (Fig. 10, Table 2) .55

The residuals Ietre randomly distributed above and below the regression

line, indicating no> bias in the fitting of the chosen model. (Fig. 10) .

Outliers wFere defined as those experimental concentrations which had

residual values, e, greater than 2 (~Eq. 3) which corresponds to a

greater than 200% deviation from the presumed best fitted value. One

such outlier in dog B at 2.5632 mg/kg (Study #3) dose of buprenorphine

was not included in the nonlinear least square curve fitting technique















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(Appendix I) .54 The outlier was included in all other pertinent

pharmacokinetic analyses and plots, such as excretion rate, sigma minrus,

and clearance plots. There were no outliers in the other studies.

Since the triexponential equation 1 adequately described the

post-intravenous bolus injection data (Figs. 4-9) a 3-compartment body

model was the simplest pharmacokinetic model for the disposition of

buprenorphine in dogs. The elimination of buprenorphine could occur from

a central compartment (C) reversibly connected with shallow (S) and

deep (D) peripheral compartments (scheme II) :53








Scheme II


The equation which describes the time course of the IV bolus

administered drug in the central compartment Cp as a function of time t

as per scheme 2 is given by53


Cp =(XO/Vc) [[(k21 -n ) (k31 -n w )/v- a) (v- 8) ] e +





where X0 (k21 -* ) (k31 w) /Vc (w- a) ( -B ) =P Eqg. 5

X0 (k21 -a ) (a -k31 ) /Vc(n a) ( a-B ) = A Eq. 6
and


10 (k21 8 ) (k31 B ) /Vc (a -B ) ( v -B ) = B


Eq. 7









where Xg = dose, Ve = volume of distribution of the central

compartment.

Validity of the terminal rate constant. Proper estimation (Appendix

II) of the terminal rate constant (and half-life) depends upon a)

analytical sensitivity; b) number of terminal plasma concentration

values, the time interval between these values, and the number of

terminal half-lives over which the samples were collected; c) selection

of the conmpartment model; and d) proper weighting of the data. Upon

acute IV bolus administration of buprenorphine in dogs at the 0.7-2.6

mg/kg doses used, the plasma concentrations of buprenorphine wee below

20 ng/ml at 1000 min (See Figs. 4-9) Thus the available analytical

sensitivity of 5 ng/ml did not permit accurate estimation of the

terminal half-life. For example, at the 2.5632 mg/kg (Study #3) IV bolus

dose of buprenorphine in dog C, the estimated terminal rate constant

obtained from a semilogarithmic plot of the terminal phase plasma data

against time (n=12) was 1.7 X 10-4 (half-life = 4040 min) + 0.70 X

10- (SE) min`- Thus the range that would include the 95% confidence

limits for this rate constant wFould be 1.48 X 10-5 (balf-life = 47000

min) to 3.3 X 10' (half-life = 2111 min) minl (See Table 3) .

The terminal half-life significantly depends upon the number of

compartments assumed. Consider dog A (Study #1) Fitting of the data

weighted by the inverse of the concentration to a 3-compartment model
gave a terminal rate constant of 7.6 X 104 mi hl-lf 1

min) When the same data were fitted to a 4Q-compartment model, the

terminal rate constant estimated by using the computer program (Appendix
I)ws3.8X1-4 09 1-4 (S) -1
I) ws 378 X10 0.84 X10 SE)min (half-life = 1840 min,

n=3; The 95% confidence limits; + s t, where t=12.71; 433 min to time


















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infinity; Fig. 4, top inset) This large range for the confidence limits

is attributable to the estimation of a terminal rate constant from few

(n=3) plasma values. The estimated terminal rate constants from the

semilogarithmic plots of the terminal plasma concentrations against time,

their respective standard errors, and 95% confidence limits are given in

Table 3.

The terminal half-life estimated for studies 1,2,4,5 and 6 have

relatively less error compared to study 3 (Table 3) Yet, estimation of

the true terminal half-life depends on the number of terminal plasma

values, the time interval between these values and the number of

terminal half-lives over which the samples were collected. T'he terminal

plasma data in studies 1,2,4,5, and 6 were not representative of the

true terminal phase since the plasma data needed to accurately estimate

the terminal half-life were below the analytical sensitivity and were

not available.

Total body clearance. Th~e total body clearance Cltot o oe

XO, can be calculated from53


Ctot 0 O/AUCoo Eq. 8

where AUC =area under the plasma concentration-time curve up to time

infinity. AUCt up to the last observed plasma point, Cpn can be
calculated by the trapezoidal rule. The area from the last plasmaL

sampling time to infinity can be estimated from Cpn / 8 where a is the

terminal rate constant. Also, AUCoo can be explicitly calculated by

integrating equation 2 between time 0 to oo, i~e.,


AUCoo = (P/w ) +(A/ a ) +(B/ B )


Eq. 9









The parameters of the above equation were obtained by fitting the

buprenorphine plasma data of dogs 1-6 to equation 2 using the computer

program of Yamaoka et. al.54 (Appendix I) The values of the parameters

of equation 9 are given in the legends of Figs. 4-9 or can be calculated

from the normalized values given in Table 2. The calculated percent

contribution of the term B/B of the terminal area to the total area in

studies 1-6 wetre 72, 68, 53, 44, 58 and 47 respectively. This

demonstrates the significance of B in the estimation of AUC, and

consequently the total body clearance derived from this value (Equation

8) Thus, uncertainties in the estimates of 8 can lead to uncertainties

in the estimates of AUCo and total body clearance. In the IV bolus
studies (#1-6) the contributions of the terms P/ x and A/a were only

about half of the total area under the plasma concentration time curve.

When equation 2 was used to fit the plasma data of buprenorphine

(Yamaoka et. al.,54 Appendix I) the P, W A, and a parameters were

estimated from the data in high range (50-5000 ng/ml) of plasma

concentrations. These data are held in greater confidence than the

estimates obtained from the low values of the terminal phase. Thus, if

the error in AUCo estimation is primarily due to the error in the

estimation of B the 95% confidence limits for AUCo and the derived

total body clearance (Equation 8) can be estimated from the terminal

rate constant, 8 and its standard error.

To estimate the error in AUCo (Equation 9) the parameters P, n ,

A, and a were obtained by fitting the complete plasma data of

buprenorphine to the tri-exponential equation 2 (using the computer

program of Yamaoka et. al.54, Appendix I) However, the parameters B

and 8 were obtained from the regressions of the semilogarithmic plots of









the terminal phase plasma data against time. The standard error value,

sm, of the terminal slope was multiplied by the t-value obtainedd from
t table for a=0.025 level of significance, two tailed for (n-2) degree
of freedom; where n is the number of terminal plasma points) The

resulting 3+ t~sm permitted the estimation of the the upper and lower

95% confidence limits for AUC, calculated in accordance with equation
9. The upper and lower limits for the Cltot were derived from the upper
and lowerI limits of AUC in accordance with equation 8. These calculated

total body clearances and the respective 95% confidence limits are

reported in Table 3 for the 6 IV bolus studies in the dogs.
Volumes of distribution of buprenorphine. The plasma concentration
of a drug in the central compartment at time zero is given by53


Cp = P+A+B = X0 c Eq. 10

when an IV bolus is administered into a 3-compartment body model. Vc is

the apparent volume of distribution of the central compartment. The

average Ve was 13.1 + 2.73 (SEM) L (Table 2) This value exceeds the
volume of blood (1.8 L) 56 and the extracellular water (4.8-6.6 L) 57 i

dogs. This indicates rapid sequestration of the drug in the
extracellular space upon bolus administration.
If the clearing organ is in the central compartment (Scheme II) ,

then the clearance from the central comrpartment, Cdc, is given by53

C1c = Vc k1 Eq. 11

If the drug is solely eliminated from the body through the central

compartment, the Cle is the total body clearance Cltot at any time
during the post-distributive phase in accordance with the equation,53










Vd 8= Ve k1 = Clto Eq. 12


where Vd is the overall apparent volume of distribution of the

equilibrated fluids of the body.

Thus ,


V = Cl / 8B Eq. 13

and


V = X /AUCo E~q. 14

If B or AUC have large errors, then the estimates of Vd have large
errors. TIhus the calculated distribution volumes in accordance with

equation 13 based on the best computer fit (Appendix I) of the plasma

data to tri-exponential equation are suspect. However, the calculated

Vd values in accordance with equation 13 (reported in Table 2) averaged
434 L, in excess of total body water in dogs (11-15 L) 5 and does

indicate a high degree of sequestration by body tissues.

Dose-independent pharmacokinetics of buprenorphine. The

pharmacokinetic parameters of a drug are dose-independent when all
distribution and elimination processes are first order with respect to

compartmental concentrations. The rate constants must be invariant at

highly varying administered doses and there must be no saturable first

pass metabolism. To establish whether or not there is dose-independency,

the drug is administered to the same animal at different doses. If the

plasma levels divided by the respective doses are superimposable, then
dose independency can be postulated.









Unfortunately dose independent pharmacokinetics of buprenorphine in

dogs could not be studied at highly varying intravenous bolus (1-100

fold) dose levels. The lowr limit of detecton (5 ng/ml) of

buprenorphine in plasma necessitated a certain minimal IV bolus dose to

adequately quantify terminal plasma concentrations. The fact that the

doses of buprenorphine in excess of 1.2-2.6 mg/kg would exhibit

significant side effects demanded an upper limit to the IV bolus dose

that could be administered.

At least two or more of the following toxic effects were observed

during a pharmacokinetic study: Defecation and muscle relaxation,

labored and forceful breathing for about 1 h after bolus dose, profuse

salivation continuing up to 4 h. The side effects observed following

rapid IV bolus injection of buprenorphine could be attributed to the

peak plasma levels (2000-5000 ng/ml, Figs. 4-9) reached immediately. All

five dogs exhibited drowsiness throughout the experiment, and the state

of general depression (characterized by lack of food intake, minimal

physical motion, lack of response to stimulus such as clapping of hands

and prolonged sleeping up to 12 h at a stretch) continued up to 1-5 days

depending upon the dose of buprenorphine. Higher doses produced longer

duration of these side effects.

To minimize the peak plasma concentrations of buprenorphine and the

associated side effects encountered upon IV bolus administration, and

yet to obtain adequate plasma concentration values in the terminal

phase, the higher doses of buprenorphine, 4.69, 3.85 and 3.741 mg/kg

dose in dog B, D and F (Study #7, 8 and 11) respectively, were

administered by constant rate infusion over a period of 3 h. However,

superimposition to validate dose independency is inoperative if the drug









is administered by two different modes (such as IV bolus and infusion) .

If a relationship can be established between the plasma levels of a drug

administered by IV bolus and by IV infusion, superimposition can be

challenged by the use of the transformed IV infusion data.

Superimposition of this transformed high dose IV infusion data on lowJ
dose IV bolus data was effected by the outlined procedure58 that

follows.

Analysis and transformation of IV infusion data. The post-infusion
data were fitted to a sum of either two (Study #7 and 8) or three (Study

#11) exp~onentials in accordance with the equation,53~

P' e- a T) + A' e-a (t-T) + B' e- tT Eq. 15


where T is the time at which infusion was stopped and t is the time

after initiating the infusion. The first term in the above expression is

set equal to zero when the post-infusion data are fitted to a

2-compartment body model. The relationship between P and P' of equations

2 and 15 respectively is53

P = P'T a / (1-e )T Eq. 16


Similarly, the relationships between A, A' and B, B' are


-aT

B = B'Tf3 / (1-e )Eq. 18


The calculated P, A, and B values were used to generate the Cpcalc

values of equation 2. These could be the calculated plasma

concentrations if the same dose was administered by IV bolus. These

estimated Cp concentrations obtained from the infusion studies were
calc









divided by the total infused dose (mg/kg) and superimposed on the

experimental values of buprenorphine (divided by the IV bolus dose in

mg/kg) obtained after low dose bolus injection in the same dog. In dog B
at three dose levels (1.64, 2.56, 4.69 mg/kg, Study #2,3 and 7) dog C

at two dose levels (1.2, 1.44 mg/kg, Study #4 and 5) dog D at two dose

levels (0.78 and 3.85 mg/kg, Study #6 and 8) and dog F at two dose

levels (0.754 and 3.741 mg/lkg, Study #17 and 11) there were no apparent

dose dependencies as demonstrated by the tests of superimposition
59
(statistically confirmed by nonparametric Kruskal-Wallis test applied

to the dose-normalised plasma concentration data. See Figs. 11-14 and

the legends, also refer to Appendix III) The parameters of equations

15-18 for IV infusion studies in dogs B, D and F are given in Table 5.

Plasma pharmacokinetics of the derived metabolite. The metabolite

(M) assayed in plasma was the acid hydrolyzable conjugate of

buprenorphine (1) .43 This buprenophine conjugate (M) upon acid

hydrolysis presumably generated the aglycone which quantitatively

rearranged to demethoxybuprenorphine (3) Rather than assaying the

buprenorphine conjugate or the aglycone directly, this rearranged

product was assayed by HPLC separation and fluorimetric detection. Other
metabolites such as norbuprenorphine or its conjugates observed in

rm338, 39 were not detectable in dog plasma with the assay sensitivity

of 5 ng/ml. The conjugate concentration in plasma was highest at the

initial sampling time, and decreased at a rate similar to that of the

parent compound (Fig. 15) The metabolite profile in 4 IV bolus studies
could be fitted by a triexponential equation (Eq. 2) The fitting was

effected by nonlinear least square regression by using the computer

program54 (Appendix I) where the metabolite concentrations in plasm~a



















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were wenighted by their inverse. The validity of triexponential equation

2 was confirmed by demonstration that the regressions in various studies

(Fig. 10) of the weighted residuals e (Eq. 3) against log Mlpcal
(estimated metabolite concentrations) gave mean residuals e slopes

and intercepts, all of which were not statistically significantly

different from zero (Table 2) The plasma concentration-time profiles of

metabolite in the IV bolus studies (1-5) are given in Figs. 16-20.

Maximum plasma concentration of the metabolite was observed at the

initial sampling time (about 1 min) Continued sampling gave

monotonically declining metabolite concentrations similar to the decay

of the parent compound. The parallel decays of buprenorphine and its

conjugate concentrations (Fig. 15) in plasma during the initial

distributive phase indicate that the rate determining step in the plasma

decay of the conjugate was its formation. During the terminal

elimination phase, the rate determining step in the plasma decay of

buprenorphine and its conjugate was the slow return of buprenorphine

from deep tissues to the central compartment where it could be

metabolized. This is the classical 'flip-flop' pharmacokinetics for the

conjugate.53




















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URINARY EXCRETION OF BUPREUNORPHINE

Sigma minus plots. If it can assumed that buprenorphine is solely

eliminated from the central compartment, the urinary excretion rate of

intact drug can be defined as

dU/dt = kU Xc Eq. 19

where kU is the urinary excretion rate constant, and Xe is the amount
of drug in the central compartment at time t. Integrating equation 19

between 0 to U (time; 0 to t) results in53

at -art Bt
E Uo EU = P"e + A"e + B"e Eq. 20

where


P" = P kU V / v Eq. 21

A" = Aki V / ar Eq. 22


and B" = B kU V / B Eq. 23

The values of P, A and B are same as in Eqs. 5,6 and 7, respectively if

constant renal clearance is presumed. Thus a plot of the logarithm of

the amount of unchanged drug remaining to be excreted versus time (sigma

minus plot) gives a straight line with a terminal slope equal to

- 8/2.303, i.e., the same terminal slope obtained from a semilogarithmic

plot of plasma concentration (Cp) versus time.

Representative examples of the sigma minus plots for the urinary

excretion of buprenorphine are given in Fig. 21. For dog A (Study #1,

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Fig. 21a),r semilogarithmic fitting of the initial data was linear only
-1
up to 100 min. The estimated apparent rate constant was 0.0176 min

(balf-life = 39 min) This corresponded to the second distributional

half-life (26 min) obtained for buprenorphine from the plasma data

(Table 2) The sigma minus plot of buprenorphine in urine of dog B (Fig.

21b) at 1.6369 mg/kg (Study #2) dose showed curvature, and could be

fitted to a sum of two exponentials. The resulting hybrid rate constants
-1
were 0.026 (half-life = 27 min) and 0.00176 (balf-life = 390 min) min

The first half-life corresponded to the second distributional half-life

of buprenorphine (39 min, Table 2) for this dog. For dog C (Fig. 21c) at

1.2023 mg/kg dose (Study #4, Table 2) the sigma minus plot of urinary

data gave an apparent terminal phase rate constant of 0.0017 min-1

(half-life = 400 min) This corresponded with the terminal half-life of

buprenorphine (673 min) obtained from the plasmai data. For the same dog

at 1.439 mg~/kg dose (Study #5) sigma minus plot (Fig. 21d) shorwed

cunrature, and could be fitted to a sum of two exponentials, and the

respective apparent rate constants were 0.00695 (half-life = 100 min)
-1
and 0.000287 (half-life = 2412 min) min The first half-life obtained

from the urine data corresponded with the second distributional

half-life (61 min, Table 2) obtained for buprenorphine from plasma data.

The sigma minus plots for the urinary excretion of buprenorphine in

other dogs showed great scattering and reasonable estimates of the

apparent rate constants were not possible.

The half-lives obtained from the various sigma minus plots shown in

Fig. 21 for the urinary excretion of buprenorphine reasonably

approximated the first and second distributional half-lives of

bulprenorphine in plasma. Since a minor fraction of the the dose was









excreted unchanged in urine (<1%) and the limit of detection of

buprenorphine was 5 ng/ml, the terminal half-life of buprenorphine in

dogs could not be readily estimated from the urinary data.

Sigma minus plots for the conjugates (M) are given in Figs. 22, 23.

For dog A (Study #1) the curve could be unexpectedly and for no obvious

reason, fitted best by a simple linear equation to indicate a constant

rate of renal elimination even with decreasing plasma concentrations of

the conjugate. The excretion rate was approximately 330 ng/min,

independent of concentration of metabolite in the central compartment

(Fig. 22a) For dog B at 1.6369 mg/kg dose (Study #2) of buprenorphine

(Table 2) the sigma minus plot gave an apparent rate constant of 0.0032
-1
min (half-life = 215 min, Fig. 22b) For dog C at 1.2023 mg/kg dose
-1
(Study #4) (Fig. 22c) an apparent rate constant of 0.0022 min

(balf-life = 312 min) was obtained, which corresponded well with the

tterminal phase half-life obtained for M from plasma data (305 min, Table

2) For dog B at 2.5632 mg/kg dose (Study #3) the sigma minus plot

(Fig. 22d) showed curvature and could be fitted to a sum of two
-1
exponentials, and the apparent rate constants were 0.023 min

(half-life = 30 min) and 0.000567 min~-1 (half-life = 1220 min)

respectively, where the second half-life corresponded with the terminal

balf-life obtained from the plasma data of M in this dog (Table 2,

half-life = 1098 min) The sigma minus plot for M in dog C at 1.439

mgJ/kg dose (Study #5) showed curvature (Fig. 23) and could be fitted to

a sum of ~two exponentials, the apparent rate constants being 0.018 main

-1 (half-life = 39 min) and 0.000812 mir -1 (half-life = 853 min) .

In dog C at 1.2023 mg/kg (Study #4) IV bolus dose of buprenorphine,

the terminal half-life of buprenorphine was estimated as 673 min (Table


















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2) The plasma data and sigma minus plot of M in this dog gave an

apparent terminal half-lives of 305 (Fig. 18) and 312 (Fig 22c)

respectively. This metabolite half-life is therefore, an apparent
distributional half-life and not the terminal half-life.

Urinary excretion rate plots. Since Xe = Vc Cp in equation 19,
substituting the value of Cp from equation 2 into equation 19,

dU/dt = Pll el +l A -a ei + f B Eq. 24


where PU = P ku VC Eq. 25

All = A k Ve Eq. 26

BU = B ku Vc Eq. 27

A semilogarithmic plot of excretion rate of unmetabolized drug

versus time according to equation 24 would yield a triexponential curve.

As with the semilogarithmic plasma concentration-time plot, the terminal

exponential phase rate constant can be obtained from the terminal slope,

- 0 /2.303, and B11 is the extrapolated intercept of the terminal
linear phase to time zero. Similar to the treatment of the plasma data

with multicompartment characteristics, the method of residuals could be

used to obtain the parameters of the distributional phases of equation
24.

Semilogarithmic plots of 1a U/ Clt (approximations of the

instantaneous excretory rate, dU/dt) finite amounts (A U) of either 1

or M excreted during a finite time interval ( 6 t) against t-mid

(mid-point of the collection interval) were highly scattered in most of
the studies. Thus only the data for dog A (Study #1) and B (Study #C2)

are reported here. In dog A (Study #1) urine data of buprenorphine up




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