Maternal-fetal cocaine metabolism, distribution, and immunotoxicology

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Maternal-fetal cocaine metabolism, distribution, and immunotoxicology
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Research   ( mesh )
Maternal-Fetal Exchange   ( mesh )
Cocaine -- metabolism   ( mesh )
Cocaine -- toxicity   ( mesh )
Cocaine -- pharmacokinetics   ( mesh )
Ethanol -- toxicity   ( mesh )
Ethanol -- metabolism   ( mesh )
Opportunistic Infections -- Child   ( mesh )
Opportunistic Infections -- Infant   ( mesh )
Immune Tolerance -- Child   ( mesh )
Immune Tolerance -- Infant   ( mesh )
Substance-Related Disorders -- Pregnancy   ( mesh )
Department of Pharmaceutics thesis Ph.D   ( mesh )
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Thesis:
Thesis (Ph.D.)--University of Florida, 1995.
Bibliography:
Bibliography: leaves 163-180.
Statement of Responsibility:
by Diane Lynne Phillips.
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Typescript.
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Vita.

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MATERNAL-FETAL COCAINE METABOLISM, DISTRIBUTION,
AND IMMUNOTOXICOLOGY














By

DIANE LYNNE PHILLIPS


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













ACKNOWLEDGMENTS


I would like to thank all of the members of my supervisory committee, Dr. lan

R. Tebbett, Dr. Hartmut Derendorf, Dr. Roger L. Bertholf, and Dr. Kathleen T.

Shiverick, for their significant contributions towards the completion of the research

for this dissertation. I would especially like to thank my advisor, mentor, and friend

Dr. lan Tebbett for all of his support through the years I have spent on my graduate

education both at the University of Florida and at the University of Illinois at

Chicago.

I would like to thank those individuals who spent their own valuable time

teaching me new techniques or supplying samples used for this work, including

C Dr. Stephen Roberts, Center for Human and Environmental Toxicology,

University of Florida, with whom we collaborated for all of the cocaine and

ethanol interaction studies in mice.

c> Dr. Robert Covert, M.D., and Dr. Samir Wassef, M.D., of the University of

Chicago for supplying samples of meconium for the comprehensive

meconium testing and for statistical analysis.

> Dr. Lucky Jain, M.D., of the University of Illinois at Chicago for supplying samples.

o Dr. Joanna Peris, Department of Pharmacodynamics, University of Florida, for

providing samples for the rat cocaine and ethanol interaction study.

ii







0 Ruth Winecker, graduate student for Dr. Bertholf, for her invaluable assistance

with sample derivitization and GC/MS.

1 Scott Masten, graduate student for Dr. Shiverick, for teaching me everything I

ever wanted to know about cell culture and for helping to critically analyze

the IM-9 cell data as experiments were completed.

0 Dr. Janet Karlix and Becky Frieberger, Department of Pharmacy Practice,

University of Florida, for additional cell culture work for the human cord blood

PBMC study.

C Kathie Wobie, Project Care, Department of Pediatrics, University of Florida, for

teaching me my "social worker skills" and for her expertise on subject

enrollment for the cord blood PBMC study.













TABLE OF CONTENTS

Rage

ACKNOW LEDGMENTS...................................................................... ii

ABSTRACT.... ........................................................................ .............. vi

CHAPTERS

1 BACKGROUND AND SIGNIFICANCE............................. ....... 1
Pharmacological and Physiological Effects of Cocaine........... 2
Cocaine Administration and Pharmacokinetics.................... 7
Cocaine Metabolism......................................................... 13
Maternal-Fetal Circulation and Pharmacokinetics................. 18
Drug Distribution to the Fetus.............................................. 21
Effects of Gestational Cocaine Exposure on the Neonate...... 24
Immunotoxicology of Cocaine......................................... 28
Cocaine and Alcohol Interactions.................................... 35
Analytical Methods for Detection of Gestational
Cocaine Exposure............... ............................. 37
Hypotheses for this Study..................................................... 41
Specific Aim s ... ................................................. .............. 41

2 METHODS
Biological Samples ............................................... ............ 42
Specific Aim #1....................................... 43
Specific Aim #2.................................................................... 57
Specific Aim #3.................................... 58
Specific Aim #4............ ............................ 62

3 RESULTS
Specific Aim #1 .................................................................. 69
Specific Aim #2................................................... ............... 93
Specific Aim #3.................................................... ............... 94
Specific Aim #4 ........... ................ ...... ................ 123

4 DISCUSSION........ .......... ................. ...... 135







APPENDICES

A CALIBRATION CURVES................................... ..................... 158
B RAW DATA FOR INTERDAY AND INTRADAY
VARIABILITY CALCULATIONS............................ ............. 160

REFERENCES.......................................... 163

BIOGRAPHICAL SKETCH.................................. ................ 181













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

MATERNAL-FETAL COCAINE METABOLISM, DISTRIBUTION,
AND IMMUNOTOXICOLOGY

By

DIANE LYNNE PHILLIPS

May 1995


Chairman: Dr. lan R. Tebbett
Major Department: Pharmaceutics

Cocaine use by women during pregnancy has prompted interest in cocaine

distribution to the mother and fetus and in long term effects of cocaine on fetal

development. Conservative estimates indicate that 11-15% of all newborns have

been exposed to cocaine during gestation. Hypotheses: the fetus, given unique

routes of exposure in utero and immature metabolism, is exposed to higher

concentrations of cocaine than the mother during maternal cocaine use in

pregnancy; the ingestion of cocaine with alcohol further increases cocaine

concentration in both mother and fetus; and cocaine and metabolites have

immunotoxicological effects thereby making the neonate more susceptible to

opportunistic infections.







Amniotic fluid, cord blood, and neonatal urine were obtained at delivery from

women who revealed cocaine use during pregnancy. Cocaine and metabolites

were extracted from body fluids using solid-phase extraction and quantified using

high performance liquid chromatography and/or gas chromatography-mass

spectrometry. Results of human and animal studies indicate that the fetus is

exposed to high cocaine concentrations in utero and eliminates the drug more

slowly than the mother. In mice, ethanol cotreatment resulted in a fivefold increase

in peak cocaine serum concentrations and a sixfold increase in peak cocaine

concentrations in the brain. Peak cocaine concentrations were reached faster in

ethanol treated mice and rats. These observations suggest that ethanol can alter

the pharmacokinetics of cocaine, which could partially account for reports of

increased toxicity of the drug when combined with alcohol. Cocaine has multiple

actions on several neurotransmitters many of which have a direct effect on immune

function. The immunotoxicologic effects of cocaine are therefore of interest. No

studies have been published examining the effect of cocaine metabolites on the

immune system. Proliferative effects of cocaine and cocaine metabolites

benzoylecgonine, norcocaine, and cocaethylene on B lymphocytes were examined

using the IM9 human cell line. Results suggest that cocaine has an inhibitory effect

on T-cells while having a stimulatory action on B-cells. This effect is similar to that

produced by the human immunodeficiency virus. Cord blood mononuclear cells

isolated from both cocaine exposed and nonexposed neonates showed similar

responsiveness to mitogenic challenge.













CHAPTER 1
BACKGROUND AND SIGNIFICANCE


Cocaine is one of the most widely abused illicit drugs in the United States.

An estimated 50 million Americans (25% of the population) have used cocaine and

5-6 million use it regularly [1,2]. The most recent population estimates on the use

of cocaine were reported by the National Institute on Drug Abuse and the

Substance Abuse and Mental Health Services Administration in the 1992 "National

Household Survey on Drug Abuse." Results indicated an estimated 5 million

Americans used cocaine in the previous year with the majority of users reporting

"crack" cocaine use [3]. As the price of cocaine has dropped in the last 20 years,

the number of users has risen significantly and the use pattern has changed from

recreational experimentation to abuse. It is estimated that 15-30% of all cocaine

users are women of childbearing age [1,3]. Recently, more attention has been

directed to this population and some hospitals perform routine screening of

newborn's urine for drugs of abuse. From such screening studies, it has been

reported that 11-15% of all pregnant women have used cocaine during pregnancy

[4-7]. However, there appears to be significant geographical variation in

prevalence. One recent study from a Chicago hospital yielded a prevalence rate

of 37% of very-low birthweight babies testing positive for cocaine and/or cocaine






2

metabolites [8], while 18% of newborns tested positive in a Boston survey and 2-3%

in a Rhode Island survey [9]. In a survey of 36 hospitals from different

geographical regions across the country, the overall prevalence rate was 11% with

reports ranging from 0.4-27% [6]. The effect of cocaine use by the mother during

pregnancy has been associated with medical complications in the newborn. Of

particular concern at this time is the effect of cocaine on immune function as over

75% of all perinatally acquired human immunodeficiency virus (HIV) infections are

associated with intravenous drug use by the mother or her sexual partner [9].


Pharmacological and Physiological Effects of Cocaine

Cocaine has multiple actions affecting dopaminergic, cholinergic,

serotonergic, and noradrenergic receptor sites. Cocaine enhances the experience

of pleasure with increased alertness, sexuality, and energy, and decreased anxiety

and social inhibition. However, while the use of low doses of cocaine is reported

to enhance feelings of pleasure, the chronic use of high doses of cocaine may lead

to irritability, aggression, paranoia, anorexia, and psychosis. The effects of cocaine

on the central nervous system (CNS) are well characterized and demonstrate a

biphasic patten of intense stimulation followed by depression. Cortical stimulation

is manifested by euphoria, restlessness and excitability. However, none of the

known neurochemical actions of cocaine singly provide a full explanation of how the

drug produces euphoria. In the CNS, cocaine blocks the reuptake of dopamine at

the dopamine transporter in the synaptic cleft thereby producing an accumulation

of dopamine at postsynaptic receptor sites (Figure 1-1). This, in turn, increases







3

neurotransmission in the mesolimbic and mesocortical dopaminergic pathways in

the brain. These pathways are termed the "reward center" of the brain and function

in producing the euphoria associated with cocaine use.





TYR


DOPA





SA BIDNG
TRANS. Nc*
PORTER


DA
No-




ATP CYCLASE CAMP



Figure 1-1: Effects of cocaine at dopaminergic synapses [10].


It is not clear, however, whether the euphoria is a direct effect of cocaine on

dopaminergic neurons or a combination effect due to simultaneous actions on other

neurotransmitters, like serotonin [10-12]. By blocking the reuptake of dopamine

with chronic cocaine use, the tissue stores of this neurotransmitter are gradually

depleted and this results in compensatory upregulation and supersensitivity of

dopamine receptors [1,11]. The blockade of dopamine reuptake is associated with

the intense reinforcing stimulus of cocaine [13,14]. Cocaine also blocks the







4
reuptake, turnover, and synthesis of serotonin (5HT) and this mechanism may be

involved in the euphoric action of cocaine as well as the onset of tolerance and

effects on sleep, appetite, and aggression [1,14].

Cocaine also blocks the reuptake of catecholamines, primarily

norepinephrine (NE), at adrenergic nerve endings potentiating sympathetically

mediated vasoconstriction, tachycardia, hyperglycemia, and hypertension [1,4,15].

Initially, in the brain, the levels of NE are elevated; however, cocaine activates the

presynaptic alpha-2 receptor which, in turn, produces feedback inhibition of NE. As

with dopamine receptors, chronic cocaine use results in compensatory upregulation

of adrenergic beta receptors in the CNS.

The toxic effects of cocaine have been the subject of a great deal of research

in recent years and several complications associated with cocaine abuse have been

identified. Pyrexia is a prominent feature of cocaine poisoning. Heat retention

occurs due to vasoconstriction, increased muscle activity, and direct effects on the

heat regulating center of the diencephalon [15]. Death by respiratory collapse has

occurred immediately after IV administration of cocaine and pulmonary edema is

a common clinical finding at autopsy following cocaine overdose. The use of crack

cocaine has been associated with characteristic pulmonary injury known as "crack

lung" which is characterized by inflammatory cell infiltration and alveolar

hemorrhage [16]. Oral and nasal ingestion have resulted in death after a symptom

free period of one hour followed by the onset of generalized seizures [17].

Cerebrovascular accidents, including seizures, intracerebral hemorrhage,







5
subarachnoid hemorrhage and cerebrovascular infarction, have also been reported.

The mechanism of hemorrhage is thought to be associated with cocaine-induced

hypertension causing the rupture of a pre-existing lesion [15,18]. Cocaine and

metabolites are also reported to be potent vasoconstrictors of cerebrovascular

arterioles [19,20]. The accumulation of catecholamines predisposes the

myocardium to arrhythmias which may compromise cardiac output, in some cases

resulting in myocardial infarction and ischemia due to the myocardial increased

demand for oxygen. Due to the vasoconstrictive action of cocaine, the myocardium

is not able to utilize compensatory autoregulatory mechanisms involving

vasodilation in response to the increased oxygen demand. Cocaine toxicity has

also been implicated in sudden arrhythmic death, aortic rupture, contraction band

necrosis, cardiomyopathy, and myocarditis [15, 21-30]. Intestinal ischemia may

result from cocaine induced catecholamine stimulation of alpha receptors in

mesenteric vasculature, causing intense vasoconstriction and reduced blood flow

[15,30]. Death from cocaine overdose usually results in 1-5 hours, and the number

of cocaine related emergency room visits have tripled in the last 10 years.

Additionally, cocaine also serves as a local anesthetic and this was one of

the earliest uses of cocaine in the United States. Cocaine has been used as a local

anesthetic in dental, nasal, and ophthalmic surgery. The pharmacologic action of

cocaine (as well as other local anesthetics) is the blockade of sodium channels in

the membranes of neuronal cell bodies, neuronal axons, and cardiac muscle

[31,32]. Sodium channel blockade prevents the propagation of action potentials to







6
neighboring cells and the nerve conducts fewer impulses. While unionized cocaine

penetrates the membrane faster, the cationic form predominates at physiological

pH and is the active form of the drug at the receptor site. When compared to other

local anesthetics, cocaine has a unique effect in that it also acts as a

vasoconstrictor at the site of application. This prolongs the local anesthetic effect

and reduces bleeding. Other local anesthetics are often administered with a

vasoconstrictor, such as epinephrine, to produce a similar effect. Since cardiac

muscle cells also contain sodium channels, these are also blocked by cocaine. This

disrupts normal cardiac pacemaker activity and conduction, which when combined

with the blockade of norepinephrine reuptake, can produce the arrhythmia and

hypertension discussed previously.

Finally, studies in rodents and non-human primates indicate cocaine also

affects normal endocrine function and alters secretion of anterior pituitary hormones

via its action on central dopamine systems [33-37]. Pituitary prolactin secretion is

under tonic inhibitory regulation by dopamine (dopamine is known as prolactin

inhibitory factor (PIF)). Dopaminergic neurons in the tuberofundibular pathway

(connecting the arcuate nuclei with the hypothalamus and posterior pituitary) are

responsible for prolactin release from the pituitary. In human studies, chronic

cocaine use has been associated with persistent hyperprolactinemia in cocaine

users [38-40]. A controlled study with 8 male human cocaine users demonstrated

that hyperprolactinemia was associated with chronic cocaine use; however, there

was no difference in the pulse frequency of prolactin release. This indicated that







7
the hyperprolactinemia may have resulted from the effect of cocaine on

dopaminergic inhibition of basal prolactin secretion [34]. A follow-up study by this

group with 42 patients undergoing treatment for cocaine addiction found that while

hyperprolactinemia was not common, its presence was a poor prognostic sign for

abstinence from cocaine use [41]. Research in this area is on-going, especially

evaluation of the role of serotonin on hypothalamic-pituitary-adrenal axis

dysregulation.

Cocaine Administration and Pharmacokinetics

Cocaine is a naturally occurring alkaloid found in the leaves of the South

American shrub Erythroxylon coca. The leaves are estimated to contain up to 2%

cocaine by weight and are harvested at 3-4 years old [4,42]. South American

natives have chewed the coca leaf for centuries for its stimulant effects, and it was

introduced to the United States in 1885 for use both as a local anesthetic and in

some over-the-counter products, including wine and Coca-Cola [1,4,13]. In the

early 1900s, as the abuse potential of cocaine became evident, laws were passed

to restrict its use. By World War II, all states had laws prohibiting cocaine use for

other than medicinal use as a local anesthetic. Presently, cocaine is placed in

Schedule 2 of the Controlled Drug Act following guidelines stipulated by the federal

government. Drugs placed in Schedule 2 have an accepted medical use, but also

have high abuse potential. Cocaine powder, or cocaine hydrochloride, is extracted

from the coca leaves using acids and an organic solvent like gasoline. This form

of cocaine is a water-soluble salt which can be administered by nasal insufflation






8

or by intravenous injection of an aqueous solution. However, since 1985, free-base

cocaine, in the form of "crack" cocaine, has become the most widely used form of

the drug. "Crack" cocaine is an impure free-base manufactured from cocaine

powder by mixing with ammonia, baking soda, and warm water. The free base

precipitates in the alkaline water and the water is then boiled away leaving hard

chunks of the drug [43,44]. "Crack" is not water-soluble but is stable at

temperatures required for pyrolysis and is administered by vaporization via smoking

in a water pipe. Crack cocaine can be obtained illicitly for as little as $10 and the

rapid, intense euphoria produced makes it highly reinforcing [45].

The pharmacokinetics of cocaine is dependent on the route of administration.

Natives of the Andes and Amazon chewed coca leaves with ash thereby forming

a coca paste. The coca paste was either held in the mouth for long periods of time

and gradually swallowed or it was mixed with tobacco and smoked. The chewing

of paste does not produce euphoria, but does provide mild stimulation and

enhancement of endurance. The chewing of powdered coca leaves which

contained 17-48mg of cocaine was reported to produce peak plasma

concentrations of 10-150pg/L within 0.4-2 hours in 6 volunteers [46]. The chewing

of 50g of coca leaves in a 3 hour period produced plasma cocaine concentrations

of 150-450ng/ml in native Peruvians [47] and similar cocaine levels were reached

much faster when coca paste was smoked [48].

Cocaine is absorbed from mucous membranes and the gastrointestinal tract,

although oral ingestion is not common. Gastrointestinal absorption is slow and







9
bioavailability of cocaine is estimated to be 30% by this route [4]. Gastrointestinal

absorption is slow since cocaine (with a pKa of 8.6) would be ionized in the stomach

(pH 2.0) and would perhaps be more readily absorbed from the more alkaline small

intestine (pH 6.0-8.0). One study using human volunteers measured plasma

cocaine concentrations after oral administration of a dose of 2mg cocaine

hydrochloride per kilogram body weight in a gelatin capsule [49]. For comparison,

the same dose was also administered topically to the nasal mucosa. Results

indicated that after oral administration, cocaine was not detected in plasma for 30

minutes. Peak plasma concentrations of 104-424ng/ml were reached in 50-90

minutes and then declined over the next 4-5 hours and the half-life was 0.9 hours.

Results of intranasal application demonstrated detectable plasma cocaine

concentrations by 15 minutes and peak concentrations of 61-408ng/ml at 60-120

minutes with a half-life of 1.3 hours. Subjective measures of "high" by the subjects

were reached faster in the intranasal applications [49].

Nasal insufflation of cocaine hydrochloride was once the predominant route

of drug administration by users [50]. After topical application of a cocaine dose of

1.5mg/kg body weight to the nasal mucosa of 13 human patients, the absorption

half-life was about 12 minutes and peak plasma levels of 100-500ng/ml were

achieved in 15-60 minutes. Plasma cocaine concentrations then decreased

gradually over the next 3-5 hours, although cocaine was still detectable on swabs

from the nasal mucosa three hours after the application [51]. In a study designed

to parallel the illicit intranasal use of cocaine, 10 human volunteers were instructed







10
to inhale 100mg of a mixture of cocaine hydrochloride powder (16-96mg) and

lactose through a 5.0 cm straw [52]. On alternate days, subjects were given 16-

32mg of cocaine hydrochloride intravenously in 1ml of saline. Plasma cocaine

concentrations were measured after dosing. After nasal insufflation, peak plasma

cocaine concentrations of 53-206ng/ml were reached in 30-60 minutes; however,

plasma cocaine concentrations persisted for a longer period of time than subjective

drug effects reported by the subjects. After intravenous administration, peak

plasma cocaine concentrations of 200-300ng/ml were reached in 5 minutes and

physiological and subjective drug effects paralleled declining cocaine

concentrations [52]. Jeffcoat et al [53] used radiolabeled cocaine to examine

disposition after nasal insufflation by human volunteers. A dose of 106mg of [4-3H]-

cocaine (equivalent to 94.6mg of cocaine) was insufflated through a glass tube.

The average bioavailability was 80%. The absorption half-life was 11.7 minutes and

the elimination half-life was 80 minutes with the mean peak plasma cocaine

concentration of 220ng/ml reached by 45 minutes.

Intravenous injection of cocaine and smoking free-base cocaine produce

similar pharmacokinetic profiles and both produce an intense euphoria within 1

minute of administration. Intravenously injected cocaine has been reported to reach

the brain in 16 seconds; however, due to efficient respiratory exchange, smoked

free-base cocaine reaches the brain faster (in 8 seconds), but has a shorter

duration of action [4]. Jeffcoat et al [53] also administered radiolabeled cocaine to

human volunteers by intravenous injection and smoking. A dose of 23mg of [4-3H]-







11
cocaine (equivalent to 20.5mg of cocaine) was dissolved in 3 ml of saline and then

injected into an antecubital vein over a 1 minute period. The mean peak plasma

cocaine concentration of 180 ng/ml was reached by 5 minutes with the distribution

half-life reported to be 11 minutes and the elimination half-life 78 minutes [53]. For

administration via smoking, a dose of 50 mg of [4-3H] cocaine free base was placed

in a glass pipe and the pipe was heated to 250"C in a hot oil bath. Subjects were

instructed to inhale vapors for 10 seconds (holding the inhaled vapor for 15

seconds) at 30 second intervals for 5 minutes. The absorption half-life was 1.1

minutes and the mean peak plasma concentration of 203ng/ml was reached by 6

minutes [53]. The average bioavailability was 57%; however, a previous study

demonstrated that most undecomposed cocaine reaches the circulation and the

lower bioavailability is related to decomposition before inhalation [54]. The

inhalation efficiency of "crack" pyrolysis has been measured as 739% and 6211%

at 170"C and 220"C respectively [55].

Plasma cocaine concentrations after multiple dosing were also reported

following intravenous and pulmonary administration of cocaine to human subjects

by Isenschmid et al [56]. Subjects were given two separate doses of cocaine

spaced 14 minutes apart by either intravenous injection (16mg or 32mg cocaine)

or by smoking (25mg or 50mg cocaine). Plasma samples were analyzed for

cocaine and metabolite concentrations and were found to be dose dependent.

Peak cocaine concentrations ranged from 210ng/ml for 25mg cocaine via smoking

to 470ng/ml for 32mg cocaine via intravenous injection. Results indicate that the






12
half-life of cocaine was 38-39 minutes regardless of dose or route of administration.

To simulate the binge cocaine use of a typical user, some subjects were given 5-7

doses of cocaine in 90 minutes using a mixture of intravenous injection and

smoking. The maximum cocaine concentration measured was 1200ng/ml from an

individual who received 16mg of cocaine by IV bolus and then smoked 50mg

cocaine 6 separate times over 90 minutes. In subjects smoking 50mg cocaine 6

separate times over 90 minutes, the average plasma cocaine concentration

achieved was 890ng/ml [56].

The pharmacokinetics of cocaine have been studied by many investigators

with most reporting the half-life to be 43-60 minutes [52,53,56-58]. The volume of

distribution of cocaine is small and reported values range from 1.2-2.7L/kg

[4,18,53,59]. When plasma levels of cocaine and urinary excretion data were

plotted together, the decline of the excretion rate plot paralleled that of the decline

in plasma indicating cocaine follows a linear pharmacokinetic model [60]. Garrett

and Seyda [61] found the plasma protein binding of cocaine to be less than 10%

and not a significant pharmacokinetic variable. However, this report is challenged

by Edwards and Bowles [62] who report that cocaine is highly bound to the serum

proteins albumin and alpha-1-acid glycoprotein. Support that this protein binding

is not a significant factor, however, comes from placental transfer studies which

found that while cocaine was 50-90% bound to serum proteins, diffusion across the

placenta was still rapid and cocaine binding to umbilical cord serum was

significantly less than to maternal serum [63].








Cocaine Metabolism

Only 1-5% of a dose of cocaine is excreted unchanged in the urine [4,60].

The remainder is metabolized by several pathways to water soluble metabolites

which are excreted in the urine (Figure 1-2):

(1) Cocaine is hydrolyzed by plasma and liver cholinesterases to ecgonine

methyl ester; in addition, non-enzymatic hydrolysis and hydrolysis by liver

carboxylesterases to benzoylecgonine also occurs. Ecgonine methyl ester is

primarily a urinary metabolite and is usually detected in the blood only in post-

mortem cases [64]. These pathways account for 70-90% of cocaine metabolism in

adults.

(2) The second pathway is via N-demethylation by mixed function oxidase

to norcocaine, a physiologically active cocaine metabolite [65]. Human cytochrome

P450 3A is the oxygenase responsible for the oxidative metabolism of cocaine.

(3) When cocaine is used simultaneously with ethanol, a transesterification

reaction occurs in the liver (catalyzed by hepatic carboxylesterase) to form

cocaethylene [66-68]. Cocaethylene is an active metabolite retaining cocaine's

pharmacologic and toxicologic properties and has a half-life of more than twice that

of the parent compound.

(4) Other, minor metabolites have been identified in the blood and urine of

cocaine users including, ecgonine, ecgonidine, norecgonine methyl ester, and

anhydroecgonine [69,70].

















CH3


SCOOCH3

SH


SCH3
N


BENZOYLECGONINE


H
NORCOCAINE


Figure 1-2: Routes of metabolism of cocaine in vivo.


CH3
N


-KZ


COCAETHYLENE






15
Benzoylecgonine and ecgonine methyl ester are water soluble metabolites

readily excreted in the urine and can be detected for 24-72 hours after a cocaine

dose. Acidification of the urine, in combination with diuretics, can increase renal

excretion of cocaine. Renal excretion of cocaine in the neonate may be slow as the

glomerular filtration rate is only 30-40% that of an adult and is directly related to

gestational age. In addition, the rate of tubular secretion in neonates is only 20-

30% that of an adult and the renal blood flow velocity is significantly lower [31,71].

Only 2-4% of a cocaine dose is excreted in the feces and bile.

As mentioned earlier, plasma and liver cholinesterases metabolize cocaine.

Therefore, any factor affecting esterase activity could have a significant effect on

cocaine metabolism. It has been reported that pregnant women, fetuses, and

people with liver disease have lower cholinesterase activity thus potentially

increasing the risk of cocaine toxicity in these groups [4,15]. In addition, due to

genetic polymorphisms, 1-13% of North Americans are considered to have little or

no cholinesterase activity [72]. The formation of norcocaine by N-demethylation

was higher in subjects with lower cholinesterase activity [65]. Ecobichon and

Stephens [73] were able to demonstrate that plasma pseudocholinesterase activity

in the neonate was 50-60% of that in the adult. Detectable levels of pseudo-

cholinesterase were observed at 28 weeks of gestation and activity increased

rapidly until 1 year of age when the neonatal levels reached those of adults. Their

observations suggested that premature neonates would have significantly different

rates of hydrolysis of ester-type drugs [73]. The activities of the fetal drug








metabolizing enzymes are only 50-70% that found in adults and the half-lives of

various drugs in the neonate are significantly longer than the half-lives in adults [31].

Aspects of drug metabolism and monoamine transport by the placenta must

also be considered in this discussion. The placenta essentially serves as a sieve

for the passage of drugs from the matemal circulation to the fetus. Lipophilic drugs

readily cross the placental barrier and only drugs of very high molecular weight or

drugs that are highly ionized are unable to cross the membranes. Cocaine and

several cocaine metabolites readily cross the placenta via passive diffusion [63,74].

However, water soluble metabolites like benzoylecgonine do not diffuse well. When

formed in the fetus, these metabolites will tend to accumulate on the fetal side due

to ionization of the drugs since the pH of fetal blood is slightly lower than the pH of

maternal blood.

The placenta also serves as a site of oxidative metabolism for some drugs

(i.e. ethanol and pentobarbital) including hydroxylation, demethylation, and

dealkylation reactions [31]. The placenta has considerable amounts of smooth

endoplasmic reticulum and expresses several families of cytochrome P450. The

cytochrome P450 family CYP3A, including 3A3,4, and 7 is responsible for a portion

of cocaine metabolism. CYP3A4 and 7 are expressed in fetal liver and are also

expressed in placental tissue [75-77]. However, in studies by Schenker et al [74]

and Krishna et al [63] with perfused human placenta there was no evidence of

placental metabolism of cocaine. Conversely, Roe et al [72], using human placental

microsomes, showed a 20% decrease in cocaine concentration over a 130 minute






17
in vitro incubation period. In addition, using human placental villus tissue

homogenates, Ahmed et al [78] were able to identify a protein binding site with high

specificity for cocaine. Acetylcholinesterase has also been identified in human

placental villus tissue homogenates indicating the placenta may have the capacity

to metabolize cocaine; however, there is evidence that the placental

acetylcholinesterase is different both genetically and functionally from plasma

cholinesterase [79].

Human placenta expresses transporters for both serotonin [80] and

norepinephrine [81]. These transporters are present on the brush border

membranes which are in contact with the maternal blood. While the purpose of

these placental transporters in fetal development has not been elucidated, in the

CNS, the disruption of monoamine transport by cocaine produces significant

physiological responses. The presence of these transporters may indicate the

placenta may actually serve as a primary target organ for cocaine and, therefore,

as an important mediator of the fetal effects of maternal cocaine use. Work with the

placental serotonin transporter has shown that cocaine is a potent competitive

inhibitor of the transporter [80]. It is speculated that serotonin, a vasoconstrictor,

may be involved in regulation of the uteroplacental circulation; thus, the action of

cocaine at this transporter would result in increased concentrations of serotonin and

vasoconstriction of the placental vasculature [80].








Materno- Fetal Circulation and Pharmacokinetics

During gestation, the placenta serves as the system for fetal respiratory gas

exchange, nutrient supply, and removal of metabolic wastes. The fetal lungs are

filled with fluid until just prior to birth. The maternal and fetal circulatory systems are

separate; however, there is diffusion between the circulations via the placenta.

Oxygenated blood enters the placenta via the maternal uterine and ovarian arteries

and the maternal spiral arteries, where the oxygen is exchanged for carbon dioxide

with the fetal blood. Drugs used by the mother during pregnancy also diffuse from

the maternal circulation through this process. The placento-fetal circulation consists

of the umbilical cord, which has 2 arteries and 1 vein. Since gas and nutrient

exchange is handled by the placenta during gestation, circulatory adaptations are

present in the fetus which are then adjusted on delivery to the circulation present

throughout postnatal life. These adaptations include the ductus venosus, foramen

ovale, and ductus arteriosus. Oxygenated blood enters the fetus via the umbilical

vein and a substantial amount is shunted through the ductus venosus to the inferior

vena cava and right atrium; however, some blood enters the fetal liver through the

portal vein. Blood is then shunted from the right atrium through 2 pathways: (1)

through the foramen ovale, to the left atrium, and then to the ascending aorta. This

pathway provides the circulation to the fetal heart, brain, and upper extremities; (2)

to the right ventricle and then to the pulmonary circulation (less than 10% due to the

high pulmonary resistance) and through the ductus arteriosus to the descending

aorta and lower body. Blood then returns to the placenta via the umbilical arteries.






19
After birth, once the umbilical cord has been clamped, the ductus venosus closes,

the respiratory center is activated, and blood flow increases through the pulmonary

veins. The foramen ovale closes as left atrial pressure rises over right atrial

pressure and the ductus arteriosus flow reverses and then constricts due to the high

oxygen tension of the blood. If the ductus arteriosus remains patent after birth, it

must be either induced to close by administration of indomethacin (which promotes

closure by inhibition of prostaglandin synthesis) or, as a last resort, it must be

surgically ligated. A recent study has found that prenatal cocaine exposure is

associated with indomethacin failure and subsequent surgical ligation of the patent

ductus arteriosus (R. F. Covert, University of Chicago, personal communication).

Complete evaluations of human maternal-fetal cocaine pharmacokinetic

relationships have proven difficult to measure due to the complex technical issues

confronted. Factors that must be considered in such pharmacokinetic

determinations would include maternal cocaine absorption, distribution, and

elimination, fetal distribution and elimination, placental transfer (bi-directional),

placental disposition, and amniotic fluid disposition. Several compartmental models

have been proposed to model maternal-fetal drug kinetics. Szeto [82] proposed a

two-compartment model, one compartment representing the mother and the second

representing the fetus. Drug elimination from both mother and fetus would be

considered. Another possible compartmental model would include a

multicompartment arrangement with compartments for the placenta and amniotic

cavity (Figure 1-3).
























Amniotic


Figure 1-3: Proposed maternal-fetal pharmacokinetic model.
(Modified from Devane [83]).


Physiological models have also been proposed [83,84]. These models

consider drug disposition to the tissues and organs of the body as a function of time

and can model altered physiological states. Luecke et al [84] have developed a

sophisticated, computer driven, physiologically based model for human pregnancy

that considers the rapid growth that occurs during pregnancy. However, since

available data from human pregnancy are generally limited to single time-point

measurements taken at delivery and since cocaine doses are only obtained from

patient self-report, most cocaine pharmacokinetic determinations have utilized

animal models. Sheep are often the animal model of choice given the relatively

large fetal size and the ability to cannulate fetal vessels and withdraw blood for






21

measurement of drug levels. This may, however, not be a good model for cocaine

metabolism and pharmacokinetics.


Druo Distribution to the Fetus

Maternal drug use during pregnancy is of great concern due to the rapid

passage of drugs across the placenta discussed earlier in this chapter. Therefore,

it would also be useful to determine to what extent the fetus is exposed to drugs

ingested by the mother. Many studies have observed that the fetal concentration

of a drug is lower than the maternal concentration. Drugs studied have included

methadone, morphine, meperidine, lidocaine, aspirin, and dilantin [82].

Information on fetal concentrations of cocaine is somewhat inconclusive.

Devane and colleagues [85] reported that the time of sample collection was an

important consideration when comparing maternal and fetal plasma cocaine

concentrations. Pregnant ewes were administered an IV bolus of cocaine in saline

at doses of 0.5-4.0mg/kg and both maternal and fetal plasma cocaine

concentrations were measured over time. Results showed higher maternal cocaine

concentrations until 4 minutes after the cocaine dose at which time the maternal

and fetal concentrations were of similar magnitude. The area-under-the-curve

(AUC) for the maternal samples was substantially greater than the fetal [85]. A

separate study also utilizing pregnant ewes found that fetal blood concentrations of

cocaine observed 5 minutes after maternal infusion were approximately 12% of the

maternal level [86].







22
In pregnant rats, following a single intraperitoneal dose of cocaine (30mg/kg),

cocaine was distributed throughout the fetus with the highest concentrations found

in the placenta, followed by the fetal liver, maternal heart, fetal brain, and maternal

brain and plasma [87]. Spear et al [88] reported fetal plasma cocaine

concentrations, following subcutaneous dosing in pregnant rats, to be 2 to 1 of

those of the dams. It was suggested that the difference could be explained by lower

plasma protein binding in the fetus. Dow-Edwards [89] administered a single dose

of cocaine (60mg/kg) by intragastric gavage to pregnant rats on day 22 of gestation

and cocaine concentrations were measured at time points for 90 minutes. Cocaine

plasma levels peaked at 15 minutes in both dams and fetuses and the mean peak

maternal concentration was 1.8 times higher than the fetal; however, maternal

cocaine levels decreased faster than fetal levels, and from 30-90 minutes fetal

levels were higher than maternal levels. Cocaine studies in pregnant mice showed

significantly lower cocaine concentrations in fetal tissues following a single cocaine

dose [90]. However, chronic cocaine dosing in rabbits demonstrated fetal cocaine

concentrations near or above maternal levels [91]. In that study, pregnant New

Zealand White rabbits received 6 doses of cocaine (0.5mg/kg IV) between days 22-

28 of gestation. Mothers were sacrificed on day 29 and the maternal and fetal

tissue cocaine concentrations were measured. Fetal brain mean cocaine

concentration was 20% higher than mean maternal brain concentration, but mean

maternal liver cocaine concentration was higher [91].







23
Chronic dosing in the pregnant guinea pig (chosen for its metabolic similarity

to humans) demonstrated a similar effect. In that study, pregnant Dunkin-Harley

guinea pigs were given a daily subcutaneous dose of cocaine (6mg/kg) from days

50-59 of gestation. Starting 1 hour after the last injection, sampling was begun as

follows: amniotic fluid collected from around each fetus, cord blood collected from

the umbilical veins, brains removed, maternal blood collected by cardiac puncture,

and maternal urine collected if possible. Maternal and fetal plasma cocaine

concentrations were not significantly different and the cocaine brain to plasma ratios

were also not significantly different. Higher benzoylecgonine concentrations were

found in maternal plasma, but fetal plasma contained higher concentrations of

benzoylnorecgonine. However, amniotic fluid cocaine concentrations were 3 times

that found in either the maternal or fetal plasma [92].

Controlled pregnancy studies are difficult to implement with non-human

primates due to their tendency to abort the fetus if it is surgically removed,

cannulated, and returned to the womb. In the only published report found, 4

pregnant baboons were studied at days 155-160 of gestation (term is 184 days).

Catheters were placed in the femoral arteries and veins of both the mothers and the

fetuses and catheters were also placed in the amniotic sac. Mother baboons were

administered a single dose of cocaine (2mg/kg IV) over a 2 minute period and then

blood and amniotic fluid concentration-time profiles were measured for 24 hours.

Serum cocaine concentrations were observed to decline in a biphasic manner in

both mother baboons and their fetuses; however, while the mean AUC for the fetal






24
samples was lower than the mean maternal AUC, the elimination half-life in fetuses

was 25% longer [93].


Effects of Gestational Cocaine Exposure on the Neonate

Given the documented physiological and toxicological effects of cocaine use

and the evidence that cocaine readily crosses the placental barrier, the effects of

maternal cocaine use in pregnancy on the fetus and newborn have been examined.

Documented labor and delivery complications associated with maternal cocaine use

have included spontaneous abortion [15,94], stillbirth [94,95], preterm labor and

delivery [96-100], premature rupture of the membranes [101,102], and abruptio

placentae [5,97,103,104]. The pathophysiological mechanism believed to be

responsible for the documented obstetric complications is the accumulation of

catecholamines in the periphery produced by cocaine. Accumulation of

catecholamines, in turn, produces vasoconstriction in the maternal and placental

circulation and increased uterine contractility which leads to placental abruption

[15,30,104]. In addition, intrauterine growth retardation has also been reported to

be significantly higher in cocaine users [102,105]. Placental insufficiency and

decreased blood flow produced by chronic cocaine use may be a major factor on

intrauterine growth regulation. Experimental evidence in pregnant sheep

administered intravenous doses of cocaine have demonstrated maternal and fetal

hypertension, decreased uterine blood flow, increased uterine vascular resistance,

elevated levels of serum catecholamines, and fetal hypoxemia [106-109]. Using

conscious fetal lambs, Covert et al [109] also reported reduced fetal cerebral blood






25

flow following cocaine administration (acute IV bolus) to the ewe, but increased

cerebral blood flow following cocaethylene administration. However, in work

involving acute IV continuous infusions of cocaine to either pregnant ewes or fetal

lambs, it has been reported that cocaine increased fetal cerebral blood flow [110].

In this work, cocaine administered to the ewe decreased fetal oxygen tension and

oxygen content; however, cerebral and myocardial blood flows were increased

which compensated for the effects of the fetal hypoxemia. Infusion directly to the

fetus did not alter cerebral blood flow [110].

On the surface, it would appear these independent reports yield opposing

results; however, there are very significant differences in the experimental

protocols which could account for these observations: (1) cocaine administration:

acute IV bolus (1mg/kg) versus acute continuous infusions lasting 30 minutes

(0.3mg/kg); (2) time of blood flow measurements: Covert et al [109] began blood

flow measurement immediately upon dosing while Burchfield et al [110] controlled

for behavioral state. Cocaine administration to either the pregnant ewe or directly

to fetal lambs has been reported to disrupt REM sleep in the fetal lambs [108,111].

Thus, Burchfield et al [110] monitored the behavioral state of the fetus and began

blood flow measurements only once the fetus had returned to REM sleep after the

infusion was stopped. Usually, a recovery time of 1.5-2 hours was required for the

fetus to resume REM sleep. REM periods were chosen for blood flow monitoring

because cerebral blood flow is as much as 35% higher during REM and REM sleep

is a metabolically active state [112].






26

Fetal hypoxemia, secondary to the placental vasoconstriction induced by

cocaine, may also be a contributing factor to the higher incidence of meconium-

stained amniotic fluid seen in pregnant women who have used cocaine during

pregnancy [113-116]. It has been suggested that meconium-stained amniotic fluid

is indicative of fetal stress. Meconium staining is associated with fetal cardiac

disturbances and aspiration of meconium by the fetus can result in respiratory

distress and infection.

Maternal cocaine use in pregnancy is also associated with adverse affects

in the neonate. These problems have included lower gestational age, lower birth

weight, length, or head circumference, a higher incidence of intrauterine growth

retardation and/or small for gestational age infants, fetal hypertension and

ventricular tachycardia, longer newborn hospital stay, and lower Apgar scores (a

measure of newborn health) with poorer general outcome [6,94,99-101,114,117].

Other medical problems reportedly associated with maternal cocaine use have

included retinopathy and ophthalmic abnormalities [118] and perinatal seizures and

cerebral infarction [8,119]. The risk of sudden infant death syndrome is reported

to be 3-7 times higher in newborns exposed to cocaine during pregnancy than in

the general population [5,96,120,121]. Case reports also indicate an increased risk

of necrotizing enterocolitis (NEC) in cocaine exposed newborns [122,123]. NEC is

a serious illness, characterized by ischemic necrosis of the intestinal tract

complicated by mixed-organism infection, with a high mortality rate [124]. The







27
illness is generally seen in small, preterm infants and develops after the onset of

feeding and would be exacerbated by the vasoconstriction produced by cocaine.

The evidence that prenatal cocaine exposure is associated with a higher rate

of congenital malformations is weak. Bingol et al [95] studied 50 cocaine using

women and 110 polydrug abusers, and noted a higher malformation rate in the

cocaine group. However, the teratogenic information reviewed to date indicates

that prenatal cocaine use is not a significant teratogen and the only consistent

teratogenic findings have been those of the genitourinary tract [102,125,126].

Despite limited reports that infants and toddlers exposed to cocaine in utero

are more jittery, irritable, and inconsolable, and less organized and interactive [119],

the long-term behavioral and neurological consequences of prenatal cocaine

exposure are unknown. In rodents and primates, gestational cocaine exposure was

associated with decreases in learning and memory [9]. The National Institute on

Drug Abuse is currently funding longitudinal studies in Seattle, Pittsburgh, Miami,

and rural Florida where cocaine exposed newborns are followed for 3-5 years to

study their neurobehavioral outcomes [9]. Results of these studies will provide

evidence for more definitive statements regarding the effect of prenatal cocaine

exposure on neurobehavioral progress.

Finally, prenatal cocaine exposure is also associated with increased fetal and

newborn deaths [96,127-129]. Fetal deaths in many cases were attributed to

placental abruption, respiratory distress, and prematurity. A majority of the mothers

in fatal cases had received no prenatal care. Two additional cases of newborn






28
death associated with cocaine using mothers, but not prenatal exposure, also

warrant attention as they are not isolated occurrences [9]. One infant who had

been born cocaine positive later died from dehydration and malnourishment

following maternal neglect and a second newborn died from acute cocaine

intoxication resulting from cocaine laced baby formula. Both cases were classified

as homicides [130].

Immunotoxicolovy of Cocaine

In addition to the toxic effects of cocaine previously discussed, evidence

exists to suggest cocaine has a marked effect on the immune system. The

pharmacologic effect of cocaine on modulation of monoamine transport would

indicate that cocaine may have a significant effect on the regulation of the

hypothalamic-pituitary-adrenal axis and therefore, a direct effect on the immune

system. If cocaine is toxic to the developing fetal immune system, maternal cocaine

use in pregnancy could potentially increase the risk of the newborn acquiring

opportunistic infections.

The immune system is characterized by mechanisms of innate and acquired

immunity that protect the body from attack by foreign substances. Innate immunity

is characterized by mechanisms always present in the body to act as barriers to

infection: mucous membranes, coughing, pH, fever, interferon, granulocytes, and

macrophages. Acquired immunity is the mechanism by which the body responds

to foreign substances (called antigens) after a previous exposure. Acquired

immunity functions through the activation of lymphocytes to respond to the antigen.






29
The cell types involved in acquired immunity include T and B lymphocytes and

macrophages. Human lymphocytes possess binding sites for dopamine as well as

adrenergic, cholinergic, and opioergic binding sites [131]. These dopamine binding

sites resembled neuronal uptake sites and dopamine binding at this site was

inhibited by cocaine and other biogenic amine inhibitors [131]. In a separate study,

cocaine and other monoamine uptake inhibitors were found to inhibit in vitro

mitogen stimulated B and T lymphocyte proliferation in rats [132]. These reports

indicate cocaine may have the potential to modulate the immune response directly

through effects on lymphocytes as well as indirectly through hypothalmic-pitutitary-

adrenal axis neuroendocrine pathways [133].

The purpose of the developing fetal immune system is to produce

functionally mature B and T lymphocytes. T cells are responsible for cellular

immunity and recognize foreign antigens in association with a major

histocompatability (MHC) bearing accessory cell (macrophage) that has processed,

and presents, the antigen to the T cell. For example, resting T cells will recognize

cells that are infected with intracellular organisms like a virus or bacteria. The virus

or bacteria is endocytosed by the accessory cell and then the antigen surface

marker proteins and MHC molecules are expressed on the cell surface. The resting

T cell is then activated by its interaction with the accessory cell and the activated

T cells are then stimulated to produce and release lymphokines. B cells constitute

humoral immunity and are responsible for synthesis and secretion of

immunoglobulins (IgG, IgA, IgM, IgE, IgD). These immunoglobulins are able to bind







30
to circulating free antigens, including bacteria and toxins. The B cell response is,

however, dependent on cooperative interactions with activated T cells. Activated

T cells release lymphokines which are known as interleukins (IL) because of their

effects on other lymphocytes as well as their action as autocrine growth factors.

These interleukins include IL2, 4, and 5 and IL-4 and 5 activate the B cell response

[134]. Additional evidence demonstrates that B cell differentiation may be

stimulated by direct cell to cell contact between B cells and T cells, specifically

CD4+ T helper cells, not just via lymphokines [135].

B and T lymphoctye development is initiated during the first trimester and

matures during the second trimester [136]. A mature B or T cell is defined as one

that expresses a surface receptor marker for recognizing specific antigens.

Important T cell markers include CD4 or T helper cells and CD8 or T

suppressor/cytotoxic cells. T cells mature and differentiate in the thymus and the

thymus develops early in gestation. Lymphocytes are recognizable by week 10 of

gestation and by week 17, the distribution of T cell surface markers is essentially

that of the adult. By week 15, fetal T cells will respond to the mitogen

phytohemagglutinin (PHA). T cell maturation occurs only during fetal development

and for a short time after birth and the T cells then populate the secondary lymphoid

organs (spleen and lymph nodes). B cells are first observed in the fetal liver by 9-

10 weeks of gestation. B cells are then observed soon after in the fetal bone

marrow, spleen, and blood. At this time in fetal development, B cells only express

IgM and synthesize no other immunoglobulins. Later in gestation, maternal IgG







31
crosses the placenta and provides the fetus and neonate with some resistance to

infection until the newborn is able to synthesize immunoglobulins. B cells reach the

numerical adult number in the cord blood by week 15 of gestation and, by the

second trimester, B cell generation shifts to the bone marrow [136]. Therefore, any

substance taken during gestation that alters this maturation process could severely

compromise the immune system of the newborn.

Although several studies have investigated the effects of cocaine on human

and animal immune functions, the relationship remains unclear. Studies evaluating

the effect of cocaine on cellular immune function have indicated that the drug

causes suppression of mitogenic responses in human and mouse T lymphocytes

[137]. These studies, however, involved incubation of the cells with relatively high

concentrations of cocaine (12.5-300pg/ml). A complicating factor of many

published studies with cultured lymphocytes has been that the dose levels far

exceed blood plasma levels found in human cocaine users [51,53]. Cocaine has

also been shown to suppress proliferation of phytohemagglutinin (PHA)-activated

human peripheral blood lymphocytes at lower drug concentrations (0.9-12.OM)

[138]. Previous work with cultured mouse splenocytes demonstrated that cocaine

induced suppression of PHA-activated cells was a biphasic process [139]. More

significantly, cocaine (at concentrations observed in human cocaine abusers) was

demonstrated in vitro to augment human T-lymphocyte proliferation stimulated by

the anti-CD3 antibody [140]. This was designed to more accurately mimic the in

vivo stimulation of T-cells via the T-cell receptor complex than is PHA activation.






32

Additional work demonstrated that the effect of cocaine stimulated T-lymphocyte

proliferation was modulated by alterations in calcium mobilization and IL-2

production [141]. Recently, it was reported that cocaine impairs early activation of

CD4+ T-cells [142]. The specific sub-population of CD4+ T-cells affected was the

population carrying a surface marker that targets the cells to migrate to the lymph

nodes. The in vivo effect of this impaired activation would potentially lead to greater

risk for the development of opportunistic infections.

In one of the more promising reports to date on the immunotoxicology of

cocaine, Ruiz et al [143] found alterations in specific T cell subpopulations in

cocaine-intoxicated individuals. Total numbers of natural killer cells, T and B

lymphocytes, and T cell sub-sets including memory CD8 T cells, activated T cells,

T helper cells, and T helperlinducer cells were counted using flow cytometry

combined with immunofluorescence staining targeted to surface marker antigens.

Results demonstrated a reduction in the total number of T cells in cocaine-

intoxicated individuals which was primarily a result of a decreased percentage of

CD4+ T helper cells. In addition, CD8+ "memory" cells were also reduced.

However, there was also a preferential stimulation of activated T cells in these

individuals. These results indicate that in cocaine-intoxicated individuals there may

be a dysfunctional immune response. In response to an immunological challenge,

a larger proportion of T cells would be activated but they would not evolve into

memory cells [143]. The effect on the immunocompetence of human fetal T-cell






33
sub-populations would be of particular concern due to the chronic exposure of the

fetus to cocaine in utero.

Cocaine has been shown to stimulate both natural killer cell activity and

natural killer cell numbers in human volunteers who received an intravenous

infusion of cocaine [144] and also in cocaine-intoxicated patients admitted to a

hospital emergency room [143]. However, an in vitro study indicated that cocaine

had no effect on natural killer cell activity and no effect on cytotoxic T lymphocytic

activity [145]. Thus, cocaine may have differential effects in vivo on recruitment

versus activation of immune cell subpopulations.

Studies of humoral immunity have produced less definitive information on the

immunomodulatory effects of cocaine. Klein et al [139] reported that the

lymphoproliferative response to a B cell mitogen was not affected by cocaine at a

concentration which suppressed T cell proliferation. In another study, rats injected

with cocaine for a 10 day period were shown to have elevated B cell responses to

helper T cell independent and dependent antigens. However, in this same study,

in vitro exposure of lymphocytes to cocaine had no effect on B cell proliferation

[146]. Previous work by Havas et al [147] in mice found that, even at lethal doses

of cocaine, the B cell response was elevated.

There is limited evidence to support indirect effects of cocaine on human B

cells. Elevated serum levels of IgG have been reported in IV drug users; however,

when human peripheral blood mononuclear cells (PBMC's) were cultured with

pokeweed mitogen (a B cell activator) and cocaine there was no effect of the







34
cocaine on IgG synthesis when compared to drug free controls [148]. This indicates

cocaine, and other IV drugs, may induce B cell activation through indirect

mechanisms; however, the elevated IgG levels may be an effect of the presence of

more infectious agents seen in the IV drug using population [148]. In a related

report in the rat, cocaine (1mg/kg) was administered IV to the jugular vein 2 times

per day for 12 consecutive days. Serum immunoglobulin levels were measured 24

hours after the last cocaine dose and compared to controls. Results demonstrated

significant increases in IgG levels in the cocaine treatment group and smaller,

although significant, decreases in serum IgA [149].

Finally, the potential role of cocaine as a co-factor in human

immunodeficiency virus (HIV) infection and in the development and progression of

acquired immunodeficiency syndrome (AIDS) has been raised [150-152]. The role

of cocaine as a co-factor in perinatal HIV transmission has also been raised [153].

Early indications are that HIV infection associated with cocaine users is a result of

intravenous drug use or sexual contact with an intravenous drug user [150,154].

In addition, female cocaine users also demonstrate increased incidence of sexual

transmitted diseases attributed to a more sexual promiscuous lifestyle [151,155].

However, after adjusting for sexual promiscuity and prostitution, the rates of

infection were still higher in cocaine using women indicating involvement of some

other factor [155]. A recent study in Baltimore of 140 IV drug users found that 43%

of females and 57% of males were HIV positive. When correlated to drug usage,

the group with the highest infection rate was cocaine-preferring females with 58%






35

HIV positive [156]. Cocaine has also been shown to potentiate the replication of the

HIV virus in human peripheral blood mononuclear cells in in vitro culture [157,158].


Cocaine and Alcohol Interactions

Many addicts report the concomitant use of ethanol and cocaine and report

a better "high" when both drugs are used [68,159]. In fact, Gorelick [160] reports

that clinical and epidemiological data indicate the co-abuse of these substances is

more common than might be expected and that the 1988 NIDA household survey

found that 80% of the cocaine users also reported ethanol use. An earlier survey

found that 30% of cocaine users drank ethanol every time they used cocaine. One

estimate indicates that as many as twelve million people may use these drugs in

combination in any one year [68]. If the combination of cocaine and ethanol were

to increase the concentration of cocaine in the systemic circulation and the brain,

the toxicity experienced by the fetus could be exacerbated.

Ingestion of cocaine and ethanol leads to the in vivo formation of the active

metabolite cocaethylene (also known as ethylcocaine or ethylbenzoylecgonine).

This metabolite has been detected in the urine of cocaine users also testing positive

for ethanol [159,161]. In vitro studies indicate the formation of CE occurs via a

transesterification reaction in the liver catalyzed by hepatic carboxylesterase

forming a 2-carboxyethyl ester in place of the 2-carboxymethyl ester of cocaine

[66,162]. This enzyme is also reported to catalyze the conversion of cocaine to

benzoylecgonine. Controlled studies in vivo in rats and mice have found that serum

benzoylecgonine concentrations are lower with ethanol pretreatment indicating








ethanol inhibits the formation of benzoylecgonine while simultaneously catalyzing

the formation of CE [162,163]. The enzymatic formation of CE has been

demonstrated utilizing human liver extracts and human, mice, and rat liver

microsomal homogenates [66,163,164]. In vitro incubation of cocaine and ethanol

with homogenates of human kidney, brain, liver, lung, and placenta show that only

liver homogenates produce cocaethylene [165].

Cocaethylene retains the pharmacologic and toxicologic properties of

cocaine yet has a half-life of 2 hours compared to the 45-60 minute half-life of

cocaine [68]. This metabolite has high affinity for the dopamine transporter in the

CNS and may enhance the euphoria experienced by cocaine users due to the

blockade of dopamine reuptake [166]. However, the effect on modulation of the

serotonin system was significantly less than cocaine indicating CE is targeted to

dopaminergic function [167]. An enhanced and prolonged euphoria was reported

by subjects administered both cocaine and ethanol. The plasma cocaine and

norcocaine concentrations were also significantly higher in the acutely intoxicated

individuals as compared to those receiving only cocaine [67]. In primates, CE was

equipotent to cocaine in maintaining self-administration [168].

Because of the high rate of the co-abuse of ethanol and cocaine, one major

effect of the combination has been an increase in cocaine related hospital

admissions and sudden death [68,169,170]. It has been reported that the co-abuse

of these drugs increases the risk of cocaine-related sudden death 18-fold [164].

Significant levels of CE have been measured in postmortem blood and brain






37
samples from cocaine related deaths [166]. In one study, urine and blood, collected

from 15 patients admitted to the hospital for trauma (motor vehicle accidents,

gunshot or stab wounds), were analyzed for cocaine and cocaine metabolites.

Cocaethylene was detected in the plasma of 13 of the 15 patients and some

samples had only cocaethylene detected, no cocaine [170]. Cocaethylene has also

been detected in the urine of newborn babies [8,169]. This cocaine metabolite is

believed to mediate cocaine related toxicity and lethality [171,172] exhibiting an

LDs in mice of 60.7mg/kg versus 93.0mg/kg for cocaine. Cocaethylene has also

been found to be toxic to liver hepatocytes [173] and ethanol has been found to

potentiate the hepatotoxicity of cocaine [174-176].

Although the cocaine/ethanol metabolite cocaethylene will be used in

immune function studies, the role of ethanol alone will not be studied as it has been

extensively reviewed [177-179].


Analytical Methods for Detection of Gestational Cocaine Exposure

Due to the potential for adverse effects of gestational cocaine exposure,

efficient and sensitive methods of detection are needed to identify those newborns

exposed. The most common analytical method for detection of drugs of abuse is

a urine screen using an immunoassay technique, followed by solid-phase extraction

of the drug from the biological matrix and structural confirmation with gas

chromatography-mass spectrometry. Immunoassay is a rapid, relatively

inexpensive screening procedure which is designed primarily to eliminate drug free

samples. However, immunoassay is fairly non-specific and any sample which tests






38

positive for a particular drug must be confirmed by a more specific chromatographic

analysis. Immunoassay is based on the reaction of an antibody with the drug for

which the sample is being tested, ultimately producing a measurable response. In

the case of cocaine, the immunoassay test involves the reaction of an antibody with

benzoylecgonine, the principle hydrolysis product of cocaine in adult urine. This

antibody is much less cross-reactive towards cocaine or other metabolites such as

norcocaine or EME. Immunoassay methods commonly used include enzyme

multiplied immunoassay (EMIT), fluorescence polarization immunoassay (FPIA),

and cloned enzyme donor immunoassay (CEDIA) which are homogeneous assays

requiring no separation step of bound drug from unbound drug.

Radioimmunoassay (RIA) is also used and is a non-homogeneous assay requiring

separation of bound from unbound drug. For urine testing, the legal and

commercially established immunoassay cut-off limit for benzoylecgonine is

generally 300ng/ml.

Chromatographic techniques used for analysis of cocaine and metabolites

have included both gas chromatography-mass spectrometry (GC-MS) and high

performance liquid chromatography (HPLC). GC-MS provides the highest degree

of specificity and is the accepted "gold standard" method of analysis for cocaine,

benzoylecgonine, and EME [56,69,180-185]. The main disadvantage to GC-MS is

that compound derivatization is routinely required prior to analysis. The use of

HPLC is becoming more common as the advent of diode-array and multiwavelength

detectors has improved the selectivity of the method giving ultraviolet (UV)







39
absorption profiles and derivative spectral data for each peak in the chromatogram.

Very sensitive HPLC methods have been developed for cocaine detection and

quantitation in a variety of biological matrices [18,92,186-189]. The main

disadvantages of HPLC are that EME is not detectable using UV detection and that

matrix interference is more common than that seen with GC-MS.

Urinalysis of newborn urine has limitations. In most cases it only indicates

recent use (within 48-72 hours) of the drug by the mother. This sample may be

highly inaccurate in newborns due to the elapsed time from presentation of the

mother for labor, delivery, and subsequent urine collection in the nursery. However,

when the urine of 70 newborns born to cocaine using women was sampled every

8 hours for 6 days after delivery, benzoylecgonine was detected in some newborns

up to 120 hours (mean 80 hours) after delivery [97]. In the case of detection of

cocaine exposure in newborns, it has been suggested that meconium, the first feces

of the baby, is a better indicator of cocaine exposure [190,191]. It is hypothesized

that drugs in the maternal-fetal circulation may accumulate in the meconium from

the time it is first produced at about week 18 of gestation. Since the initial reports

by Ostrea et al [190], other investigators have confirmed that meconium was a

better biological matrix than urine for detection of gestational cocaine exposure

[192-195]. However, a recent report employing structured drug use interviews

found that meconium testing did not improve detection of cocaine use occurring in

early to mid-gestation; in fact, overall results found testing of urine (maternal or

fetal) and meconium produced equivalent results when the same analytical methods






40
were used. Cocaine use had to have occurred in the three weeks prior to delivery

to elicit a positive cocaine test using meconium [196].

Amniotic fluid may also be a good biological matrix for detection of prenatal

cocaine exposure. Reports from cocaine studies using guinea pigs, sheep, and

humans indicate amniotic fluid often contains high concentrations of cocaine and/or

cocaine metabolites [92,109,129,197-199]. Since the concentrations in amniotic

fluid are often much higher than in maternal urine or serum, cocaine and

metabolites may accumulate in this medium. Since the fetus is exposed to the

contents of the amniotic fluid, both orally via swallowing and transdermally, during

the gestational period, the determination of the presence and concentration of

compounds in this fluid may be significant as it would prolong fetal exposure to the

toxic effects of cocaine. Using esophageal ligation in fetal lambs, Mahone [198]

demonstrated cocaine and metabolites were still detected in the fetal plasma when

cocaine was infused into the amniotic fluid. It has been reported that the fetus

swallows between 200-450ml of amniotic fluid per day and urinates as much as

500ml per day into the amniotic cavity [200]. Since the fetus urinates into the

amniotic cavity, the presence of benzoylecgonine in the amniotic fluid is indicative

of cocaine metabolism by the fetus since benzoylecgonine does not cross the

placenta from mother to fetus [74,109].

The above review of the literature indicates the following: that fetal

metabolism and elimination of cocaine differs from that of adults, the concomitant

use of alcohol with cocaine may exacerbate the toxic effects of cocaine, and







41
cocaine has a modulatory effect on the immune system. This research was

therefore designed to investigate the following hypotheses.



Hvyotheses for this Study

(1) The main hypothesis is that the fetus is exposed to higher concentrations of

cocaine than the mother during maternal cocaine use in pregnancy.

(2) The ingestion of cocaine with concurrent ethanol use further increases

cocaine concentration. In pregnancy, cocaine concentration is increased in

mother and fetus.

(3) Cocaine and its metabolites have an adverse effect on the immune

system (in vitro).



Specific Aims


1. To develop and validate analytical methods for the analysis of cocaine and

cocaine metabolites benzoylecgonine (BZE), norcocaine (NC), and

cocaethylene (CE) in biological samples.

2. To characterize maternal/fetal cocaine metabolism and distribution using

both human samples and animal models.

3. To characterize cocaine metabolism and distribution with the concurrent use

of ethanol using animal models.

4. To examine the effect of cocaine, benzoylecgonine, norcocaine, and

cocaethylene on T and B lymphocyte proliferation (in vitro).













CHAPTER 2
METHODS


Biological Samples

Human biological fluid and tissue samples from were provided by subjects

identified by neonatologists at the University of Illinois Hospital (Chicago, Illinois),

the University of Chicago Hospitals (Chicago, Illinois) or at Shands Hospital

(Gainesville, Florida). The use of these samples was approved by the Institutional

Review Boards at all participating universities. Animal studies were conducted at

the University of Florida Gainesville and approved by the Animal Care Committee.

Amniotic fluid (1-5ml) was collected at the time of membrane rupture,

delivery, or amniocentesis. Upon arrival in the nursery, a urine bag was placed on

the infant to collect a sample of the first voided urine (1-5ml) and before 24 hours

of age. Urine samples were then transferred to a plastic sample container. The

newbom's meconium was collected from the diaper and placed in a plastic sample

container. All samples were stored frozen at -20C until analysis. Cord blood (5-

20ml) was obtained after delivery and placed in a heparinized blood collection tube.

Serum and tissue samples from animal studies were stored at -20*C until analysis.








Specific Aim #1

To develop and validate analytical methods for the analysis of cocaine and

cocaine metabolites benzoylecgonine, norcocaine, and cocaethylene.


Basic Materials

Methanol, acetonitrile, and potassium phosphate monobasic (all HPLC

grade) were purchased from Fisher (Fair Lawn, NJ). Diethylamine, butylamine,

chloroform, isopropanol, sodium hydrogen carbonate, sodium hydroxide, acetic

acid, hydrochloric acid, ammonium hydroxide, and tris buffer were reagent grade

(Fisher, Fair Lawn, NJ). Lipase was reagent grade (Sigma, St. Louis, MO).

Cocaine hydrochloride, benzoylecgonine, and bupivacaine were obtained from

Sigma (St. Louis, Mo). Cocaethylene and norcocaine were obtained from the

National Institute on Drug Abuse (NIDA, Rockville, MD).


Fluorescence Polarization Immunoassav Screening

Instrumentation, materials, and calibration

An Abbott Laboratories ADT fluorescence polarization immunoassay

instrument (Abbott Park, IL) was used for screening of urine, amniotic fluid, and

meconium extracts. This technology uses specific antibodies in the reagent

systems that react with free drug (benzoylecgonine) in the sample to form an

antibody-drug complex. Therefore, when analyzed by this method, a "positive"

sample indicated the presence of cocaine metabolite. The reagent kit contained all

components needed for the reaction of the antibody with any free drug in the






44
sample which included: antibody, antigen-tracer, and a pretreatment which

prepared the sample for analysis. Kits for this system have been developed by the

manufacturer for analysis of either urine or serum. Dilution buffer, reagent kits,

calibrators, and controls for benzoylecgonine were purchased from Abbott (Abbott

Park, IL).

The ADTM was calibrated using 6 calibration solutions prepared in human

urine with a benzoylecgonine concentration range of 0-5000ng/ml. Low, medium,

and high control solutions of benzoylecgonine were included in all runs for quality

control monitoring. All calibrators, controls, and samples were diluted with an equal

volume of buffer (pH 7.7) during sample analysis.

Cocaine hydrolysis experiment

Since the ADxM screens samples only for the presence of benzoylecgonine,

any sample which contained only cocaine would screen "negative". Previous work

however indicated high concentrations of the parent drug (cocaine) in meconium

and amniotic fluid. This experiment was therefore designed to find the optimal

conditions at which cocaine (spiked into urine) was completely hydrolyzed to

benzoylecgonine allowing its subsequent detection by immunoassay techniques.

Drug free urine was obtained from a volunteer. A 100lg/ml sample of cocaine

hydrochloride was prepared by solubilizing 0.01g cocaine hydrochoride in 100ml of

drug free human urine. Standards were prepared by dilution of the stock solution

with human urine to yield cocaine concentrations of 1000ng/ml and 100ng/ml.

Samples were incubated over a range of pH values of 8.1-10.4 (the pH adjusted







45
using concentrated NaOH), for time periods from 15-60 minutes, in erlenmeyer

flasks placed in a warm water bath (50C). After the incubation period, the pH of

the sample was immediately adjusted using 6M HCI to a final pH range of 8.1-10.4

for analysis by ADTM. Samples were analyzed by ADTM and reported values of

benzoylecgonine were compared to the known concentrations.

Clinical samples of urine and meconium

Subsequently, a series of actual clinical samples of newborn urine and

meconium were screened by both the direct and the hydrolysis method to

determine if hydrolysis provided improved detection of gestational cocaine use. Ten

samples of urine and 5 samples of meconium were used. One aliquot of the urine

sample was analyzed directly by ADTM. A second aliquot of urine (400pl) was

adjusted to pH>9.5 with concentrated NaOH, incubated for 60 minutes at 50"C, and

then adjusted to pH 8.0 for analysis by ADx. The following procedure was used

for meconium analysis by ADx: (1) The meconium sample was weighed and

placed in a test tube (12X75mm); (2) Either 100% methanol (2ml) was added (for

direct analysis) or 3% ammoniacal methanol (2ml) was added (for hydrolysis); (3)

The mixture was vortexed and centrifuged for 10 minutes at 1000g; (4) Followed by

incubation for 60 minutes at 50C; (5) The supernatant was then transferred to a

clean test tube and evaporated to dryness; (6) The residue was reconstituted in

ADTM buffer (0.5ml) prior to analysis by ATM Benzoylecgonine concentrations

measured using both methods were then compared.








Solid Phase Extraction (SPEI Procedures for Biological Samples

Meconium

Meconium (0.5-1.0g) was extracted by suspension and vortex mixing with

3ml of methanol. After centrifuging for 10 minutes at 1000g, the remaining

methanolic supernatant was transferred to a stoppered tube and vortexed with

0.025M potassium phosphate buffer (1ml) at pH 3 and 10pl of internal standard

(bupivacaine 100pg/ml). This extract was then applied to a Strong Cation

Exchange column (SCX) (Varian, Harbor City, CA.) with a capacity of iml, which

had been conditioned under vacuum on a Vac Elut manifold (Varian) with methanol

(2ml), water (Iml) and 0.25M phosphate buffer (1ml). After application of the

sample, the column was air dried for approximately 30 seconds and then washed

with phosphate buffer (1ml) and 0.1M acetic acid (0.5ml). The column was again

air dried for 30 seconds before eluting the adsorbed drugs with 3% ammoniacal

methanol (2ml). The final extract was evaporated to dryness under nitrogen.

Urine

An aliquot of urine (0.5-1ml) was mixed with an equal volume of 0.025M

potassium phosphate buffer at pH 3 and also with 10pl of internal standard

(bupivacaine 100pg/ml) and then applied directly to an SCX extraction cartridge and

extracted as described above for meconium. Extracts were evaporated to dryness

under nitrogen.








Whole blood or cord blood

Due to the lipophilic nature of whole blood samples, a more polar extraction

column was utilized for the successful extraction of cocaine and metabolites from

this matrix. Whole blood or umbilical cord blood (0.5-1ml) was extracted using the

following procedure: Bond Elut (Varian, Harbor City, CA.) columns, containing C2

packing material with a capacity of 3ml, were positioned on a Vac Elut vacuum

manifold and conditioned with methanol (3ml), followed by 0.1M sodium hydrogen

carbonate (3ml) at pH 8.5. The blood sample was diluted with Iml of carbonate

buffer (pH 8.5) and 10pl of internal standard (bupivacaine 100pg/ml) and then

applied to the extraction column and allowed to dry for 30 seconds before it was

washed with carbonate buffer (3ml), followed by 1ml of 5% v/v methanol in water.

The absorbed drugs were then eluted from the column with 6 X 0.250 ml of

chloroform : isopropanol (4:1). The extracts were evaporated to dryness under

nitrogen.

Amniotic fluid

The viscosity of amniotic fluid required the use of a high flow extraction

cartridge as follows: Amniotic Fluid (1-5 ml) was added to phosphate buffer (1-5 ml,

pH 6) and 10pl of internal standard (bupivacaine 100pg/ml). The mixture was

applied to an X-TracT column (United Chemical Technologies, Horsham, PA), with

a capacity of 15ml, previously conditioned with methanol (5ml), water (3ml) and

phosphate buffer (3ml) at pH 6. The sample was drawn through under vacuum and

the column air dried for 30 seconds. The column was then washed with water






48
(2ml), 100mM hydrochloric acid (2mL), and methanol (3ml). The column was again

allowed to dry before eluting adsorbed compounds with 10ml of a mixture of

chloroform : isopropanol : ammonium hydroxide (78:20:2). The eluent was

evaporated to dryness under nitrogen.

Brain tissue

Two methods were used to extract drugs from brain tissue. Due to its

lipophilic nature, brain tissue must be digested prior to extraction. In method 1, the

brain sample was weighed in a sample vial. Then, 3ml of Tris buffer (0.2M) was

added to the vial along with 1mg of lipase and 10pl of internal standard

(bupivacaine 100pg/ml). The mixture was homogenized using a mechanical

homogenizer and then incubated at 60C for 2.5 hours. The sample was then

extracted using Bond Elut (Varian) columns containing C2 packing material and with

a capacity of 1 ml as described above for whole blood. In method 2, the brain tissue

was weighed in a large, conical tube. Then 3ml of 0.025M potassium phosphate

buffer at pH 3 and 10pl of internal standard (bupivacaine 100pg/ml) were added.

The sample was homogenized using a mechanical homogenizer and centrifuged

for 10 minutes at 1500g. The supernatant was then extracted using SCX columns

with a 1ml capacity as described above for urine. Extraction recovery was

determined using both for comparison.

Serum or plasma

Serum and plasma from humans or animals was extracted following the

method described above for urine.








Placenta and liver

Placental and liver tissue were extracted using method 2 described above

for brain tissue and using C2 extraction columns with a Iml capacity.


High Performance Liquid Chromatoaraphv (HPLC)

Mobile phase

The initial mobile phase used was that of Browne et al [192] which consisted

of 0.025M potassium phosphate buffer: acetonitrile (85:15) containing diethylamine

(25ml/L) with the final pH adjusted to 2.9 with concentrated orthophosphoric acid.

Mobile phase 2 consisted of 0.025M potassium phosphate buffer (with 2%

butylamine): acetonitrile (78:22) with the final pH adjusted to 3.0. Mobile phase 3

consisted of 0.025M potassium phosphate buffer : acetonitrile : butylamine

(78:20:2) with the final pH adjusted to 3.0. Mobile phase 4 consisted of 0.025M

potassium phosphate buffer : acetonitrile : butylamine (81:18:1) with the pH

adjusted to 3.0 with concentrated orthophosphoric acid. Solvents used were of

HPLC grade and were degasssed by bubbling with helium prior to use.

Analytical column

Two analytical columns were utilized throughout this research. Column 1

was a Waters (Marlborough, MA) Novapak C18, 4um, ODS column (15cm x 3.9mm

ID). Mobile phase was delivered to this column at 0.5 ml/min. Column 2 was an

Alltech (Deerfield, IL) Lichrosorb RP18 10um column (25cm X 4.6mm ID). Mobile

phase was delivered to this column at 1.5 ml/min.








Instrumentation

Analysis was performed using two separate systems. System 1 used a

Waters (Marlborough, MA) Model 510 pump to deliver mobile phase to the

analytical column. A C18 Novapak Guard Pak precolumn (Waters, Milford, MA)

was used to protect the analytical column. The sample was injected using a Waters

model UK6 universal liquid chromatograph injector equipped with a 50pI sample

loop. The detector was a Spectra Physics Focus multiwavelength detector (Thermo

Separation Products, Winter Park, FL) with an IBM Personal System/2 data system

for acquisition, integration, and processing. The eluent was monitored at 230, 255

and 275nm and full spectra were recorded from 190 to 400nm for each peak.

Quantitative analysis was achieved by comparison of peak areas of unknowns with

extracted standards. All peak areas were reported as relative peak areas by

comparison to the peak area of the internal standard (bupivacaine 1 pg/ml). Each

determination was taken as the mean of duplicate injections in most cases. The

calibration curve was produced over the range 0.05-2pg/ml. Method validation

studies were completed as described below.

HPLC system 2 used two Waters Model 501 pumps to deliver mobile phase

to the analytical column. A Waters C18 Novapak Guard pak precolumn was used

to protect the analytical column. The sample was injected using a Waters WISP

7108 autosampler with a capacity for 48 samples. The sample size injected was

50pl. The detector was a Waters model 486 tunable absorbance detector and the

eluent was monitored at 230nm. Instrument control, data acquisition, processing,







51
and custom reporting was handled using a NEC Powermate 386 computer and

Waters Millennium 2010 (version 2.0) Chromatography Manager software (Waters,

Marlborough, MA). In addition, this system also included Waters Millennium

System Suitability software which provided for trend plotting, quality control, and

method validation following GMP/GLP regulatory protocols. Therefore, in addition

to the method validation procedures described below and performed with System

1, a GLP method validation program was developed for the analysis of cocaine and

metabolites via HPLC. With this program, the chromatographic system was

validated prior to unknown sample analysis to insure that samples were analyzed

on a validated method. If the method failed the validation test, problems were

corrected before analysis of unknowns.


Method Validation and Quality Control

Standard analytical procedures

Standard curves for quantitative analysis were constructed using bupivacaine

as the internal standard for both HPLC analysis and for GC-MS. In each case the

range of the standard curve, precision and accuracy, specificity and sensitivity were

determined using the following standard procedures.

Extraction recovery

Extraction recovery of cocaine, benzoylecgonine, norcocaine, and

cocaethylene was determined by comparison of the peak area observed for a

nonextracted standard solution in an appropriate solvent injected directly onto the

HPLC to the peak area observed with a solution prepared at the same






52
concentration in the appropriate biologic matrix and injected onto the HPLC

following extraction. Extraction recovery was determined from all biological

matrices used with five replicates at each concentration.

Range of standard curve and control samples

The standard curve was constructed so that the maximum calibrator was

>20% of the expected maximum concentration and the minimum calibrator was at

least 10% > than the minimum quantifiable value (10 x baseline noise). A 6-8 point

calibration curve was prepared in the range 100-1750ng/ml for each compound

(cocaine, benzoylecgonine, norcocaine, and cocaethylene) with a constant

bupivacaine concentration. Control samples were included in the analysis of

unknown samples by injection of a control for every 5-10 unknown samples

injected. Stock solutions of 1mg/ml of cocaine, norcocaine, benzoylecgonine,

and bupivacaine and 1.5mg/ml of cocaethylene were prepared in methanol. A

mixed standard stock solution containing 10pg/ml of COC, BZE, NC, and CE was

then prepared in methanol. Calibration standards in the range 100-1750ng/ml of

COC, BZE, NC, and CE and 1000ng/ml of bupivacaine were prepared in methanol

and evaporated to dryness. Standards were reconstituted in the HPLC mobile

phase (for direct injection) or in the biological matrix (in preparation for solid-phase

extraction).

Linearity and precision of standard curves

The standard curve was determined from the calibrators by a linear least

squares fit to the equation y = mx + b, where x = concentration and y = ratio of drug







53

peak area to internal standard peak area. The regression line was not forced

through zero. An "r" value between 0.97 to 1.0 was considered to represent

acceptable linearity. Precision was assessed from the slope of replicate standard

curves run on the same day and over a minimum two week period. The y-intercept

must be less than 25% of the value of the minimum standard curve calibrator.

Throughout the study period, changes in curve slope, y-intercept and residuals were

assessed to check for occurrence of proportional or determinate error.

Accuracy and precision

Intraday and interday precision were determined on the standards. Replicate

(n = 7) analysis of control samples was performed on the same day and over a

minimum period of two weeks. Precision is expressed as the relative standard

deviation of the intraday and interday replicate analysis for each control sample.

Accuracy is expressed as the relative difference between the actual value and the

mean measured intraday and interday value for each control. Minimal acceptable

accuracy and precision criteria are defined based on the specific requirements of

each study and for this study the minimal acceptable variation was 15% for interday

replicate analysis.

Sensitivity

The limit of detection of each assay procedure was defined as three times

the signal to noise ratio and the limit of quantitation as five times the signal to noise

ratio determined by repetitive analyses of a drug free specimen. Sensitivity was

documented by replicate analysis of blank (drug free) samples and samples







54
containing drug at minimal detectable and minimal quantifiable concentration in the

appropriate biological matrix.

Specificity

Samples of the appropriate biological matrix known to be drug free were

analyzed both with and without a known quantity of drug and/or metabolites added

to the sample to demonstrate lack of interference from endogenous materials.

Resolution of chromatographic peaks of drug, metabolites, internal standard and/or

other eluting compounds was determined.


Gas Chromatography-Mass Spectrometry / HPLC Comparison Experiment

Instrumentation

A Finnigan MAT Incos 50 quadruple mass spectrometer (San Jose, CA),

Hewlett-Packard Model 7673A autosampler and Hewlett-Packard Model 5890 gas

chromatograph (Palo Alto, CA) were used for quantitative analysis of CO, BZE, NC,

BUP, and CE. The compounds were separated on a 30 m X 0.32 mm i.d. fused

silica capillary column coated with a 0.25pm film thickness of bonded-phase methyl

silicone (DB-1) (J&W Scientific, Folsom, CA). The injector was operated in the

splitless mode at 2800C, and helium was used as carrier gas at a column head

pressure of approximately 6psi and flow rate of 1ml/sec. Specimens were injected

at 500C, the split valve opened after 1 minute, and the temperature was increased

to 2800C at a rate of 33C/min. The ion source was operated at 1800C, with an

accelerating voltage of 70eV, 750pA filament current, and 1 kV electron multiplier

voltage. Ion current was acquired at the following masses for cocaine and







55
benzoylecgonine: CO: 82, 182, 303 and BZE: 300, 316, 421. A multiple ion

descriptor was determined for the other metabolites before other studies were done.

The total scan time was 0.439 seconds.

Multiple ion descriptor

A multiple ion descriptor was determined for all compounds, including the

internal standard. Using 100pg of drug diluted in 100pl of dimethylformamide

(DMF), each sample was chromatographed and the full range was scanned. The

multiple ion descriptor was selected based on the fragmentation pattern of the

compounds. A minimum of three ions were chosen. Individual metabolites (except

BZE) were also subjected to a derivitization procedure as described below to

determine if the norcocaine, bupivacaine, or cocaethylene would derivitize.

Derivatization

Each sample for GC/MS was transferred to a silanized Reacti-vialTM(Varian,

Harbor City, CA) following SPE and the eluting buffer was evaporated at 60-700C

under a gentle nitrogen sweep. To this residue, 50pL of pentafluoropropionic

anhydride (Aldrich, Milwaukee, WI) and 25pL of pentafluoropropanol (Aldrich) was

added. The vial was tightly capped, vortex-mixed, and the derivatization reaction

allowed to proceed for 20 minutes at 780C, after which time the reagent was

evaporated under a nitrogen sweep. The residue was reconstituted in 50pL of

dimethylformamide prior to GC/MS analysis.









Experimental

A six point calibration curve was prepared in urine. A sample of drug-free

urine was obtained from a volunteer. The urine was spiked with cocaine,

benzoylecgonine, norcocaine, and cocaethylene to prepare a stock solution

(10pg/ml). Bupivacaine (0.5pg/ml), the internal standard, was added to individual

standards and samples prior to SPE. Concentrations were extracted in the range

of 0-1500ng/ml. Samples were extracted via the solid-phase extraction method

described above for urine. The SPE eluant was then evaporated to dryness under

nitrogen, reconstituted in 100pl of methanol, and split into 2x50pl samples. These

samples were then evaporated to dryness and one sample was reconstituted in

50pl HPLC mobile phase and subjected to HPLC analysis. The second sample was

derivitized as described above, reconstituted in 50pl DMF, and subjected to GC/MS

analysis.

A calibration curve for each compound was prepared on three separate days

over a 2 week period using this method. Then, 10 urine samples identified as

known cocaine positive were also extracted and analyzed by the procedure

described for standards. Finally, interday variability was also determined by

replicate analysis of 7 standard samples, split and analyzed according to the above

procedure. The HPLC used for this experiment was System 2 described above.

A statistical comparison of the GC/MS and HPLC data was performed for

comparison of sensitivity, precision, quantitation, and reproducibility using paired T-

tests and a standard error of the estimate determination.








Specific Aim #2

To characterize cocaine metabolism and distribution in the maternal-fetal unit

using both human samples and animal models.


Human Studies Meconium and Urine

Two studies were done. In study 1, samples of infant urine and meconium

were collected from 22 consecutive live births. Urine samples were screened using

the Abbott ADT analyzer described above. Any positive samples were confirmed

by HPLC, following solid-phase extraction, as described above under methods for

specific aim #1. Meconium samples were analyzed by HPLC only (following solid-

phase extraction (SPE)). In the second study, comprehensive meconium testing

was compared to targeted urine immunoassay screening. For this study, all

newborns delivered at a large, urban community hospital over a one month period

and all newborns delivered at a large, suburban community hospital over a one

month period were included. The total number of newborns studied was 312.

Meconium was collected from all newborns and analyzed by HPLC following SPE.

Newborn urine samples were collected only from those deliveries considered as

high risk for gestational cocaine exposure. Risk factors identified were history of

cocaine use, history of other illicit drug use, preterm labor, no prenatal care,

unexplained hypertension, and abruptio placentae. Targeted urines were screened

by immunoassay. The incidence of cocaine exposure was determined by each

method and the test sensitivity was then calculated for each method based on the

total number of newborns subsequently assigned to the "cocaine exposed" group.






58

Incidence and sensitivity were evaluated for statistically significant differences using

chi-squared analysis.

Human Study Amniotic Fluid. Cord Blood. and Newborn's Urine

Samples of amniotic fluid, cord blood, and infant urine were obtained from

14 women who either reported a history of cocaine use at some time during

pregnancy or who were strongly suspected of such use. These individuals were

identified by the neonatologists. Samples were collected as described above at the

beginning of the methods chapter. Cord blood (0.5-1ml) was analyzed by HPLC

only (following SPE). Urine samples were screened using the Abbott ADTM

analyzer and any positive samples were confirmed by HPLC (following SPE). All

amniotic fluid samples were analyzed by HPLC (following SPE); however, these

samples were also screened with the Abbott ADxT analyzer for method

development purposes. All samples were analyzed for detection and quantification

of cocaine and cocaine metabolites.


Specific Aim #3

To characterize cocaine metabolism and distribution with the co-ingestion of

alcohol.


High Dose Cocaine--Mice

Two groups of male ICR mice (Harlan, Indianapolis,IN) with 18 mice per

group were used. Group 1 mice were dosed with cocaine (55mg/kg IP). Group 2

mice were dosed with cocaine (55mg/kg IP) and pretreated with ethanol (3g/kg






59

gavage) 30 minutes prior to the cocaine dose. Mice were euthanized with CO2

asphyxiation at time points of 15, 30, 60, 90, 120, and 180 minutes with 3 mice

sacrificed at each time point. After sacrifice, blood was collected by cardiac

puncture and the serum (300-500pl) was isolated. The brain was also harvested.

Samples were stored frozen at -20C until SPE and HPLC analysis and quantitation.

Statistical analysis was performed using a one-way ANOVA to compare the mean

drug concentration at each time point between groups 1 and 2.

Low Dose Cocaine--Mice

Two groups of male ICR mice (Harlan) with 18 mice per group were used.

This experiment was designed as under "high dose" above except the cocaine

dosage was lower and designed to more closely approximate human physiological

attainable cocaine concentrations. Group 1 mice were dosed with cocaine

(10mg/kg IP). Group 2 mice were dosed with cocaine (10mg/kg IP) and pretreated

with ethanol (2g/kg gavage) 30 minutes prior to the cocaine dose. Mice were

euthanized with CO2 asphyxiation at time points of 15, 30, 60, 90, 120, and 180

minutes with 3 mice sacrificed at each time point. After sacrifice, blood was

collected by cardiac puncture and the serum (300-500pl) was isolated. The brain

was also harvested. In addition, serum ethanol concentrations were measured in

a 10pl aliquot using a commercially available kit (Sigma, St. Louis, MO). Samples

were stored frozen at -20C until SPE and HPLC analysis and quantitation. Area-

under-the curves for the time versus concentration plots were calculated using the

trapezoidal rule and compared for statistically significant differences using a one-






60

way ANOVA. The time to peak concentration (Tmax) and the peak concentration

(Cmax) were determined for each group from the plot of time versus concentration.

Pregnant Mice

Two groups of female ICR timed-pregnant mice (Harlan) were used with 15

mice per group. On day 17 of gestation, Group 1 mice were dosed with cocaine

(5mg/kg tail vein IV ). On day 17 of gestation, Group 2 mice were dosed with

cocaine (5mg/kg tail vein IV) and pretreated with ethanol (2g/kg gavage) 30 minutes

prior to the cocaine dose. Mice were euthanized with CO2 asphyxiation at time

points of 15, 30, 60, and 90 minutes with 4 mice sacrificed at each time point. After

sacrifice, maternal blood was collected (300-500pl) by cardiac puncture and the

maternal brain was harvested. The fetal brains were also harvested. All fetal

tissues from the same dam were pooled and then split into 2 separate samples if

sufficient tissue had been collected. Samples were stored frozen at -20C until SPE

and HPLC analysis and quantitation. Statistical analysis was performed between

the groups using a one-way ANOVA to compare maternal and fetal brain cocaine

concentrations and maternal blood cocaine and benzoylecgonine concentrations.

In addition, elimination rates for cocaine were calculated on brain cocaine

concentrations over time using a semi-log plot of time versus cocaine concentration.

Elimination rates were evaluated by independent t-tests for statistically significant

differences.








Benzovlecaonine Dosed Mice

To examine BZE transport across the blood brain barrier, male ICR mice

(Harlan) were divided into 2 groups as described above for cocaine dosing. Group

1 mice were dosed with BZE (10mg/kg IP). Group 2 mice were dosed with BZE

(10mg/kg IP) and pretreated with ethanol (2g/kg gavage) 30 minutes prior to the

BZE dose. Mice were euthanized with CO2 asphyxiation at time points of 15, 30,

60, 90, 120, and 180 minutes with 3 mice sacrificed at each time point. After

sacrifice, blood was collected by cardiac puncture and the serum (300-500pl) was

isolated. The brain was also harvested. Samples were stored frozen at -200C until

SPE and HPLC analysis and quantitation. Statistical analysis was performed using

a one-way ANOVA to compare the mean drug concentration at each time point

between groups 1 and 2.

Rat Acute and Chronic Dosing with Cocaine / Ethanol

Four groups of Sprague Dawley male rats were used for this experiment and

with five rats per group. Groups 1 and 2 were "acute" groups. Group 1 received

a single dose of cocaine (10mg/kg IP). Group 2 received a single dose of cocaine

(10mg/kg IP) and of ethanol (1.5g/kg IP). Blood samples were collected at 15, 30,

60, 120, and 180 minutes. The brain was harvested after the last blood collection

and the hypothalamus was sectioned from the rest of the brain. Samples were

stored frozen at -20C until extraction and analysis and quantitation by HPLC.

Groups 3-4 rats were dosed daily for 24 days and were considered "chronic"

groups. Group 3 was dosed with cocaine (10mg/kg IP) and saline (1.5g/kg IP).






62

Group 4 was dosed with cocaine (10mg/kg IP) and ethanol (1.5g/kg IP). Blood

samples were collected at 15, 30, 60, 120, and 180 minutes. The brain was

harvested after the last blood collection and the hypothalamus was sectioned from

the rest of the brain. Samples were stored frozen at -20C until extraction and

analysis and quantitation by HPLC. Area-under-the curves for the time versus

concentration plots were calculated and compared for statistically significant

differences. In addition, the time to peak concentration (Tmax) and the peak

concentration (Cmax) were determined for each group from the plot of time versus

concentration.


Specific Aim #4

To examine the effect of cocaine, norcocaine, benzoylecgonine, and

cocaethylene on T and B cell proliferation.


IM-9 B-Lvmphoblastoid Human Cell Line

Cells

IM-9 cells (CCL#159, ATCC, Rockville,MD) were used to study the effect of

cocaine and metabolites on B cell function. These cells have receptor sites for

human growth hormone, IGFII, insulin, and calcitonin. The cells were cultured in

suspension in a complete medium consisting of 90% RPMI1640 (supplemented with

100pg/ml streptomycin, 100 units/ml penicillin, and 2mmol L-glutamine) and 10%

fetal bovine serum. The cell suspension was replenished every 48 hours.








Materials

Cocaine hydrochloride, benzoylecgonine, and phorbol 12-myristate 13-

acetate (PMA) were obtained from Sigma (St. Louis, MO). Cocaethylene and

norcocaine were obtained from the National Institute on Drug Abuse (NIDA,

Rockville, MD). Drug solutions were sterile filtered through 0.2pm syringe acrodisc

filters (Gelman Sciences, Ann Arbor, MI). RPMI 1640 medium (Gibco, Grand

Island, NY) was supplemented with streptomycin (100pg/ml; Gibco), penicillin

(100units/ml; Gibco), and L-glutamine (2mM; Gibco). IM-9 cells (CCL#159) were

obtained from ATCC (Rockville, MD) and fetal bovine serum (FBS) from Hyclone

Laboratories. [3H]-thymidine (5.0 Ci/mmol) was obtained from Amersham Life

Science, scintillation fluid from Research Products Division (Costa Mesa,CA), and

cell culture plates were from Corning (Marietta, GA).

Drug Preparation

Cocaine hydrochloride, benzoylecgonine, and cocaethylene were dissolved

in sterile phosphate buffered saline (PBS) at concentrations of 40X final well

concentration. Norcocaine was dissolved in a PBS solution containing 1% methanol

to aid solubility, then diluted to 40X final concentration in PBS. Then, all drug

solutions were sterile filtered through 0.2pm syringe acrodisc filters. Solutions were

then diluted just prior to use in complete RPMI serum-free medium (described

under materials) to 4X final well concentration. Final well concentrations of drug

solutions were in the range 0.01 pg/ml 10.Opg/ml with four concentrations of each






64

drug. Phorbol 12-myristate 13-acetate (40nM) was used as a negative control in

proliferation studies.

[3H] Thymidine Incorporation Assays

Proliferation assays were performed when the cells were in early log-phase

growth. Cells were cultured in either RPMI 1640 serum-free medium or in RPMI

1640 medium supplemented with 2% fetal bovine serum (final well concentration)

for 24 and 48 hours in a humidified, 37C atmosphere with 5%CO2 in air.

Cocaine, benzoylecgonine, norcocaine, and cocaethylene (50pl, 4X final

well concentration) in serum-free RPMI 1640 medium were added in triplicate to the

96 well plate for final well concentrations of 0.01-10.0pg/ml. Drug-free controls and

PMA controls were included. IM-9 cells were collected by centrifugation, washed

once with serum-free medium, and resuspended at 2X105 cells/ml (100pl added to

each well). This resulted in 2X104 cells per well. Viability and cell density were

determined by trypan blue exclusion. Cells were cultured in 96 well plates in a final

volume of 200pl. The cells were pulsed with 1 pCi of [3H]-thymidine for the last four

hours of incubation. The cells were harvested onto glass fiber filters using a

Brandel automatic cell harvester. The filters were dried overnight and then placed

into scintillation vials with 4ml of scintillation fluid. The amount of incorporated

tritiated thymidine (in counts per minute (CPM)) was determined for both the

controls and the drug treated groups using standard liquid scintillation counting.

Preliminary experiments were completed to determine the best incubation

conditions for experiments. Using the methods described, IM-9 cells were






65
incubated in varied conditions. Using, (1) Two cell types: Cells cultured prior to the

experiments in RPMI 1640 serum-free medium or in complete medium; (2) Four

medium types for experiments: In either RPMI 1640 serum-free medium or in RPMI

1640 medium supplemented with fetal bovine serum producing a final serum well

concentration of 1, 2, or 5%; (3) Two time periods: 24 and 48 hours; (4) Three cell

densities: 1X104 cells/well, 2X104 cells/well, or 5X104 cells/well.

In addition, a series of experiments were conducted to find external controls

to be included during the proliferation experiments with cocaine and metabolites.

Compounds investigated as controls were tumor necrosis factor (TNFa),

transforming growth factor (TGFP,), interleukin 6 (considered a B cell stimulating

factor), dexamethasone, human growth hormone, and phorbol 12-myristate 13-

acetate (PMA). Experimental conditions were: 4 concentrations of each compound,

2X104 cells/well, 48 hour incubation with a 4 hour [H]-thymidine pulse, and in either

2%FBS RPMI 1640 medium or in RPMI 1640 serum-free medium. Cell proliferation

responses were evaluated in order to choose a suitable control for cocaine and

metabolite experiments.

Drug stability during the incubation period

Since cocaine and cocaethylene, and norcocaine are subject to hydrolysis

to benzoylecgonine and norbenzoylecgonine respectively, drug stability for all

compounds was monitored over the incubation period to insure that any effects

observed were due to the specific drug studied and not a degradation product.

Drug concentrations were also verified by HPLC prior to use. The HPLC system







66
used was System 1 described above. Stability of the drugs in medium, under

incubation conditions, was determined by incubation of two concentrations of each

drug in both serum-free and 2%FBS RPMI 1640 medium and both with or without

cells for 48 hours. After 48 hours, the medium was removed from the wells with a

pipet and stored frozen at -20C pending HPLC analysis. Medium from wells

containing cells was centrifuged (1000g, 10 minutes) and the supernatant removed

for analysis by HPLC.

Statistics

The amount of incorporated tritiated thymidine (in CPM) was determined for

both the controls and the drug treated groups. Treated cells were then expressed

as the percentage of control for the respective treatment condition (incubation time

and media type). Means and standard errors for each drug and each drug

concentration were calculated using the percentage of control values from three

separate experiments. One-way ANOVA was performed at each drug

concentration.


Cord Blood PBMCs (Peripheral Blood Mononuclear Cells)

Sample collection

Cord blood (20ml) from both cocaine exposed (n=5) and non-exposed (n=5)

newborns was collected in heparinized blood collection tubes immediately after

delivery. Women who used cocaine during pregnancy were identified by chart

review upon their arrival for labor and delivery. Cord blood mononuclear cells were

isolated and stimulated with a mitogen to compare proliferative responses between






67

the cocaine exposed and non-exposed groups. Phytohemagglutinin was selected

as the mitogen for these experiments because it stimulates the first signal

transduction pathway in T cell activation.

Materials

Human albumin, histopaque 1077, phytohemagglutinin (PHA-L), RPMI 1640

medium, and ['H]-thymidine were purchased from Sigma (St. Louis, MO).

Scintillation fluid was from National Diagnostics (Atlanta, GA) and cell culture plates

were from Corning (Marietta, GA).

PBMC culture and p3Hl-thvmidine incorporation assays

Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll

Hypaque density gradient centrifugation. Cord blood (up to 20ml) was diluted to

40ml with RPMI 1640 medium supplemented with 5% human albumin. Histopaque

density gradient (10ml) was then layered under the blood. The sample was

centrifuged for 30 minutes at 1200g to isolate the PBMCs. After centrifugation, the

PBMCs form a layer between the histopaque density gradient and excess RPMI

medium. This layer was removed with a sterile transfer pipet, diluted to 50ml with

RPMI 1640 medium supplemented with 5% human albumin, and centrifuged for 10

minutes at 1200g. The cell pellet was resuspended at 1X106 cells/ml (100pl pipeted

to each well). This resulted in 1X105 cells per well. Viability and cell density were

determined by trypan blue exclusion. Cells were cultured in 96 well plates in a final

volume of 200pl.








The PBMCs (1x105 cells/well) were then cultured with phytohemagglutinin

(5pg/ml) and RPMI 1640 medium supplemented with 5% human albumin for 72

hours in a 37C humidified atmosphere with 5% CO2. PBMCs were also cultured

without PHA to serve as unstimulated controls for the PHA stimulated cells. The

cells were pulsed with 1 pCi of [3H]-thymidine for the last 24 hours of incubation.

The cells were harvested onto glass fiber filters using a cell harvester. The filters

were dried overnight and then placed into scintillation vials with 4ml of scintillation

fluid. The amount of incorporated tritiated thymidine (in CPM) was determined for

both the controls and the drug treated groups using standard liquid scintillation

counting.

Statistics

The differences in cell proliferation, in response to stimulation by PHA,

between the cocaine exposed versus non-exposed cord blood lymphocytes was

evaluated using a one-way ANOVA.












CHAPTER 3
RESULTS

Specific Aim #1

Non-enzymatic Hydrolysis of Cocaine in Human Urine Analysis by AD.

Results (Tables 3-1, 3-2, 3-3) indicate cocaine spiked into human urine can

be adequately hydrolyzed to benzoylecgonine for analysis by immunoassay. Ideal

hydrolysis conditions were: hydrolysis pH 9.5 or greater, incubation time of at least

30 minutes at 50"C, and the sample adjusted to pH 8.0 in preparation for analysis

by immunoassay. The next objective was to examine whether this procedure could

be followed for analysis of actual clinical samples.


Table 3-1: Initial experimental conditions for hydrolysis.

Sample Description Concentration reported by AD, (ng/ml)
Medium Control 1440
Blank human urine 0
Cocaine spiked urine (1000ng/ml) 13
Cocaine spiked urine @ pH 8 and 82
left at room temp. for 10min
Cocaine spiked urine @ pH 5.7 22
and left at room temp. for 10min
Cocaine and BZE spiked urine High (>5000ng/ml)
(5pg/ml)
BZE spiked urine (5pg/ml) High (>5000ng/ml)








Table 3-2: Hydrolysis experiment 2 of cocaine to benzoylecgonine at varying pH
after incubation for 60 minutes at 50"C.

Sample Hydrolysis AD, pH b Concentration
pH" (ng/ml) C
Blank urine d -- Low
Blank urine --- --- Low
hydrolyzedd"
COC spike no --- 2
hydrolysis
Medium Control -- 1417
Spike 1000ng/ml COC 9.5 7.1 1021
Spike 1000ng/ml COC 9.8 6.2 1071
Spike 1000ng/ml COC 10.2 6.4 932
Spike 1000ng/ml COC 10.2 7.9 962
Spike 1000ng/ml COC 10.5 8.1 765
Spike 1000ng/ml COC 10.5 6.6 679
BZE spike --- High

Hydrolysis pH is the pH to which the urine sample is adjusted with NaOH
prior to hydrolysis incubation.

b AD, pH is the pH to which the urine sample is adjusted with 6M HCI prior
to analysis after the incubation period.

c Concentration reported is not quantitative due to sample dilution produced
by pH adjustments. "Positive" results would indicate the sample would
be submitted for confirmatory analysis and quantitation.


d ---- indicates not applicable to this sample.







71
Table 3-3: Final experiment to determine ideal cocaine hydrolysis conditions at
50*C.

Sample Hydrolysis pH Hydrolysis AD, pH Concentration
Time (min) (ng/ml)
Blank urine unhydrolyzed ---- Unchanged 6
Blank urine 10.1 60 8.0 9
1000ng/ml unhydrolyzed ---- Unchanged 65
100ng/ml unhydrolyzed ---- Unchanged 5
Low control unhydrolyzed ---- Unchanged 494


1000ng/ml 8.1 15 8.1 209
1000ng/ml 8.0 15 8.0 223
1000ng/ml 8.9 30 7.8 623
1000ng/ml 8.7 30 7.8 585
1000ng/ml 8.5 60 8.5 893
1000ng/ml 8.7 60 8.0 802
1000ng/ml 9.7 15 9.7 NA
1000ng/ml 9.2 30 9.2 843
1000ng/ml 9.8 15 8.3 623
1000ng/ml 9.4 30 7.9 909
1000ng/ml 10.1 15 7.4 574
1000ng/ml 10.4 30 10.4 862


100ng/ml 8.9 30 8.3 34
100ng/ml 8.5 60 8.5 69
100ng/ml 10.0 15 7.8 61
100ng/ml 9.7 30 7.0 96
100ng/ml 10.2 15 7.9 57
100noml 10.2 30 8.3 93









Clinical Samples of Fetal Urine and Meconium


The presence of unmetabolized cocaine in newborn urine and meconium

samples would limit the use of sensitive, rapid immunoassay screening. The results

of experimental non-enzymatic hydrolysis of infant urine and meconium to improve

detection of gestational cocaine exposure are shown in Table 3-4 and 3-5.


Table 3-4: BZE detection in meconium by immunoassay following hydrolysis.

Sample I.D. BZE Concentration by the BZE concentration by the
"direct" method (ng/g) hydrolysiss" method (ng/g)
M0472 0 74
M0474 Low Low
M0476 1 4
M0478 3 Low
M0479 287 281
Low control 485 --
cocaine spiked 89 952
meconium (1pg)_ ___







73
Table 3-5: BZE detection in newborn urine by immunoassay following hydrolysis.

Sample I.D. BZE Concentration by the BZE Concentration by the
"direct" method (ng/g) hydrolysiss" method (ng/g)
U0439 185 34
U0440 0 4
U0443 2 3
U0446 2 10
U0448 4935 1830
U0449 1 5
U0450 6 1
U0451 6 5
U0455 22 169
U0456 158 59
High Control 2832 ---


Results of the hydrolysis experiments found that this method did not improve

detection of gestational cocaine exposure in actual clinical samples. The method

followed to complete the hydrolysis experiments was as labor-intensive as the solid-

phase extraction and HPLC analysis described in Chapter 2, yet did not improve

detection. For these reasons, this method was discontinued.


Solid-Phase Extraction Procedures for Biological Samples

Solid-phase extraction was the analytical method used to extract cocaine and

metabolites from the biological matrices. Methods used for SPE are as written in

Chapter 2. The percentage of recovery of drug from each biological matrix is






74
summarized in Tables 3-6 and 3-7 Extraction recovery was satisfactory with all

methods. Extraction recovery from placenta and liver tissue was variable and

requires the use of an internal standard.


Table 3-6: Extraction recovery of cocaine and metabolites (1pg) from biological
matrices.

Matrix BZE" COCO NC" CE" BUP"


Meconium 8111 879 887 917 915
Urine 849 924 864 936 894
Amniotic Fluid 6212 868 ---- -- 885
Whole Blood 758 864 903 918 854
Cord Blood 8011 888 --- -- 856
Serum 839 936 927 966 933

a Recovery is reported as % recovery % std. dev. of recovery from n=5
determinations.

b --- indicates recovery not determined for this compound.

Table 3-7: Extraction recovery of cocaine and metabolites (1 pg) from brain tissue.

Extraction BZE" COC" NC" CE' BUP'
Method
1--Digested 858 896 ----b ---- 904


2--Undigested 816 917 886 905 855

Recovery is reported as % recovery % std. dev. of recovery from n=5
determinations for method 1 and n=3 determinations for method 2.

b ---- indicates recovery not determined for this compound.






75
Results of extraction of cocaine and metabolites from brain tissue indicated

that digestion was not required. Therefore, solid-phase extraction method 2 was

utilized for all brain extractions for this project. In brief, brain tissue was

homogenized with phosphate buffer (3ml) and internal standard (10 pl), centrifuged

for 10 minutes, and the supernatant extracted using SCX columns. However, fetal

brain tissue was sufficiently fluid such that the uncentrifuged homogenate could be

extracted directly without blocking the SCX column. This method was followed for

extraction of fetal brain tissues under Specific Aim#3.


High Performance Liquid Chromatoaraphv (HPLC)

The HPLC mobile phase providing the best resolution of drugs and

endogenous matrix interference was Mobile phase 4 which consisted of 0.025M

potassium phosphate buffer : acetonitrile : butylamine (81:18:1) with the final pH

adjusted to 3.0 with concentrated orthophosphoric acid. This mobile phase

provided excellent chromatographic performance with both analytical columns used

throughout the project. A typical HPLC chromatogram from HPLC system 1 is

shown in Figure 3-1. In addition, system 1 employs a multiwavelength detector

which allows for acquiring full spectra from 190-400nm (Figure 3-2) and the

generation of the ultra-violet (UV) and derivative spectrum for each peak in the

chromatogram (Figure 3-3 and Figure 3-4).










0.0200


0.0159


0.0118


0.0077


0.0036


-0.0005 1
0.00 8.00 16.00 24.00 32.00

Chromatogram Display: \COCAINE6.BFF

Figure 3-1: HPLC chromatogram of cocaine and metabolites at 230nm (system 1).













220
240
260
280
300
320
0 6.00 12.00 18.00 24.00 30.00

Chromatogram Display: \FOCUS\STDMIX.BFF



Figure 3-2: Full spectrum of standard solution of cocaine and metabolites.
























Spectra Analysis: \FOCUS\STD1.BFF

Figure 3-3: UV spectrum of a standard solution of cocaine.






+









230
220 240 260 280 300


Spectra Analysis: \FOCUS\STD1.BFF

Figure 3-4: UV derivative spectrum of a standard solution of cocaine.







78

HPLC system 2 employs a tunable absorbance detector which is operated

at 230nm for detection of cocaine and metabolites. A typical HPLC chromatogram

is shown in Figure 3-5. System 2 also employs computer software to manage data

acquisition and processing as well as quality control monitoring to evaluate

chromatographic performance prior to and during sample analysis. Method

validation information acquired after analysis of a set of standard samples for

method calibration of cocaine and metabolites is shown in Figure 3-6 and Figure 3-

7. This data was evaluated after the calibration to insure that the variations in

column efficiency, resolution, and retention times were less than 10% and therefore

indicating suitable chromatographic performance for the analysis of unknown

samples.
0.014 1


5.00 10.00 15.00 20.00
Figure 3-5: HPLC chromatogram of cocaine and metabolites (system 2).
Figure 3-5: HPLC chromatogram of cocaine and metabolites (system 2).



























Mille -niu Reults Report: S3 Peak Plot Proc Chan: 486 Page 1
For Sample: 1750 Vial: 1 Inj: 1 Chan: 486 Date Processed 10/07/93 05:03 PM
Channel Descr: WATWS5 486







nnill n uium Sa .pl. Inf oration


Project Name:
Sample Name:
Vial:
Injection:
Channel:
Date Acquired:
Scale Factor:
Acq Meth Set:
Processing Method:


COCAINE
1750
1
1
486
10/03/93 03:28 PM
1.00
COCAINE
COCAINE


Suaple Type:
Volume:
Run Time:
Date Processed:
Dilution:


Standard
50.00
22.0 min
10/07/93 05:03 PI
1.00000


Peak: BEE, Fi, sia Width of 0.758
0.02 .. t



0o0oi 0 5 ia 1012.






5.00 0.00 7.00
MtiaitB*


Mat..


Figure 3-6: Validation report to monitor the chromatographic system.

































































H
0.
C


Figure 3-7: Method validation bar graph indicating with-in run variability.







81
The linearity and precision of standard curves of cocaine, benzoylecgonine,

norcocaine, and cocaethylene with bupivacaine as an internal standard were

routinely determined throughout the course of the project. A representative

calibration curve is shown in Figure 3-8 for cocaine with the data shown in Table 3-

8. Representative calibration curves for the metabolites are in Appendix A.


Table 3-8: Representative standard calibration data for cocaine.

Concentration Average Standard n
(ng/g) Response Deviation
100 320 10 2
250 650 10 2
500 1440 10 2
750 1770 20 2
1000 2400 10 2
1250 2910 0 2
1500 3490 20 2
1750 4030 10 2


The data is fit to the equation y=b + mx where m=slope and b=intercept.

Results of this calibration show: slope = 2.223
Intercept =68

The correlation coefficient (r) = 0.9984
























2000




1000





0 500 1000 1500 2000

Concentration (ng/ml)




Figure 3-8: Representative calibration curve for cocaine.


Throughout the study period, changes in curve slope, y-intercept, and

correlation coefficient were assessed (Table 3-9). Results indicate that the average

deviation of slope for all compounds was less than 25% over a two-year period.

These values were also determined utilizing two different HPLC systems, minor

alterations in mobile phase, and two different types of analytical columns.








Table 3-9: Variation in slope, y-intercept, and correlation coefficient over time.

BENZOYLECGONINE COCAINE
Date HPLC slope y-int r slope y-int" r
System
3/30/92 1 3.71 -25 0.9825 2.75 15 0.9891
5/26/93 1 _b 3.88 40 0.9927
6/3/93 1 4.60 -24 0.9901 2.40 -70 0.9954
6/10/93 1 4.77 -31 0.9960 2.99 -16 0.9810
6/24/93 1 5.03 -33 0.9962 3.04 -26 0.9897
7/20/93 1 3.99 10 0.9976 2.01 7 0.9995
7/22/93 1 4.21 114 0.9669 2.83 -1 0.9996
12/1/93 1 4.73 75 0.9830 3.28 82 0.9952


7/13/93 2 3.36 1 0.9991 2.65 -33 0.9996
8/13/93 2 3.56 -2 0.9977 2.68 11 0.9930
10/4/93 2 3.92 33 0.9990 2.90 33 0.9989
2/21/94 2 3.89 48 0.9989 2.23 68 0.9984
5/20/94 2 4.80 -52 0.9896 3.80 -39 0.9974
8/10/94 2 4.82 -22 0.9998 3.51 9 0.9993
12/21/94 2 4.80 -17 0.9993 3.54 2 0.9993
10/26/94 2 5.61 -76 0.9923 4.41 -76 0.9944



y-intercept values are reported in units of concentration.

b indicates determination not made on this date.









Table 3-9--Continued.


NORCOCAINE COCAETHYLENE
Date HPLC slope y-int" r slope y-inta r
System
3/30/92 1 b --- ----
5/26/93 1 --- --- ---- -- --

6/3/93 1 1.90 -13 0.9959 3.20 -138 0.9898

6/10/93 1 4.22 7 0.9840 3.14 -143 0.9764
6/24/93 1 4.66 -67 0.9889 3.23 -156 0.9897

7/20/93 1 1.73 49 0.9957 2.49 -118 0.9759

7/22/93 1 3.70 -15 0.9995 2.63 -113 0.9913

12/1/93 1 3.42 111 0.9962 2.69 58 0.9930


7/13/93 2 2.93 -24 0.9990 2.38 -33 0.9997

8/13/93 2 2.79 20 0.9951 1.71 -25 0.9962

10/4/93 2 3.26 18 0.9988 2.23 -53 0.9988
2/21/94 2 3.14 26 0.9990 2.01 49 0.9977

5/20/94 2 4.03 -47 0.9972 2.90 -100 0.9959

8/10/94 2 4.36 -21 0.9998 2.82 -113 0.9984

12/21/94 2 4.33 -14 0.9996 2.76 -93 0.9982

10/26/94 2 4.37 -101 0.9919 2.69 -93 0.9944

a y-intercept values are reported in units of concentration.


b --- indicates determination not made on this date.










Intraday and interday quantiation variability were also assessed throughout

the course of the study for both HPLC systems to determine the precision of the

method (Tables 3-10, 3-11, 3-12). Raw data points are tabulated in Appendix B.


Table 3-10: Interday variability for HPLC system 1 at 1000ng/ml.

Benzoylecgonine Cocaine Norcocaine Cocaethylene
mean 1033 1044 1037 1005
Std.Dev. 106 94 114 140
%RSD 10.3 9.0 11.0 13.9

n 9 10 8 8


Table 3-11: Interday variability for HPLC system 2 at 1000ng/ml.

Benzoylecgonine Cocaine Norcocaine Cocaethylene
mean 1012 968 1065 991
Std.Dev. 29 34 89 115
%RSD 2.9 3.5 8.4 11.6
n 13 13 13 13


Table 3-12: Intraday variability for HPLC system 2 at 1000ng/ml.

Benzoylecgonine Cocaine Norcocaine Cocaethylene
mean 993 901 1050 976
Std.Dev. 11 24 33 27
%RSD 1.1 2.7 3.1 2.8
n 11 11 11 11






86

Method sensitivity and specificity were determined to demonstrate the limit

of detection and the lack of interference with endogenous materials in the biological

matrix. The HPLC methods provided adequate specificity for all compounds of

interest; however, GC/MS demonstrated the best specificity of all analytical

methods used.


Table 3-13: Limits of detection (LOD) and limits of quantitation (LOQ).

LIMITS (ng/ml)
Analytical BZE Cocaine Norcocaine CE
Method
Method LOD LOQ LOD LOQ LOD LOQ LOD LOQ

HPLC System 1 1 5 1 5 1 5 4 20


HPLC System 2 1 5 1 5 1 5 2 10


GC/MS 1 5 2 10 10 50 1 5



Gas Chromatoaraohv-Mass Spectrometrv / HPLC Comparison

Results of the experiment to compare HPLC analysis of cocaine and

metabolites to the "gold standard" GC/MS indicate that HPLC is a valid method of

analysis. However, in situations where structural identity is required for confirmation

purposes (for example, in the analysis of unknown urine samples for drugs of

abuse), GC/MS is the preferred method of analysis. In the research laboratory,

HPLC using photodiode array or multiwavelength detection can provide limited

specificity via UV and derivative spectra and can be used for analysis of samples







87
from designed experiments (for example, when mice are injected with a known

quantity of cocaine). The multiple ion descriptors for mass spectral analysis are

illustrated in Table 3-14. Each compound, except the internal standard, was

described by three major ions each of which must be present for a positive

identification. Calibration results are compiled in Table 3-15 and include mean

deteminations of slope, y-intercept, and correlation coefficient for the three separate

calibrations by each method. Sensitivity of the analytical methods was compared

by paired T-tests on the mean slope determinations (Table 3-16). Precision was

evaluated by a paired T-test on the interday variabilities of the methods (Table 3-

17). Quantitation of benzoylecgonine from human urine samples, previously

identified as positive by a certified drug testing laboratory, showed excellent

correlation between the methods (Figure 3-9). Results indicate that GC/MS

demonstrated better precision than HPLC, but the methods provide equivalent

sensitivity.

Table 3-14: Multiple ion descriptors for GC/MS analysis.

Compound m/z monitored
Benzoylecgonine derivitized 300 316 421
Cocaine 182' 198 303
Cocaethylene 196' 272 317
Bupivacaine 140"
Norcocaine derivitized 194' 313 435
Norcocaine 1368 168 289

SMass used for regression analysis








Table 3-15: Calibration curve comparisons for GC/MS and HPLC (n=3 calibrations).

Compound Slope Intercept (ng/ml) r

Mean SD Mean SD Mean SD
BZE 6.37 0.22 -12 15 0.9972 0.0019
o Cocaine 3.90 0.15 4 18 0.9972 0.0034
a-
I NOR 5.31 0.21 -41 8 0.9970 0.0074
CE 9.86 0.295 3 4 0.9972 0.0035


W D-BZE 6.63 1.30 27 70 0.9954 0.0111

SCocaine 6.07 0.85 25 62 0.9896 0.0168
D-NOR 1.48 1.59 -34 86 0.9980 0.1009
CE 8.37 0.49 51 76 0.9942 0.0068


Table 3-16: Comparison of method precision.


* p < 0.05 using Paired T-test


Table 3-17: Comparison of method sensitivity.

Benzoylecgonine Cocaine Norcocaine Cocaethylene

Equivalent GC/MS" HPLC Equivalent

** p < 0.05 using Paired T-test









The standard error of the estimate (Table 3-18) was calculated at the 95%

confidence interval for each drug. Three calibrations were done using each method

(HPLC and GC/MS) for each drug. The slopes and intercepts were determined by

linear regression (y=mx+b). Then, using the calculated values of slope and

intercept, the "calculated" concentration was determined. The "calculated" values

were than plotted on the y-axis and the "actual" concentrations (50-1500ng/ml) were

plotted on the x-axis. A linear regression of these plotted data yielded the

calculation of the standard error of the estimate.


Table 3-18: Standard error of the estimate (ng/ml) for the calculated versus
actual calibration concentrations (residuals) by GC/MS and HPLC.
Calculated concentrations determined by solving the y=mx+b equation
following method calibration.

Method and Cocaine BZE Norcocaine Cocaethylene
Calibration #


GC/MS #1 142.46 7.56 -" 43.57
HPLC # 1 62.48 62.91 38.05 33.38


GC/MS #2 50.24 106.78 --- 86.42
HPLC #2 62.05 47.37 17.31 51.98


GC/MS #3 97.16 16.88 57.30 91.78
HPLC #3 28.88 48.05 85.81 68.58

---- indicates value not calculated.







IPLE IRIFIE CONTROL 1589 WVnL
CO~S., El
RANGEC 1,129 L9ELI N I, 4.0 AM> A 1, 1.0 J 0 W U 29. 3 9
5193731.
lee.O 219025 .

rorocaine







HZE
841
RIC 2532. 933.
15 2171 8. 237151 .
S361 7428.



Ib~ffa" JT





stN
11483.




5l51 6.13 6,35 6,457 7.H I,41 *u IIM


Figure 3-9: Gas chromatogram of cocaine and metabolites after derivitization.






11S5 SFECTRUII
81/27': 94 8:42:! + 7:34
SaIPLE: COCAETHYLENE, I UG/SI
COINS.: SCA141 50-500
TEMPl 264 DEG. C







196







272

51 122 212





JJ, .230. 1. 2tn [ '$1 302
M/Z 58 108 150 200 251 3E0


Figure 3-10: Mass spectrum of cocaethylene extracted from human urine.











3000 --





2500





S 2000





1500





1000





500 -





0
0 500 1000 1500 2000 2500 3000

GCIMS Concentration (nglml)



Figure 3-11: Method comparison for BZE quantitation in urine from human cocaine
users.








Specific Aim #2

Human Studies -- Meconium and Urine

Results of analyses of newborn urine and meconium are in agreement with

previously published studies indicating meconium is a better matrix for detection of

prenatal cocaine exposure (Tables 3-19). In addition, the procedure of "targeting"

women or their newborns for urine toxicology testing was evaluated against

comprehensive meconium testing for identifying the incidence of cocaine exposed

newborns (Table 3-20).


Table 3-19: Analysis of 22 paired samples of newborn meconium and urine.

Both Negative Both Positive -ve urine/+ve +ve urine/-ve
I meconium meconium
18 2 2 0
Total positive = 4/22 = 18.2%


Of 312 infants tested by comprehensive meconium testing or targeted urine

immunoassay screening, 62 were identified as cocaine exposed by either maternal

report of drug use or detection by either method of analysis. When the cocaine

exposed group was evaluated for presence of clinical risk factors, it was found that

69% presented no risk factor. Of the 53 positive meconium samples, cocaine was

detected in 40 (75%), benzoylecgonine in 36 (68%), norcocaine in 12 (23%), and

cocaethylene in 1 (2%). Cocaine concentrations ranged from 140-3498ng/g, BZE

from 92-8433ng/g, and norcocaine from trace-1357ng/g.









Table 3-20: Targeted urine screening versus comprehensive meconium testing.

Targeted Urine Comprehensive Meconium pa
(EMIT) (HPLC)
Incidence 10/312 (3.2%) 53/312 (17.0%) 0.0001
Sensitivity 10/62 (16.1%) 53/62 (85.5%) 0.0001

Total cocaine exposed (by both methods)= 62/312 = 19.9%

a p values were determined by chi-squared analysis.


Human Studies -- Amniotic Fluid and Cord Blood


Analyses of newborn urine, cord blood, and amniotic fluid from cocaine

exposed newborns and their mothers indicate the highest concentrations of

benzoylecgonine (the major cocaine metabolite) are found in amniotic fluid. The

fetus is continually exposed to amniotic fluid throughout gestation. Results also

used in evaluation of hypothesis (1) are reported under results for specific aim #3.

Table 3-21: Human study using biological fluids from 14 women with a history of
cocaine use during pregnancy

Fluid Type # Confirmed cocaine BZE Concentration
exposed Range (ng/ml)
Amniotic Fluid 10/14a 420 3100 ng/ml
Cord Blood 6/8" 120 770 ng/ml
Infant urine 5/12" 100 -1110 ng/ml
4/14 individuals had all fluids confirmed cocaine unexposed.
3/12 individuals had a negative newborn urine but a positive amniotic fluid.
Unmetabolized cocaine was detected in 4/14 amniotic fluid samples.

Refers to the number exposed/the number of samples available for testing.




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