Pharmacokinetics, Metabolism, and

Pharmaceutics of

Drugs of Abuse

Editors:

Rao S. Rapaka, Ph.D.

Division of Basic Research

National Institute on Drug Abuse

Nora Chiang, Ph.D.

Medications Development Division

National Institute on Drug Abuse

and

Billy R. Martin, Ph.D.

Department of Pharmacology

Virginia Commonwealth University

NIDA Research Monograph 173

1997

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

National Institutes of Health

National Institute on Drug Abuse

Division of Clinical Research

5600 Fishers Lane

Rockville, MD 20857
ii

ACKNOWLEDGMENT

This monograph is based on the papers from a

technical review on "Metabolism, Pharmacokinetics,

and Pharmacodynamics of Drugs of Abuse" held on

November 4-5, 1994. The review meeting was

sponsored by the National Institute on Drug Abuse.

COPYRIGHT STATUS

The National Institute on Drug Abuse has obtained

permission from the copyright holders to reproduce

certain previously published material as noted in the

text. Further reproduction of this copyrighted

material is permitted only as part of a reprinting of

the entire publication or chapter. For any other use,

the copyright holder's permission is required. All

other material in this volume except quoted passages

from copyrighted sources is in the public domain and

may be used or reproduced without permission from

the Institute or the authors. Citation of the source is

appreciated.

Opinions expressed in this volume are those of the

authors and do not necessarily reflect the opinions or

official policy of the National Institute on Drug Abuse

or any other part of the U.S. Department of Health

and Human Services.

The U.S. Government does not endorse or favor any

specific commercial product or company. Trade,

proprietary, or company names appearing in this

publication are used only because they are considered

essential in the context of the studies reported herein.

National Institute on Drug Abuse

NIH Publication No. 97-4141

Printed 1997

NIDA Research Monographs are indexed in the Index Medicus. They are

selectively included in the coverage of American Statistics Index,

BioSciences Information Service, Chemical Abstracts, Current Contents,

Psychological Abstracts, and Psychopharmacology Abstracts.
iii

Table of Contents

Click on page number or title to go to that section

Introduction......................................................................................1

Rao S. Rapaka, Nora Chiang, and Billy R. Martin

The Potential Role of the Cytochrome P-450 2D6 Pharmacogenetic

Polymorphism in Drug Abuse............................................................6

Edward M. Sellers, S. Victoria Otton, and Rachel F. Tyndale

Human Liver Cocaine Carboxylesterases..........................................27

William F. Bosron, Robert A. Dean, Monica R. Brzezinski, and

Evgenia V. Pindel

Effects of Ethanol on Cocaine Metabolism and

Disposition in the Rat......................................................................35

Robert A. Dean, William F. Bosron, Frederick M. Zachman, Jing

Zhang, and Monica R. Brzezinski

Individual Differences in Nicotine Kinetics and

Metabolism in Humans....................................................................48

Neal L. Benowitz and Peyton Jacob III

Inhibitors of Anandamide Breakdown..............................................65

Dale G. Deutsch and Alexandros Makriyannis

Metabolic Bioactivation Reactions Potentially Related to

Drug Toxicities................................................................................85

Neal Castagnoli, Jr. and Kay P. Castagnoli

The Role of Iminium-Enamine Species in the Toxication and

Detoxication of Cyclic Tertiary Amines........................................106

Lawrence M. Sayre, David A. Engelhart, Durgesh V. Nadkarni,

M.K. Manoj Babu, Ann Marie Flammang, and G. David McCoy

Neurotoxicity of Amphetamines and Their Metabolites................128

James W. Gibb, Michel Johnson, Ikram Elayan,

Heng Keang Lim, Lisa Matsuda, and Glen R. Hanson
iv

Potential New Insights Into the Molecular Mechanisms of

Methamphetamine-Induced Neurodegeneration.............................146

Monika Z. Wrona, Zhaoliang Yang, Fa Zhang, and

Glenn Dryhurst

Immunotoxicology of Opioids, Inhalants, and

Other Drugs of Abuse.....................................................................175

Robert V. House, Peter T. Thomas, and Hemendra N. Bhargava

Inhalation Studies With Drugs of Abuse.........................................201

Yun Meng, Aron H. Lichtman, D. Troy Bridgen, and

Billy R. Martin

Biocatalysts in Detoxication of Drugs of Abuse.............................225

John R. Cashman

Antibodies as Pharmacokinetic and Metabolic Modifiers of

Neurotoxicity................................................................................259

S. Michael Owens

Toxicokinetics in the National Toxicology Program.....................273

Thomas J. Goehl
1

Introduction

Rao S. Rapaka, Nora Chiang, and Billy R. Martin

Drugs of abuse represent a wide array of chemical classes. Therefore,

it is not surprising that the consequences of drug abuse are highly

dependent upon the chemical in question and vary considerably across

drug classes. During the past decade, understanding of the actions of

these drugs at the molecular and cellular levels has increased

dramatically, thereby providing insight into developing new strategies

for the treatment of drug dependence. However, the interaction of a

drug with a particular biochemical system is only one factor to be

considered when evaluating the pathological impact of substance

abuse. The route of administration, the pharmacokinetics of the drug,

conversion to toxic metabolites, and interindividual responsive-ness

are just a few factors. The objective of this monograph is to evaluate

the current status of knowledge of metabolism, pharmacokinetics, and

pharmacodynamics of drugs of abuse. The overall goal is to identify

innovative approaches for the management and treatment of the

adverse consequence produced by drugs of abuse.

One of the most important challenges of drug abuse research has been

providing an explanation for why all individuals do not respond or

react in a similar fashion to a drug. Metabolism plays a critical role in

the pharmacological and toxicological consequences of drug exposure.

It is well known that the rate of formation of active, inactive, or

toxic metabo-lites varies among individuals. The past decade has been

marked by the characterization of substrate specificity for a large

number of P-450 enzymes and the development of biochemical tools

for quantitation of these enzyme levels. These advances have allowed

determination of the role of metabolic enzymes in susceptibility to

drug dependence, metabolic tolerance, and variability in the toxic

consequence of exposure.

The future holds great promise for utilizing a "fingerprint" of P-450

enzymes as genetic markers for predisposition to either enhanced or

diminished drug dependence. It is also becoming increasingly clear

that brain metabolism has a more prominent role than previously

thought. Notable examples include opioid, amphetamine, nicotine,

and cocaine metabolism by specific isozymes to varying degrees in

different organs. Both synthetic and degradative enzymes for

neurotransmitters and endogenous ligands, including the putative

endogenous cannabinoid ligand anandamide, deserve attention. Future
2

efforts should be directed toward characterization of additional

isozymes responsible for metabolism of drugs of abuse, understanding

regulation of gene expressions resulting in polymorphism,

understanding induction of enzymes, development of selective

substrates and inhibitors, and development of kinetic models, to name

a few. Selective enzyme inhibitors hold promise for ameliorating the

toxicity produced by metabolism of some drugs and diminishing drug

use of other agents which are converted to potent compounds. The

development of animal models with selective enzymatic profiles could

serve to assess risk and aid in development of potential therapeutic

agents for treatment of drug abuse.

While there has always been concern that metabolic transformation

of agents to reactive adducts could result in neurotoxicity, 1-methyl4-phenyl-1,2,6-tetrahydropyridine (MPTP) provided dramatic

evidence. There is direct evidence, as well as considerable indirect

evidence, for formation of reactive adducts for neurotransmitters,

including dopamine and serotonin, and for exogenous amines such as

methamphetamine, phencyclidine, and nicotine. It is now well

established that certain dose regimens of methamphetamines produce

neurodegeneration of dopaminergic and serotonergic neurons in

several species, an effect which could also occur in humans. Several

lines of evidence suggest that methamphetamine itself is not directly

responsible for this neuronal damage, but rather that it produces

oxygen radicals which in turn oxidize the neurotransmitters serotonin

and dopamine to toxic metabolites that destroy their respective

neurons. At present, the mechanism by which methamphetamine

produces its neuronal toxicity has not been fully explained and is

worthy of pursuit. If aberrant metabolites of dopamine and serotonin

are found to be responsible for methamphetamine-induced toxicity, it

is essential that these agents be identified and the mechanisms for

their formation and action at the neuron be elucidated.

There is speculation that long-term exposure to cyclic tertiary amines

such as cocaine, phencyclidine, and phenothiazines may result in

biochemical lesions through the formation of reactive metabolites.

This premise is supported by the induction of a parkinsonian state by

MPTP. Although the initial product of microsomal oxidation is an

electrophilic endocyclic iminium intermediate which is thought to be

the reactive species primarily responsible for neurotoxicity, there is

now evidence that the iminium is in equilibrium with the endocyclic

enamine and that the latter is transformed to reactive species. The

presence of iminium- detoxifying enzymes in cytosolic and

microsomal fractions suggests that rapid inactivation of the iminium
3

species averts toxicity by minimizing formation of the iminium. If

the premise is correct, future efforts should be devoted to designing

drugs which have a higher affinity for the iminium-detoxifying

enzymes. Characterizing these enzymes and identifying the reactive

species of both iminium-enamine equilibrium products will provide a

better understanding of the mechanisms involved in the neurotoxicity

of cyclic amines and the development of strategies for minimizing

their occurrence. The localization of these enzymes and

establishment of substrate specificity would be of considerable

interest.

Metabolism also plays a critical role in the pharmacology of cocaine. The

rapid hydrolysis of cocaine via two different pathways leads to its rapid

inactivation/detoxification. This rapid metabolism has been a major

determinant in the methods and modes of cocaine abuse. Identification and

characterization of these hydrolytic enzymes would be useful in that

selective induction of these enzymes offers a potential treatment strategy

for dealing with cocaine overdose. It is conceivable that long-term

elevation of the enzyme or enzymatic activity could be used in conjunction

with maintenance therapy for cocaine addicts. Hydrolases or esterases are

also responsible for the transesterfication of cocaine. The pharmacological effect of cocaine is prolonged and enhanced when cocaine is used

in conjunction with ethanol. A carboxylesterase catalyzes an ethyl

transeterification of cocaine to cocaethylene, which is biologically active.

In addition, ethanol inhibits cocaine metabolism. The increased levels of

cocaine and cocaethylene may contribute to the prolonged and enhanced

effects of cocaine. Characterization of the responsible enzymes would be

important for developing substrate specificity and other means for attenuating this process. An alternative approach to detoxification involves

biocatalysts. Theoretically, these agents would be devoid of pharmacological properties themselves, a feature that is not always present in

therapeutic compounds. In principle, the biocatalysts should work in an

exponential fashion which would enable them to detoxify large quantities of

the drug. While biocatalysts have tremendous potential, there are many

unanswered questions. In addition, development of appropriate models for

evaluating biocatalysts is essential.

It seems highly improbable that either in vitro model systems or computer

simulations will be introduced in the near future that would give researchers

the confidence to administer drugs to humans without prior testing in other

animal species. Most likely animal models will continue to be used as

predictors for pharmacokinetics and toxicity of drugs in humans.

Improvements in allometric models, or the correlation of pharmacokinetics

among different species, is to be encouraged. Also, hair has been identified
4

as an excellent repository for detection of abused drugs. While there is

relatively little controversy regarding hair analysis for the purpose of

establishing the occurrence of drug use, the reliability of monitoring the

incidence of prior use is far less certain. It remains to be determined

whether a pharmacokinetic model can be developed which has a high degree

of predictability for temporal incorporation of drugs in hair.

The primary focus of the adverse consequences of drugs of abuse has been

directed toward the central nervous system (CNS), with justification. These

drugs are abused because of their actions on the brain, and they serve as

important probes for exploring brain function as it relates to cognition,

pain perception, pleasure, and so forth. Most of the adverse consequences

of drug exposure emanate from the CNS, either through impairment during

intoxication or long-term behavioral changes. Increasingly, attention is

being directed to the adverse effects of drugs of abuse on the entire

organism rather than merely the brain. The National Toxicology Program

examines the pathological consequences of long-term exposure to any drug.

An important aspect of this program is the attempt to establish the

relevance of the animal results with human exposure.

The acquired immunodeficiency syndrome (AIDS) epidemic has served to

increase awareness that the adverse consequences of drug abuse extend

beyond the acute and chronic effects produced by these drugs on the brain.

The immune system has received considerable attention because both opioid

and cannabinoid receptors have been identified in spleen cells. Additionally,

specific effects of several drug classes on immune function are now well

documented. These studies not only provide important insights into the

potential harm that drugs of abuse inflict on a compromised immune

system; they provide opportunities to elucidate the mechanism of action

these drugs.

Lastly, the mode by which individuals abuse drugs has become an

increasingly important factor. When the primary mode of cocaine abuse

switched from nasal insufflation to inhalation, a transformation in the abuse

of cocaine occurred. The ease by which cocaine free base could be

volatilized and inhaled, coupled with savvy marketing strategies of drug

pushers, have made a lasting impact on the manner in which drugs are

abused. Although methamphetamine has been smoked for many years in

some cultures, the recent rise in heroin inhalation underscores this point.

The question arises as to whether drugs are more addictive when inhaled.

While this questions remains to be answered, it is clear that inhalation and

smoking represent highly efficient means of abusing some drugs. There is

now concern as to whether the pattern and frequency of use of other drugs

might change as dramatically as that of cocaine if the route of
5

administration changes to volatilized and inhaled. Furthermore, establishing

the pharmacokinetics of drugs after inhalation should serve to determine

whether sensitivity is enhanced via inhalation.

There is a need for new strategies in providing direct evidence for the

formation of reactive intermediates and the identification of adducts. Since

the formation of adducts may be without adverse consequence, it is

important to establish models for assessing the outcome, particularly with

regard to qualitative and quantitative analysis of adduct formation, measurement of neuronal impairment, and pathological relevance. Current treatment approaches for dealing with life-threatening conditions arising from

acute drug intoxication stress amelioration of symptomatic consequences.

New therapies in the future may well include highly selective enzyme

inhibitors, alterations in the activity of metabolic enzymes, use of

biocatalysts, and so forth. Additionally, establishing pharmacogenetic

polymorphism may well explain individual sensitivity and vulnerability to

CNS-acting drugs. Adding new research tools is necessary for meaningful

advances in the management of drug abuse treatment.

AUTHORS

Rao S. Rapaka, Ph.D.

Chief

Basic Neurobiology and Biological Systems Branch

Division of Basic Research

Nora Chiang, Ph.D.

Chemistry and Pharmaceutics Branch

Medications Development Division

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, MD 20857

Dr. Billy Martin

Professor

Department of Pharmacology

Box 613, MCV Station

Medical College of Virginia

Virginia Commonwealth University

Richmond, VA 23298-0613
6

The Potential Role of the

Cytochrome P-450 2D6

Pharmacogenetic Polymorphism in

Drug Abuse

Rao S. Rapaka, Nora Chiang, and Billy R. Martin

INTRODUCTION AND BACKGROUND

The Cytochrome CYP2D6 Genetic Polymorphism

Cytochromes P-450 (P450s) are enzymes involved in the oxidative

metabolism of a wide array of endogenous and exogenous molecules

including steroids, plant metabolites, prostaglandins, biogenic amines,

drugs, and chemical carcinogens. This broad spectrum of reactions is

due to multiple P-450 isozymes with differing but overlapping

substrate specificities. The mammalian P-450 superfamily consists of

at least 12 families and over 400 individual genes (Nelson et al.

1993). Genetic variants of P-450 have been discovered because of

atypical clinical responses in individuals who were subsequently shown

to have a reduced ability to metabolize a drug. The most widely

studied P-450 polymorphism was first revealed with the

antihypertensive drug debrisoquin (Mahgoub et al. 1977). This drug is

4-hydroxylated by a P-450 enzyme called cytochrome P-450 2D6

(abbreviated CYP2D6) that is encoded by the CYP2D6 gene.

At least 30 drugs, many of them derived from plant alkaloids, have

subsequently been shown to be oxidized by CYP2D6, including

tricyclic antidepressants, some neuroleptics, beta blockers, and

antiarrhythmic agents such as perhexiline, flecainide, and encainide.

Several drugs of abuse including codeine (Chen et al. 1988),

hydrocodone (Otton et al. 199 a), oxycodone (Otton et al., 3

unpublished observation), dextromethorphan (Schmid et al. 1985),

and p-methoxyamphetamine (PMA) (Kitchen et al. 1979) are known

to be metabolized by this enzyme. CYP2D6-mediated metabolism of

these drugs is known to be a major source of pharmaco-kinetic

variation and variation in drug effect.
7

Functional and Molecular Characterization of CYP2D6

The activity of CYP2D6 is measured using a subclinical dose of debrisoquin or

another probe drug such as dextromethorphan. Using dextromethorphan, the

amounts of unchanged drug and metabolite excreted in urine over zero to 8

hours are measured and expressed as an O-demethylation ratio (ODMR) (i.e.,

the ratio of dextromethorphan/dextrorphan or (dextromethorphan + 3-

methoxymorphinan)/(dextrorphan + 3- hydroy- morphinan)). The

frequency distribution of the logarithm of these ratios is bimodal (figure 1),

with 7 to 10 percent of the population comprising the upper mode (called

poor metabolizers [PMs]). The frequency shows considerable interethnic

variation (Kalow 1991); for example, the frequency among African

Americans is about 2 percent and among Chinese less than 1 percent. The

remainder of the population, who perform this reaction to varying degrees,

are called extensive metabolizers (EMs). ODMR values are constant over

time and are not influenced by gender, smoking, and classical P-450 inducers

such as phenobarbital and rifampicin (Eichelbaum et al. 1986; Vincent-Viry et

al. 1991).

In addition to functional assays for CYP2D6, polymerase chain reaction

(PCR) amplification assays have been developed for the detection of specific

mutations in the CYP2D6 gene (table 1). Additional genotyping
8

assays use restriction fragment length polymorphisms (e.g., Xbal

haplotypes) or allele-specific endonuclease digestion of PCR products

to genotype the CYP2D6 locus. These assays can correctly detect >

95 percent of all mutant PM alleles found in caucasians (Broly et al.

1991). The CYP2D6 gene is part of a highly homologous gene

cluster on chromosome 22 that includes CYP2D7 and CYP2D8P

(Kimura et al. 1989). CYP2D8P contains several gene-disrupting

insertions, deletions, and termination codons, indicating that this is a

pseudogene. CYP2D7 is normal except for the presence of an

insertion in exon 1 which disrupts the reading frame. The transcript

for CYP2D7 has not been isolated, and it is unclear what, if any, the

metabolic profile for this gene might be. The genotyping assays using

PCR technology have an initial amplification that is specific for

CYP2D6 introns in order to exclude the very similar CYP2D7 and

CYP2D8P genes.

TABLE 1. Frequencies of EMs and PMs among caucasians.

Phenotype Genotype Frequency(%)

EM - rapid wt/wt 54

EM - intermediate wt/A, B, or D 39

PM - slow A, B, or D/A, B, or D 7

Multiple CYP2D6 alleles that contain point mutations or codon

deletions have been isolated (table 2). These mutant alleles, in

addition to the CYP2D6 gene deletion (CYP2D6D), result in absent

or impaired enzyme function (Broly and Meyer 1993; Gaedigk et al.

1991; Hanioka et al. 1990; Johansson et al. 1993, 1994; Kagimoto et

al. 1990; Meyer 1994; Tyndale et al. 1991a). If present in the

homozygous state, defective alleles result in a PM phenotype.

Heterozygous combinations of mutant and wild type (wt) alleles tend

to result in decreased CYP2D6 activity, although no functional means

are available to fully distinguish between these heterozygous people

and those who are homozygous wt/wt. In addition, CYP2D6 gene

duplications that result in ultra-rapid enzyme function have recently

been identified (CYP2L2&12) (table 2; Johansson et al. 1993).
9

TABLE 2. Summary of CYP2D6 alleles, Xbal haplotypes, and their

phenotypic consequences.

Allele Metabolic activity Xbal haplotype (kb)

CYP2D6-wt normal 29

CYP2D6-L normal 29

(CYP2D6-L)2 ultra-rapid 42

(CYP2D6-L)12 ultra-rapid 175

CYP2D6-C slightly decreased 29

CYP2D6-J slightly decreased 29, 44

CYP2D6-W slightly decreased 29, 44

CYP2D6-Ch1 slightly decreased 29, 44

CYP2D6-A absent 29

CYP2D6-B absent 29, 44, 9 + 16

CYP2D6-D absent 11.5, 13

CYP2D6-E absent 29

CYP2D6-F absent 29

CYP2D6-G absent 29

KEY: kb = kilobase.

While the majority of alleles in caucasians have now been isolated,

the frequency of the CYP2D6 alleles listed in table 2 varies

dramatically between ethnic groups. In addition, it is likely that many

other alleles remain unidentified in other ethnic groups. Based on an

understanding of the variants found in caucasians, one might predict

that the unidentified variants might be unable to form a functional

enzyme and have altered substrate specificity, selectivity, or turnover

of the enzyme. The clinical importance of such variants is generally

unknown; however, it is feasible that some ethnoracial differences in

drug abuse patterns may be explainable by such differences.

The Rat Models of the Polymorphism

Rats have five CYP2D genes, CYP2D1 through CYP2D5, although

CYP2D1 is the predominant enzyme expressed in rat liver. In the

female Dark Agouti (DA) rat, the CYP2D1 gene is not expressed

(Matsunaga et al. 1989) and these animals exhibit impaired oxidation

of human CYP2D6 substrates (e.g., debrisoquin (Al-Dabbagh et al.

1981), dextromethorphan (Zysset et al. 1988)) compared to SpragueDawley (SD) rats. However, unlike the human deficiency, the

deficiency in the CYP2D1 is due to a regulatory difference, not a

defective gene for the enzyme. Nevertheless, the DA female rat may
10

be used as an animal counterpart of the human PM, and the SD female

rat as the EM counter-part. Although females of these two rat strains

are useful animal models, the rat CYP2D1 is not functionally identical

to human CYP2D6. For example, quinidine is a very potent and

long-lasting inhibitor of the activity of CYP2D6, both in vitro and in

vivo, and its diastereoisomer quinine is 200 times less potent (Otton

et al. 1984). However, quinine is the more potent inhibitor of rat

CYP2D1 (Kobayashi et al. 1989).

CYP2D Enzyme in Brain

Catalytic, pharmacological, immunological, and molecular criteria

have been used to identify cytochrome CYP2D in mammalian brain

(Fonne-Pfister et al. 1987; Niznik et al. 1990; Tyndale et al. 1991b).

The initial observation of CYP2D in dog brain was made during

screening of central and peripheral tissues with tritiated GBR-12935,

which labels the dopamine transporter protein and the so-called

piperazine acceptor site or mazindol-insensitive site in brain tissue

(Niznik et al. 1990). High concentrations of the piperazine acceptor

site were found in liver microsomes. The similarity between

amphetamine derivatives that inhibited both GBR-12935 striatal

binding and hepatic CYP2D6 activity prompted further studies that

demonstrated correlations between the inhibitor profile at the

piperazine acceptor site purified from dog striata and the inhibition

constant (Ki) for human hepatic CYP2D6 (r = 0.85).

Immunoprecipitation and Western blotting experiments confirmed

the presence of CYP2D in dog striata.

Subsequent studies focused on the catalytic activity of CYP2D in

canine striata (Tyndale et al. 1991b). One of the classic CYP2D6

substrates, sparteine, was used as the marker for CYP2D activity in

dog striata. High (r > 0.95) correlations were observed between

inhibition of sparteine oxidation (Ki values) in canine striata and in

human hepatic microsomes, and in human CYP2D6 expressed in

HepG2 cells (r = 0.93). (-)-Cocaine was found to have particularly

high inhibitory potency (Ki = 74 nanomolars (nM) for canine striatal

CYP2D), and a high degree of overlap was found between compounds

binding to the dopamine transporter and striatal CYP2D. The

distribution of CYP2D in dissected regions of dog brain demonstrated

a fortyfold range in activity, with the highest level being found in

supraorbital cortex and parietal cortex, and the lowest in the

cerebellum.
11

An uneven distribution of CYP2D activity was also observed in

preliminary studies of dissected monkey (C. Aethiops) brain (Tyndale

et al., unpublished data). Subcellular preparations of the nucleus

accumbens, amygdala, and parietal cortex oxidized sparteine at rates

of 300 to 600 picomoles per milligram of protein per hour (pmol/mg

protein/hour). (By contrast, the rate in monkey hepatic microsomes

was 62,000 pmol/mg protein/hour). The rate in striatum,

hippocampus, and temporal and olfactory cortex was approximately

75 pmol/mg protein/hour. The spinal cord, frontal cortex, midbrain,

medulla, and cerebellum had negligible activity. CYP2D activity in

monkey brain displayed the stereoselective inhibition by quinidine and

quinine that is characteristic of monkey liver CYP2D and human liver

CYP2D6. Regional variation in the distribution of CYP2D messenger

ribonucleic acid (mRNA) in rat and human brain has also been

observed (Tyndale et al., unpublished observations). There is

molecular evidence for CYP2D6 mRNA in human caudate (Tyndale

et al. 1991b). The polymerase chain reaction was used to amplify a

complementary deoxyribonucleic acid (cDNA) fragment (513 base

pains) from a human caudate lambda-gt11 library. One hundred

percent nucleotide identity to the CYP2D6 mRNA was found.

Relevance of CYP2D6 to Drug Abuse

A number of drugs of abuse are known substrates (e.g., codeine,

hydrocodone, p-methoxyamphetamine, amphetamine) or inhibitors

(e.g., (-)-cocaine, pentazocine) of CYP2D6. For some of these drugs,

the pharmacokinetic differences due to the polymorphism will be so

profound that they are likely to exceed pharmacodynamic sources of

variation in response. For other drugs (e.g., hydrocodone to

hydromorphone, codeine to morphine, oxycodone to oxymorphone),

CYP2D6 may not contribute importantly to the overall clearance of

the drug, but may catalyze the formation of highly active metabolites.

The consequences of absent or inhibited CYP2D6 for any particular

drug depend on the relative activity of the parent drug and its various

metabolites. In some cases, the pharmacology of the metabolite is

qualitatively similar to the parent drug (e.g., hydrocodone to

hydromorphone, codeine to morphine); in other cases, the

pharmacology is different (e.g., dextro-methorphan to dextrorphan);

more usually, it is not properly understood (e.g., PMA to 4-

hydroxyamphetamine). In addition, CYP2D6 occurs within the

central nervous system (CNS) (Fonne-Pfister et al. 1987; Tyndale et

al. 1991b). Its role in the brain is unknown, but potential formation

of active drug metabolites at their site of action makes the presence
12

of CYP2D6 here of great functional significance. A computer

simulation study of interregional differences in CNS drug metabolism

has provided a model that could account, in part, for large intersubject

variability in the pharmacodynamic effects of psychoactive drugs

(Britto and Wedlund 1992).

The authors hypothesize the following:

• The genetically determined activity of CYP2D6

that results in EMs (90 percent) and PMs (10 percent) of some

drugs of abuse is both an important risk factor and protective

factor in drug abuse and toxicity from drugs of abuse.

• Certain inhibitors of CYP2D6 or the PM state

itself result in unexpected toxicity from drugs of abuse (e.g.,

PMA) or may have utility in the treatment of drug dependence

(e.g., preventing activation of a pro-drug such as codeine or

oxycodone).

• CYP2D6 may play an important neuroregulatory

role in the brain, and may modulate brain functions important

in drug-reinforced behavior or neurotoxicity.

CYP2D6-deficient individuals (PMs) should have a much

decreased probability of abusing a drug converted to an active

metabolite capable of maintaining drug-taking behavior (e.g., codeine,

oxycodone, hydrocodone, dextromethorphan). In EMs, the

probability is increased and is propor-tionate to the individual’s

genotype (homozygous versus heterozygous) and absolute CYP2D6

activity. Conversely, PMs should experience greater risk of abuse and

of toxicity to a drug that is inactivated by CYP2D6 (e.g., PMA,

methamphetamine); EMs should have a lesser risk.

Amphetamines and Phenethylamines. Deficiency in the phydroxylation of amphetamine was one of the observations that led

to the discovery of the CYP2D6 polymorphism (Dring et al. 1970;

Smith 1986). A single oral administration of the radiolabelled

enantiomers of amphetamine to three volunteers with subsequent

analysis of urine indicated that about 5 percent of (+)-amphetamine

was converted to p-hydroxyamphetamine in two subjects but to a

much less extent in the third subject, who was later found to have

CYP2D6 deficiency (Smith 1986). The main excretion product was

unchanged amphetamine (although the extent of excretion is known

to be pH dependent), and the major metabolites were products of side
13

chain deamination (i.e., benzoic and hippuric acids). Total recovery

of the radiolabelled dose in urine after 4 days was around 90 percent

(Dring et al. 1970).

The hallucinogen PMA is O-demethylated by CYP2D6 to form 4-

hydroxyamphetamine (Kitchen et al. 1979). A PM was observed to

excrete 4.4 percent of a 5 milligram (mg) oral dose of PMA as 4-

hydroxyamphetamine, compared with 50 to 65 percent excretion of

this metabolite (free and conjugated) in EMs. Shortly after it made its

appearance in Ontario in the early 1970s, PMA was associated with a

number of deaths (Sellers et al. 1979). A cardinal feature of these

deaths was cardiovascular excitation and hyperthermia; it had been

noted earlier to produce marked and sustained elevation in blood

pressure in some people.

Urinary excretion data of human subjects indicate that 4-

hydroxylation of methamphetamine is much more extensive than

that of amphetamine; the metabolic ratio (total

hydroxymethamphetamine/methamphetamine) in urine averaged

about 15 with individual variations of approximately fiftyfold

(Shimosato 1988), suggesting considerable importance of CYP2D6

polymorphism in the fate of methamphetamine. On the other hand,

there seems to be no information on the further metabolism of phydroxymethamphetamine as is available for phydroxyamphetamine.

p-Hydroxyamphetamine has been used as a drug (paredrine) in

ophthalmology. Like amphetamine, it is known to release

norepinephrine from postgan-glionic sympathetic nerve endings;

unlike amphetamine, its use is associated with few if any CNS effects,

presumably because its relative water solubility slows its passage

through the blood-brain barrier (Burde and Thompson 1991). As with

amphetamine, the releasing action of p-hydroxyamphetamine is

thought to be a consequence of its inhibition of the reuptake

mechanism and of monoamine oxidase activity, leading to a

postsynaptic accumulation of not only dopamine but also of 5-

hydroxy-tryptamine (Arai et al. 1990; Cho et al. 1975).

p-Hydroxyamphetamine is metabolized to pharmacologically

active secondary products. Its biotransformation by dopamine-betahydroxylase leads to p-hydroxynorephedrine, a false neurotransmitter

(Coutts and Baker 1989; Dougan et al. 1986; Smith 1986) with

similar biochemical activities as p-hydroxyamphetamine (Arai et al.

1990). p-Hydroxy amphetamine is also metabolized by a neuronal
14

P450 to alpha-methyl-dopamine and further to alpha-methylnoradrenaline, a CNS-active hypotensive agent that also may

function as a false transmitter (Hoffman et al. 1979). Dougan and

colleagues (1986) described the stereoselective neuronal accumulation

of hydroxyamphetamine and hydroxynorephedrine in rat striatum

and found them to have half-lives of 1.5 and 2.5 days, respectively, at

this location.

d-Amphetamine hydroxylation has been observed in whole brain

and in striatal preparations in both microsomal and mitochondrial

membranes (Liccione and Maines 1989); manganese pretreatment

increased amphetamine hydroxylation in both membranes. After

administration of amphetamine, p-hydroxyamphetamine (also called

alpha-methyl-p-tyramine), p-hydroxynorephedrine (also called alphamethyl-p-octopamine), alpha-methyl-dopamine, and alpha-methylnoradrenaline have been found in brain (studies mostly done in rat

brain). Formation, or lack of formation, of these metabolites may be

catalyzed by CYP2D, with the p-hydroxylation of amphetamine as

the first step.

Suzuki and colleagues (1986, 1987) have tested the effects of

altering methamphetamine p-hydroxylation in rats; it appeared that

inhibition enhanced the methamphetamine-induced stereotyped

behavior. One might conjecture that an equivalent effect could occur

in humans with inborn CYP2D6 deficiency if a toxic dose of

methamphetamine were applied. Unfortunately, studies of animals do

not necessarily help; p-hydroxyamphetamine is the main metabolite

of amphetamine in rats but not in human liver (see above), and most

studies on brain metabolism have not been conducted in human tissue.

Matsuda and colleagues (1989) have shown that centrally acting

amphet-amine metabolites may have different effects on different

parts of the brain when applied locally or systemically. Thus the

locus of formation of a metabolite within the brain could be critical,

and metabolite formation in the liver might be of limited relevance.

Dextromethorphan. Dextromethorphan has the opposite steric

configuration to codeine and morphine, is devoid of analgesic effects,

but is as potent an antitussive as codeine. It is the most commonly

used antitussive worldwide. Systematic studies concerning its abuse

liability are few. In the earliest report (Isbell and Fraser 1953), no

abuse liability was found with oral doses up to 100 mg. However,

there are many case reports of dextromethorphan abuse and endemic

use principally among young people (McElwee and Veltri 1990).
15

Monkeys and rats can be trained (with difficulty) to self-administer

dextromethorphan, but do so more easily for its active metabolite

dextrorphan. They also recognize dextrophan in particular as a

discriminative stimuli, and generalize from and to phencyclidine

(PCP) (Holtzman 1980). Interest in dextrorphan continues because

of its similarity to PCP in mechanism of action and behavioral

pharmacology, the potential role of N-methyl-D-aspartate (NMDA)

antagonists in neuroprotection, and the evolving CNS pharmacology

of dextromethorphan and dextrorphan (Musacchio et al. 1988;

Tortella et al. 1989). The elucidation of the pharmacogenetic

control of dextromethorphan kinetics, metabolism, and effects as a

representative of the class could be important (Schadel et al. 1995).

In human volunteers the kinetics of dextromethorphan in EM and

PM subjects are very different (Schadel et al. 1995). Hence, in a

clinical practice the pharmacologic effects and consequences should be

very different in the EM and PM phenotypes.

Prescription Opiates. Historically, methadone has evolved as the

only widely accepted and used pharmacologial adjunct for the

treatment of opiate abuse. Because methadone has strong reinforcing

properties and produces physical dependence, it has generally been

reserved for the management of heroin abuse and dependence. From

a public health perspective, the misuse and abuse of opiate-containing

prescription medications is a poorly understood and largely

unaddressed treatment issue. Prescription analgesics including opioids

(e.g., codeine, oxycodone, and hydrocodone) alone and in

combination are extensively used worldwide and are always within the

major classes of drugs prescribed in all countries. While most users

have a legitimate need for these medications, there is also substantial

evidence of misuse, abuse, and dependence since the frequency of use

appears to exceed the frequency of acute and chronic pain in the

population.

Canada has the dubious distinction of having the world's largest

per capita consumption of precursor opiate-containing compounds

(e.g., codeine, oxycodone, and hydrocodone) (Korcok 1979). A drug

utilization review of opiate use in Canada from 1978 to 1989 shows

continued increase in the use of prescribed codeine combination

products. The defined daily dose (DDD)/1000 inhabitants/day for

prescribed codeine-acetaminophen products has increased from 3.3

DDD/1000 inhabitants/day in 1982 to 8.1 DDD/1000 inhabitants/day

in 1989. Oxycodone-acetaminophen containing products have also

been increasing from 0.04 DDD/1000 inhabitants/day in 1978 to 0.21
16

DDD/1000 inhabitants/day in 1989 (Seto, unpublished data).

Intercontinental Medical Statistics Canada reports that

acetaminophen with codeine was the most frequently dispensed

prescription product in Canada from 1989 to 1992 and accounted for

56 percent of all new prescriptions in 1992 (Theirriault 1992). The

situation is similar in the United States, with codeine being the second

most frequently dispensed chemical entity according to a 1987 drug

utilization review (Tomita et al. 1988). Acetaminophen with codeine

ranked as the fifth and fourth leading prescription product dispensed

in 1985 and 1986 respectively (Baum et al. 1987). In 1992 and

1993, as determined by chart review and index drug report by

patients, 423 patients have presented to the Clinical Research and

Treatment Institute of the Addiction Research Foundation with

codeine (60 percent), oxycodone (35 percent), or hydrocodone (5

percent) as the primary problem substance (authors’ unpublished

data). This data likely underestimate the extent of the problem

because reseachers have only recently been systematically focusing on

these drugs. The clinical impression is that at least half of all

polydrug users have lifetime prescription opiate drug abuse or

dependence.

In Vitro Oxidation of Drugs of Abuse by CYP2D6

Identification of Substrates/Inhibitors of Human Hepatic

CYP2D6.

The authors have been conducting studies to identify drugs of

abuse that interact with CYP2D6 enzyme in human liver microsomes.

The results provide drug candidates for further studies aimed at

determining whether they are CYP2D6 substrates (e.g., PMA, 3, 4-

methylenedioxyamphetamine (MDA), 3,4-

methylenedioxymethamphetamine (MDMA)) or inhibitors of

CYP2D6 (e.g., fluoxetine, (-)-cocaine). In table 3, competitive

inhibitors of CYP2D6-catalyzed formation dextrorphan from

dextrometh-orphan are identified with their Ki value. The lower the

Ki value, the more potent the inhibitor (higher affinity for CYP2D6).

These in vitro results identify compounds that interact with

hepatic CYP2D6, and compounds from this list become the

candidates for the authors’ investigations of the biological importance

of this enzyme activity.
17

TABLE 3. A selected list of drugs tested for an interaction

with human hepatic CYP2D6.

Ki (M)

Phenethylamines

(+)-Amphetamine1 28

(-)-Amphetamine no interaction

(+)-Methamphetamine1 25

4-Methoxyamphetamine (PMA)1 18

3,4-Methylenedioxyamphetamine (MDA)1 3

Opioids

Hydrocodone1 55

Oxycodone1 55

Morphine 150

Nalorphine 35

Methadone 3

Stimulants

(-)-Cocaine20.74

Miscellaneous

Nicotine no interaction

Ketamine no interaction

Caffeine no interaction

KEY: 1 = identified by further studies in vitro as a

CYP2D6 substrate; 2 = identified by further studies in vitro

not to be a CYP2D6 substrate.

Findings that have stemmed from this screening include the

following:

1. The establishment that CYP2D6 converts hydrocodone to its

active metabolite hydromorphone in vivo. This drug was

subsequently selected as the prototypical pro-drug opiate used to

study the role of CYP2D6 activity variants in opiate plasma

kinetics and in responses related to abuse (Otton et al. 1993a).

2. A possible clinical consequence of the potent (Ki = 0.18

micromolar (M)) inhibition of CYP2D6 by fluoxetine and its

major metabolite norfluoxetine. The authors described a case

report of a profound increase in the dose of oxycodone required

for analgesia after the initiation of fluoxetine therapy. This led
18

to the observation that oxycodone is converted to its active

metabolite oxymorphone via CYP2D6, and that therapeutic doses

of fluoxetine inhibit CYP2D6 activity in vivo (Otton et al.

1993b). The kinetic profile of fluoxetine and its normetabolite

favor its use as a long-acting CYP2D6 inhibitor; hence the drug

might be used to inhibit the activation of codeine, hydrocodone,

or oxycodone (also called producing a phenocopy because the

urine metabolites look the same as in PMs) in abusers and thereby

reduce the reinforcing properties of the medication.

3. In vitro inhibition of CYP2D6 activity by methadone (Ki = 3

M) predicted drug interactions in vivo. This finding was

confirmed in 42 abusers of oral opiates undergoing treatment with

methadone (Wu et al. 1993a).

4. The O-demethylation of the hallucinogen PMA to 4-

hydroxyamphetamine is catalyzed in vitro by CYP2D6 (Wu et al.

1994). This confirms earlier data from three subjects (one a PM)

that this reaction was catalyzed by CYP2D6 (Kitchen et al.

1979). Methamphetamine's major metabolite, 4-

hydroxyamphetamine, is formed via CYP2D6 (as determined by

incubations of methamphetamine with microsomes prepared from

the yeast transformed with an expression plasmid containing fulllength human CYP2D6 cDNA (a gift of Dr. S.W. Ellis and Dr.

M.S. Lennard, Sheffield, U.K.).

5. Because of (-)-cocaine's extremely high affinity for hepatic

CYP2D6 (Ki = 0.07 M), this drug is not metabolized by

CYP2D6. The authors incubated (-)-cocaine with cloned human

CYP2D6 enzyme expressed in yeast. Using a gas

chromatography/mass spectrometry assay, no detectable

ecgonine, ecgonine methyl ester, ecgonidine, ecgonidine methyl

ester, norecgonidine methyl ester, norecgonine methyl ester,

benzoylecgonine, o-m-p-hydroxycocaine, or norcocaine was

formed during the incubations (Otton et al., unpublished

observations).

In Vitro Comparison of Monkey Hepatic CYP2D6-Like Activity

With Human CYP2D6. The monkey would be a valuable model for

assessing the role of CYP2D6 enzyme activity in drug reinforcement

and response. Catalytic, immunologic, and electrophoretic

investigations indicated that the enzymes were indistinguishable

(Otton et al. 1992). However, the conclusion after more extensive

examination of the inhibitor specificity of the two enzymes was that
19

they were functionally homologous, but not identical (Wu et al.

1993b). These studies also predicted that in monkeys an established

CYP2D inhibitor, budipine, will more readily produce PM

phenocopies than quinidine.

Dextromethorphan Disposition in Rats and Monkeys

Kinetics of Dextromethorphan and Metabolites in Rat Plasma

and Brain. Because of dextrorphan's higher affinity for NMDA/PCP

sites than dextromethorphan, the abuse characteristics of

dextromethorphan are likely due to its CYP2D1-mediated conversion

to dextrorphan. Time course studies of this and other metabolites

after an intraperitoneal (IP) dose of dextromethorphan indicate that

conjugated dextrorphan is the predominant metabolite in plasma, and

that only free dextrorphan is present in the brain (Wu et al. 1995).

Brain dextrorphan levels were correlated with free dextrorphan in

plasma (r = 0.84), but not with conjugated plasma metabolite. Similar

studies of the disposition of dextrometh-orphan and metabolites

following different routes of administration suggest that each will be

associated with a different pharmacology (Wu et al. 1995).

FUTURE RESEARCH GOALS AND DIRECTION

Areas which would be important for future research include the

following:

1. Identify and characterize more drugs of abuse, particularly

phenethylamines and related compounds, that are substrates

or inhibitors of the genetically polymorphic human drug

metabolizing enzyme cytochrome P-450 2D6 (CYP2D6).

2. Define the localization, catalytic specificity, and regulation of

CYP2D forms in rat, monkey, and human brain.

3. Using animal models, determine the importance of CYP2D

deficiency or high catalytic activity to the toxicity and

behavioral consequences of amphetamines, as models of

active drugs of abuse with metabolites of different

pharmacologic activity.

4. Determine and compare the clinical consequences of CYP2D6

genotype to the metabolism, kinetics, pharmacologic effects,

and abuse liability of methamphetamine, d-amphetamine, and

dextromethorphan.
20

CONCLUSIONS

The authors expect these studies will identify some drugs of abuse

for which the EM state, the PM state, or CYP2D6 inhibitors can

modify the risk of abuse; explain why some drugs of abuse are so

attractive or so toxic to some individuals; explain why some patterns

of drug abuse are endemic and rarely epidemic (e.g., PMA,

dextromethorphan, smoked methamphetamine, phenethylamine

designer drugs); identify the location of CYP2D in the brain and

determine its role and importance for variation in drug response to

drugs of abuse; justify the incorporation of CYP2D6 screening as part

of regulatory or scheduling requirements; and establish animal models

for the study of cytochrome P-450s as risk and protective factors in

drug abuse.

This work can also result in needed new therapeutic strategies for

the treatment of drug abuse. Such issues are particularly important for

management of dependence on prescription opiates, which is the third

largest drug dependence problem in North America (after nicotine and

alcohol) and the least studied and understood.

With respect to treatment, depending on the particular

biotransformation pattern and the activity of the metabolites, several

therapeutic approaches are evident. For example, EMs (93 percent

of the caucasian population) could receive an inhibitor as part of a

therapeutic regimen. Such inhibition will alter the kinetics, toxicity,

drug-reinforcing properties, and physical dependence liability of some

drugs of abuse (e.g., hydrocodone, oxycodone, codeine) and make the

drug less pharmacologically attractive.

From a broader scientific perspective, the authors’ studies of the

drug- metabolizing activity of the brain will contribute to

psychopharmacological approaches to the treatment of mental

disorders and to researchers’ understanding of brain function

involving endogenous neurotransmitters, exogenous drugs,

neurosteroids, and neuroactive steroids.
21

REFERENCES

Al-Dabbagh, S.G.; Idle, J.R.; and Smith, R.L. Animal modeling of

human polymorphic drug oxidation: The metabolism of

debrisoquine and phenacetin in rat inbred strains. J Pharm

Pharmacol 33:161-164, 1981.

Arai, Y.; Kim, S.Y.; Kinemuchi, H.; Tadano, T.; Satoh, S.; and Satoh,

N. Inhibition of brain type A monoamine oxidase and 5-

hydroxytryptamine uptake by two amphetamine metabolites, phydroxyamphetamine and p-hydroxynorephedrine. J Neurochem

55:403-408, 1990.

Baum, C.; Kennedy, D.L.; Knapp, D.E.; Faich, G.A.; and Anello, C.

"Drug Utilization in the U.S.-1986." Eighth annual review.

Rockville, MD: Food and Drug Administration, 1987.

Britto, M., and Wedlund, P.J. Cytochrome P-450 in the brain.

Potential evolutionary and therapeutic relevance of localization of

drug-metabolizing enzymes. Drug Metab Dispos 20(3):446-450,

1992.

Broly, F., and Meyer, U.A. Debrisoquine oxidation polymorphism:

Phenotypic consequences of a 3-base-pair deletion in exon 5 of the

CYP2D6 gene. Pharmacogenetics 3(3):123-130, 1993.

Broly, F.; Gaidigk, A.; Heim, M.; Eichelbaum, M.; Marike, K.; and

Meyer, U.A. Debrisoquine/sparteine hydroxylation genotype and

phenotype: Analysis of common mutations and alleles of CYP2D6

in a European population. DNA Cell Biol 10:545-558, 1991.

Burde, R.M., and Thompson, H.S. Hydroxyamphetamine. A good drug

lost? Am J Ophthalmol 111:100-102, 1991.

Chen, Z.R.; Somogyi, A.A.; and Bochner, F. Polymorphic Odemethylation of codeine. Lancet 2:914-915, 1988.

Cho, A.K.; Schaeffer, J.C.; and Fischer, J.F. Accumulation of 4-

hydroxyamphetamine by rat striatal homogenates. Biochem

Pharmacol 24:1540-1542, 1975.

Coutts, R.T., and Baker, G.B. Metabolic implications of chiral centres

in psychotropic drugs. Prog Neuropsychopharmacol Biol Psychiatry

13:405-417, 1989.

Dougan, D.F.; Duffield, A.M.; Duffield, P.H.; and Wade, D.N. Stereoselective accumulation of hydroxylated metabolites of

amphetamine in rat striatum and hypothalamus. Br J Pharmacol

88:285-290, 1986.

Dring, L.G.; Smith, R.L.; and Williams, R.T. The metabolic fate of

amphetamine in man and other species. Biochem J 116:425-435,

1970.
22

Eichelbaum, M.; Mineshita, S.; Ohnhaus, E.E.; and Zekorn, C. The

influence of enzyme induction on polymorphic sparteine oxidation.

Br J Clin Pharmacol 22:49-53, 1986.

Fonne-Pfister, R.; Bargetzi, M.J.; and Meyer, U.A. MPTP, the

neurotoxin inducing Parkinson’s disease, is a potent competitive

inhibitor of human and rat cytochrome P450 isozymes (P450bufI,

P450db1) catalyzing debrisoquine 4-hydroxylation. Biochem

Biophys Res Commun 148:1144-150, 1987.

Gaedigk, A.; Blum, M.; Gaedigk, R.; Eichelbaum, M.; and Meyer, U.A.

Deletion of the entire cytochrome P-450 CYP2D6 gene as a cause

of impaired drug metabolism in poor metabolizers of the

debrisoquine/ sparteine polymorphism. Am J Human Gen 48:943-

950, 1991.

Hanioka, N.; Kimura, S.; Meyer, U.; and Gonzalez, F.J. The human

CYP2D6 locus associated with a common genetic defect in drug

oxidation: A G1934 to A base change in intron 3 of a mutant

CYP2D6 allele results in an aberrant 3' splice recognition site. Am J

Human Gen 47:994-1001, 1990.

Hoffman, A.R.; Rama-Sastry, B.V.; and Axelrod, J. Formation of

alpha- methyldopamine ("catecholamphetamine") from phydroxyamphetamine by rat brain microsomes. Pharmacology

19:256-260, 1979.

Holtzman, S.G. Phencyclidine-like discriminative effects of opioids in

the rat. J Pharmacol Exp Ther 214:614-619, 1980.

Isbell, H., and Fraser, H.F. Actions and addiction liabilities of

Dromoran derivaties in man. J Pharmacol Exp Ther 107:524-530,

1953.

Johansson, I.; Lundqvist, E.; Bertilsson, L.; Dahl, M.L.; Sjoqvist, F.;

and Ingelman-Sundberg, M. Inherited amplification of an active

gene in the cytochrome P450 CYP2D locus as a cause of ultrarapid

metabolism of debrisoquine. Proc Natl Acad Sci U S A

90(24):11825-11829, 1993.

Johansson, I.; Oscarson, M.; Yue, Q.Y.; Bertilsson, L.; Sjoqvist, F.; and

Ingelman-Sundberg, M. Genetic analysis of the Chinese cytochrome

P4502D locus: Characterization of variant CYP2D6 genes present

in subjects with diminished capacity for debrisoquine hydroxylation.

Mol Pharmacol 46(3):452-459, 1994.

Kagimoto, M.; Heim, M.; Kagimoto, K.; Zeugin, T.; and Meyer, U.A.

Multiple mutations of the human cytochrome P-450IID6 gene in

poor metabolizers of debrisoquine: Study of the functional

significance of individual mutations by expression of chimeric

genes. J Biol Chem 265:7201-7214, 1990.

Kalow, W. Interethnic variation of drug metabolism. Trends Pharm

Sci 12:102-107, 1991.
23

Kimura, S.; Umeno, M.; Skoda, R.C.; Meyer, U.A.; and Gonzalez, F.J.

The human debrisoquine 4-hydroxylase (CYP2D) locus. Sequence

and identification of the polymorphic CYP2D6 gene, a related

gene, and a pseudogene. Am J Human Gen 45:889-904, 1989.

Kitchen, I.; Tremblay, J.; Andres, J.; Dring, L.G.; Idle, J.R.; Smith,

R.L.; and Williams, R.T. Interindividual and interspecies variation

in the metabolism of the hallucinogen 4-methoxyamphetamine.

Xenobiotica 9:397-404, 1979.

Kobayashi, S.; Murray, S.; Watson, D.; Sesardic, S.; Davies, D.S.; and

Boobis, A.R. The specificity of inhibition of debrisoquine 4-

hydroxy-lase activity by quindine and quinine in the rat is inverse of

that in man. Biochem Pharmacol 38:2795-2799, 1989.

Korcok, M. Canadian government study shows big rise in Canadian use

of several narcotics. Can Med Assoc J 120:1270-1271, 1979.

Liccione, J.J., and Maines, M.D. Manganese-mediated increase in the

rat brain mitochondrial cytochrome-P-450 and drug metabolism

activity. Susceptibility of the striatum. J Pharmacol Exp Ther

248:222-228, 1989.

Mahgoub, A.; Idle, J.R.; Dring, L.G.; Lancaster, R.; and Smith, R.L.

Polymorphic hydroxylation of debrisoquine in man. Lancet 2:584-

586, 1977.

Matsuda, L.A.; Hanson, G.R.; and Gibb, J.W. Neurochemical effects of

amphetamine metabolites on central dopaminergic and serotonergic

systems. J Pharmcol Exp Ther 251:901-908, 1989.

Matsunaga, E.; Zanger, U.M.; Hardwick, J.P.; Gelboin, H.V.; Meyer,

U.A.; and Gonzalez, F.J. The CYP2D gene subfamily: Analysis of

the molecular basis of the debrisoquine 4-hydroxylase deficiency in

DA rats. Biochemistry 28:7349-7355, 1989.

McElwee, N.E., and Veltri, J.C. International abuse of

dextromethorphan (DM) products: 1985 to 1988 statewide data. Vet

Human Toxicol 32:355, 1990.

Meyer, U.A. "Molecular Genetics of Human Xenobiotic Metabolizing

Cytochromes P450." Proceedings of the 10th International

Symposium on Microsomes and Drug Oxidation, Toronto, Canada,

July 18-21, 1994.

Musacchio, J.M.; Klein, M.; and Santiago, L.J. High affinity dextromethorphan binding sites in guinea pig brain: Further

characterization and allosteric interactions. J Pharmacol Exp Ther

247:424-431, 1988.

Nelson, D.R.; Kamataki, T.; Waxman, D.J.; Guengerich, F.P.; and

Estabrook, R.W. The P450 superfamily: Update on new sequences,

gene mapping, accession numbers, early trivial names of enzymes,

and nomenclature. DNA Cell Biol 12(1):1-51, 1993.
24

Niznik, H.B.; Tyndale, R.F.; Sallee, F.R.; Gonzalez, F.J.; Hardwick,

J.P.; Inaba, T.; and Kalow, W. The dopamine transporter and

cytochrome P450IID1 (debrisoquine 4-hydroxylase) in brain:

Resolution and identification of two distinct [3H]GRB-12935

binding proteins. Arch Biochem Biophys 276:424-432, 1990.

Otton, S.V.; Inaba, T.; and Kalow, W. Competitive inhibition of

sparteine oxidation in human liver by beta-adrenoceptor antagonists

and other cardiovascular drugs. Life Sci 34:73-80, 1984.

Otton, S.V.; Tyndale, R.F.; Wu, D.; Inaba, T.; Kalow, W.; and Sellers,

E.M. Catalytic and immunologic similarities between monkey and

human liver cytochrome P-450db1 (human cytochrome P450

2D6). Drug Metab Dispos 20(1):1-5, 1992.

Otton, S.V.; Schadel, M.; Cheung, S.W.; Kaplan, H.L.; Busto, U.E.; and

Sellers, E.M. CYP2D6 phenotype determines the metabolic

conversion of hydrocodone to hydromorphone. Clin Pharmacol

Ther 54(5):463-472, 1993a.

Otton, S.V.; Wu, D.; Joffe, R.T.; Cheung, S.W.; and Sellers, E.M.

Inhibition by fluoxetine of cytochrome P450 2D6 activity. Clin

Pharmacol Ther 53(4):401-409, 1993b.

Schadel, M.; Wu, D.; Otton, S.V.; Kalow, W.; and Sellers, E.M.

Pharmacokinetics of dextromethorphan and metabolites in humans:

Influence of the CYP2D6 phenotype and quinidine inhibition. J

Clin Psychopharmacology 15(5):263-269, 1995.

Schmid, B.; Bircher, J.; Preisig, R.; and Kupfer, A. Polymorphic

dextromethorphan metabolism: Co-segregation of oxidative Odemethylation with debrisoquine hydroxylation. Clin Pharmacol

Ther 38:618-624, 1985.

Sellers, E.M.; Martin, P.R.; Roy, M.L.; and Sellers, E.A.

Amphetamines. In: Lomax, P., and Schonbaum, E., eds. Modern

Pharmacology- Toxicology, Volume 16 - Body Temperature:

Regulation, Drug Effects and Therapeutic Implications. New York:

Marcel Dekker, Inc., 1979. pp. 461-498.

Shimosato, K. Urinary excretion of p-hydroxylated methamphetamine

metabolites in man. II. Effect of alcohol intake on

methamphetamine metabolism. Pharmacol Biochem Behav

29:733-740, 1988.

Smith, R.L. Introduction: Human genetic variations in oxidative drug

metabolism. Xenobiotica 16:361-365, 1986.

Suzuki, T.; Chiang, H.-J.F.; Yanaura, S.; Yoshida, T.; and Kuroiwa, Y.

Studies on the mechanism of interaction between

methamphetamine and quinine in rats. J Pharmacobio-Dyn 9: 234-

238, 1986.
25

Suzuki, T.; Chiang, H.-J.F.; Misawa, M.; and Yanaura, S. Effects of

quinidine and cimetidine on methampethamine stereotypy in rats. J

Pharmacobio-Dyn 10:152-155, 1987.

Theirriault, I. The Pharmacy Practice 200: An exclusive IMS report

on the top 200 drugs of 1992. Pharm Practice 8(10):27-34, 1992.

Tomita, D.K.; Baum, C.; Kennedy, D.L.; Knapp, D.E.; and Perry, Z.A.

"Drug Utilization in the U.S.-1987." Ninth annual review.

Rockville, MD: Food and Drug Administration, 1988.

Tortella, F.C.; Pellicano, M.; and Bowery, N.G. Dextromethorphan

and neuromodulation: Old drug coughs up new activities. Trends

Pharmacol Sci 10:501-507, 1989.

Tyndale, R.F.; Aoyama, T.; Broly, F.; Matsunaga, T.; Inaba, T.;

Kalow, W.; Gelboin, H.V.; Meyer, U.A.; Gonzalez, F.J.

Identification of a new variant CYP2D6 allele lacking the codon

encoding Lys-281: Possible association with the poor metabolizer

phenotype. Pharmacogenetics 1:26-32, 1991a.

Tyndale, R.F.; Sunahara, R.; Inaba, T.; Kalow, W.; Gonzalez, F.J.; and

Niznik, H.B. Neuronal cytochrome P450IID1

(debrisoquine/sparteine type): Potent inhibition of activity by (-)

cocaine and nucleotide sequence identity to human hepatic P450

gene CYP2D6. Mol Pharmacol 40:63-68, 1991b.

Vincent-Viry, M.; Muller, J.; Fournier, B.; Galteau, M.M.; and Siest, G.

Relation between debrisoquine oxidation phenotype and

morphological, biological, and pathological variables in a large

population. Clin Chem 37(3):327-332, 1991.

Wu, D.; Otton, S.V.; Sproule, B.A.; Busto, U.; Inaba, T.; Kalow, W.;

and Sellers, E.M. Inhibition of human cytochrome P450 2D6

(CYP2D6) by methadone. Br J Clin Pharmacol 35:30-34, 1993a.

Wu, D.; Otton, S.V.; Morrow, P.M.; Inaba, T.; Kalow, W.; and Sellers,

E.M. Human hepatic cytochrome P450 2D6-like activity in nonhuman primates: Catalytic characterization in vitro. J Pharmacol

Exp Ther 266(2):715-719, 1993b.

Wu, D.; Otton, S.V.; Ellis, S.W.; Lennard, M.S.; Tucker, G.T.; Inaba,

T.; Kalow, W.; and Sellers, E.M. Identification of amphetamine

analogs as substrates and inhibitors of human cytochrome P450

2D6 (CYP2D6) in vitro. Abstracts of College on Problems of Drug

Dependence, Inc. 56th Annual Scientific Meeting (Palm Beach,

Florida), 1994.

Wu, D.; Otton, S.V.; Kalow, W.; and Sellers, E.M. Effects of route of

administration on dextromethorphan pharmacokinetics and

behavioral response in the rat. J Pharmacol Exp Ther 274(3):1431-

1437, 1995.
26

Zysset, T.; Zeugin, T.; and Kupfer, A. In vivo and in vitro

dextromethorphan metabolims in SD an DA rats. An animal model

of the debrisoquine-type polymorphic oxidation in man. Biochem

Pharmacol 37:3155-3160, 1988.

ACKNOWLEDGMENTS

This chapter and the research described were supported by NIDA

grant R01-DA06889, the University of Toronto, and the intramural

grants program of the Addiction Research Foundation of Ontario.

AUTHORS

Edward M. Sellers, M.D., Ph.D., F.R.C.P.C.

Professor

Pharmacology, Medicine and Psychiatry

S. Victoria Otton, Ph.D.

Professor

Rachel F. Tyndale, Ph.D.

Professor

Department of Pharmacology

University of Toronto

33 Russell Street

Toronto, Ontario, Canada M5S 2S1
27

Human Liver Cocaine

Carboxylesterases

William F. Bosron, Robert A. Dean, Monica R. Brzezinski, and

Evgenia V. Pindel

The stimulant effects of cocaine are relatively short lived, as

evidenced by the increase in heart rate that peaks at about 60 minutes

after cocaine administration and declines thereafter (Farre et al.

1993). This duration of effect is largely due to the rapid hydrolysis of

cocaine to two major deesterified metabolites that appear in serum

and urine, benzoylecgonine and ecgonine methyl ester (figure 1).

Neither of these deesterified metabolites is active as a stimulant when

administered peripherally, even at high doses (Spealman et al. 1989;

Misra et al. 1975). The rapid distribution half-life of cocaine from an

intravenous (IV) dose is about 10 minutes and the elimination half-life

of cocaine is 50 to 80 minutes (Jeffcoat et al. 1989). Hence, the

duration of the stimulant action of cocaine is limited by its rate of

hydrolysis to inactive metabolites.

The time course for toxic effects of cocaine also appears to be

dependent on drug metabolism. For example, almost one-third of the

deaths related to cocaine overdose occur about 2 to 5 hours after drug

administration (Wang and Carpentier 1994), a time period equal to

about two half-lives of cocaine in serum. Sudden cardiac death is one

of the acute toxic effects of cocaine overdose, but the mechanism is

not fully understood. Direct effects of cocaine on the

electrophysiological properties of isolated heart muscle preparations

have been shown in vitro (Wang and Carpentier 1994). Cocaine and

some of its metabolites have also been shown to be potent

vasoconstrictors. Hence, decreased blood flow and hypoxia of

cerebral arterioles may be important contributors to cerebral

infarction, hemorrhage, and developmental abnormalities, especially

in newborn infants exposed to cocaine in utero (Kutrh et al. 1993;

Covert et al. 1994). Cocaine-induced hepatotoxicity has been

reported in both humans and rodents and the extent of hepatic

necrosis appears to be dependent, in part, on the content of

microsomal cocaine-metabolizing enzymes (Roth et al. 1992).

Hence, the duration and magnitude of the desired stimulant effects, as

well as the undesired toxic effects of cocaine use, appear to depend on

the content and activity of enzymes responsible for cocaine

metabolism.
28

The hydrolysis of the benzoyl ester group of cocaine to ecgonine

methyl ester and benzoate (figure 1) is catalyzed by serum

cholinesterase (Stewart et al. 1977), also called pseudocholinesterase

or butyrylcholines-terase, and a human liver carboxylesterase (Dean

et al. 1991). The hydro-lysis of the methyl ester group of cocaine to

benzoylecgonine and methanol (figure 1) occurs spontaneously in

aqueous solutions at neutral to alkaline pH. However, the half-life of

cocaine at pH 7.2 and 40% is about 5 hours (Garrett and Seyda 1983),

which is substantially greater than the half-life of cocaine in vivo,

about 1 hour (Jeffcoat et al. 1989). A carboxylesterase was recently

identified in human liver that catalyzes the hydrolysis of this methyl

ester of cocaine to form benzoylecgonine and methanol (Dean et al.

1991). In the presence of ethanol, the enzyme also catalyzes the

ethyl transesterification of cocaine to form cocaethyl-ene plus

methanol (Dean et al. 1991).
29

CHARACTERIZATION OF A HUMAN LIVER COCAINE BENZOYL

ESTER HYDROLASE

An enzyme that catalyzes the hydrolysis of cocaine to ecgonine

methyl ester and benzoate was purified from human autopsy liver by

ion-exchange and affinity chromatography and gel filtration. The

enzyme was purified approximately 8,700-fold and about 150

micrograms (g) of enzyme were obtained from 70 grams (g) of

human liver. The enzyme catalyzed the hydrolysis of cocaine to

ecgonine methyl ester and benzoate and the hydrolysis of

methylumbelliferylacetate to methylumbelliferone. The Michaelis

constant (KM) for cocaine was 0.7 millimolars (mM) and the turnover

number (kcat) was 7.6 min-1. The enzyme was inhibited by

phenylmethylsulfonyl fluoride, diazinon, and eserine. The enzyme is

a monomer of approximately 60 kilodalton (kDa) subunit mass. It

has an isoelectric point (pI) of approximately 4.9 and is a

glycoprotein.

The physiological levels of cocaine can reach a maximum of about

0.3 micromolar (M) in serum immediately after cocaine

administration (Jeffcoat et al. 1989). These levels are much less than

the KM of the liver benzoyl esterase (0.7 mM). Hence, the enzyme

will obey first-order kinetics, where activity equals kcat/KM, called the

catalytic efficiency, times the cocaine concentration. The catalytic

efficiency of the liver benzoyl esterase, 11 min-1 mM-1, is similar to

human serum cholinesterase, 7 min-1 mM -1, another enzyme that

catalyzes the hydrolysis of the benzoyl group of cocaine. The

content and kinetic properties of serum cholinesterase and liver

benzoyl esterase need to be evaluated to determine which enzyme has

the greater capacity for hydrolysis of cocaine in human.

The catalytic efficiency of the liver cocaine benzoyl esterase and

serum cholinesterase is about 50 times that of a catalytic antibody

prepared to an analog of the putative cocaine benzoyl esterase

transition-state structure, 0.22 min-1 mM-1 (Landry et al. 1993). The

objective for preparing the catalytic antibody was to provide a

reagent that could bind and enhance the clearance of cocaine (Morell

1993). It was proposed that such a reagent could be used to blunt the

desire to use cocaine. However, it is not clear whether a cocaine

benzoyl esterase that is more efficient than the native serum or liver

enzymes can be created by catalytic antibody technology. Perhaps

the clearance of cocaine might be increased by inducing the native

microsomal benzoyl esterases present in liver.
30

CHARACTERIZATION OF A HUMAN LIVER COCAINE METHYL

ESTER HYDROLASE

An enzyme that catalyzes the hydrolysis of the methyl ester of

cocaine (figure 1) was purified to homogeneity by ionexchange and

hydrophobic interaction chromatography and gel filtration

(Brzezinski et al. 1994). Approximately 3 milligrams (mg) of

enzyme were purified from 70 g of human liver obtained at autopsy.

The purified carboxylesterase catalyzes the hydrolysis of cocaine to

benzoylecgonine and methanol and the hydrolysis of

methylumbelliferyl acetate to methylumbelliferone and acetate. The

enzyme is a trimer of approximately 59 kDa subunits. It has a pI

value of 5.8 and it is a glycoprotein. Fourteen different tryptic and S.

aureus V8 peptides were purified by high performance liquid chromatography (HPLC) and their amino acid sequences were determined.

After searching the amino acid and translated DNA sequences

deposited in GenBank, two identical matches were found that

correspond to nonspecific carboxylesterase cDNAs from human liver

and lung (Long et al. 1991; Riddles et al. 1991; Munger et al. 1991).

In addition to the hydrolysis of cocaine, the purified human liver

cocaine methyl ester hydrolase also catalyzed the ethyl

transesterification of cocaine with ethanol to form cocaethylene and

methanol as shown in figure 1 (Dean et al. 1991; Brzezinski et al.

1994). Both the hydrolytic and the ethyl transesterification

reactions increased as the two activities were analyzed in protein

fractions obtained during the enzyme purifi-cation by column

chromatography. This suggests that the separate activities are

catalyzed by the same enzyme. The KM values for cocaine and

ethanol of the purified enzyme at pH 7.3 were 116 M and 43 mM,

respectively. The carboxylesterase also catalyzes the formation of

ethyloleate from oleic acid and ethanol (Tsujita and Okuda 1992;

Brzezinski et al. 1994). Other hydrolases or ester transferases have

been reported to catalyze similar substrate "ethylation" reactions.

For example, an isozyme of glutathione-S-transferase will also

catalyze the fatty acid ethyl-ester synthase reaction, leading to the

formation of ethyloleate from oleic acid and ethanol (Bora et al.

1989). Also, phospholipase D catalyzes the transphosphatidylation

of phosphatidylcholine with ethanol to form phosphatidylethanol

(Kobayashi and Kanfer 1987). The active site requirements and

kinetics of the hydrolases or transferases that catalyze these

ethylation reactions are not well understood. The elucidation of

mechanisms and active site structures for enzyme-catalyzed
31

ethylation reactions should contribute to the basic understanding of

alcohol-drug interactions.

The coabuse of cocaine and alcohol is all too common (Grant and

Harford 1990), and is thought to result from the enhanced euphoria

and sense of well-being experienced when cocaine is taken with

alcohol (Farre et al. 1993). Unfortunately, coabuse of alcohol and

cocaine results in an increased health risk (Farre et al. 1993). The

formation of cocaethylene under defined experimental conditions in

rats after administration of alcohol and cocaine and the cellular and

organ toxicity of cocaethylene are discussed by Dean and colleagues

elsewhere in this volume.

STRUCTURE-REACTIVITY RELATIONSHIPS FOR COCAINE

CARBOXYLESTERASES

The human liver cocaine methyl and benzoyl carboxylesterases

catalyze the hydrolysis of the acetyl group of 4-methylumbelliferyl

acetate with specific activities of 6.8 and 140 mol/(min ß mg

protein), respectively. With cocaine as substrate, however, the

cocaine methyl and benzoyl estereases exhibit lower specific

activities, but show absolute specificity for the methyl ester group and

the benzoyl ester group, respectively. A high specificity for binding

of cocaine derivatives to proteins or catalytic specificity for

metabolic reactions is frequently observed. For example, Gatley

reported that the benzoyl group of benzoylecgonine was not

hydrolyzed by serum cholinesterase (Gatley 1991). Additionally,

Gatley observed that serum cholinesterase exhibited about a 2,300-

fold higher relative activity with the unnatural (+) isomer of cocaine

than the natural (-) isomer (Gatley 1991).

The determination of structure-reactivity relationships of cocainemetabolizing enzymes and binding proteins represents an important

direction for research in cocaine metabolism. Binding site structures

could be determined directly by protein X-ray crystallography and

computerized modeling of cocaine binding. Such structure-reactivity

studies will provide valuable information regarding potential drug or

metabolite interactions with these important cocaine metabolizing

enzymes and binding proteins.
32

REFERENCES

Bora, P.S.; Spilburg, C.A.; and Lange, L.G. Metabolism of ethanol and

carcinogens by glutathione transferases. Proc Natl Acad

Sci U S A 86:4470-4473,1989.

Brzezinski, M.R.; Abraham, T.R.; Stone, C.L.; Dean, R.A.; and Bosron,

W.F. Purification and characterization of a human liver

cocaine carboxylesterase that catalyzes the production of

benzoylecgonine and the formation of cocaethylene from

alcohol and cocaine. Biochem Pharmacol 48(9):1747-

1755, 1994.

Covert, R.F.; Schreiber, M.D.; Tebbett, I.R.; and Torgerson, L.J.

Hemodynamic and cerebral blood flow effects of cocaine,

cocaethylene and benzoylecgonine in conscious and

anesthetized fetal lambs. J Pharmacol Exp Ther

270(1):118-126, 1994.

Dean, R.A.; Christian, C.D.; Sample, R.H.B.; and Bosron, W.F. Human

liver cocaine esterases: Ethanol-mediated formation of

ethylcocaine. FASEB J 5:2735-2739, 1991.

Farre, M.; Torre, R.D.L.; Llorente, M.; Lamas, X.; Ugena, B.; Segura, J.;

and Cami, J. Alcohol and cocaine interactions in humans.

J Pharmacol Exp Ther 266(3):1364-1373, 1993.

Garrett, E.R., and Seyda, K. Prediction of stability in pharmaceutical

preparations XX: Stability, evaluation and bioanalysis of

cocaine and benzoylecgonine by high-performance liquid

chromatography. J Pharm Sci 72:258-271, 1983.

Gatley, S.J. Activities of the enantiomers of cocaine and some related

compounds as substrates and inhibitors of plasma

butyrylcholinesterase. Biochem Pharmacol 41:1249-

1254, 1991.

Grant, B.F., and Harford, T.C. Concurrent and simultaneous use of alcohol

with cocaine: Results of national survey. Drug Alcohol

Depend 25:97-104, 1990.

Jeffcoat, A.R.; Perez-Reyes, M.; Hill, J.M.; Sadler, B.M.; and Cook, C.E.

Cocaine disposition in humans after intravenous injection,

nasal insufflation (snorting), or smoking. Drug Metab

Dispos 17:153-159, 1989.

Kobayashi, M., and Kanfer, J.N. Phosphatidylethanol formation via

transphosphatidylation by rat brain synaptosomal

phospholipase D. J Neurochem 48(5):1597-1603, 1987.

Kutrh, C.D.; Monitto, C.; Albuquerque, M.L.; Feuer, P.; Anday, E.; and

Shaw, L. Cocaine and its metabolites constrict cerebral

arterioles in newborn pigs. J Pharmacol Exp Ther

265(2):587-591, 1993.
33

Landry, D.W.; Zhao, K.; Yang, G., X.-Q.; Glickman, M.; and Georgiadis,

T.M. Antibody-catalyzed degradation of cocaine. Science

259(26):1899-1901, 1993.

Long, R.M.; Calabrese, M.R.; Martin, B.M.; and Pohl, L.R. Cloning and

sequencing of a human liver carboxylesterase isoenzyme.

Life Sci 48:PL43-49, 1991.

Misra, A.L.; Nayak, P.K.; Bloch, R.; and Mule, S.J. Estimation and

disposition of [3H]benzoylecgonine and pharmacological

activity of some cocaine metabolites. J Pharm

Pharmacol 27:784-786, 1975.

Morell, V. Enzymes may blunt cocaine's action. Science 259(26):1828,

1993.

Munger, J.S.; Shi, G.-P.; Mark, E.A.; Chin, D.T.; Gerard, C.; and Chapman,

H.A. A serine esterase released by human alveolar

macrophages is closely related to liver microsomal

carboxylesterases. J Biol Chem 266:18832-18838, 1991.

Riddles, P.W.; Richards, L.J.; Bowles, M.R.; and Pond, S.M. Cloning and

analysis of a cDNA encoding a human liver

carboxylesterase. Gene 108:289-292, 1991.

Roth, L.; Harbison, R.D.; James, R.C.; Tobin, T.; and Roberts, S.M.

Cocaine hepatotoxicity: Influence of hepatic enzyme

inducing and inhibiting agents on the site of necrosis.

Hepatology 15:934-940, 1992.

Spealman, R.D.; Madras, B.K.; and Bergman, J. Effects of cocaine and

related drugs in nonhuman primates. II. Stimulant effects

on schedule-controlled behavior. J Pharmacol Exp Ther

251:142-149, 1989.

Stewart, D.J.; Inaba, T.; Tang, B.K.; and Kalow, W. Hydrolysis of cocaine

in human plasma by cholinesterase. Life Sci 20:1557-

1564, 1977.

Tsujita, T., and Okuda, H. Fatty acid ethyl ester synthase in rat adipose

tissue and its relationship to carboxylesterase. J Biol

Chem 267:23489-23494, 1992.

Wang, J., and Carpentier, R.G. Electrophysiologic in-vitro effects of

cocaine and its metabolites. Int J Cardiol 46:235-42,

1994.

ACKNOWLEDGMENTS

This chapter was prepared with support from the National Institute

on Drug Abuse grant DA-06836.
34

AUTHORS

William F. Bosron, Ph.D.

Professor

Department of Biochemistry and Molecular Biology

Department of Medicine

Robert A. Dean, Ph.D., M.D.

Associate Professor

Department of Pathology and Laboratory Medicine

Department of Biochemistry and Molecular Biology

Monica R. Brzezinski, B.S.

Graduate Student

Department of Biochemistry and Molecular Biology

Evgenia V. Pindel, Ph.D.

Postdoctoral Fellow in Biochemistry and Molecular Biology

Indiana University School of Medicine

Medical Sciences Building

635 Barnhill Drive

Indianapolis, IN 46202-5122
35

Effects of Ethanol on Cocaine

Metabolism and Disposition

in the Rat

Robert A. Dean, William F. Bosron, Frederick M. Zachman, Jing

Zhang, and Monica R. Brzezinski

COABUSE OF COCAINE AND ALCOHOL

Abuse of cocaine in combination with other drugs is a widespread

practice (Washton and Gold 1987) and the coabuse of alcohol is

particularly common, with 99 percent of cocaine addicts reporting

excessive use of ethanol in one study (Newcombe and Bentler 1987).

Simultaneous ingestion of ethanol also is common, as reported by 77

percent of cocaine users (Grant and Harford 1990). Thirty percent of

cocaine users ingest alcohol during almost every episode of cocaine

use (Jones 1987).

Why these two drugs are so frequently coadministered is not clearly

understood. Potentiation of cocaine-related euphoria by alcohol

ingestion may be the basis for this behavior and is consistent with

increased plasma ethanol concentrations observed when cocaine is

administered after alcohol ingestion (Farré et al. 1993; Perez-Reyes

and Jeffcoat 1992). Alcohol ingestion also is reported to diminish

undesirable side effects, such as cocaine-induced migraine-like

headaches (Weiss et al. 1988). Additionally, coadministration may be

influenced by the settings in which cocaine and alcohol are abused.

Regardless of the motive for combined use of cocaine and ethanol,

this practice is of clinical concern, as it increases the risk of cocainerelated morbidity (Adams et al. 1987; Kreek 1987; Kreek and

Stimmel 1984) and mortality (Kreek 1987; Rose et al. 1990).

Epidemiological data indicate that simultaneous alcohol ingestion may

increase the risk of cocaine-related sudden death by eighteenfold

(Rose et al. 1990).

COCAINE METHYL ESTERASE AND ETHYL TRANSFERASE

The rapid clearance of cocaine is in large part mediated by hydrolysis

of the methyl ester group. This reaction produces benzoylecgonine

(figure 1), a metabolite devoid of cocaine-like psychomotor activity
36

(Misra et al. 1975; Spealman et al. 1989). While hydrolysis of

cocaine to benzoylecgonine occurs spontaneously (Stewart et al.

1979), the slow rate of this reaction at physiological pH (Taylor et

al. 1976) does not account for the relatively large percentage of

cocaine recovered from urine as benzoylecgonine (Cook et al. 1985).

A nonspecific carboxylesterase with cocaine methyl esterase activity

was identified in human liver (Dean et al. 1991) and was subsequently

purified and characterized (Bosron, this volume; Brzezinski et al.

1994). Under in vitro conditions using purified human liver

carboxylesterase, ethanol was found to inhibit cocaine methyl esterase

activity, decreasing hydrolysis to benzoylecgonine (Brzezinski et al.

1994; Dean et al. 1991). In the presence of ethanol, this same

carboxylesterase catalyzed the ethyl transesterification of cocaine to

cocaethylene (benzoylecgonine ethyl ester) (figure 1) (Bosron, this

volume; Brzezinski et al. 1994; Dean et al. 1991).

EFFECTS OF ETHANOL ON THE DISTRIBUTION OF COCAINE AND

METABOLITES

Cocaine metabolism and disposition following acute ethanol

administration were studied in the rat to determine if the in vitro

effects of ethanol on cocaine methyl esterase and ethyl transferase

activities had significance in vivo (Zachman et al. 1993). The rat was

used as it possesses both ethyl transferase and methyl esterase

activities, is frequently employed for behavioral and toxicity studies

of cocaine, and the size provides sufficient tissue for analytical work.

This study was designed to address three questions. First, do

significant concentrations of cocaethylene form and accumulate in

tissues with controlled coadministrations of cocaine and alcohol?

Second, does ethanol administration significantly diminish the

hydrolysis of cocaine to benzoylecgonine and methanol, as occurs in

vitro when cocaine and ethanol are coincubated with purified human

liver carboxylesterase (Brzezinski et al. 1994; Dean et al. 1991)?

Third, does ethanol inhibition of cocaine methyl ester hydrolysis

increase the N-oxidative metabolism of cocaine, as noted when

rodents are pretreated with nonspecific esterase inhibitors (Thompson

et al. 1979)?

Male Wistar rats were pretreated with 2.5 grams (g) ethanol per

kilogram (kg) bodyweight in water or an equal volume of water via an

indwelling intragastric catheter. Sixty minutes after pretreatment, 10

milligrams (mg)/kg cocaine was infused through an indwelling

intravenous (IV)
37

catheter. Animals were sacrificed 2.5 to 60 minutes after cocaine

administration and serum and solid tissues were harvested for

measurement of cocaine, cocaethylene, benzoylecgonine, norcocaine,

norcocaethylene, and benzoylnorecgonine.
38

ALCOHOL-DEPENDENT FORMATION OF COCAETHYLENE

Cocaethylene was detected in all tissues from alcohol-pretreated

animals, as previously observed following intraperitoneal (IP)

coadministration of cocaine and alcohol (Dean et al. 1992). Based on

area under the concentration curves (AUCs) from 2.5 to 60 minutes,

the amount of cocaethylene in liver and lung was 14 and 11 percent

of the measured cocaine, respectively. Peak cocaethylene

concentrations in liver, lung, and kidney were noted within 2.5

minutes after cocaine administration, the earliest time point for

specimen collection. These observations answered the first question,

demonstrating cocaethylene formation sufficient to contribute to the

combined effects of cocaine and ethanol. Cocaethylene AUCs in

heart, brain, spleen, and serum were less than 2 to 4 percent of the

cocaine AUCs, and peak cocaethylene concentrations in these tissues

occurred approximately 10 minutes after cocaine administration.

The relatively high AUCs and rapid peak for cocaethylene in liver,

lung, and kidney suggest that cocaethylene formation occurs

predominantly in these tissues with subsequent distribution to other

tissues. This conclusion was supported by direct measurement of

cocaine ethyl transferase activity in tissue homogenate supernatants.

Ethyl transferase activity, determined by measuring cocaethylene

formation in the presence of saturating levels of cocaine and ethanol,

was confirmed in rat liver, lung, heart, and kidney. When normalized

for protein content in tissue homogenate supernatants, the relative

activity in these tissues decreased in the order listed.

The relative distribution of rat cocaine ethyl transferase activity was

consistent with the distribution of immunoreactive protein as

determined by gel electrophoresis and Western blot analysis using

rabbit anti-human cocaine ethyl transferase antibody. No ethyl

transferase activity was identified in brain, spleen, or serum. When

rat cocaine ethyl transferase activity was normalized for whole organ

volume, liver exhibited activity that was 33-fold greater than that in

lung and in excess of 600-fold greater than that in kidney and heart.

Formation and accumulation of cocaethylene in liver is of potential

concern; this ethylated metabolite undergoes N-demethylation to

norcocaethylene and produces cocaine-like cytotoxicity in cultured

rat hepatocytes (Boelsterli et al. 1993; Dean et al. 1992) and the

intact mouse (Roberts et al. 1992).
39

Formation and accumulation of cocaethylene is thought to alter the

subjective effects and enhance the toxicity of cocaine when used in

combination with beverage alcohol. Support for this hypothesis is

based on the following evidence. First, cocaethylene has been

identified in urine, blood, brain, and liver obtained at autopsy from

individuals succumbing after combined exposure to cocaine and

ethanol (Hearn et al. 1991a; Hime et al. 1991; Jatlow et al. 1991;

Rafla and Epstein 1979; Smith 1984). This metabolite was also

detected in the blood of emergency room patients seeking treatment

after combined use of cocaine and ethanol (Jatlow et al. 1991).

Similarly, ethanol-dependent accumulation of cocaethylene in serum

and plasma was noted following controlled coadministration of

cocaine and ethanol, when the cocaine was known to be free of

contaminants, including cocaethylene (de la Torre et al. 1991; Farré

et al. 1993; McCance-Katz et al. 1991; Perez-Reyes and Jeffcoat

1992; Perez-Reyes 1994). Second, when administered under

controlled conditions, cocaethylene produced similar but milder and

more pleasurable subjective effects than cocaine (Peres-Reyes 1993).

Comparable affinity of cocaethylene and cocaine for the dopamine

transporter in human striatal membranes may explain similarities in

the subjective effects of these two drugs (Hearn et al. 1991a), while

lower cocaethylene affinity for serotonin uptake sites and the

norepinephrine transporter might explain differing subjective effects.

Third, controlled administration of cocaethylene in humans produced

tachycardia comparable to that induced by cocaine (Perez-Reyes

1993). However, cocaethylene is a more potent blocker of cardiac

sodium channels in guinea pig ventricular myocytes (Xu et al. 1994)

and of muscarinic receptor-stimulated phosphoinositide metabolism in

the rat (Tan and Costa 1994), and has a greater negative ionotropic

effect in isolated ventricular myocytes from ferret (Qiu and Morgan

1993). If true in humans, cocaethylene may increase the risk of

conduction and contractile disturbances in the heart. Finally, the

median lethal dose for cocaethylene in the mouse was significantly

less than that for cocaine (Hearn et al. 1991b; Katz et al. 1992).
40

ALCOHOL INHIBITION OF BENZOYLECGONINE FORMATION

Ethanol pretreatment of rats dramatically decreased the AUCs for

benzoylecgonine in all tissues and serum. This in vivo effect is

consistent with in vitro ethanol inhibition of carboxylesterasecatalyzed cocaine hydrolysis to benzoylecgonine and methanol.

Although ethanol decreased AUCs for benzoylecgonine in all tissues,

the time to peak benzoyl-ecgonine concentrations was variable. In

liver, peak concentrations occurred 2.5 minutes after cocaine

administration, suggesting high hepatic methyl esterase activity. By

contrast, benzoylecgonine levels in serum continued to rise over the

entire 60 minutes of the experiment and likely reflect redistribution

of this polar metabolite. The distribution of cocaine methyl esterase

activity in rat tissues was determined by directly measuring the

enzymatic hydrolysis (total minus spontaneous) of cocaine to

benzoylecgonine in homogenate supernatants. Methyl esterase

activity was identified in kidney, liver, lung, heart, and brain. When

normalized for tissue protein, the relative amount of cocaine methyl

esterase activity decreased in the order listed. When normalized for

whole organ volume, hepatic cocaine methyl esterase activity was 15-

fold to 100-fold greater than the activity in other tissues. No such

activity was detected in spleen or serum. The presence of cocaine

methyl esterase activity and absence of ethyl transferase activity in

brain, plus poor correlation between these two activities in other

tissues, suggest that more than one enzyme catalyzes these two

reactions in the rat. Although a single carboxylesterase is known to

catalyze both reactions in humans (Brzezinski et al. 1994; Dean et al.

1991), it is not known if other human enzymes possess cocaine

methyl esterase or ethyl transferase activities.

Although lacking psychomotor activity, benzoylecgonine recently

was shown to be a more potent vasoconstrictor of cerebral arteries in

cat and sheep than cocaine (Covert et al. 1994; Madden and Powers

1990; Schreiber et al. 1994). Similarly, benzoylecgonine increased

mean blood pressure with no effect on heart rate or QRS duration in

the anesthetized rat (Erzouki et al. 1993). By contrast, cocaine

decreased mean blood pressure and heart rate and increased the QRS

duration. These different cardiovascular effects of cocaine and

benzoylecgonine appear to reflect differing activity within the

autonomic nervous system. Unlike cocaine, which produces

vasoconstriction by blocking catecholamine reuptake,

benzoylecgonine-induced vasoconstriction appears to be mediated

predominantly by stimulation of 1-adrenergic receptors (Schreiber et

al. 1994). Although cause and effect have yet to be established, a

temporal relationship exists between recurrent coronary artery
41

vasoconstriction and increasing blood benzoylecgonine

concentrations in humans (Brogan et al. 1992). Similarly, increasing

benzoylecgonine concentrations might contribute to the migraine-like

vasospastic headaches observed in cocaine users (Satel and Gawin

1989). If so, alcohol ingestion might diminish vasoconstriction of

cerebral, coronary, and possibly other vascular beds by inhibiting

cocaine methyl esterase activity and benzoylecgonine production.

This effect might well contribute to combined use of cocaine and

ethanol.

ALCOHOL EFFECT ON N-OXIDATIVE METABOLISM OF COCAINE

In humans, N-demethylation of cocaine to norcocaine is catalyzed by

a cytochrome P-450 enzyme either directly or following oxidation of

cocaine to cocaine N-oxide by a flavin-adenine dinucleotide (FAD)-

containing mono-oxygenase (figure 1, 4) (Kloss et al. 1983). The Ndemethylation of benzoylecgonine to benzoylnorecgonine was

demonstrated in the rat following administration of radiolabeled

benzoylecgonine (figure 1, 5)(Misra et al. 1975).

In animals pretreated with ethanol, metabolism of cocaine to

norcocaine and benzoylnorecgonine increased, as reflected by higher

tissue AUCs, as compared with those receiving water. Ethanol

pretreatment also resulted in measurable levels of norcocaethylene in

liver and lung. These observations are consistent with the increased

hepatotoxicity (presumably due to enhanced N-oxidative metabolism)

observed when mice were exposed to cocaine or cocaethylene and the

esterase inhibitor diazinon (Roberts et al. 1992; Thompson et al.

1979). This shift toward N-oxidative metabolism provides a

mechanism to explain potentiation of cocaine hepatotoxicity by

ethanol (Jover et al. 1991). Detection of norcocaethylene in

ethanol- pretreated rats is consistent with norcocaethylene detected

in the hair of heavy cocaine users, suggesting common pathways

including hydrolysis, transesterification, and N-demethylation (figure

1) (Cone et al. 1991).

ALCOHOL EFFECTS ON COCAINE DISPOSITION

Ethanol pretreatment had variable effects on AUC curves for cocaine

from tissue to tissue. Ethanol increased the AUC for cocaine in liver

and decreased the AUC for cocaine in serum and the heart. Ethanol

pretreatment had little or no effect on the AUCs for cocaine in brain,

lung, kidney, and spleen. While lower tissue benzoylecgonine AUCs
42

support the notion that alcohol inhibits cocaine methyl esterase

activity, variable effects of ethanol on cocaine AUCs suggest that the

effects of ethanol are not fully explained by inhibition of methyl

esterase activity and simultaneous initiation of ethyl transferase

activity. The increased AUC for liver and decreased AUC for serum

might be explained by increased hepatic extraction of cocaine when

coadministered with ethanol. It is not clear how this might occur, but

ethanol-mediated vasodilation could increase hepatic blood flow

(Orrego et al. 1988; Shaw et al. 1977), which is typically limited by

the vasoconstricting effects of cocaine (Garhart et al. 1989).

Alternatively, oxidative metabolism of ethanol might decrease the

intracellular pH and enhance trapping of cocaine in hepatocytes.

CONCLUSION

It is not known why coabuse of cocaine and ethanol is so common.

Similarly, the specific mechanisms by which ethanol potentiates

cocaine-related morbidity and mortality are not clearly defined. The

studies reviewed in this chapter suggest that multiple effects of

ethanol on cocaine metabolism may be contributory. In particular,

ethanol inhibition of cocaine methyl esterase may slow normally

rapid inactivation of cocaine to benzoylecgonine while simultaneously

initiating ethyl transesterification to cocaethylene and increasing Ndemethylation to norcocaine. Although acute ethanol exposure did

not uniformly increase serum and tissue cocaine AUCs, the collective

increase in the levels of the psychoactive compounds cocaine,

cocaethylene, norcocaine, and norcocaethylene may enhance or

prolong cocaine-related euphoria and toxicity. Additionally, unique

and/or relative differences in the activities of these metabolites may

contribute to the effects of ethanol on the pharmacokinetics and

pharmacodynamics of cocaine. More extensive characterization of

the enzymes responsible for cocaine metabolism and the development

of pharmacokinetic and pharmacodynamic models for cocaine will

facilitate efforts to understand the interactions of this drug with

alcohol.
43

REFERENCES

Adams, E.H.; Gfroerer, J.C.; Rouse, B.A.; and Kozel, N.J. Trends in

prevalence and consequences of cocaine use. In: Gold,

M.S.; Galanter, M.; and Stimmel, B., eds. Cocaine:

Pharmacology, Addiction and Therapy. New York:

Haworth Press, 1987. pp. 49-71.

Boelsterli, U.A.; Wolf, A.; and Göldlin, C. Oxygen free radical production

mediated by cocaine and its ethanol-derived metabolite,

cocaethylene, in rat hepatocytes. Hepatology

18(5):1154-1161, 1993.

Brogan, W.C.; Lange, R.A; Glamann, D.B; and Hillis, L.D. Recurrent

coronary vasoconstriction caused by intranasal cocaine:

Possible role for metabolites. Ann Intern Med 116:556-

561, 1992.

Brzezinski, M.R.; Abraham, T.L.; Stone, C.L.; Dean, R.A.; and Bosron,

W.F. Purification and characterization of a human liver

cocaine carboxylesterase that catalyzes the production of

benzoylecgonine and the formation of cocaethylene from

alcohol and cocaine. Biochem Pharmacol 48:1747-1755,

1994.

Cone, E.J.; Yousefnejad, D.; Darwin, W.D.; and Maguire, T. Testing

human hair for drugs of abuse. II. Identification of unique

cocaine metabolites in hair of drug abusers and evaluation

of decontamination procedures. J Anal Toxicol 15:250-

255, 1991.

Cook, C.E.; Jeffcoat, A.R.; and Perez-Reyes, M. Pharmacokinetic studies

of cocaine and phencyclidine in man. In: Barnett, G., and

Chiang, C.N., eds. Pharmacokinetics and

Pharmacodynamics of Psychoactive Drugs. Foster City,

CA: Biomedical Publications, 1985. pp. 48-74.

Covert, R.F.; Schreiber, M.D.; Tebbett, I.R.; and Torgerson, L.J.

Hemodynamic and cerebral blood flow effects of cocaine,

cocaethylene and benzoylecgonine in conscious and

anesthetized fetal lambs. J Pharmacol Exp Ther 270:118-

126, 1994.

Dean, R.A.; Christian, C.D.; Sample, R.H.B.; and Bosron, W.F. Human

liver cocaine esterases: Ethanol-mediated formation of

ethylcocaine. FASEB J 5:2735-2739, 1991.

Dean, R.A.; Harper, E.T.; Dumaual, N.; Stoeckel, D.A.; and Bosron, W.F.

Effects of ethanol on cocaine metabolism: Formation of

cocaethylene and norcocaethylene. Toxicol Appl

Pharmacol 117:1-8, 1992.

de la Torre, R.; Farrè, M.; Ortuño, J.; Camì, J.; and Segura, J. The

relevance of urinary cocaethylene following simultaneous
44

administration of alcohol and cocaine. J Anal Toxicol

15:223, 1991.

Erzouki, H.K.; Baum, I.; Goldberg, S.R.; and Schindler, C.W. Comparison

of the effects of cocaine and its metabolites on

cardiovascular function in anesthetized rats. J Cardiovasc

Pharmacol 22:557-563, 1993.

Farré, M.; de la Torre, R.; Llorente, M.; Lamas, X.; Ugena, B.; Segura, J.;

and Camí, J. Alcohol and cocaine interactions in humans.

J Pharmacol Exp Ther 266:1364-1373, 1993.

Garhart, C.A.; Anwer, M.S.; and Shuster, L. Cocaine-induced changes in

perfusion pressure and bile flow in perfused rat livers.

Pharmacology 38:2139-2145, 1989.

Grant, B.F., and Harford, T.C. Concurrent and simultaneous use of alcohol

with cocaine: Results of national survey. Drug Alcohol

Depend 25:97-104, 1990.

Hearn, W.L.; Flynn, D.D.; Hime, G.W.; Rose, S.; Confino, J.C.; ManteroAtienza, E.; Wetli, C.W.; and Mash, D.C. Cocaethylene:

A unique cocaine metabolite displays high affinity for the

dopamine transporter. J Neurochem 56:698-701, 1991a.

Hearn, W.L.; Rose, S.; Wagner, J.; Ciarleglio, A.; and Mash, D.C.

Cocaethylene is more potent than cocaine in mediating

lethality. Pharmacol Biochem Behav 39:531-533, 1991b.

Hime, G.W.; Hearn, W.L.; Rose, S.; and Cofino, J. Analysis of cocaine and

cocaethylene in blood and tissues by GC-NPD and GC-ion

trap mass spectrometry. J Anal Toxicol 15:241-245,

1991.

Jatlow, P.; Elsworth, J.D.; Bradberry, C.W.; Winger, G.; Taylor, J.R.;

Russell, R.; and Roth, R.H. Cocaethylene: A

neuropharmacologically active metabolite associated with

concurrent cocaine-ethanol ingestion. Life Sci 48:1787-

1794, 1991.

Jones, R.T. Psychopharmacology of cocaine. In: Washton, A.M., and

Gold, M.S., eds. Cocaine: A Clinician's Handbook. New

York: Guilford Press, 1987. pp. 55-72.

Jover, R.; Ponsoda, X.; Gómez-Lechón, M.J.; Herrero, C.; del Pino, J.; and

Castell, J.V. Potentiation of cocaine hepatotoxicity by

ethanol in human hepatocytes. Toxicol Appl Pharmacol

107:526-534, 1991.

Katz, J.L.; Terry, P.; and Witkin, J.M. Comparative behavioral

pharmacology and toxicology of cocaine and its ethanolderived metabolite, cocaine ethyl ester (cocaethylene).

Life Sci 50:1351-1361, 1992.

Kloss, M.W.; Rosen, G.M.; and Rauckman, E.J. N-Demethylation of

cocaine to norcocaine. Evidence for participation by
45

cytochrome P-450 and FAD-containing monooxygenase.

Mol Pharmacol 23:482-485, 1983.

Kreek, M.J. Multiple drug abuse patterns and medical consequences. In:

Meltzer, H.Y., ed. Psychopharmacology: The Third

Generation of Progress. New York: Raven Press, 1987.

pp. 1597-1604.

Kreek, M.J., and Stimmel, B. Alcoholism and Polydrug Use. New York:

Haworth Press, 1984. pp. 1-6.

Madden, J.A., and Powers, R.H. Effect of cocaine and cocaine metabolites

on cerebral arteries in vitro. Life Sci 47:1109-1114, 1990.

McCance-Katz, E.F.; Price, L.H.; McDougle, C.J.; Marek, G.J.; Kosten,

T.R.; and Jatlow, P. Cocaethylene formation following

sequential administration of cocaine and ethanol to

humans: Pharmacological, physiological, and behavioral

studies. Soc Neurosci Abs 17:890, 1991.

Misra, A.L.; Nayak, P.K.; Bloch, R.; and Mule, S.J. Estimation and

disposition of 3H-benzoylecgonine and pharmacological

activity of some cocaine metabolites. J Pharm

Pharmacol 27:784-786, 1975.

Newcombe, M.D., and Bentler, P.M. Cocaine use among young adults. In:

Gold, M.S.; Galanter, M.; and Stimmel, B., eds. Cocaine:

Pharmacology, Addiction and Therapy. New York:

Hawthorn Press, 1987. pp. 73-96.

Orrego, H.; Carmichael, F.J.; Saldivia, V.; Giles, H.G.; Sandrin, S.; and

Israel, Y. Ethanol-induced increase in portal blood flow:

Role of adenosine. Am J Physiol 254:495-501, 1988.

Perez-Reyes, M. The order of drug administration: Its effects on the

interaction between cocaine and ethanol. Life Sci 55:541-

550, 1994.

Perez-Reyes, M. Subjective and cardiovascular effects of cocaethylene in

humans. Psychopharmacology 113:144-147, 1993.

Perez-Reyes, M., and Jeffcoat, A.R. Ethanol/cocaine interaction: Cocaine

and cocaethylene concentrations and their relationship to

subjective and cardiovascular effects. Life Sci 51:553-563,

1992.

Qiu, Z., and Morgan, J.P. Differential effects of cocaine and cocaethylene

on intracellular Ca2+ and myocardial contraction in cardiac

myocytes. Br J Pharmacol 109:293-298, 1993.

Rafla, F.K., and Epstein, R.L. Identification of cocaine and its metabolites

in human urine in the presence of ethyl alcohol. J Anal

Toxicol 3:59-63, 1979.

Roberts, S.M.; Roth, L.; Harbison, R.D.; and James, R.C. Cocaethylene

hepatotoxicity in mice. Biochem Pharmacol 43:1989-

1995, 1992.
46

Rose, S.; Hearn, W.L.; Hime, G.W.; Wetli, C.V.; Ruttenber, A.J.; and

Mash, D.C. Cocaine and cocaethylene concentrations in

human post mortem cerebral cortex. Neurosci Abs

16(11.6), 1990.

Satel, S.L., and Gawin, F.H. Migrainelike headache and cocaine use. JAMA

261:2995-2996, 1989.

Schreiber, M.D.; Madden, J.A.; Covert, R.F.; and Torgerson, L.J. Effects of

cocaine, benzoylecgonine, and cocaine metabolites on

cannulated pressurized fetal sheep cerebral arteries. J Appl

Physiol 77:834-839, 1994.

Shaw, S.; Heller, E.A.; Friedman, H.S.; Baraona, E.; and Lieber, C.S.

Increased hepatic oxygenation following ethanol

administration in the baboon. Proc Soc Exp Biol Med

156:509-513, 1977.

Smith, R.M. Ethyl esters of arylhydroxy- and

arylhydroxymethoxycocaines in the urines of

simultaneous cocaine and ethanol users. J Anal Toxicol

8:38-42, 1984.

Spealman, R.D.; Madras, B.K.; and Bergman, J. Effects of cocaine and

related drugs in nonhuman primates. II. Stimulant effects

on schedule-controlled behavior. J Pharmacol Exp Ther

251:142-149, 1989.

Stewart, D.J.; Inaba, T.; Lucassen, M.; and Kalow, W. Cocaine metabolism:

Cocaine and norcocaine hydrolysis by liver and serum

esterases. Clin Pharmacol Ther 25:464-468, 1979.

Tan, X.X., and Costa, L.G. Inhibition of muscarinic receptor-stimulated

phosphoinositide metabolism by cocaine, norcocaine and

cocaethylene in rat brain. Develop Brain Res 79:132-135,

1994.

Taylor, D.; Estevez, V.S.; Englert, L.F.; and Ho, B.T. Hydrolysis of

carbon-labeled cocaine in human serum. Res Comm Chem

Path Pharmacol 14:249-257, 1976.

Thompson, M.L.; Shuster, L.; and Shaw, K. Cocaine-induced hepatic

necrosis in mice—The role of cocaine metabolism.

Biochem Pharmacol 28:2389-2395, 1979.

Washton, A.M., and Gold, M.S. Recent trends in cocaine abuse: A view

from the national hotline, "800-cocaine." In: Gold, M.S.;

Galanter, M.; and Stimmel, B., eds. Cocaine:

Pharmacology, Addiction and Therapy. New York:

Hawthorn Press, 1987. pp. 31-47.

Weiss, R.D.; Mirin, S.M.; Griffin, M.L.; and Michael, J.L.

Psychopathology in cocaine abusers: Changing trends. J

Nerv Ment Dis 176:719-725, 1988.

Xu, Y-Q.; Crumb, W.J.; and Clarckson, C.W. Cocaethylene, a metabolite

of cocaine and ethanol, is a potent blocker of cardiac
47

sodium channels. J Pharmacol Exp Ther 271:319-325,

1994.

Zachman, F.M.; Dean, R.A.; Dumaual, N.G.; and Bosron, W.F. The effects

of ethanol on cocaine metabolism and disposition.

Toxicologist 13:1083, 1993.

ACKNOWLEDGMENTS

This chapter was prepared with support from National Institute on

Drug Abuse grant DA-08215.

AUTHORS

Robert A. Dean, Ph.D., M.D.

Associate Professor

Department of Pathology and Laboratory Medicine

Department of Biochemistry and Molecular Biology

William F. Bosron, Ph.D.

Professor

Department of Biochemistry and Molecular Biology

Department of Medicine

Frederick M. Zachman, B.S.

Medical Student

Jing Zhang, B.S.

Graduate Student

Department of Biochemistry and Molecular Biology

Monica R. Brzezinski, B.S.

Graduate Student

Department of Biochemistry and Molecular Biology

Indiana University School of Medicine

Medical Sciences Building

635 Barnhill Drive

Indianapolis, IN
48

Individual Differences in Nicotine

Kinetics and Metabolism in Humans

Neal L. Benowitz and Peyton Jacob III

INTRODUCTION

Cigarette smoking remains the major preventable cause of premature

disability and death in developed countries (Peto et al. 1992).

Cigarette smoking is maintained by addiction to nicotine. Nicotine

addiction develops in most people before the age of 20 (Department

of Health and Human Services 1994). Many youth experiment with

cigarettes, but only about 25 percent of high school seniors become

addicted smokers (Escobedo et al. 1993). Thus, there appears to be

individual variability in susceptibility to nicotine addiction.

In support of the idea of individual variability to nicotine addiction

are twin studies showing genetic linkages for never smoking, for

quitting (i.e., former smoker status), and even for being a light versus

a heavy smoker (Carmelli et al. 1992). The basis for individual

differences in susceptibility to addiction is unknown. Possible factors

include differences in pharmacokinetics and metabolism of nicotine,

pharmaco-dynamic differences, and factors related to personality,

including affective disorders and, of course, environmental influences

(Benowitz 1992). Of note is an apparent shared inheritance in

susceptibility to nicotine addiction and alcohol abuse (Swan et al.

1990).

NICOTINE METABOLISM AND SMOKING BEHAVIOR

This chapter considers individual differences in the pharmacology of

nicotine. While there is evidence of genetic difference in

pharmacologic response to nicotine in rodents (Marks et al. 1991),

there has been very little research into individual differences in

pharmacodynamics in humans. Individual differences in

pharmacokinetics and metabolism have been much better

documented, and are the major focus of this discussion.

Individual differences in nicotine kinetics and metabolism could affect

smoking behavior in two ways. First, an individual’s rate of nicotine

metabolism could affect how much a person smokes. Smokers tend to
49

adjust their smoking to maintain particular levels of nicotine in the

body. A person who metabolizes nicotine quickly may need to smoke

more to achieve a particular level of nicotine than does a person who

metabolizes nicotine more slowly. The phenomenon of regulation

has been demon-strated experimentally in a study in which the rate of

nicotine elimination was increased by acidification of the urine

(Benowitz and Jacob 1985). In conditions of urinary acidification,

smokers consumed 18 percent more nicotine per day from cigarettes,

compensating by about 50 percent for the excess loss of nicotine by

increased renal clearance.

A second mechanism by which individual differences in metabolism

could affect nicotine addiction is through the pattern of metabolites

generated. Some nicotine metabolites may be pharmacologically

active. These include nicotine iminium ion, -nicotyrine, cotinine,

and nornicotine. Nicotine iminium ion is an intermediate in the

metabolism of nicotine to cotinine (figure 1). Nicotine iminium ion

can covalently bind to macro-molecules (Shigenaga et al. 1988) and

may thereby produce tissue injury and/or promote carcinogenesis. -

nicotyrine is a minor metabolite of nicotine that has been shown to

inhibit nicotine metabolism in vitro (Shigenaga et al. 1989). Cotinine

is the major proximate metabolite of nicotine. Cotinine is inactive

toward nicotinic cholinergic receptors, but does appear to affect a

number of enzyme systems, including those involved in steroid

synthesis (Benowitz 1994). Cotinine may also have central nervous

system (CNS) activity, reportedly modifying nicotine withdrawal

symptoms in abstinent smokers (Keenan et al. 1994). The site or

mechanism of CNS action of cotinine is unknown, but if there is CNS

activity, cotinine, which is present at 15 times the concentration of

nicotine, could contribute significantly to nicotine addiction.

Nornicotine is a minor metabolite of nicotine as well as a component

of tobacco itself. It is as potent in pharma- cologic activity and

toxicity as nicotine (Risner et al. 1988). Thus, considering the

activity of various metabolites, individual differences in the amount

of various metabolites generated could influence differential

susceptibility to nicotine addiction and/or toxic effects of tobacco use.
50

INDIVIDUAL VARIABILITY IN NICOTINE METABOLISM

Nicotine is metabolized via cytochrome P450 (CYP 450) to nicotine

iminium ion, and then by aldehyde oxidase to cotinine (figure 1).

Cotinine is the major proximate metabolite of nicotine, with 70 to 80

percent of nicotine metabolized to cotinine in most smokers

(Benowitz and Jacob 1994). Cotinine is, in turn, metabolized to

trans-3'-hydroxy-cotine, which is the most abundant nicotine

metabolite in the urine of most smokers. Nicotine-N’-oxide is

formed via a flavoprotein enzyme and is a minor metabolite,

averaging about 4 percent of the systemic nicotine dose (Benowitz et

al. 1994). Nicotine, cotinine, and trans-3'-hydroxycotinine are also

conjugated (Benowitz et al. 1994; Byrd et al. 1992). Nicotine and

cotinine form quaternary N-glucuronides, whereas trans-3'-

hydroxycotinine forms an O-glucuronide (figure 1). Figure 2 shows

the average pattern of nicotine metabolism and urinary recovery,

based on a study in individuals receiving nicotine at steady state via

transdermal nicotine patches. While this figure illustrates the average

pattern, there is individual variability as shown in figure 3. Thus, for
51

most individuals, trans-3'-hydroxycotinine is the most abundant

metabolite in the urine, but cotinine is more abundant in others.

There is considerable variability in the extent of conjugation. Of

note, the extent of conjugation of nicotine and cotinine within

subjects is highly correlated, whereas there is no relationship between

nicotine or cotinine conjugation and the extent of conjugation of

trans-3'-hydroxy-cotinine. These data suggest that nicotine and

cotinine are conjugated by the same enzyme, while trans-3'-

hydroxycotinine is conjugated by a different enzyme.

These data show that there are considerable individual differences in

the metabolism of nicotine. If metabolites contribute to nicotine

addiction, individual variability in pattern of metabolism could explain

some of the individual variability in susceptibility to nicotine

addiction.
52

INDIVIDUAL VARIATION IN NICOTINE AND COTININE KINETICS

As expected by analogy to other weak bases that are extensively

metabolized, there is considerable individual variability in the

clearance of nicotine. Early studies on nicotine kinetics were

performed by infusing nicotine in smokers who were abstinent from

tobacco (Benowitz et al. 1982; Rosenberg et al. 1980). However, it is

most relevant to investigate the disposition kinetics of a drug in the

chemical environment where the drug is normally used. Using labeled

compounds, one can study the metabolism and kinetics of nicotine in

smokers while they are smoking. To do so, deuterium-labeled analogs

of nicotine (3',3'-dideuteronicotine, nicotine-d2) and cotinine

(2,4,5,6-tetra-deuterocotinine, cotinine-d4), both with the natural (S)-

configurations, have been synthesized. Concentrations of natural and

labeled nicotine and cotinine, as well as their metabolites, are

measured by gas chromatography/mass spectrometry (GC/MS).

Comparing the pharmacokinetics of labeled and natural compounds,

the absence of an isotope effect was demonstrated, validating their use
53

in studies of nicotine and cotinine metabolic disposition (Benowitz

and Jacob 1994; Jacob et al. 1991).

NICOTINE DISPOSITION IN SMOKERS AND NONSMOKERS

Cigarette smoke contains a variety of chemicals, including polycyclic

aromatic hydrocarbons, that may affect the metabolism of various

other drugs. For example, smokers are well known to have increased

metabolic activity of liver CYP 1A2, which results in the accelerated

metabolism of caffeine, theophylline, and other drugs (Dawson and

Vestal 1982). Earlier research had suggested that smokers metabolize

nicotine more rapidly than nonsmokers (Kyerematen et al. 1982,

1990). If true, this might be a significant factor in the natural history

of tobacco addiction as a mechanism of metabolic tolerance. That is,

the longer a person smoked, the faster nicotine would be metabolized;

therefore, one would have to smoke more to maintain a desired

nicotine level in the body.

The stable isotope technique described above was used to compare

nicotine kinetics in smokers and nonsmokers (Benowitz and Jacob

1993). Labeled (S)-(-)-nicotine was infused intravenously for 30

minutes, and blood and urine samples were collected for 96 hours.

Smokers and nonsmokers received the same low dose of nicotine (0.5

micrograms per kilogram per minute (g/kg/min)), and on another

day the smokers also received a higher dose of nicotine (2.0

g/kg/min) that resulted in plasma nicotine concentrations similar to

those they achieve with smoking. Nonsmokers are unable to tolerate

this dose due to toxicity.

As shown in figure 4, nicotine levels were similar in smokers and

nonsmokers. Pharmacokinetic analysis revealed that clearance was

slightly but significantly greater in nonsmokers than smokers, while

the steady-state volume of distribution and half-lives were similar

among groups (figure 5). These findings indicate that smokers do not

metabolize nicotine more rapidly than nonsmokers. In fact, the

reverse appears to be true; cigarette smoking appears to inhibit the

metabolism of nicotine. In any case, metabolic tolerance does not

appear to be a factor in the natural history of tobacco addiction.
54

INDIVIDUAL DIFFERENCES IN THE METABOLISM OF NICOTINE TO

COTININE

By simultaneously infusing labeled nicotine-d2 and cotinine-d4, and by

measuring levels of cotinine-d2 generated from nicotine-d2, the

fractional conversion of nicotine to cotinine can be determined

(Benowitz and Jacob 1994). An example of data generated by such a

study is shown in figures 6a and 6b. Using this approach in 20

smokers, it was determined that on average 72 percent of nicotine is

converted to cotinine (range 55 to 92 percent) (figure 7). No

differences in the clearances of nicotine or cotinine or the percentage

of nicotine conversion to cotinine were seen
55

when comparing data from men and women. As expected, clearances

did vary among individuals, with coefficients of variation of 25

percent and 27 percent for clearances of nicotine and cotinine,

respectively. The extent of individual variability and the percentage

of nicotine conversion to cotinine was less, with a coefficient of

variation of 12 percent.

Data on the fractional conversion of nicotine to cotinine and the

clearance of cotinine for an individual can be used to compute a factor

(K) that converts the steady-state plasma cotinine concentration to

the intake of nicotine from smoking per day. The equation is:

Dnic (mg/24h) = K x (plasma COT) (ng/mL)

On average, K = 0.08 with a range of 0.47 to 0.102. The K factor,

along with plasma cotinine levels, can be used to estimate daily intake

of nicotine from active or passive smoking.
56
57

Of note in the above study was the finding that the clearance of

nicotine and the fractional conversion of nicotine to cotinine were

significantly correlated (r = 0.59). This correlation suggests that

cotinine is the most rapid or rate-limiting pathway for nicotine

metabolism. Thus, people who metabolize nicotine via pathways

other than those to cotinine are likely to have slower elimination of

nicotine in general.

DEFICIENT C-OXIDATION OF NICOTINE

While most people metabolize nicotine extensively into cotinine, a

few individuals have been identified who generate very little cotinine.

One such person, a 57-year-old woman, was identified in a smoking

cessation trial. The subject was found to have unexpectedly low

plasma concentrations of cotinine, but normal concentrations of
58

nicotine (Benowitz et al. 1995b), both while smoking and while using

nicotine patches. This individual was studied using a dual infusion of

labeled nicotine and cotinine. As seen in figures 8a and 8b, little

cotinine was generated from nicotine. The subject was found to

convert only 9 percent of nicotine to cotinine, in contrast to the

average 72 percent seen in the study described previously (Benowitz

and Jacob 1994). This individual’s clearance of nicotine was unusually

low (6.5 mL/min/kg versus 17.2 mL/ min/kg in 20 controls), the halflife was abnormally long (348 versus 138 min), and the formation

clearance of cotinine for this individual was exceedingly low (0.4

mL/min/kg) versus that seen in controls (12.1 mL/min/kg). The

clearance and half-life of cotinine were, however, normal in this

subject.

Thus, an individual with markedly deficient C-oxidation of nicotine

has been identified. The liver enzymes responsible for C-oxidation of

nicotine have not been fully characterized. In vitro studies suggested

a role for CYP 2A6, 2D6, 2E1, and/or 2C9 (Cashman et al. 1992;

Flammang et al. 1992; McCracken et al. 1992). Cholerton and

colleagues (1994) reported five subjects with unusually high

nicotine/cotinine ratios in the urine after oral nicotine who were

genotypically homozygous for 2D6 mutations. They suggested that

2D6 is an important enzyme for nicotine metabolism. However, the

subject described above was a normal metabolizer of

dextromethorphan, and therefore a phenotypically normal

metabolizer via CYP 2D6. Studies are ongoing to identify which

enzymatic defects are responsible for deficient C-oxidation of

nicotine.

The biological significance of deficient C-oxidation of nicotine is

unclear, but could be considerable. Slow metabolizers of nicotine

such as this subject might be expected to smoke fewer cigarettes

and may be at less risk for smoking-related diseases linked to the

consumption of cigarette smoke. On the other hand, the long halflife of nicotine may mean that nicotine levels persist at higher levels

when the smoker is not smoking, and could lead to more severe

physical dependence. In addition, if cotinine has significant

biological activity that contributes to the pharmacologic effects of

nicotine, people who do not generate cotinine will experience a

different profile of pharmacologic effects from nicotine.
59
60

ETHNIC DIFFERENCES IN NICOTINE AND COTININE METABOLISM

Ethnic differences in nicotine metabolism have been hypothesized to

contribute to differences in health effects and/or susceptibility to

addiction in blacks versus whites (Henningfield et al. 1990). The

intriguing observation has been made that cotinine levels per cigarette

smoked were significantly higher in blacks versus whites

(Wagenknecht et al. 1990). In contrast, plasma levels of

thiocyanate, a marker of exposure to cigarette smoke in general, were

similar. There is also evidence that blacks have higher rates of lung

cancer for any given level of cigarette smoking compared with whites

(Satariona and Swanson 1988). Ethnic differences in the metabolism

of nicotine or cotinine could help explain these observations.

To examine this issue, dual-labeled nicotine and cotinine infusions

were administered to 40 black and 39 white smokers matched for age,

gender, and self-reported cigarette consumption (Benowitz et al.

1995a). The clearance of nicotine and percentage of nicotine

conversion to cotinine were similar for blacks and whites. However,

the clearance of cotinine was significantly slower (0.56 versus 0.69

mL/min/kg) and the half-life of cotinine slightly longer (1,064 versus

950 min) in blacks versus whites. These data clarify at least in part

the observation of higher cotinine levels when normalized for

cigarette consumption in blacks. The implications of differences in

cotinine metabolism regarding susceptibility to nicotine addiction or

health consequences of smoking are still unclear.

SUMMARY AND CONCLUSION

Individual differences in susceptibility to nicotine addiction, the

likelihood of successful smoking cessation, and the development of

adverse health effects of smoking are well recognized. The basis for

these individual differences is as yet unknown. This chapter examines

individual differences in the metabolism and kinetics of nicotine as a

possible factor.

Rare individuals appear to be deficient metabolizers of nicotine.

Individual differences are described both in the pattern and rates of

nicotine metabolism. Ethnic differences in cotinine metabolism have

also been observed. However, the enzymes responsible for nicotine

metabolism and their genetic regulation have not been fully

characterized. Understanding the basis for individual differences in

nicotine kinetics and metabolism, and linking these differences to
61

pharmacodynamic studies, may provide important clues for the

prevention and treatment of nicotine and possibly other drug

addictions.

REFERENCES

Benowitz, N.L. The genetics of drug dependence: Tobacco addiction. N

Engl J Med 327:881-883, 1992.

Benowitz, N.L. Human pharmacology of nicotine and its metabolites.

Supplement. J Smoking Related Dis 5:129-133, 1994.

Benowitz, N.L., and Jacob, P., III. Nicotine renal excretion rate influences

nicotine intake during cigarette smoking. J Pharmacol

Exp Ther 234:153-155, 1985.

Benowitz, and Jacob, P., III. Nicotine and cotinine elimination

pharmacokinetics in smokers and nonsmokers. Clin

Pharmacol Ther 53:316-323, 1993.

Benowitz, N.L., and Jacob, P., III. Metabolism of nicotine to cotinine

studied by a dual stable isotope method. Clin Pharmacol

Ther 56:483-493, 1994.

Benowitz, N.L.; Jacob, P., III; Fong, I.; and Gutta, S. Nicotine metabolic

profile in man: Comparison of cigarette smoking and

transdermal nicotine. J Pharmacol Exp Ther 268:296-

303, 1994.

Benowitz, N.L.; Jacob, P., III; Jones, R.T.; and Rosenberg, J.

Interindividual variability in the metabolism and

cardiovascular effects of nicotine in man. J Pharmacol

Exp Ther 221:368-372, 1982.

Benowitz, N.L.; Perez-Stable, E.; Herrera, B.; and Jacob, P., III. African

American-Caucasian differences in nicotine and cotinine

metabolism (Abstract). Clin Pharmacol Ther 57:159,

1995a.

Benowitz, N.L.; Sachs, D.; and Jacob, P., III. Deficient C-oxidation of

nicotine. Clin Pharmacol Ther 57:590-594, 1995b.

Byrd, G.D.; Chang, K.; Greene, J.M.; and deBethizy, J.D. Evidence for

urinary excretion of glucuronide conjugates of nicotine,

cotinine, and trans-3'hydroxycotinine in smokers. Drug

Metab Dispos 20:192-197, 1992.

Carmelli, D.; Swan, G.E.; Robinette, D.; and Fabsitz, R. Genetic influence

on smoking—A study of male twins. N Engl J Med

237:829-833, 1992.
62

Cashman, J.R.; Park, S.B.; Yang, Z.C.; Wrighton, S.A.; Jacob, P., III; and

Benowitz, N.L. Metabolism of nicotine by human liver

microsomes: Stereoselective formation of trans-nicotineN’-oxide. Chem Res Toxicol 5:639-646, 1992.

Cholerton, S.; Arpanahi, A.; McCracken, N.; Boustead, C.; Taber, H.;

Johnstone, E.; Leathart, J.; Daly, A.K.; and Idle, J.R. Poor

metabolisers of nicotine and CYP 2D5 polymorphism.

Lancet 343:52-63, 1994.

Dawson, G.W., and Vestal, R.E. Smoking and drug metabolism. Pharmacol

Ther 15:207-221, 1982.

Department of Health and Human Services, Public Health Service.

Preventing Tobacco Use Among Young People. A Report

of the Surgeon General. Washington, DC: Supt. of Docs.,

U.S. Govt. Print. Off., 1994.

Escobedo, L.G.; Marcus, S.E.; Holtzman, D.; and Giovino, G.A. Sports

participation, age at smoking initiation, and the risk of

smoking among U.S. high school students. JAMA

269:1391-1395, 1993.

Flammang, A.M.; Gelboin, H.V.; Aoyama, T.; Gonzalez, F.J.; and McCoy,

G.D. Nicotine metabolism by cDNA-expressed human

cytochrome P-450s. Biochem Arch 8:1-8, 1992.

Henningfield, J.E.; Cohen, C.; and Giovino, G.A. Can genetic constitution

affect the ‘objective’ diagnosis of nicotine dependence?

Am J Public Health 80:1040-1041, 1990.

Jacob, P., III; Yu, L.; Wilson, M.; and Benowitz, N.L. Selected ion

monitoring method for determination of nicotine,

cotinine, and deuterium-labeled analogs. Absence of an

isotope effect in the clearance of (S)-nicotine-3'3'-d2 in

humans. Biol Mass Spectrometry 20:247-252, 1991.

Keenan, R.M.; Hatsukami, D.K.; Pental, P.R.; Thompson, T.; and Grillo,

M.A. Pharmacodynamic effects of cotinine in abstinent

cigarette smokers. Clin Pharmacol Ther 55:581-590,

1994.

Kyerematen, G.A.; Damiano, M.D.; Dvorchik, B.H.; and Vesell, E.S.

Smoking-induced changes in nicotine disposition:

Application of a new HPLC assay for nicotine and its

metabolites. Clin Pharmacol Ther 32:769-780, 1982.

Kyerematen, G.A.; Morgan, M.L.; Chattopadhyay, B.; deBethizy, J.D.; and

Yesell, E.S. Disposition of nicotine and eight metabolites

in smokers and nonsmokers: Identification in smokers of

two metabolites that are longer lived than cotinine. Clin

Pharmacol Ther 48:641-651, 1990.
63

Marks, M.J.; Campbell, S.M.; Romm, E.; and Collins, A.C. Genotype

influences the development of tolerance to nicotine in the

mouse. J Pharmacol Exp Ther 259:392-402, 1991.

McCracken, N.W.; Cholerton, S.; and Idle, J.R. Cotinine formation by

cDNA-expressed human cytochromes P450. Med Sci Res

20:877-878, 1992.

Peto, R.; Lopez, A.D.; Boreham, J.; Thun, M.; and Heath, C., Jr. Mortality

from tobacco in developed countries: Indirect estimation

from national vital statistics. Lancet 339:1268-1278,

1992.

Risner, M.E.; Cone, E.J.; Benowitz, N.L.; and Jacob, P., III. Effects of the

stereoisomer of nicotine and nornicotine on schedulecontrolled responding and physiological parameters of

dogs. J Pharmacol Exp Ther 244:807-813, 1988.

Rosenberg, J.; Benowitz, N.L.; Jacob, P., III; and Wilson, K.M. Disposition

kinetics and effects of intravenous nicotine. Clin

Pharmacol Ther 28:516-522, 1980.

Satariona, W.A., and Swanson, G.M. Racial differences in cancer incidence:

The significance of age-specific patterns. Cancer

62:2640-2653, 1988.

Shigenaga, M.K.; Kim, B.H.; Caldera-Munoz, P.; Cairns, T.; Jacob, P., III;

Trevor, A.J.; and Castagnoli, N., Jr. Liver and lung

microsomal metabolism of the tobacco alkaloid -

nicotyrine. Chem Res Toxicol 2:282-287, 1989.

Shigenaga, M.K.; Trevor, A.J.; and Castagnoli, N., Jr. Metabolism

dependent covalent binding of (S)-[5'-3H]-nicotine to

liver and lung microsomal macromolecules. Drug Metab

Disp 16:397-402, 1988.

Swan, G.E.; Carmelli, D.; Rosenman, R.H.; Fabsitz, R.R.; and Christian, J.C.

Smoking and alcohol consumption in adult male twins:

Genetic heritability and shared environmental influences.

J Subst Abuse 2:39-50, 1990.

Wagenknecht, L.E.; Cutter, G.R.; Haley, N.J.; Sidney, S.; Manolio, T.A.;

Hughes, G.H.; and Jacobs, D.R. Racial differences in serum

cotinine levels among smokers in the coronary artery risk

development in (young) adults study. Am J Public Health

80:1053-1056, 1990.

ACKNOWLEDGMENT

This chapter was prepared with support from the National Institute

on Drug Abuse grants DA02277 and DA01696. Research described
64

herein was conducted at the General Clinical Research Center of San

Francisco General Hospital with support of NIH grant RR-00083.

AUTHORS

Neal L. Benowitz, M.D.

Professor of Medicine

Division of Clinical Pharmacology and Experimental

 Therapeutics

and

Departments of Medicine and Psychiatry

University of California, San Francisco

San Francisco General Hospital

Building 30, Room 3220

1001 Potrero Avenue

San Francisco, CA 94110

Peyton Jacob III, Ph.D.

Research Chemist

Departments of Medicine and Psychiatry

University of California, San Francisco

San Francisco General Hospital

Building 100, Room 235

1001 Potrero Avenue

San Francisco, CA 94110
65

Inhibitors of Anandamide Breakdown

Dale G. Deutsch and Alexandros Makriyannis

INTRODUCTION

9

-Tetrahydrocannabinol (THC), the psychoactive marijuana

plant- derived cannabinoid, and numerous synthetic derivatives have

been shown to bind to a specific brain receptor, cannabinoid receptor

1 (CB1) (Howlett et al. 1990; Matsuda et al. 1990; Herkenham et al.

1990; Mailleux and Vanderhagen 1992). Arachidonoyl ethanolamide

(anandamide), homo--linolenyl ethanolamide, and docosatetraenyl

ethanolamide are naturally occurring brain constituents that bind to

CB1 and as a class are called the anandamides (Mechoulam et al.

1994; Devane et al. 1992; Hanus et al. 1993; Felder et al. 1993;

Devane 1994). Anandamide behaves as a cannabimimetic compound

in vitro, stimulating receptor-mediated signal transduction that leads

to the inhibition of forskolin-stimulated adenylate cyclase (Vogel et

al. 1993; Childers et al. 1993)1. In a neuroblastoma cell line,

anandamide causes partial inhibition of N-type calcium currents via a

pertussis toxin-sensitive guanosine triphosphate binding protein (Gprotein) pathway, independently of cyclic adenosine monophosphate

(cAMP) metabolism (Mackie et al. 1993). Using a series of

behavioral tests to evaluate cannabinoid analogs, anandamide has been

shown to be a cannabinoid receptor agonist exhibiting

pharmacological activity in mice parallel to that of other

psychotropic cannabinoids (Fride and Mechoulam, 1993; Crawley et

al. 1993; Smith et al. 1994; Abadji et al. 1994).

Soon after the discovery of anandamide, enzymatic activities

responsible for its degradation (an amidase, also called an

amidohydrolase) and synthesis (synthase) were described (DiMarzo et

al. 1994; Desarnaud et al. 1995; Ueda et al. 1995; Deutsch and Chin

1993; Kruszka and Gross 1994; Devane and Axelrod 1994).

Phenylmethylsulfonyl fluoride (PMSF) was discovered to be a potent

inhibitor of the enzymatic breakdown of arachidonoylethanolamide

(Deutsch and Chin 1993). A series of anandamide analogs was

synthesized (trifluoromethyl ketone, -keto ester, and -keto amide

derivatives) and tested in vitro and in intact cells as amidase inhibitors

(Koutek et al. 1994). The trifluoromethyl ketones (e.g.,

arachidonyltrifluoromethyl ketone) were found to be potent

inhibitors in the low micromolar (M) range. Most recently, a

potent irreversible inhibitor of anandamide hydrolysis (AM 374) has
66

been synthesized and found to be effective in the low nanomolar (nM)

range.

ANANDAMIDE SYNTHESIS

An enzymatic activity has been identified thatcatalyzes the synthesis

of arachidonoylethanolamide (Deutsch and Chin 1993) as shown

below:

Arachidonic Acid + Ethanolamine _ Arachidonoyl Ethanolamide +

H2O

This catalytic activity for the biosynthesis of anandamide requires

ethanolamine and arachidonic acid and is readily detected in

incubations of rat brain homogenates. When [3H]-arachidonic acid

was employed as the label, addition of exogenous ethanolamine was

necessary to observe anandamide synthesis, indicating that

ethanolamine is limiting in the brain homogenate. It was found that

anandamide synthesis increased with increasing amounts of

ethanolamine so that 35 percent of the [3H]-arachidonate counts

were incorporated into anandamide with 7 millimolar (mM) unlabeled

ethanolamine (Deutsch and Chin 1993). (Subsequently, one of the

authors (DGD) found that synthesis may be observed with as little as

0.1 mM ethanolamine.) When labeled ethanolamine was employed,

the apparent increased rate of anandamide synthesis, in the presence

of PMSF, was an artifact caused by the reaction of PMSF with

ethanolamine to form the corresponding ethanolamide, which had the

same mobility on the thin layer chromatography (TLC) plate as

anandamide (W. Devane, personal communication, July 1994).

Interestingly, Devane and Axelrod (1994) observed that PMSF was a

potent inhibitor of the synthase. Recently, it was postulated that the

amidase and synthase are the same enzyme. This is based upon the

observation that the amidase and synthase activities

cochromatograph during purification and that they exhibit the same

behavior towards inhibitors, in their pH dependence, and in their heat

inactivation profiles (Ueda et al. 1995). Synthesis of anandamide has

also been demonstrated in subcellular fractions. The highest activity

occurred in the synaptic vesicles, myelin, and microsomal and

synaptosomal membranes (Devane and Axelrod 1994). The reaction

was selective for arachidonic acid as the aliphatic constituent and

ethanolamine as the polar moiety. Furthermore, it was clearly

demonstrated that this reaction occurs through an adenosine

triphosphate (ATP)- and CoA-independent process (Kruszka and
67

Gross 1994). It is interesting to note that Bachur and Udenfriend

(1966) described a rat liver microsomal system utilizing aliphatic

fatty acids, but not arachidonate, and several amines as substrates for

the synthesis of fatty acid amides.

Burstein and Hunter (1995) observed that THC stimulated the

biosynthesis of anandamide in neuroblastoma cells employing either

ethanolamine or arachidonic acid as the label. Anandamide biosynthesis

has also been shown to occur in primary cultures of rat brain neurons

labelled with 3[H]-ethanolamine when stimulated with ionomycin, a Ca++

ionophore (Di Marzo et al. 1994). These authors proposed an alternate

model for the biosynthesis of anandamide in which N-arachidonoyl

phosphatidyl ethanolamine is cleaved by a phospholipase D activity to

yield phosphatidic acid and ararchidonoylethanolamide. This model is

based upon extensive studies undertaken by Schmid and collaborators

(1990), who have shown that fatty acid ethanolamide formation results

from the N-acylation of phosphatidyl ethanolamine by a transacylase to

form N-acyl phosphatidylethanolamine. Possibly resulting from

postmortem changes, this compound is subsequently hydrolyzed to the

fatty acid ethanolamide and the corresponding phosphatide by a

phosphodiesterase, phospholipase D.

ANANDAMIDE AMIDASE

The reaction for the degradation of anandamide to arachidonic acid and

ethanolamine is shown below.

Arachidonoyl Ethanolamide + H2O _ Arachidonic Acid + Ethanolamine

The distribution of this amidase activity was characterized in tissues from

the rat using a TLC assay with [arachidonoyl 5,6,8,9,11,12,14,15- 3H]-

ethanolamide as the substrate (Deutsch and Chin 1993). This enzymatic

reaction was expressed in homogenates from brain, liver, kidney, and

lung (figure 1). Under these conditions, the main degradation product

detected was arachidonate. Barely detectable activity was present in

homogenates of rat heart and skeletal muscle (Deutsch and Chin 1993;

Desarnaud et al. 1995). In addition to the main arachidonic acid

degradation product, lung and liver produced an unknown second

catabolite that may be an oxidized form of anandamide. Recently, it has

been shown that anandamide may be hydroxylated by the cytochrome

P450s (Bornheim et al. 1993) and this may account, in part, for the

additional metabolite. When the ethanolamides of a series of fatty
68
69

acids was tested as substrates for the amidase, the highest substrate

specificity was for arachidonoyl ethanolamide (Desarnaud et al. 1995;

Ueda et al. 1995).

CELL CULTURE EXPERIMENTS AND ANANDAMIDE AMIDASE

When 3[H]- anandamide ([arachidonoyl 5,6,8,9,11,12,14,15- 3H]-

ethanolamide) was incubated with neuroblastoma (N18TG2) or glioma

(C6) cells, there was a time-dependent decrease of its levels in the

media (Deutsch and Chin 1993). Anandamide was taken up by the

cells immediately, but it did not accumulate since it was converted to

arachidonate and other lipids containing arachidonate that migrate on

TLC near the phospholipids, triglycerides, and cholesterol esters

(figure 2). After 1 hour of incubation, only about 1 percent of the

total radioactivity detected in all the cell fractions was from

anandamide. Arachidonylethanolamide amidase was not expressed in

all cell lines. Activity was found in neuroblastoma, glioma, and

nonsmall-cell lung carcinoma cells, but not in HeLa cells, larynx

epidermoid carcinoma (Hep2), and hepatocellular carcinoma (HepG2)

cells (data not shown). The uptake and degradation of anandamide

was recently confirmed in primary cultures of rat brain (Di Marzo et

al. 1994).

SUBCELLULAR FRACTIONATION AND BRAIN LOCALIZATION

When the neuroblastoma and glioma cells were fractionated into

membrane and soluble fractions, the enzymatic activity for the

degradation of anandamide resided mainly in the mitochondrial and

microsomal membrane fractions (figure 3). Within the central

nervous system, the distribution of the amidase activity correlated

well with the distribution of the cannabinoid receptor. The highest

activity was found in the globus pallidus, hippocampus, substantia

nigra, cerebral cortex, and cerebellum, and the lowest activity was

found in the brain stem and medulla where cannabinoid receptors are

sparse (Desarnaud et al. 1995; Hillard et al. 1995).
70

A RAPID ASSAY FOR ANANDAMIDE AMIDASE

The assays (see above) for arachidonoyl ethanolamide hydrolysis

(anandamide amidase) employed 3H-anandamide as the substrate
71
72

(Deutsch and Chin 1993; Koutek et al. 1994). Using this assay, a

TLC step is required to separate labeled arachidonic acid product from

anandamide substrate. A new assay employing arachidonoyl

ethanolamide labeled in the ethanolamine portion of anandamide

(arachidonoyl ethanolamide-[1,2-14C]) has recently been developed

which obviates the need for the TLC step (Omeir et al. 1995). After

incubation of the enzyme with this substrate, the reaction mixture is

stopped by the addition of organic solvent. The radiolabeled

anandamide substrate partitions into the organic phase while the

product (ethanolamine- [1,2-14C]) conveniently partitions into the

aqueous phase which is subsequently measured by liquid scintillation

counting. Employing this assay, it was found that the reaction

proceeded linearly for at least 30 minutes. Anan-damide amidase

exhibits maximal activity between pH 8 and pH 9 with a steep decline

in activity at pH values below pH 6 and above pH 10. Arachidonoyl

ethanolamide-[1,2-14C] was found to be a good substrate to assay the

amount of anandamide amidase from 10 to approximately 100

micrograms (g) of protein in brain homogenate (figure 4). Under

the conditions of this assay, less than 10 percent of the substrate is

hydrolyzed. Defatted serum albumin is included in the assays to bind

the fatty acid product, thus preventing product inhibition. The results

of a representative experiment for the initial velocity of anandamide

degradation versus anandamide substrate concentration, when plotted

as a rectangular hyperbole of the steady-state Michaelis-Menten

equation, yields a Michaelis constant (Km) of 30 Å 7 M and a

maximal velocity (Vmax) of 198 Å 13 nanomoles (nmoles)

ethanolamine formed per hour per mg protein homogenate. The Km

and Vmax values calculated from the saturating hyperbola must be

considered approximate in view of the fact that the interfacial

enzyme reaction occurs in an impure preparation, whose substrate and

product have the potential to form micelles, which in turn may affect

the enzyme activity. Recently Hilliard and colleagues (1995),

Desarnaud and colleagues (1995), and Ueda and colleagues (1995)

reported values of Km and Vmax for the hydrolysis of anandamine as

follow: Km 3.4, 12.7, and 60M and V max 0.132, 0.337, and 28.8

moles/hr/mg protein, respectively.

The enzyme showed some specificity when two chiral

methanandamide stereoisomers (Abadji et al. 1994) were tested as

competitors of anandamide (figure 5). At low concentration the (R)-

methanandamide stereoisomer did not affect the enzyme activity,

while the (S)-meth-anandamide isomer interacted weakly. At higher

concentrations this effect disappeared, with both compounds giving

some inhibition of
73

the anandamide-amidase reaction. These results may be interpreted in

terms of one stereoisomer (at low concentrations) being more

effective at the substrate binding site on the enzyme.

Interestingly, the enzymatic activities found for anandamide amidase

may be related to those reported in the literature for the hydrolysis of

other fatty acid amides. Bachur and Udenfriend (1966) and Schmid

and colleagues (1990) described a rat liver microsomal enzyme that

hydrolyzed fatty acid ethanolamides. Natarajan and colleagues (1984)

described activity in a dog brain microsomal fraction that hydrolyzed
74

palmitoylethanolamide with an apparent Km of 53 M and a V max of

667 nmole/h/mg.

PUTATIVE TRANSITION-STATE INHIBITORS OF ANANDAMIDE

AMIDASE

As shown in figure 3, PMSF was discovered to be a potent inhibitor of

the enzymatic breakdown of arachidonoylethanolamide in

neuroblastoma and glioma cellular fractions (Deutsch and Chin 1993).

It was also found that 1.5 mM PMSF (which was originally added to

prevent proteolytic degradation of the enzymes in the subcellular

fractions) completely abolished the amidase activity in rat brain and

other tissue homogenates. This is consistent with the observation

that inclusion of PMSF in receptor binding assays increased the

apparent potency of anandamide and cogeners susceptible to the

amidase (Abadji et al. 1994; Childers et al. 1994). The anandamide

amidase activity was not inhibited by aprotinin, benzamidine,

leupeptin, chymostatin, or pepstatin, suggesting that it is distinct

from some of these common proteases that are susceptible to the

tested inhibitors (Deutsch and Chin 1993). The amidohydrolase did

not hydrolyze substrates for plasmin, aminopeptidase, elastase, or

chymotrypsin (Ueda et al. 1995). The detailed mechanism by which

PMSF inhibited this activity remains to be elucidated, although PMSF

is known to inhibit serine proteases, some thiol proteases, and

nonprotease enzymes such as erythrocyte acetylcholinesterease.

Based on the behavior of anandamide with receptor preparations in

the presence of PMSF, Childers and colleagues (1994) postulated that

the enzyme mechanism may involve an active-site serine hydroxyl.

To further explore the question of the mechanism of anandamide

amidase inhibition, analogs of anandamide were synthesized (Koutek

et al. 1994). These anandamide analogs (figure 6) represent three

classes of putative transition-state inhibitors: trifluoromethyl ketone

(4), -keto ester (3), and -keto amide derivatives (2). The general

strategy of this study was based upon the hypothesis that polarized

carbonyls, such as those in trifluoromethyl ketones and -keto

carboxylate derivatives, may form stabilized hydrates or enzyme

adducts that mimic the tetrahedral
75
76

intermediates formed during the reaction between the nucleophilic

residue (e.g., an active-site serine hydroxyl in the hydrolytic enzyme)

and the carbonyl group of anandamide (figure 7). They were tested

for inhibition of anandamide hydrolysis in vitro, in cell culture, and as

ligands for CB1 (Koutek et al. 1994). When tested for their ability to

inhibit the hydrolysis of anandamide in vitro, the most effective

classes of compounds were the trifluoromethyl ketones (4a-e) and -

keto esters (3a-c). The trifluoromethyl ketones and -keto esters

showed nearly 100 percent inhibition of anandamide hydrolysis in

vitro in the presence of 7.5 M inhibitor and 27.7 M anandamide.

Arachidonyl trifluoromethyl ketone (4a) and ethyl 2-oxostearate (3b)

were the most active members of these groups, yielding nearly 100

percent inhibition of the enzyme. The inhibition of anandamide

amidase by arachidonyl trifluoromethyl ketone was reversible with

increasing concentrations of anandamide. Arachidonyl

trifluoromethyl ketone is also a potent inhibitor of the synthase

(Ueda et al. 1995). The least potent inhibitors were the -keto

amides (2a-c) and the saturated analogs of anandamide (1b-d). When

incubated with neuroblastoma (N18TG2) cells, anandamide is taken up

by the cells and rapidly hydrolyzed to arachidonate, which is then

incorporated into other lipids containing arachidonate (figure 8).

However, in the presence of arachidonyl trifluoromethyl ketone (4a),

there is an approximately fivefold increase of anandamide levels at

7.8 M arachidonyl trifluoromethyl ketone (figure 8). The amount

of anandamide in the experimental cells increases to a twelvefold

maximum, relative to the control cells, at approximately 12 M

arachidonyl trifluoromethyl ketone (figure 8, inset). The mechanism

apparently involves inhibition of the amidase rather than increased

uptake of anandamide, since preloading the cells with labeled

anandamide and then treating with 4a also resulted in a dramatic

increase in anandamide levels in the cells.

The series of fatty acid derivatives was each tested, at 10 M, for

their ability to displace [3H]CP-55940 ([1, 2 ( R), 5] - (-) - (1,1-

dimethyl- heptyl) -2 - [5-hydroxypropylcyclohexyl] - phenol))

binding to the THC receptor in rat brain membranes (CB1).

Arachidonoyl trifluoromethyl ketone was the only synthetic

compound in this series of fatty acid derivatives to significantly

displace [3H]CP-55940 binding to CB1 with an inhibition constant

(Ki) of 0.65 M. This represents approximately a fortyfold lower

affinity from that of arachidonoyl ethanolamide.

Arachidonyl trifluoromethyl ketone was reported to be a slow tightbinding inhibitor of a novel 85 kilodalton-(kDa) cytosolic human
77

phospholipase A2 (Street et al. 1993; Trimble et al. 1993). Some -

keto acid derivatives have been shown to act as inhibitors of serine

and cysteine proteinases (Ocain et al. 1992; Peet et al. 1990), and

effective inhibition of cathepsin B and papain by peptidyl -keto

esters, -keto amides, -diketones, and -keto acids has also been

demonstrated (Hu and Abeles 1990). The disparity between the

activity of ethyl 2-oxo-stearate 3b in vitro and in cell culture may be

due to its susceptibility to enzymatic degradation in cell culture. It is

not known if arachidonyl trifluoromethyl ketone is metabolized in

the neuroblastoma cells employed in this study, but when incubated at

10 M with monocytic cells in culture for 10 min, 10 percent is

converted to the corresponding alcohol (Riendeau et al. 1994).
78
79
80

A POTENT IRREVERSIBLE INHIBITOR OF ANANDAMIDE AMIDASE

Very recently the authors developed a novel anandamide amidase

inhibitor, AM374, whose potency in vitro and in neuroblastoma cells

significantly exceeds that of other compounds developed to date as

well as PMSF. In intact neuroblastoma cells, AM374 was found to

dramatically increase the level of undegraded anandamide 55-fold at

10 nM. Interestingly, its affinity for the CB1 receptor was

approximately tenfold weaker than anandamide (Deutsch and

Makriyannis, unpublished data).

Ideally, a selective amidase inhibitor should antagonize the enzyme at

concentrations that fail to appreciably bind to cannabinoid receptors.

Furthermore, unlike PMSF, an inhibitor should not be toxic to the

cells. Many of the synthetic compounds in this study fulfill these

criteria; they do not bind significantly to CB1 at concentrations that

inhibit amidase activity by greater than 90 percent in cell-free

preparations and appear to have low toxicity toward the cells. The

role that these inhibitors play in different tissues such as spleen, where

a peripheral receptor (CB2) exists (Munro et al. 1993), or as

inhibitors of the cytosolic phospholipase A2 in brain and N18TG2

cells, remains to be elucidated. Furthermore, the most successful

inhibitors will be subjected to in vivo testing. The development of

inhibitors that block the breakdown of anandamide may be of value in

any of the therapeutic applications in which THC (Mechoulam 1986)

or anandamide (Crawley et al. 1993; Fride and Mechoulam 1993;

Smith et al. 1994) has been shown to be potentially useful including

analgesia, mood elevation, nausea, appetite, sedation, locomotion,

glaucoma, and immune function.

REFERENCES

Abadji, V.; Lin, S.; Taha, G.; Griffin, G.; Stevenson, L.A.; Pertwee, R.G.;

and Makriyannis, A. (R)-methanandamide: A chiral novel

anandamide possessing higher potency and metabolic

stability. J Med Chem 37(12):1889-93, 1994.

Bachur, N.R., and Udenfriend, S. Microsomal synthesis of fatty acid

amides. J Biol Chem 241(6):1308-13, 1966.

Bornheim, L.M.; Kim, K.Y.; Chen, B.; and Correia, M.A. The effect of

cannabidiol on mouse hepatic microsomal cytochrome

P450-dependent anandamide metabolism. Biochem

Biophys Res Comm 197(2):740-6, 1993.
81

Burstein, S.H., and Hunter, S.A. Anandamide biosynthesis in N-18TG2

neuroblastoma cells is stimulated by THC. Biochem

Pharmacol 49(6):855-858, 1995.

Childers, S.R.; Sexton, T.; and Roy, M.B. Effects of anandamide on

cannabinoid receptors in rat brain membranes. Biochem

Pharmacol 47(4):711-5, 1994.

Childers, S.R.; Pacheco, M.A.; Bennett, B.A.; Edwards, T.A.; Hampson,

R.E.; Mu, J.; and Deadwyler, S.A. Cannabinoid receptors:

G-protein-mediated signal transduction mechanisms.

Biochem Soc Symposia 59:27-50, 1993.

Crawley, J.N.; Corwin, R.L.; Robinson, J.K.; Felder, C.C.; Devane, W.A.;

and Axelrod, J. Anandamide, an endogenous ligand of the

cannabinoid receptor, induces hypomotility and

hypothermia in vivo in rodents. Pharmacol Biochem

Behav 46(4):967-72, 1993.

Desarnaud, F.; Cadas, H.; and Piomelli, D. Anandamide amidohydrolase

activity in rat brain microsomes. J Biol Chem

279(11):6030-6035, 1995.

Deutsch, D.G., and Chin, S.A. Enzymatic synthesis and degradation of

anandamide, a cannabinoid receptor agonist. Biochem

Pharmacol 46(5):791-6, 1993.

Devane, W.A. New dawn of cannabinoid pharmacology. Trend Pharmacol

Sci 15(2):40-1, 1994.

Devane, W.A., and Axelrod, J. Enzymatic synthesis of anandamide, an

endogenous ligand for the cannabinoid receptor, by brain

membranes. Proc Nat Acad Sci U S A 91(14):6698-701,

1994.

Devane, W.A.; Hanus, L.; Breuer, A.; Pertwee, R.G.; Stevenson, L.A.;

Griffin, G.; Gibson, D.; Mandelbaum, A.; Etinger, A.; and

Mechoulam, R. Isolation and structure of a brain

constituent that binds to the cannabinoid receptor.

Science 258(5090):1946-1949, 1992.

Di Marzo, V.; Fontana, A.; Cadas, H.; Schinelli, G.C.; Schwarz, J.; and

Piomelli, D. Formation and inactivation of endogenous

cannabinoid anandamide in central neurons. Nature

372:686-91, 1994.

Felder, C.C.; Briley, E.M.; Axelrod, J.; Simpson, J.T.; Mackie, K.; and

Devane, W.A. Anandamide, an endogenous

cannabimimetic eicosanoid, binds to the cloned human

cannabinoid receptor and stimulates receptor-mediated

signal transduction. Proc Nat Acad Sci U S A

90(16):7656-7660, 1993.
82

Fride, E., and Mechoulam, R. Pharmacological activity of the cannabinoid

receptor agonist, anandamide, a brain constituent. Eur J

Pharmacol 231(2):313-4, 1993.

Hanus, L.; Gopher, A.; Almog, S.; and Mechoulam, R. Two new

unsaturated fatty acid ethanolamides in brain that bind to

the cannabinoid receptor. J Med Chem 36(20):3032-

3034, 1993.

Herkenham, M.; Lynn, A.B.; Little, M.D.; Johnson, M.R.; Melvin, L.S.; de

Costa, B.R.; and Rice, K.C. Cannabinoid receptor

localization in brain. Proc Nat Acad Sci U S A

87(5):1932-1936, 1990.

Hillard, C.J.; Wilkison, D.M.; Edgemond, W.S.; and Campbell, W.B.

Characterization of the kinetics and distribution of Narachidony-lethanolamine (anandamide) hydrolysis by rat

brain. Biochem Biophy Acta 1257:249-256, 1995.

Howlett, A.C.; Bidaut-Russell, M.; Devane, W.A.; Melvin, L.S.; Johnson,

M.R.; and Herkenham, M. The cannabinoid receptor:

Biochemical, anatomical and behavioral characterization.

Trend Neurosci 13(10):420-423, 1990.

Hu, L.Y., and Abeles, R.H. Inhibition of cathepsin B and papain by

peptidyl alpha-keto esters, alpha-keto amides, alphadiketones, and alpha-keto acids. Arch Biochem Biophys

281(2):271-274, 1990.

Koutek, B.; Prestwich, G.D.; Howlett, A.C.; Chin, S.A.; Salehani, D.;

Akhavan, N.; and Deutsch, D.G. Inhibitors of

arachidonoyl ethanolamide hydrolysis. J Biol Chem

269(37):22937-22940, 1994.

Kruszka, K.K., and Gross, R.W. The ATP- and CoA-independent synthesis

of arachidonoylethanolamide. A novel mechanism

underlying the synthesis of the endogenous ligand of the

cannabinoid receptor. J Biol Chem 269(20):14345-

14348, 1994.

Mackie, K.; Devane, W.A.; and Hille, B. Anandamide, an endogenous

cannabinoid, inhibits calcium currents as a partial agonist

in N18 neuroblastoma cells. Mol Pharmacol 44(3):498-

503, 1993.

Mailleux, P., and Vanderhaeghen, J.J. Distribution of neuronal cannabinoid

receptor in the adult rat brain: A comparative receptor

binding radioautography and in situ hybridization

histochemistry. Neuroscience 48(3):655-668, 1992.

Matsuda, L.A.; Lolait, S.J.; Brownstein, M.J.; Young, A.C.; and Bonner,

T.I. Structure of a cannabinoid receptor and functional

expression of the cloned cDNA. Nature 346(6284):561-

564, 1990.
83

Mechoulam, R., ed. Cannabinoids as Therapeutic Agents. Boca Raton, FL:

CRC Press, Inc., 1986. pp. 1-186.

Mechoulam, R.; Hanus, L.; and Martin, B.R. Search for endogenous ligands

of the cannabinoid receptor. Biochem Pharmacol

48(8):1537-1544, 1994.

Munro, S.; Thomas, K.L.; and Abu-Shaar, M. Molecular characterization

of a peripheral receptor for cannabinoids. Nature

365(6441):61-65, 1993.

Natarajan, V.; Schmid, P.C.; Reddy, P.V.; and Schmid, H.H. Catabolism of

N-acylethanolamine phospholipids by dog brain

preparations. J Neurochem 42(6):1613-1619, 1984.

Ocain, T.D., and Rich, D.H. Alpha-Keto amide inhibitors of

aminopeptidases. J Med Chem 35(3):451-456, 1992.

Omeir, R.L.; Chin, S.; Hong, Y.; Ahern, D.G.; and Deutsch, D.G.

Arachidonyl ethanolamide-[1,2-14C] as a substrate for

anandamide amidase. Life Sci 56(23/24):1999-2005,

1995.

Peet, N.P.; Burkhart, J.P.; Angelastro, M.R.; Giroux, E.L.; Mehdi, S.; Bey,

P.; Kolb, M.; Neises, B.; and Schirlin, D. Synthesis of

peptidyl fluoromethyl ketones and peptidyl alpha-keto

esters as inhibitors of porcine pancreatic elastase, human

neutrophil elastase, and rat and human neutrophil

cathepsin G. J Med Chem 33(1):394-407, 1990.

Riendeau, D.; Guay, J.; Weech, P.K.; Laliberte, F.; Yergey, C.I.; Desmarais,

S.; Perrier, H.; Liu, S.; Nicoll-Griffith, D.; and Street, I.P.

Arachidonyl trifluoromethyl ketone, a potent inhibitor of

85-kDa phospholipase A2, blocks production of

arachidonate and 12-hydroxyeicosatetraenoic acid by

calcium ionophore-challenged platelets. J Biol Chem

269(22):15619-15624, 1994.

Schmid, H.H.; Schmid, P.C.; and Natarajan, V. N-acylated

glycerophospholipids and their derivatives. [Review].

Prog Lipid Res 29(1):1-43, 1990.

Smith, P.B.; Compton, D.R.; Welch, S.P.; Razdan, R.K.; Mechoulam, R.;

and Martin, B.R. The pharmacological activity of

anandamide, a putative endogenous cannabinoid, in mice.

J Pharmacol Exp Ther 270(1):219-227, 1994.

Street, I.P.; Lin, H.K.; Laliberte, F.; Ghomashchi, F.; Wang, Z.; Perrier,

H.; Tremblay, N.M.; Huang, Z.; Weech, P.K.; and Gelb,

M.H. Slow- and tight-binding inhibitors of the 85-kDa

human phospholipase A2. Biochemistry 32(23):5935-

5940, 1993.

Trimble, L.A.; Street, I.P.; Perrier, H.; Tremblay, N.M.; Weech, P.K.; and

Bernstein, M.A. NMR structural studies of the tight
84

complex between a trifluoromethyl ketone inhibitor and

the 85-kDa human phospholipase A2. Biochemistry

32(47):12560-12565, 1993.

Ueda, N.; Kurahashi, Y.U.; and Tokunaga, T. Partial purification and

characterization of the porcine enzyme brain hydrolyzing

and synthesizing anandamide. J Biol Chem

270(40):23823-23827, 1995.

Vogel, Z.; Barg, J.; Levy, R.; Saya, D.; Heldman, E.; and Mechoulam, R.

Anandamide, a brain endogenous compound, interacts

specifically with cannabinoid receptors and inhibits

adenylate cyclase. J Neurochem 61(1):352-325, 1993.

ACKNOWLEDGMENTS

This work was supported by National Institute on Drug Abuse grants

to D.G.D. (DA7318 and DA9374) and A.M. (DA7215).

AUTHORS

Dale G. Deutsch, Ph.D.

Associate Professor of Biochemistry and Cell Biology

Department of Biochemistry and Cell Biology

State University of New York at Stony Brook

Stony Brook, NY 11794-5215

Alexandros Makriyannis, Ph.D.

Professor of Medicinal Chemistry

School of Pharmacy, U-92

University of Connecticut

372 Fairfield Road

Storrs, CT 06269-2092

 
85

Metabolic Bioactivation Reactions

Potentially Related to Drug Toxicities

Neal Castagnoli, Jr. and Kay P. Castagnoli

The majority of metabolic transformations that xenobiotics undergo

in mammalian systems leads to more polar and, in general, less

pharmaco-logically and toxicologically potent products. This

generalization has led to the proposal that evolutionary factors

influencing the emergence of the principal enzymes responsible for

the biotransformations of xenobiotics have a link to the improved

survival potential of those individuals equipped with enzyme systems

capable of converting otherwise toxic substances produced by plantand animal-based food sources to nontoxic metabolites (Jakoby and

Ziegler 1990).

The types of biotransformation reactions that many xenobiotics

undergo have been organized into two principal classes: Phase I

transformations that, for the most part, are oxidative in nature and

generally introduce a hydroxy group into the substrate molecule; and

phase II transformations that convert the newly introduced hydroxy

group to polar conjugates such as glucuronides and sulfate esters

(Caldwell 1986; Guengerich and Ziegler 1990). In the case of

carboxylic acids, polar amido esters derived from various amino acids

are formed (Killenberg and Webster 1980). Since many toxic natural

products are lipophilic organic molecules, the metabolic conversion of

these substances in land-bound animals to polar conjugates makes

teleological sense in that polar compounds partition with difficulty

across cell membranes but are readily filtered through the nephron and

thus are more readily eliminated from the body through renal

excretion. Depending on the structures of the molecules, however,

the same metabolic events, on occasion, can generate chemically

reactive and toxic metabolites (Mulder et al. 1986; Parke 1987;

Korzekwa and Jones 1993; Guengerich 1994; Gonzalez and Gelboin

1994). Consequently, an important part of drug development focuses

on the characterization of the metabolic profile of candidate drugs in

an attempt to avoid structural features that may lead to the formation

of toxic metabolites.

This chapter is concerned with the metabolic fate of cyclic tertiary

amines and, in particular, a consideration of potential bioactivation
86

pathways that may lead to toxic metabolites. Cyclic tertiary amines

form an important class of compounds in the area of drug abuse since

a variety of psychoto-mimetic agents such as cocaine (figure 1,

structure 1), lysergic acid diethylamide (LSD 25) (structure 2) and

phencyclidine (structure 3), as well a large group of centrally acting

compounds such as the neuroleptic phenothiazine chlorpromazine

(structure 4), the central nervous system (CNS) stimulant mazindol

(structure 5), the narcotic analgetic fentanyl (structure 6), and the

antidepressant imipramine (structure 7) either have been used to treat

CNS disorders and/or have the potential for abuse. These types of

compounds undergo extensive oxidative metabolic transformations.

The discussion that follows focuses on selected examples of metabolic

transformations that may be linked to the formation of potentially

neurotoxic metabolites. Although the number of well-characterized

examples of such biotransformations is relatively few, it may be

reasonable to speculate that the neurological disorders associated with

long-term exposure to substances of abuse and some behaviormodifying medications may involve biochemical lesions mediated by

chemically reactive metabolites. Thus, it may be important when

attempting to assess the possible significance of metabolic

bioactivation processes to appreciate that the chemical instability of

reactive metabolites which can make the identification and

characterization of their biological properties difficult.

The most important metabolic transformation that cyclic amines

undergo is -carbon oxidation which generates the corresponding

iminium products (Koymans et al. 1993). This reaction is catalyzed

by members of the cytochrome P450 superfamily of hemoproteins

(Nelson et al. 1993). As discussed below, recent studies have

documented that the outer mitochondrial membrane bound

flavoproteins monoamine oxidase A and B (MAO-A and MAO-B) are

efficient catalysts for the -carbon oxidation of a specific class of

cyclic tertiary amines, namely 4-substituted 1-methyl-1, 2, 3, 6-

tetrahydropyridines (Kalgutkar et al. 1995). The most generally

accepted catalytic pathway (figure 2) for this reaction assumes an

initial single electron transfer (SET) step from the amine substrate

(structure 8) nitrogen lone pair to generate an aminyl radical cation

(structure 9) which, following loss of an -proton, is converted to a

highly reactive carbon-centered radical (structure 10). A second

single- electron oxidation of the carbon-centered radical gives, in the

case of the cytochrome P450-catalyzed reaction, the carbinolamine

(structure 11),
87

which is in equilibrium with the iminium ion product (structure 12).

In the MAO-catalyzed reaction, intermediate structure 10 is

converted directly to structure 12. As shown in figure 2, the electron

acceptor for the cytochrome P450-catalyzed reaction is the perferryl

oxo species (FeVO), while the electron acceptor for the MAOcatalyzed reaction is the oxidized flavin moiety (FAD).

Although the experimental evidence supporting the SET mechanism

for the cytochrome P450-catalyzed (Miwa et al. 1983; Hanzlik et al.

1984; Guengerich and Macdonald 1984; Macdonald et al. 1989) and

the MAO-catalyzed (Silverman 1992) reactions is extensive, it has

been challenged by results from several laboratories. These include

results obtained with deuterium kinetic isotope effects (Peterson et al.

1987; Peterson and Castagnoli 1988; Dinnocenzo et al. 1993; Walker

and Edmondson 1994), model chemical reactions (Kim et al. 1993,

1995), and the unexpected substrate properties (Kuttab et al. 1994) of

certain tertiary cyclopropyl- amine derivatives, that according to the

SET pathway, would be expected to act as enzyme inactivators only

(Silverman 1984). These results have led some investigators to

propose a direct hydrogen atom abstraction pathway (structure 8 –>

structure 12) that bypasses the aminyl radical cation intermediate

(structure 9), for both the cytochrome P450-catalyzed (Dinnocenzo

et al. 1993) and the MAO-catalyzed pathways (Walker and

Edmondson 1994; Ottoboni et al. 1989), and a polar (2-electron)
88

pathway for MAO catalysis that proceeds via an amine-FAD adduct

(Kim et al. 1993, 1995). The metabolic processes in figures 2 to 9

are described below.

As illustrated in figure 2, these -carbon oxidations lead to the

formation of iminium ion products which undergo spontaneous

hydrolysis to the corresponding aldehyde (structure 13) and secondary

amine (structure 14),

the net outcome being N-dealkylation. Cyclic tertiary amines (figure 3,

structure 15) also undergo oxidative N-dealkylation, via hydrolysis of

the enzyme generated exocyclic iminium intermediate (figure 3,

structure 16), to yield an aldehyde (structure 13) and the cyclic

secondary amine (figure 3, structure 17). The corresponding oxidation

of a ring -carbon atom generates the cyclic iminium intermediate

(figure 3, structure 18). Unlike the acyclic regioisomer structure 16,

hydrolysis of structure 18 to the aminoaldehyde structure 19 is

reversible, giving rise to the possible further metabolic processing of

structure 18. These intermediary metabolites are often oxidized to the

biologically less active lactams (structure 20) in a reaction that is

catalyzed by the liver cytosolic enzyme aldehyde oxidase (Bielawski et

al. 1987). If special structural features are present in the substrate

molecule or if the cyclic iminium metabolite is generated in

extrahepatic tissues lacking aldehyde oxidase, these reactive
89

intermediates may undergo alternative chemical transformations that

can produce toxic products.

An example of the metabolic conversion of a 5-membered

azaheterocylic system to toxic metabolites is summarized in figure 4

with the hepatotoxic and carcinogenic pyrrolizidine alkaloids

(Mattocks 1986). Initial cyto-chrome P450-catalyzed, -carbon

oxidation of the bicyclic parent tertiary amine (structure 21)

generates the iminium intermediate (structure 22) that, upon loss of a

proton, forms the pyrrolic derivative (structure 23). However,

compound 23 is unstable because of the presence of the leaving groups

in the side chains. Departure of the RCOO- group generates the

highly reactive electrophilic intermediate (structure 24) that is

attacked by a nucleophilic group present on biomacromolecules to

generate an adduct (structure 25). The pyrrolizidine alkaloids are

bisalkylating agents because of the presence of the second

strategically positioned leaving group (R.COO-) with the resulting

formation of a crosslinked biopolymer (structure 26).

The dramatic toxicity of the pyrrolizidine alkaloids and related highly

toxic compounds has led investigators to focus their attention on the
90

underlying biochemical mechanisms. More subtle toxicities, however,

may be associated with compounds that undergo similar oxidative

conversions. The authors have been interested in the potential

metabolic bioactivation of the tobacco alkaloid (S)-nicotine (figure 5,

structure 27), a compound that is metabolically bioactivated to

reactive intermediates that form covalent bonds to

biomacromolecules (Shigenaga et al. 1988). The principal oxidative

pathway for this compound also proceeds via cytochrome P450-

catalyzed oxidation to form the corresponding iminium metabolite

(structure 28). When generated in the presence of liver aldehyde

oxidase, structure 28 is rapidly converted to the nontoxic lactam (S)-

cotinine (structure 29). In the absence of aldehyde oxidase, however,

the iminium ion (structure 28), presumably via the corres-ponding

free enamine base (structure 30), can be oxidized to the pyrrolic

metabolite -nicotyrine (structure 31) in a reaction that is catalyzed

by MAO-B (Shigenaga 1989). -nicotyrine is an electron-rich

heterocyclic aromatic system that undergoes rapid cytochrome P450-

catalyzed conver- sion to the pyrrolinones (structures 35 and 36),

which in turn autoxidize to the 5-hydroxypyrrolinone (structure 38)

(Shigenaga et al. 1989).

The proposed reaction pathway leading to these products is depicted

in figure 5. The electron-rich pyrrole ring system is oxidized to the

reactive arene oxide (structure 32) which rearranges to the

zwitterionic species
91

 (structure 33). Proton loss generates the anion structure 34 which

leads to an equilibrium mixture of pyrrolinones (structures 35 and 36).

Alternatively, the anion (structure 34) undergoes autoxidation,

leading to formation of superoxide radical anion (O2

.-) and the

resonance stabilized radical, structure 37a <—> 37b, which eventually

is converted to the final product (structure 38). The possible

toxicological significance of this metabolic pathway remains to be

documented. Since human exposure to tobacco products occurs over

the course of many years, even low-level exposures to reactive

intermediates (such as structure 37) could have a cumulative effect

that may contribute to the degenerative processes linked to tobacco

use. In this regard, the efficient conversion of (S)-nicotine to the

corresponding iminium metabolite (Shigenaga et al. 1988) by lung

cytochrome P450 may be particularly significant since the levels of

aldehyde oxidase in this tissue are likely to be very low or absent

(Huff and Chaykin 1967; Beedham 1985).

A major impetus for considering the bioactivation of cyclic tertiary

amines is derived from studies on the parkinsonian-inducing

nigrostriatal neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-

tetrahydropyridine (MPTP) structure 39. Extensive metabolic,
92

biochemical, and toxicological investigations have documented that

the neurodegenerative properties of MPTP are mediated by a

mitochondrial neurotoxin that is formed according to the reaction

sequence shown in figure 6 (Sayre 1989; Maret et al. 1990; Kopin

1992; Tipton et al. 1993; Tipton and Singer 1993; Singer et al.

1993). The substrate amine (structure 39) undergoes C-6 (allylic) ring

-carbon oxidation to form the cyclic iminium metabolite, the 1-

methyl-4-phenyl-1,2-dihydropyridinium species (MPDP+) (structure

40). Structure 40, although stable as its solid perchlorate salt (Chiba

et al. 1985), is too unstable to isolate from incubation mixtures

(Weissman et al. 1985). Since structure 40 is an excellent substrate

for aldehyde oxidase, it is rapidly converted in whole liver

homogenates to the corresponding lactam structure 41 (Wu et al.

1988). In the absence of the aldehyde oxidase, however, structure 40

(< 50 millimolars (mM)) in pH 7.4 buffer undergoes slow autoxidation

to yield the 1-methyl-4-phenyl-pyridinium product MPP+ depicted in

structure 42 (Wu et al. 1988). At higher con- centrations, this

dihydropyridinium metabolite also may participate in two alternative

reactions (figure 7). The first is a bimolecular dispropor-tionation

reaction in which the free base structure 43 derived from structure 40

functions as a hydride donor, while structure 40 serves as a hydride

acceptor. The net result is the formation of stoichiometric amounts

of MPTP and MPP+ (Wu et al. 1988). A second and more complex

reaction sequence involves the net consumption of three moles of the

dihydropyridinium metabolite (structure 40) that eventually yields the

isoquinoline system (structure 44), a mole of MPP+, and a mole of

methyl- amine (figure 7) (Leung et al. 1989). The extent to which

these or similar reactions involving endogenous reactants occur in

vivo is not known.

In vitro metabolic studies with rodent and human liver microsomal

prepara- tions have established that MPTP undergoes both oxidative

N-demethylation and C-6 (allylic) oxidation in reactions that are -

nicotinamide adenine dinucleotide phosphate (NADPH) dependent

and therefore likely to be cytochrome P-450 catalyzed (Weissman et

al. 1985; Ottoboni et al. 1990). Although the latter transformation

can lead to the toxic pyridinium metabolite MPP+, the cytochrome

P450-catalyzed pathway is unlikely to contribute significantly to the

neurotoxicity of MPTP. As mentioned above, liver aldehyde oxidase

diverts the inter-mediate dihydropyridinium metabolite away from

pyridinium ion formation by catalyzing the conversion of structure

40 to the nontoxic lactim structure 41. Further-more, even if formed

in the periphery, the polar pyridinium metabolite would have limited

access to the central nervous system (CNS). The low
93
94

concentrations of the P450s in the brain (Warner et al. 1993)

preclude the in situ formation of toxic levels of MPP+ within the CNS

by this pathway.

Continued interest in the possible metabolic activation of MPTP led

to brain tissue homogenate studies that initially established

bioactivation in these tissues by MAO (Chiba et al. 1984). Later

studies demonstrated the unexpected and excellent (kcat/KM = 1400

min-1mM-1 at 37%C) MAO-B substrate properties of MPTP (Kuttab

et al. 1994). Subsequent studies employing a monkey model of

MPTP-induced parkinsonism established the role of MAO-B in the

mediation of the nigrostriatal toxicity of MPTP. The critical

experiment showed that the selective MAO-B inhibitor (R)-deprenyl

(structure 45, figure 8) completely protects against MPTP's toxicity

in this model (Langston et al. 1984; Heikkila et al. 1984). MPTP

also is a substrate for MAO-A (kcat/KM = 47 min-1mM-1 at 30%C)

(Singer et al. 1986). However, since pretreatment with the MAO-A

selective inactivator clorgyline (structure 46) does not protect against

its neurotoxicity, this form of the enzyme does not appear to

contribute to MPTP's neuro-degenerative properties.

The selective toxicity of MPTP is remarkable, particularly since

there is no evidence for the presence of MAO-B in the susceptible

dopaminergic nigrostriatal neurons (which do, however, contain

MAO-A) (Moll et al. 1990). This apparent dilemma has been

resolved by the demonstration that MPP+ is a substrate for the

dopamine transporter (Javitch et al. 1985). Once localized

intraneuronally, MPP+ is concentrated further within the inner

mitochondrial membrane (Youngster et al. 1989a; Davey et al.

1992), where it inhibits electron transport (Nicklas et al. 1985, 1987)

leading to adenosine triphosphate (ATP) depletion (Di Monte et al.

1986) and cell death.

The ability of MPTP to cause a lesion that parallels in many ways the

characteristic lesion of idiopathic Parkinson's disease has stimulated

efforts to identify possible environmental and endogenous compounds

that may possess MPTP-type properties (Ikeda et al. 1992).

Intracerebral microdialysis studies that estimate irreversible neuronal

degeneration have provided evidence that a variety of pyridinium and

related quaternized azaheteroaromatic systems are toxic to

dopaminergic neurons (Rollema et al. 1990, 1994). Nevertheless,

relatively few compounds meet all of the characteristics required for
95

an MPTP-type neurotoxin; these characteristics include the in situ

MAO-B-catalyzed biotransformation in the brain to a pyridinium

metabolite. The metabolite is actively transported into the

nigrostriatal nerve terminals and then into the inner mitochondrial

membrane, where it must inhibit electron transport. Extensive studies

have documented that only 1-methyl-1,2,3,6- tetrahydropyridine

derivatives bearing selected substituents at C-4 are good substrates for

MAO-B (Maret et al. 1990; Sablin et al. 1994; Youngster et al.

1989b; Kalgutkar et al. 1994). Furthermore, various types of

tetrahydropyridine derivatives that are good substrates for MAO-B do

not display MPTP-type activity when tested in vivo since, for

various structural reasons, the intermediate dihydropyridinium

metabolites are not converted to the corresponding pyridinium

species (Naiman et al. 1990; Dalvie et al. 1992).

The MAO-catalyzed bioactivation of MPTP to MPDP+, leading to

the neurotoxic MPP+ pyridinium metabolite, is dependent on the

allylamine unit present in the tetrahydropyridine moiety. A similar

reaction sequence, however, may occur with piperidine derivatives

lacking the double bond but at the same oxidation state as the

tetrahydropyridine. One compound of particular interest that fits this

description is haloperidol (figure 9, structure 47), a potent neuroleptic

agent that, like other members of this pharmacological class, causes

severe extrapyramidal side effects including parkinsonism and tardive

dyskinesias (Tarsy and Baldessarini 1986). This 4-piperidinol

derivative resembles MPTP in that it bears an aryl group at C-4 of

the piperidinol. Dehydration of HP structure 47, a reaction that is

reported to occur in microsomal incubations (Fang and Gorrod 1991),

gives the corresponding 1,2,3,6-tetrahydropyridine derivative HPTP,

structure 50. HP 47 and HPTP 50 are not substrates for MAO-B, but
96

evidence obtained with the aid of mass spectral techniques and a

sensitive high-performance liquid chromatography (HPLC)

fluorescence assay have documented the conversion of HP (in

humans) and both HP and HPTP (in rodents) to the pyridinium

product HPP+ (structure 52) (Subramanyam et al. 1990, 1991a,

1991b; Igarashi and Castagnoli 1992; Van der Schyf et al. 1994).

The proposed metabolic sequence for the oxidation of HPTP to

HPP+ (figure 9) proceeds via the dihydropyridinium intermediate

structure 51 followed by autoxidation of structure 51 to structure 52

(Subramanyam et al. 1991b). The oxidation of HP is thought to

proceed via initial formation of the iminium inter-mediate structure

48 which, via the aminoenol structure 49, is converted to the

dihydropyridinium species structure 51. These ring -carbon

oxidations parallel the pathway outlined in figure 3 for cyclic tertiary

amines in general. Since oxidative N-dealkylation (analogous to the

sequence 8 –> 12 –> 13 + 14, figure 2) is a major metabolic pathway

for HP (Forssman and Larsson 1977), it is not surprising to observe

ring - carbon oxidation as a competing pathway.

The metabolic pathways leading to the production of these urinary

pyridinium metabolites are likely to be mediated by one or more

forms of liver cytochrome P450. In vitro metabolic studies with

rodent (Igarashi et al., unpublished results) and human (Usuki et al.,

submitted) microsomal preparations have demonstrated the NADPHdependent oxidation of both HP and HPTP to HPP+

. Ongoing studies

in the authors' laboratory have shown that HPP+ and related

pyridinium metabolites are present in brain tissues obtained from C57

black mice that had been treated with HPTP (Van der Schyf et al.

1994). Additionally, results obtained from intra-cerebral

microdialysis, mitochondrial respiration, and rat embryonic

mesencephalic cell culture studies suggest that HPP+ possesses MPP+

type neurotoxic properties (Rollema et al. 1992, 1994; Bloomquist et

al. 1994).

The critical question concerns the neurotoxic potential of HP in the

human. Since the development of drug induced tardive dyskinesias

often requires months or even years of drug exposure (Gerlach and

Casey 1988; Casey 1991), the demonstration of toxin-induced lesions

in experimental animals may be difficult. Furthermore, in view of the

dramatic species selectivity of MPTP (Singer et al. 1987; Giovanni et

al. 1994a, 1994b), the absence of a detectable anatomical lesion in

HP- or HPTP-treated rodents may not provide a definitive answer to

the question
97

of the neurotoxic potential of HP. It is hoped that this issue will be

resolved by an ongoing study in baboon (Van der Schyf et al.,

unpublished observations).

The potential neurotoxicity of pyridinium and related quaternary

azaheterocyclic species and the possible formation of such

metabolites from six-membered azaheterocyclic systems are of

interest because of the many drugs, including many CNS-acting agents,

that contain such structural features. Reports on the metabolism of

compounds such as LSD (structure 3) morphine (structure 4),

phencyclidine (structure 5), and fentanyl (structure 6) have not

provided evidence for pyridinium ion formation. However, the

proposed metabolites are quaternary cations and might not be readily

detected without appropriate analytical tools. Investigations of the

possible bioactivation of cyclic tertiary amines to neurotoxic

quaternary cationic azaheterocyclic metabolites could provide

valuable information that might lead to a better understanding of the
98

drug-related CNS disorders associated with long-term exposure to

certain abused substances.

REFERENCES

Beedham, C. Molybdenum hydroxylases as drug-metabolizing enzymes. Drug

Metab Rev 16(1&2):119-156, 1985.

Bielawski, J.; Brandange, S.; and Rodrigues, B. Kinetic resolution in the oxidation

of iminium ion to lactam catalyzed by aldehyde oxidase. Acta

Chem Scand [B] 41(3):198-201, 1987.

Bloomquist, J.; King, E.; Wright, A.; Mytilineou, C.; Kimura, K.; Castagnoli, K.;

and Castagnoli, N., Jr. 1-Methyl-4-phenylpyridinium- like

neurotoxicity of a pyridinium metabolite derived from

haloperidol: Cell culture and neurotransmitter uptake studies. J

Pharmacol Exp Ther 270(2):822-830, 1994.

Caldwell, J. Conjugation mechanisms of xenobiotic metabolism: Mammalian

aspects. In: Paulson, G.D.; Caldwell, J.; Hutson, D.H.; and Menn,

J.J., eds. Xenobiotic Conjugation Chemistry. Washington, DC:

American Chemical Society, 1986. pp. 2-28.

Casey, D.E. Neuroleptic drug-induced extrapyramidal syndromes and tardive

dyskinesia. Curr Opin Psychiatry 4:86-89, 1991.

Chiba, K.; Trevor, A.J.; and Castagnoli, N., Jr. Metabolism of the neurotoxic

tertiary amine MPTP by brain monoamine oxidase. Biochem

Biophys Res Comm 120(2):574-578, 1984.

Chiba, K.; Peterson, L.A.; Castagnoli, K.P.; Trevor, A.J.; and Castagnoli, N., Jr.

Studies on the molecular mechanism of bioactivation of the

selective nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine (MPTP). Drug Metab Disp 13(3):342-347,

1985.

Dalvie, D.; Zhao, Z.; and Castagnoli, N., Jr. Characterization of an unexpected

product from a monoamine oxidase B generated 2,3-

dihydropyridinium species. J Org Chem 57:7321-7324, 1992.

Davey, G.P.; Tipton, K.F.; and Murphy, M.P. Uptake and accumulation of 1-

methyl-4-phenylpyridinium by rat liver mitochondria measured

using an ion-selective electrode. Biochem J 288:439-443, 1992.

Di Monte, D.; Jewell, S.A.; Ekstrom, G.; Sandy, M.S.; and Smith, M.T. 1-

Methyl-1-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-

methyl-4-phenyl-pyridine (MPP+) cause rapid ATP depletion in

isolated hepatocytes. Biochem Biophys Res Commun 137:310-

315, 1986.
99

Dinnocenzo, J.P.; Karki, S.B.; and Jones, J.P. On isotope effects for the

cytochrome P-450 oxidation of substituted N,Ndimethylanilines. J Am Chem Soc 115(16):7113-7116, 1993.

Fang, J., and Gorrod, J.W. Dehydration is the first step in the bioactivation of

haloperidol to its pyridinium metabolite. Toxicol Lett 59:117-

123, 1991.

Forsman, A., and Larsson, M. On the metabolism of haloperidol in man. Curr

Ther Res 21(4):606-617, 1977.

Gerlach, J., and Casey, D.E. Tardive dyskinesia. Acta Psychiatr Scand 77:369-

378, 1988.

Giovanni, A.; Sieber, B.-A.; Heikkila, R.E.; and Sonsalla, P.K. Studies on species

sensitivity to the dopaminergic neurotoxin 1-methyl-4- phenyl1,2,3,6-tetrahydropyridine. Part 1: Systemic administration. J

Pharmacol Exp Ther 270(3):1000-1007, 1994a.

Giovanni, A.; Sieber, B.-A.; Heikkila, R.E.; and Sonsalla, P.K. Studies on species

sensitivity to the dopaminergic neurotoxin 1-methyl-4- phenyl1,2,3,6-tetrahydropyridine. Part 2: Central administration of 1-

methyl-4-phenylpyridinium. J Pharmacol Exp Ther

270(3):1008-1014, 1994b.

Gonzalez, F.J., and Gelboin, H.V. Role of human cytochromes P450 in the

metabolic activation of chemical carcinogens and toxins. Drug

Metab Rev 26(1-2):165-183, 1994.

Guengerich, F.P. Catalytic selectivity of human cytochrome P450 enzymes:

relevance to drug metabolism and toxicity. Toxicol Lett

70(2):133-138, 1994.

Guengerich, F.P., and Macdonald, T.L. Chemical mechanisms of catalysis by

cytochromes P-450: A unified view. Acc Chem Res 17:9-16,

1984.

Guengerich, F.P., and Ziegler, D.M. Enzymatic oxidation of xenobiotic

chemicals. Biochem Molec Biol 25(2):97-153, 1990.

Hanzlik, R.P.; Hogberg, K.; and Judson, C.M. Microsomal hydroxylation of

specifically deuterated monosubstituted benzenes. Evidence for

direct aromatic hydroxylation. Biochemistry 19(23):3048-

3055, 1984.

Heikkila, R.E.; Manzino, L.; Cabbat, F.S.; and Duvoisin, R.C. Protection against

the dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,5,6-

tetrahydropyridine by monoamine oxidase inhibitors. Nature

311:467-469, 1984.

Huff, S.D., and Chaykin, S. Genetic and androgenic control of N.-

methylnicotinamide oxidase activity in mice. J Biol Chem

242(6):1265-1270, 1967.

Igarashi, K., and Castagnoli, N., Jr. Determination of the pyridinium metabolite

derived from haloperidol in brain tissue, plasma, and urine by
100

high performance liquid chromatography with fluorescence

detection. J Chromatog Biomed App 579(2):277-283, 1992.

Ikeda, H.; Markey, C.J.; and Markey, S.P. Search for neurotoxins structurally

related to 1-methyl-4-phenylpyridine (MPP+) in the

pathogenesis of Parkinson's disease. Brain Res 575(2):285-298,

1992.

Jakoby, W.B., and Ziegler, D.M. The enzymes of detoxication. J Biol Chem

265(34):20715-20718, 1990.

Javitch, J.A.; D'Amato, R.J.; Strittmatter, S.M.; and Snyder, S.H. Parkinsonisminducing neurotoxin, N-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine: Uptake of the metabolite N-methyl-4-

phenylpyridine by dopamine neurons explains selective

toxicity. Proc Natl Acad Sci U S A 82(7):2173-2177, 1985.

Kalgutkar, A.; Testa, B.; and Castagnoli, N., Jr. Selective inhibitors of

monoamine oxidase (MAO-A and MAO-B) as probes of its

catalytic site and mechanism. Med Res Rev 15(4):325-388,

1995.

Kalgutkar, A.; Castagnoli, K.; Hall, A.; and Castagnoli, N., Jr. Novel 4-

(aryloxy)tetrahydropyridine analogs of MPTP as monoamine

oxidase A & B substrates. J Med Chem 37(7):944-949, 1994.

Killenberg, P.G., and Webster, L.T., Jr. In: Jakoby, W.B., ed. Enzymatic Basis of

Detoxification. Vol. 2. New York: Academic Press, 1980. pp.

141-167.

Kim, J.-M.; Bogdan, M.A.; and Mariano, P.S. Mechanistic analysis of the 3-

methyllumiflavin-promoted oxidative deamination of

benzylamine. A potential model for monoamine oxidase

catalysis. J Am Chem Soc 115:10591-10595, 1993.

Kim, J.-M.; Hoegy, S.E.; and Mariano, P.S. Flavin chemical models for

monoamine oxidase inactivation by cyclopropylamines, -

silylamines, and hydrazines. J Am Chem Soc 117:100-105,

1995.

Kopin, I.J. Features of the dopaminergic neurotoxin MPTP. Ann N Y Acad Sci

648:96-104, 1992.

Korzekwa, K.R., and Jones, J.P. Predicting the cytochrome P450 mediated

metabolism of xenobiotics. Pharmacogenetics 3(1):1-18, 1993.

Koymans, L.; Donne-op den Kelder, G.M.; Koppele Te, J.M.; and Vermeulen,

N.P. Cytochromes P450: Their active-site structure and

mechanism of oxidation. Drug Metab Rev 25(3):325-387,

1993.

Kuttab, S.; Kalgutkar, A.; and Castagnoli, N., Jr. Mechanistic studies on the

monoamine oxidase B catalyzed oxidation of 1,4-disubstituted

tetrahydropyridines. Chem Res Tox 7:740-744, 1994.
101

Langston, J.W.; Irwin, I.; Langston, E.B.; and Forno, L.S. Pargyline prevents

MPTP-induced parkinsonism in primates. Science 225:1480-

1482, 1984.

Leung, L.; Ottoboni, S.; Oppenheimer, N.; and Castagnoli, N., Jr.

Characterization of a product derived from the 1-methyl-4-

phenyl-2,3- dihydropyridinium ion, a metabolite of the

nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine. J Org Chem 54(5):1052-1055, 1989.

Macdonald, T.L.; Gutheim, W.G.; Martin, R.B.; and Guengerich, F.P. Oxidation

of substituted N,N-dimethylanilines by cytochrome P-450:

Estimation of the effective oxidation-reduction potential of

cytochrome P-450. Biochemistry 28(5):2071-2077, 1989.

Maret, G.; Testa, B.; Jenner, P.; Nabil, E.T.; and Carrupt, P.-A. The MPTP

story: MAO activates tetrahydropyridine derivatives to toxins

causing parkinsonism. Drug Metab Rev 22(4):291-332, 1990.

Mattocks, A.R. Chemistry and Toxicology of Pyrrolizidine Alkaloids. Orlando,

FL: Academic Press, Inc., 1986.

Miwa, G.T.; Walsh, J.S.; Kedderis, G.L.; and Hollenberg, P.F. The use of

intramolecular isotope effects to distinguish between

deprotonation and hydrogen atom abstraction mechanisms in

cytochrome-P450- and peroxidase-catalyzed N-demethylation

reactions. J Biol Chem 258(23):14445-14449, 1983.

Moll, G.; Moll, R.; Riederer, P.; Gsell, W.; Heinsen, H.; and Denney, R.M.

Immunofluorescence cytochemistry on thin frozen sections of

human substantia nigra for staining of monoamine oxidase A

and monoamine oxidase B: A pilot study. In: Riederer, P., and

Youdim, M.B.H., eds. Amine Oxidases and Their Impact on

Neurobiology. Springer-Verlag. 4th International Amine

Oxidases Workshop, Wurzburg, Federal Republic of Germany, J

Neural Transm S 32:67-77, 1990.

Mulder, G.J.; Meerman, J.H.H.; and van den Goorbergh, A.M. Bioactivation of

xenobiotics by conjugation. In: Paulson, G.D.; Caldwell, J.;

Hutson, D.H.; and Menn, J.J., eds. Xenobiotic Conjugation

Chemistry. Washington, DC: American Chemical Society, 1986.

pp. 282-300.

Naiman, N.A.; Rollema, H.; Johnson, A.; and Castagnoli, N., Jr. Studies on 4-

benzyl-1-methyl-1,2,3,6-tetrahydropyridine, a non-neurotoxic

analog of the parkinsonian inducing agent 1-methyl-4-phenyl1,2,3,6- tetrahydropyridine. Chem Res Toxicol 3(2):133-138,

1990.

Nelson, D.R.; Kamataki, T.; Waxman, D.J.; Guengerich, F.P.; Estabrook, R.W.;

Feyereisen, R.; Gonzalez, F.J.; Coon, M.J.; Gunsalus, I.C.; Gotob,

O.; Okuda, K.; and Nebert, D.W. The P450 superfamily: Update
102

on new sequence, gene mapping, accession numbers, early trivial

names of enzymes, and nomenclature. DNA Cell Biol 12:1-51,

1993.

Nicklas, W.J.; Youngster, S.K.; Kindt, M.V.; and Heikkila, R.E. MPTP, MPP+

and mitochondrial function. Life Sci 40(8):721-729, 1987.

Nicklas, W.J.; Vyas, I.; and Heikkila, R.E. Inhibition of NADH-linked oxidation

in brain mitochondria by 1-methyl-4-phenyl-pyridine, a

metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-

tetrahydro-pyridine. Life Sci 2503-2508, 1985.

Ottoboni, S.; Carlson, T.; Castagnoli, K.; Trager, W.F.; and Castagnoli, N., Jr.

Studies on the cytochrome P-450 catalyzed ring a-carbon

oxidation of the nigrostriatal toxin 1-methyl-4-phenyl1,2,3,6-tetrahy-dropyridine (MPTP). Chem Res Tox 3(5):423-

427, 1990.

Ottoboni, S.; Caldera, P.; Trevor, A.; and Castagnoli, N., Jr. Deuterium isotope

effect measurements on the interactions of the neurotoxin 1-

methyl-4-phenyl-1,2,3,6-tetrahydropyridine with monoamine

oxidase B. J Biol Chem 264:13684-13688, 1989.

Parke, D.V. Activation mechanisms to chemical toxicity. Arch Toxicol 60:5-15,

1987.

Peterson, L.A., and Castagnoli, N., Jr. Regio- and stereochemical studies on the

a-carbon oxidation of (S)-nicotine by cytochrome P-450 model

systems. J Med Chem 31(3):637-640, 1988.

Peterson, L.A.; Trevor, A.; and Castagnoli, N., Jr. Stereochemical studies on the

cytochrome P-450 catalyzed oxidation of (S)-nicotine to the

(S)-nicotine 1'(5')-iminium species. J Med Chem 30:249-254,

1987.

Rollema, H.; Subramanyam, B.; Skolnik, M.; D'Engelbronner, J.; and Castagnoli,

N., Jr. MPP+-like neurotoxicity of HPP+, a pyridinium

metabolite of haloperidol: A microdialysis study. In: Monitoring

Molecules in Neuroscience. Groningen, The Netherlands:

University Center for Pharmacy, 1992. pp. 367-372.

Rollema, H.; Skolnik, M.; d'Engelbronner, J.; Igarashi, K.; Usuki, E.; and

Castagnoli, N., Jr. MPP+-like neurotoxicity of a pyridinium

metabolite derived from haloperidol: In vivo microdialysis and

in vitro mitochondrial studies. J Pharmacol Exp Ther

268(1):380-387, 1994.

Rollema, H.; Johnson, E.A.; Booth, R.G.; Caldera, P.; Lampen, P.; Youngster,

S.K.; Trevor, A.J.; Naiman, N.; and Castagnoli, N., Jr. In vivo

intracerebral microdialysis studies in rats of MPP+ analogues and

related charged species. J Med Chem 33:2221-2230, 1990.

Sablin, S.O.; Krueger, M.G.; Singer, T.P.; Bachurin, S.O.; Khare, A.B.; Efange,

S.M.N.; and Tkachenko, S.E. Interaction of
103

tetrahydrostilbazoles with monoamine oxidase A and B. J Med

Chem 37:151-157, 1994.

Sayre, L.M. Biochemical mechanism of action of the dopaminergic neurotoxin

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

Toxicol Lett 48(2):121-149, 1989.

Shigenaga, M.K. Studies on the Metabolism and Bioactivation of (S)-Nicotine

and Beta-nicotyrine. Ph.D. dissertation, University of

California, San Francisco. 1989.

Shigenaga, M.; Trevor, A.; and Castagnoli, N., Jr. Metabolism-dependent

covalent binding of (S)[5-3H]nicotine to liver and lung

microsomal macromolecules. Drug Met Disp 16(3):397-402,

1988.

Shigenaga, M.K.; Kim, B.H.; Caldera-Munoz, P.; Cairns, T.; Jacob, P., III;

Trevor, A.J.; and Castagnoli, N., Jr. Liver and lung microsomal

metabolism of the tobacco alkaloid -nicotyrine. Chem Res

Tox 2(5):282-287, 1989.

Silverman, R.B. Effect of a-methylation on inactivation of monoamine oxidase

by N-cyclopropylbenzylamine. Biochemistry 23:5206-5213,

1984.

Silverman, R.G. In: Mariano, P.S., ed. Advances in Electron Transfer

Chemistry.Vol. 2. Greenwich, CT: Academic Press, 1992. pp.

177-213.

Singer, T.P.; Salach, J.I.; Castagnoli, N., Jr.; and Trevor, A. Interaction of the

neurotoxic amine, MPTP, with monoamine oxidases A and B in

relation to Parkinsonism. Biochem J 235:785-789, 1986.

Singer, T.P.; Ramsay, R.R.; Sonsalla, P.K.; Nicklas, W.J.; and Heikkila, R.E.

Biochemical mechanisms underlying MPTP-induced and

idiopathic parkinsonism. New vistas. Adv Neurol 60:300-305,

1993.

Singer, T.P.; Trevor, A.J.; and Castagnoli, N., Jr. Biochemistry of the

neurotoxic action of MPTP: Or how a faulty batch of 'designer

drug' led to parkinsonism in drug abusers. Trends Biochem Sci

12:266-270, 1987.

Subramanyam, B.; Pond, S.; Eyles, D.; Whiteford, H.; Fouda, H.; and Castagnoli,

N., Jr. Identification of a potentially neurotoxic pyridinium

metabolite in the urine of schizophrenic patients treated with

haloperidol. Biochem Biophys Res Comm 181(2):573-578,

1991a.

Subramanyam, B.; Woolf, T.; and Castagnoli, N., Jr. Studies on the in vitro

conversion of haloperidol to a potentially neurotoxic

pyridinium metabolite. Chem Res Tox 4(1):123-128, 1991b.

Subramanyam, B.; Rollema, H.; Woolf, T.; and Castagnoli, N., Jr. Identification

of a potentially neurotoxic pyridinium metabolite of
104

haloperidol in rats. Biochem Biophys Res Comm 166(1):238-

244, 1990.

Tarsy, D., and Baldessarini, R.J. In: Shah, N., and Donald, A., eds. Movement

Disorders. New York: Plenum Press, 1986. pp. 240-243.

Tipton, K.F.; McCrodden, J.M.; and Sullivan, J.P. Metabolic aspects of the

behavior of MPTP and some analogues. Adv Neurol 60:186-

193, 1993.

Tipton, K.F., and Singer, T.P. Advances in our understanding of the mechanisms

of the neurotoxicity of MPTP and related compounds. J

Neurochem 61(4):1191-1206, 1993.

Usuki, E.; Pearce, R.; Parkinson, A.; and Castagnoli, N., Jr. "Studies on the

Conversion of Haloperidol and its Tetrahydrophyridine

Dehydration Product to Potentially Neurotoxic Pyridinium

Metabolites by Human Liver Microsomes." Manuscript

submitted for publication.

Van der Schyf, C.J.; Pond, S.; and Castagnoli, N., Jr. Studies in progress.

Van der Schyf, C.J.; Castagnoli, K.; Usuki, E.; Fouda, H.G.; Rimoldi, J.M.; and

Castagnoli, N., Jr. Metabolic studies on haloperidol and its

tetrahydropyridine analog in C57BL/6 Mice. Chem Res Toxicol

7(3):281-281, 1994.

Walker, M.C., and Edmondson, D.E. Structure-activity relationships in the

oxidation of benzylamine analogues by bovine liver

mitochondrial monoamine oxidase B. Biochemistry

33(23):7088-7098, 1994.

Warner, M.; Stromstedt, M.; Wyss, A.; and Gustafsson, J.A. Regulation of

cytochrome P450 in the central nervous system. J Steroid

Biochem Mol Biol 47(1-6):191-194, 1993.

Weissman, J.; Trevor, A.; Chiba, K.; Peterson, L.; Caldera, P.; Castagnoli, N.,

Jr.; and Baillie, T. Metabolism of the nigrostriatal toxin 1-

methyl-4-phenyl-1,2,3,6-tetrahydropyridine by liver

homogenate fractions. J Med Chem 28(8):997-1001, 1985.

Wu, E.; Shinka, T.; Caldera-Munoz, P.; Yoshizumi, H.; Trevor, A.; and

Castagnoli, N., Jr. Metabolic studies on the nigrostriatal toxin

MPTP and its MAO-B generated dihydropyridinium metabolite

MPDP+. Chem Res Tox 1(3):186-194, 1988.

Youngster, S.K.; Sonsalla, P.K.; Sieber, B.-A.; and Heikkila, R.E. Structureactivity study of the mechanism of 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine (MPTP)-induced neurotoxicity. I.

Evaluation of the biological activity of MPTP analogs. J

Pharmacol Exp Ther 249:820-828, 1989b.

Youngster, S.K.; Nicklas, W.J.; and Heikkila, R.E. Structure-activity study of the

mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

(MPTP)-induced neurotoxicity. II. Evaluation of the biological
105

activity of the pyridinium metabolites formed from the

monoamine oxidase-catalyzed oxidation of MPTP analogs. J

Pharmacol Exp Ther 249(3):829-835, 1989a.

AUTHORS

Neal Castagnoli, Jr., Ph.D.

Peters Professor of Chemistry

Kay P. Castagnoli, B.A.

Senior Research Associate

Harvey W. Peters Center

Department of Chemistry

Virginia Polytechnic Institute and State University

107 Davidson Hall

Blacksburg, VA 24061
106

The Role of Iminium-Enamine

Species in the Toxication and

Detoxication of Cyclic Tertiary

Amines

Lawrence M. Sayre, David A. Engelhart, Durgesh V.

Nadkarni, M.K. Manoj Babu, Ann Marie Flammang, and

G. David McCoy

INTRODUCTION AND BACKGROUND

Aliphatic cyclic tertiary amines constitute a major class of naturally

occurring and synthetic drugs directed at central biogenic amine

receptors. Microsomal metabolism of these amines is known to be

associated with low levels of covalent binding and/or suicide

inactivation of the pertinent metabolizing P-450 isozymes; two of

the more notorious examples are phencyclidine (1-(1-

phenylcyclohexyl)piperidine) (PCP) (Hoag et al. 1984) and nicotine

(Shigenaga et al. 1988).

Covalent binding was initially believed to result from alkylation of

protein-based nucleophiles by the respective endocyclic iminium

metabolite arising from cytochrome P-450 mediated two-electron

oxidation (Hoag et al. 1984; Nguyen et al. 1979; Overton et al. 1985;

Shigenaga et al. 1988; Ward et al. 1982). These iminium species are

generated in equilibrium with a carbinolamine, an endocyclic enamine,

and a ring-opened aminocarbonyl compound (figure 1). In contrast to

acyclic tertiary amines, where the iminium intermediates are readily

hydrolyzed via carbinolamines to secondary amines and aldehydes (or

ketones) (figure 2), iminium hydrolysis for cyclic tertiary amines

remains reversible, ensuring the persistence of all equilibrium species

in figure 1. This explains why covalent binding is seen only for cyclic

tertiary amines.

Support for the notion that the iminium electrophiles could be the

covalent binding species is found in their ready formation of stable

cyanide adducts and the fact that the presence of cyanide during

metabolism of the parent amines protected against covalent binding

(Hoag et al. 1984; Nguyen et al. 1979; Shigenaga et al. 1988; Ward et

al. 1982). However, cyanide is a special nucleophile (it forms a C-C

bond) that may not be representative of most physiologic
107

nucleophiles. Also, although nicotine- 1.(5.)-iminium forms a pmethylthiophenol adduct (Brandange and Lindblom 1979a), the

iminium does not bind to nucleophilic polyamino acids except

polycysteine, and this only in the presence of O2 (Obach and Van

Vunakis 1988). Unpublished studies from the authors’ laboratory

indicate that simple cyclic iminium species form stable covalent

adducts with typical bionucleophiles only under special conditions, and

no evidence has been obtained for the persistent binding of iminium

species directly under physiologic conditions.

The protection against covalent binding by cyanide trapping of the

initial iminium metabolite could be equally well explained if a species

derived from metabolism beyond the iminium stage were the

responsible culprit. In recent years, increasing evidence has arisen

that most covalent binding does in fact arise from some type of

advanced metabolite (Hoag et al. 1984, 1987; Osawa and Coon 1989;

Sayre et al. 1991). For example, recent studies on the independently

synthesized PCP iminium demonstrate little (Hoag et al. 1987) or no

(Osawa and Coon 1989) P-450 inactivation in the absence of

NADPH (the reduced form of nicotinamide-adenine dinucleotide

phosphate). If the iminium species itself is not directly responsible

for covalent binding, then it becomes possible to explain why

glutathione (GSH) and related thiols can protect against metabolismdependent covalent binding even though their iminium adducts only

form reversibly (Hoag et al. 1984; Obach and Van Vunakis 1988;

Ward et al. 1982), viz, GSH is evidently trapping the advanced

reactive metabolite(s).
108

-Aryl-substituted amines such as PCP and nicotine possess pK as

about 1 unit lower than normal tertiary amines, and enamines have

pKas about 1 unit lower than the corresponding amines (Cook 1988).

Thus, cyclic enamines corresponding to PCP and nicotine, for

example, should have pKas of about 8.5, suggesting that the enamines

could reach a concen- tration of nearly 10 percent that of the

iminium at physiologic pH. Although higher pH also favors the

carbinolamine (Brandange and Lindblom 1979a), the enamine would

be favored over the more hydrophilic carbinolamine in a biological

compartment of low dielectric constant such as the cytochrome P450 active site. Such biological compartment will not only favor

enamine relative to carbinolamine, but will also decrease the effective

iminium pKa on account of favoring charge minimization. In fact,

the enamine appears to be the form of the iminium that elutes under

reverse phase chromatographic conditions (Hallstrom et al. 1983;

Herber et al. 1991; Mattammal et al. 1987).

The electron-rich enamines should be excellent substrates for

processing by enzymes involved in oxidative metabolism, and could be

giving rise to second-generation metabolites with reactive properties.

A potentially important role of endocyclic enamines in xenobiotic

metabolism has not been widely appreciated. It is proposed here that

the enamine, rather than the ring-opened aminoaldehyde, is the

iminium-derived species giving rise to metabolism-dependent P-450

inactivation. This hypothesis suggests that there should be a

correlation between the degree of inactivation and the tendency of

various heterocyclic ring sizes to exist in ring-closed versus ringopened form. Such correlation is supported by a study on analogs of

PCP, which revealed a rank order of 6 > 5 >> 7 in ring size for loss of

both benzphetamine demethylase activity and heme CO binding

(Brady et al. 1987). As expected, N,Ndiethylphenylcyclohexylamine, the acyclic version of the 5-

membered ring PCP analog, exhibited no significant loss of heme.
109

Detoxication at the endocyclic iminium stage of metabolism is

traditionally considered to involve iminium conversion to lactams by

the action of the cytosolic molybdoenzyme aldehyde oxidase (AO),

the prototype reaction for which is the conversion of pyridinium

compounds to 2- and 4-pyridones (Felsted et al. 1973). As far as

xenobiotics are concerned, the best example has been the conversion

of nicotine 1.(5.)- iminium to the major human metabolite cotinine

(figure 3) (Brandange and Lindblom 1979b). Some additional

examples of lactam formation have been reported in the literature

(Hammer et al. 1968; Lin et al. 1993; Wall and Baker 1989).

However, although the generality of this transformation has been

assumed, a consistent detoxication of iminium species by this pathway

has never been demonstrated. In the key case of PCP, no lactam

metabolite analogous to cotinine has ever been reported.

A preliminary report by Obach and Van Vunakis (1990) claimed that

cotinine could also be formed by a microsomal nicotinamide adenine

dinucleotide (NAD)+-dependent dehydrogenase (abbreviated MND).

Inhibitor studies suggested that MND is not a typical aldehyde

dehydrogenase. The presence of this activity in rabbit microsomes

was confirmed in the authors’ laboratories (Flammang 1994). The

rate of cotinine production from the 1.(5.) -iminium by this route was

found to be comparable to the rate of conversion of nicotine to the 1.(5.) -iminium (as assessed by forma

this enzyme might play in xenobiotic metabolism in general. It is

expected that the substrate-structure dependence of MND will be quite

different from that of cytosolic AO.

Detoxication at the iminium/enamine/carbinolamine stage also occurs

through oxidation and reduction of the ring-opened aminoaldehyde by

aldehyde and alcohol dehydrogenases, respectively, which have wide

subcellar distribution. Significant differences are not expected in

metabolic rates for various acyclic aldehydes arising from

carbinolamine dissociation; detoxication via such routes should depend

mainly on the equilibrium concentration of the ring-opened as

opposed to ring-closed forms, and overall may play a minor role.

Overall, the iminium/enamine stage of metabolism (figure 1) is viewed

by the authors as the critical determinant between toxic activation

and detoxication. Inefficient conversion to lactams by cytosolic AO

and/or MND appears to correlate with increasing levels of covalent

binding.
110

In an effort to elucidate the nature of endocyclic iminium-derived

species that give rise to metabolism-dependent covalent binding and

to clarify what factors govern the balance between toxic activation

and detoxication
111

at the iminium stage of cyclic tertiary amine metabolism, the authors

initiated a series of studies to examine the effects of systematic

structural changes at the level of simple piperidine and pyrrolidine

derivatives as prototypes for PCP and nicotine, respectively. These

compounds permit a focus on nitrogen-directed metabolism and the

role of the iminium
112

species, since other metabolic pathways are eliminated. For example,

study of 1-benzylpiperidine (1-BP) as a structurally abbreviated form

of PCP would give information about metabolic

toxication/detoxication uncomplicated by the numerous cyclohexyl

ring-derived PCP metabolites (Gole et al. 1988; Holsztynska and

Domino 1985). The expectation that 1-BP would exhibit the

endocyclic metabolism of interest and not just N-debenzylation was

based on the report of metabolism-dependent covalent binding for its

homolog, 1-benzylpyrrolidine (Ho and Castagnoli 1980).

Using phenobarbital-induced rabbit liver microsomes and an NADPH

regenerating system, data was obtained on metabolic rates and

products, cytochrome P-450 inactivation, and the effect of CNå

trapping on these parameters (Engelhart 1994). The methods have

been described in an initial report (Sayre et al. 1991), and are not

duplicated here. Synthetic procedures for the amines and iminium

compounds (figure 4) are described separately.

RESULTS AND DISCUSSION

Metabolic Profiles of Parent Amines

The metabolic rates and major product profiles for the tertiary

amines studied are shown in table 1. The first three amines listed

undergo oxidation principally at the N-C bond, leading to the

endocyclic (major) and exocyclic (minor) iminium intermediates, the

latter dissociating to a secondary amine (not quantified) and

benzaldehyde. The preference for encyclic over exocyclic oxidation

was also seen previously for 1-benzyl-pyrrolidine (Ho and Castagnoli

1980). Exocyclic N-dealkylation is blocked in the case of PCP and

,-DMBP. Cytochrome P-450 binding constants obtained for 1-

BP, 2,6-DMBP, and PCP were 92, 37, and 10 micromolars (M),

respectively, and appear to reflect the differences in metabolic rates

and the concentrations needed to observe maximal metabolism.

Although PCP was oxidized more rapidly than 1-BP and 2,6-DMBP,

the fraction of metabolism resulting in formation of the PCP-Im+ was

lower, consistent with the fact that PCP additionally gives rise to

cyclohexyl ring-derived metabolites. Interestingly, 4,4-DMBP

exhibited a higher rate than did 1-BP and 2,6-DMBP, but a smaller

fraction of the total metabolism was accounted for by the

corresponding iminium. For 1-BP, 1-BP-Im+ 1 was not observed

directly, but the THA dimer 3 was observed instead (figure 5). Dimer

3 is the expected
113

TABLE 1. Substrate activity of tertiary amines for cytochrome P-450

using microsomes from phenobarbital-induced rabbits.

Substrate Metabolic

rate

(nmol/min/

mg)

Major products formed during

initial metabolic phasea

1-BP

1-BP (1mM

KCN)

1-BP-THA dimer

 55 (10

mM)

 52 (10

mM)

 40 (10

mM)

C -C .dimer (50%), PhCHO

(1.5%)

-cyano (25%), PhCHO

(1.0%)

2,6-DMBP

2,6-DMBP

(1mM KCN)

 65 (10

mM)

 59 (10

mM)

iminium (71%), PhCHO

(0.5%)

-cyano (58%), PhCHO

(0.5%)

2-MBP 54 (10

mM)

iminium C -C . dimer,

PhCHOb

4,4-DMBP

4,4-DMBP

(1mM KCN)

4,4-DMBP-Im+

112 (10

mM)

 31 (10

mM)

 44 (1 mM)

iminium (12%), PhCHO

(3.2%)

PhCHO (7.1%)

PCP

PCP (1mM KCN)

PCP-Im+

 89 (1 mM)

 80 (1 mM)

 80 (1 mM)

iminium (30%)

-cyano (11%)

,-DMBP

,-DMBP

(0.5 mM KCN)

,-DMBP-Im +

 37 (1 mM)

 42 (1 mM)

 70 (1 mM)c

iminium (89%)

iminium (37%), -cyano

(50%)

KEY: a = Represented as percentage of the total starting material

consumed; b = yields not quantified because the authors did not

distinguish between the two possible iminiums and the various

possible C -C .dimers; the PhCHO was found in only trace amounts,

however. c = estimated as a lower limit. PhCHO = benzaldehyde;

KCN = potassium cyanide; THA = tetrahydro-anabasine. Other

abbreviations are per figure 4.
114

outcome of iminium-enamine C -C . coupling (Leonard and Hauck

1957). Control studies on the authentic iminium perchlorate 1

established the short lifetime of this species under the incubation

conditions (pH 7.4, 37%C). The endocyclic iminium could, however,

be trapped as the cyano adduct 4 when metabolism was conducted in

the presence of 1 mM KCN (no dimer 3 was detected in this case).

For the other amines, where ring methyl substitution was expected to

sterically inhibit iminium-enamine coupling (Leonard and Hauck

1957), the corresponding iminiums were observed directly. Control

studies on the independently prepared iminium salts (figure 4)

established their relatively long-term stability under both high

performance liquid chromatography (HPLC) assay and incubation

conditions, with the exception of 6-MBP-Im+, which is analogous to

1-MBP-Im+. On account of the instability of 6-MBP-Im+, no

attempt was made to distinguish the isomer distribution of the

iminium metabolite arising from 2-MBP. As previously reported in

the case of PCP and 1-benzylpyrrolidine (Hoag et al. 1984; Ho and

Castagnoli 1980; Ward et al. 1982), the iminium metabolites could be

trapped as the -cyano adducts in the presence of 1 mM cyanide,

though this trapping was incomplete in some cases.

P-450 Inactivation

Data on the ability of various amines and metabolic intermediates to

inhibit microsomal benzphetamine demthylase activity are listed in

table 2. This is the standard assay for the phenobarbital-inducible P450
115

TABLE 2.Inhibition of benzphetamine N-demethylase activity.

Substrate (1 mM) % activity

direct assay

% activity after

pelleting/resuspension

1-BP, - G6PD

1-BP, + G6PD

1-BP, + G6PD (0.5 mM KCN)

99b

57 Å 5

73 Å 4

 99 Å 2

 65 Å 7

 77 Å 3

2-MBP, - G6PD

2-MBP, + G6PD

2-MBP, + G6PD (0.5 mM KCN)

2-MBP-Im+, - G6PD

2-MBP-Im+, + G6PD

6-MBP-Im+, - G6PD

6-MBP-Im+, + G6PD

99 Å 1

57 Å 5

75 Å 9

96 Å 3

85 Å 3

81 Å 4

43 Å 3

105 Å 3

 58 Å 2

 81 Å 2

101 Å 3

 89 Å 8

2,6-DMBP, -G6PD

2,6-DMBP, + G6PD

2,6-DMBP-Im+, - G6PD

2,6-DMBP-Im+, + G6PD

98b

71 Å 3

96 Å 4

54 Å 2

 98 Å 3

 80 Å 2

108 Å 2

 63 Å 1

4,4-DMBP, - G6PD

4,4-DMBP, + G6PD

4,4-DMBP, + G6PD (0.5 mM KCN)

4,4-DMBP-Im+, - G6PD

4,4-DMBP-Im+, + G6PD

95 Å 2

 85 Å 11

 83 Å 11

99b

75 Å 6

104 Å 3

 93 Å 4

 86 Å 4

102 Å 3

 86 Å 3

PCP, - G6PD

PCP, + G6PD

PCP, + G6PD (0.5 mM KCN)

PCP-Im+, - G6PD

PCP-Im+, + G6PD

98b

58 Å 6

78 Å 5

80 Å 2

36 Å 2

104 Å 3

 57 Å 9

 88 Å 4

 78 Å 4

 27 Å 1

,-DMBP, - G6PD

,-DMBP, + G6PD

,-DMBP, + G6PD (0.5 mM KCN)

,-DMBP-Im +, - G6PD

,-DMBP-Im +,+ G6PD

95 Å 3

43 Å 3

75 Å 2

91 Å 7

37 Å 4

104 Å 3

 45 Å 5

 75 Å 5

 97 Å 4

 46 Å 3

KEY: a = Incubated for 30 minutes, 37%C, pH 7.4 with microsomes from PBinduced rabbits. Values are percent of control activity Å S.E.M. of at least 3

determinations; b = Single determination; c = 0.5 mM concentration.
116

isoform (2B4) that is the most active in aliphatic amine metabolism.

No inhibition by the parent tertiary amines was seen in the absence of

metabolism (the - G6PD data). However, all parent amines exhibited

a metabolism-dependent inactivation of cytochrome P-450 that was

predominantly irreversible, as shown by the data obtained after

pelleting/resuspension of the microsomes. The rank order of

inhibitory potency was ,-DMBP > PCP ~ 2-MBP > 1-BP > 2,6-

DMBP > 4,4-DMBP. The authors’ interpretation of the increased

level of metabolism-dependent inactivation seen for PCP and ,-

DMBP relative to 1-BP is that removal of the exocyclic C-N

dehydrogenation pathway (1-BP metabolism gives substantial Ndebenzylation) results in an increased endocyclic metabolism, which

apparently is the source of enzyme inactivation. In fact, for ,-

DMBP, the level of inactivation is virtually the same as that seen for

the parent amine, implying that essentially all parent amine is

metabolized through endocyclic C-N dehydrogenation. The finding

that ,-DMBP-Im +and PCP-Im+ are equipotent inactivators

confirms that the weaker inactivation of PCP relative to ,-DMBP

at the parent amine stage reflects the alternate ability of PCP to be

metabolized on the cyclohexyl ring.

If the iminium species is on the pathway of metabolism-dependent

inactivation of P-450 by the parent amine, the independently

prepared iminium should be a more potent inactivator. In an earlier

study, no attempt was made to evaluate the P-450 inactivating

potential of 1-BP-Im+ because of its rapid "dimerization" to 3 (Sayre

et al. 1991). However, the iminium species corresponding to the

parent amines 2,6-DMBP, 4,4-DMBP, and PCP were evaluated

directly and were found to exert a greater inhibitory potency than the

parent amines. The finding that most (see below) of the increased

inhibition was dependent on the presence of G6PD (which again was

not reversed by pelleting/resuspen-sion) indicates that the main

inactivating potential arises not from the iminiums themselves but

from their further metabolism.

In the case of 2-MBP, the finding that the more substituted iminium

2-MBP-Im+ is a weaker inactivator than the parent amine, whereas

the less substituted iminium 6-MBP-Im+ is a more potent inactivator,

indicates that endocyclic C-N metabolism on the less-substituted side

is primarily responsible for inactivation by the parent amine.

The finding that PCP-Im+ elicits significant irreversible inactivation

in the a absence of G6PD (a similar result was obtained for 6-MBPIm+

) is consistent with the earlier report by Hoag and colleagues
117

(1987) suggesting a possible direct binding of this iminium to the

enzyme. However, the G6PD-independent inactivation of the

iminiums may represent residual NADPH in the microsomal

preparations, because Osawa and Coon (1989) observed essentially no

G6PD-independent activity loss for PCP-Im+ using purified rabbit P450 forms 2, 3b, and 6 in a reconstituted system.

Based on the ability to trap, at least partially, the various iminium

metabolites using cyanide in the incubation medium, it was expected

that cyanide would inhibit the loss of cytochrome P-450 activity if

the iminium species were an obligatory intermediate in the

inactivation process. As indicated in table 2, partial protection in the

presence of 0.05 mM (or less) KCN was observed in the case of 1-BP,

2-MBP, ,-DMBP, and, consistent with earlier findings (Hoag et al.

1984), PCP. However, the presence of higher concentrations of

KCN (1 mM and above) resulted in either no apparent protection

against inactivation or an actual enhancement of inactivation by the

parent amines (data not shown). The latter finding is contrary to the

expectation that higher [CN'] would result in more efficient trapping

of the iminium species, and even at [CN'] = 1 mM, trapping of a

,-DMBP-Im + (table 1) and especially 2,6-DMBP-Im+ (not shown)

was incomplete. The apparent discrepancies in the cyanide

protection experiments may be explained in part by the observation

that the corresponding -cyano adducts (independently synthesized)

are themselves rather potent inhibitors of the enzyme (data not

shown). Another complicating factor is that 1 mM KCN causes a

significant degree of inhibition of benzphetamine demethylase

activity in its own right. Further work will be needed to arrive at a

fully satisfactory explanation for the cyanide results.

Enamine-Derived Metabolites

Evidence for oxidative metabolism of the endocyclic enamines has

been obtained for both 1-BP and PCP. In a previously reported

(Masumoto et al. 1991) preparative scale metabolism of 1-BP, it was

found that in addition to the C -C . coupled dimer 3, small amounts

of 1-benzyl-3- piperidol (1-BP-3-ol), 1-benzyl-4-piperidol (1-BP-4-

ol), and 1-benzyl-3-piperidone (1-BP-3-one) were obtained (figure 5).

It was further found that cyanide reduced greatly the levels of 1-BP-3-

one metabolite but not the 1-BP-3-ol metabolite, suggesting that 1-

BP-3-one is generated principally from the initial metabolite (1-BPIm+

) rather than from oxidation of 1-BP-3-ol (Masumoto et al. 1991;

Sayre et al. 1991). Consistent with this notion is the finding of

relatively large amounts of the corresponding 3-one in the
118

microsomal metabolism of PCP under conditions where the 4-ol is

observed exclusively of any 4-one (Masumoto et al. 1989). A

plausible mechanism for cytochrome P-450 metabolism of endocyclic

enamines consistent with the generation of 3-piperidones is shown in

figure 6.

Also shown in figure 6 is an alternate route of enamine metabolism

that would lead to a 2-en-4-ol and, after subsequent oxidation, to a 2-

en-4-one. Although no 2-en-4-one was detected in the microsomal

metabolism of 1-BP (Sayre et al. 1991), the corresponding 2-en-4-

one was found by Hoag and colleagues (1988) as a major product

resulting from microsomal metabolism of PCP-Im+. Also of interest

is the report by Zhao and colleagues (1991) that exposure of PCPIm+

 to rat brain and liver mitochondria resulted in the isolation of a

3-formyl derivative that can be rationalized in terms of formylation

of PCP-enamine by a tetrahydrofolic acid formyl donor. These

iminium/enamine metabolic pathways for PCP are summarized in

figure 7. At this time, the information available on the fate of

metabolism of the endocyclic enamines derived from 1-BP and PCP

does not permit an assessment of the actual species responsible for

covalent binding.

Detoxication

As mentioned above, detoxication at the

enamine/iminium/carbinolamine stage occurs in large part through

oxidation to a lactam by both a cystosolic AO and a newly discovered

MND. Although nicotine- 1.(5.)-iminium is readily converted to

cotinine by both enzymes, the lack of any reported lactam metabolite

in the case of PCP suggests that the exocyclic -branching in this

case was sterically interfering with oxidation at the endocylic -

position. Using the rabbit liver postmicro- somal supernatant as

source of AO, it was confirmed that PCP-Im+ and ,-DMPB-Im +

are not converted to lactam (2-one) metabolites (independently

synthesized). However, additional preliminary studies indicate that

even some sterically unencumbered iminium species are poor

substrates for AO (see table 3). The substrate structure-activity

factors which control iminium metabolism by this enzyme have never

been thoroughly investigated, and deserve further research. One

obvious factor may be the effect of ring size on the equilibrium

between iminium, carbinolamine, and ring-opened aldehyde forms.

As far as metabolism by MND is concerned, no substrate other than

nicotine 1.(5.)-iminium has been identified to date. Evidence that at
119

least some interaction with the enzyme can be obtained, however, is

indicated by the ability of at least one iminium species to partially

inhibit the conversion of nicotine 1.(5.)-iminium to cotinine (table 3).

Nonetheless, general iminium ion metabolism is certainly not the

case.
120

In studies designed to begin characterization of MND (Flammang

1994), it was found that MND activity could be destroyed by

delipidation of the microsomes (exposure to detergent), and that

activity of the protein pellet could be restored by addition of dilauroyl

phosphatidyl choline. Kinetic studies revealed activities associated

with both a low- and high-affinity binding of NAD+ but with a singular

binding affinity of the iminium (Flammang 1994). Also, microsomes

obtained from
121
122

phenobarbital-induced rabbits exhibited a less efficient

dehydrogenase activity, in contrast to what is observed for

aldehyde oxidase-mediated cotinine formation. Clearly, much more

work will be needed to elucidate the nature of MND, its

endogenous substrates and physiological role, and its substratestructure profile.

CONCLUSIONS

The most telltale data in terms of mechanism of inactivation of

cytochrome P-450 by the methylated 1-BP analogs is provided by the

results on ,-DMBP and 4,4-DMBP. In the first case, the fact that

the parent amine and iminium intermediate exhibit the same level of

enzyme inactivation provides compelling evidence for a metabolic

route through the endocyclic enamine. In the case of 4,4-DMBP, one

might be tempted to explain the weak inactivation by the parent

amine on the basis of a low level of endocyclic iminium formation.

However, the independently prepared 4,4-DMBP-Im+ is itself a weak

inactivator. The decreased inactivation seen for 4,4-DMBP-Im+ can

be interpreted in terms of figure 6, wherein 4,4-dimethyl substitution

would preclude functionalization at C-4 and would also sterically

hinder enzymatic oxygenation at C-3. The fact that 4,4-DMBP-Im+

still exhibits some inactivation is thus more consistent with C-3

oxygenation being the pathway leading to inactivation. This is also

compatible with the decreased inactivation caused by the C-2 methyl

group in 2-MBP-Im+ and 2,6-DMBP-Im+. Thus, although one cannot

deduce at this time the nature of the reactive species generated,

cytochrome P-450 oxygenation at C-3 of the enamine appears to be

the most reasonable path leading to enzyme inactivation.

The significance of the covalent binding that accompanies the

metabolism of nicotine and PCP is uncertain. Although there is

presently no direct evidence for acute toxic effects specifically

associated with such covalent binding, a role in the toxicologic effects

of these or related drugs cannot be excluded at present. Any drug in

clinical use has a long list of contra-indications. For any given drug,

there is always a percentage of users whose individual biochemical

metabolism and/or other drug use creates a situation that causes an

adverse response to the new drug. The reasons for this adverse

reaction are often not apparent. If drugs with associated covalent

binding potential inactivate a sufficiently high percentage of a

particular metabolic isozyme, then adverse drug interaction scenarios
123

may arise. This possibility has been recognized recently in the case of

PCP (Owens et al. 1993).

A key focus of this chapter has been on the iminium/enamine stage of

metabolism (figure 1) as the critical point governing the balance

between toxic activation and detoxication pathways. Thus, although

AO and MND efficiently intercept the 1.(5.)-iminium derived from

nicotine, PCP-Im+ is not a substrate for either enzyme. It seems quite

significant that the lack of aldehyde oxidase substrate potential in this

case correlates with increased levels of covalent binding of PCP

relative to nicotine.

The balance between toxication and detoxication may also vary

considerably between different tissues. For example, whereas

microsomal rates of conversion of nicotine to the 1.(5.)-iminium

ion for rabbit liver and lung are comparable on a per weight basis,

nicotine is oxidized at a higher rate by lung microsomes than by liver

microsomes when one corrects for the much smaller P-450 content

of lung microsomal protein (McCoy, unpublished data). The higher

rate of nicotine metabolism in the rabbit lung results from the

enrichment in P-450 isozymes which preferentially metabolize

nicotine to the 1.(5.)-iminium ion, for example, CYP2B4 and

CYP4B1, which account for 90 percent of lung P-450 content

(Serabjit-Singh et al. 1979) but less than 20 percent of the liver total

P-450 content (Lu and West 1980). Further studies (Flammang

1994) demonstrate very low aldehyde oxidase activity in rabbit lung

compared to liver, and also very low MND activity in lung compared

to liver, as also reported by Obach and Van Vunakis (1990). Thus

because of the high nicotine oxidation rate and the poor

iminiumøcotinine activity in lung tissue, the potential exists for the

intracellular concentration of nicotine 1.(5.)-iminium to be much

greater in lung than in liver. Covalent binding to tissue macromolecules (Williams et al. 1990) resulting from high intracellular

concentrations of the 1.(5.)-iminium or its metabolites may thus be

especially problematic in the lung.

Many naturally occurring and synthetic drugs directed at biogenic

amine receptors contain a basic tertiary amine center. It is common

practice in the development of new central nervous system (CNS)-

active pharma-ceuticals to incorporate pyrrolidino, piperidino, or

morpholino moieties (in addition to dimethylamino) in structureactivity investigations. A significant number of cyclic tertiary amine

drugs have thus appeared (and continue to appear) in the marketplace

despite the fact that this structural construct appears to be especially
124

prone to generating reactive intermediates during oxidative

metabolism.

In some instances, avoiding reactive intermediate generation in the first

place may be possible. If one has the choice between two

pharmacologically equivalent drug analogs, only one of which has a

covalent binding potential, it is clear which analog would be more desirable.

The work summarized here was aimed at elucidating any patterns that might

permit strategic elimination of metabolism-dependent covalent binding

potential. It may be a simple matter of using 4,4-dimethylpiperidino

instead of piperidino, for example, as long as the desired biological activity

is not compromised. Information leading to prevention of potential

toxicity should be quite welcome.

REFERENCES

Brady, J.F.; Dokko, J.; Di Stefano, E.W.; and Cho, A.K. Mechanism-based

inhibition of cytochrome P-450 by heterocyclic analogues of

phencyclidine. Drug Metab Dispos 15(5):648-652, 1987.

Brandange, S., and Lindblom, L. Synthesis, structure and stability of nicotine 

1.(5.)-iminium ion, an intermediary metabolite of

nicotine. Acta Chem Scand B 33:187-191, 1979a.

Brandange, S., and Lindblom, L. The enzyme "aldehyde oxidase" is an iminium

oxidase. Reaction with nicotine 1.(5.)-iminium ion.

Biochem Biophys Res Commun 91(3):991-996, 1979b.

Cook, A.G. Structure and physical properties of enamines. In: Cook, A.G., ed.

Enamines: Synthesis, Structure, and Reactions. 2nd ed. New

York: Marcel Dekker, 1988. pp. 1-102.

Engelhart, D.A. "Part I. Metabolic Activation of Cyclic Tertiary Amines. Part

II. Neurotoxic Activation of -.-iminodipropionitrile

(IDPN)." Ph.D. thesis, Case Western Reserve University, 1994.

Felsted, R.L.; Chu, A.E.-Y.; and Chaykin, S. Purification and properties of the

aldehyde oxidases from hog and rabbit livers. J Biol Chem

248(7):2580-2587, 1973.

Flammang, A.M. "Relative Rates of Hepatic and Pulmonary Enzyme Systems

Involved in the Metabolic Fate of Nicotine." Ph.D. thesis, Case

Western Reserve University, 1994.

Gole, D.J.; Pirat, J.-L.; Kamenka, J.-M.; and Domino, E.F. Hydroxy metabolites

of phencyclidine. Drug Metab Disp 16(3):386-391, 1988.

Hallstrom, G.; Kammerer, R.C.; Nguyen, C.H.; Schmitz, D.A.; Di Stefano, E.W.;

and Cho, A.K. Phencyclidine metabolism in vitro. The

formation of a carbinolamine and its metabolites by rabbit liver

preparations. Drug Metab Disp 11(1):47-53, 1983.
125

Hammer, W.; Holmstedt, B.; Karlen, B.; Sjoqvist, F.; and Vessman, J. The

metabolism of tremorine. Identification of a new biologically

active metabolite, N-(4-pyrrolidino-2-butynyl)--aminobutyric

acid. Biochem Pharmacol 17:1931-1941, 1968.

Herber, R.; Perrin, R.; Ziegler, J.-M.; Villoutreix, J.; Minn, A.; and Siest, G.

Identification of novel phencyclidine metabolites formed in

vitro by rabbit microsomal metabolism. Xenobiotica

21(11):1493-1499, 1991.

Ho, B., and Castagnoli, N., Jr. Trapping of metabolically generated electrophilic

species with cyanide ion: Metabolism of 1-benzylpyrrolidine. J

Med Chem 23:133-139, 1980.

Hoag, M.K.P.; Trevor, A.J.; Asscher, Y.; Weissman, J.; and Castagnoli, N., Jr.

Metabolism-dependent inactivation of liver microsomal

enzymes by phencyclidine. Drug Metab Dispos 12(3):371-375,

1984.

Hoag, M.K.P.; Trevor, A.J.; Kalir, A.; and Castagnoli, N., Jr. Phencyclidine

iminium ion. NADPH-dependent metabolism, covalent binding

to macromolecules, and inactivation of cytochrome(s) P-450.

Drug Metab Dispos 15(4):485-490, 1987.

Hoag, M.K.P.; Schmidt-Peetz, M.; Lampen, P.; Trevor, A.; and Castagnoli, N.,

Jr. Metabolic studies on phencyclidine: Characterization of a

phencyclidine iminium ion metabolite. Chem Res Toxicol

1:128-131, 1988.

Holsztynska, E.J., and Domino, E.F. Biotransformation of phencyclidine. Drug

Metab Rev 16(3):285-320, 1985.

Leonard, N.J., and Hauck, F.P., Jr. Unsaturated amines. X. The mercuric acetate

route to substituted piperidines, 2-tetrahydropyridines

and 2-tetrahydroanabasines. J Am Chem Soc 79:5279-5292,

1957.

Lin, G.; Hawes, E.M.; McKay, G.; Korchinski, E.D.; and Midha, K.K. The

metabolism of piperidine-type phenothiazine antipsychotic

agents. Xenobiotica 23(1):37-52, 1993.

Lu, A.Y.J., and West, S.B. Multiplicity of mammalian microsomal cytochromes

P-450. Pharmacol Rev 31(4):277-295, 1980.

Masumoto, H.; Takeuchi, K.; Ohta, S.; and Hirobe, M. Application of chemical

P450 model systems to studies on drug metabolism. I.

Phencyclidine: A multi-functional model substrate. Chem

Pharm Bull 37(7):1788-1794, 1989.

Masumoto, H.; Ohta, A.; and Hirobe, M. Application of chemical cytochrome

P-450 model systems to studies on drug metabolism. IV.

Mechanism of piperidine metabolism pathways via an iminium

intermediate. Drug Metab Dispos 19(4):768-780, 1991.

Mattammal, M.B.; Lakshmi, V.M.; Zenser, T.V.; and Davis, B.B. Lung

prostaglandin H synthase and mixed-function oxidase
126

metabolism of nicotine. J Pharmacol Exp Ther 242(3):827-

832, 1987.

Nguyen, T.-L.; Gruenke, L.D.; and Castagnoli, N., Jr. Metabolic oxidation of

nicotine to chemically reactive intermediates. J Med Chem

22(3):259-263, 1979.

Obach, R.S., and Van Vunakis, H. Non-metabolic covalent binding of nicotine1.(5.)-iminium ion to liver microsomes and sulfhydrylcontaining polyamino acids. Biochem Pharacol 37(24):4601-

4604, 1988.

Obach, R.S., and Van Vunakis, H. Nicotinamide adenine dinucleotide (NAD)-

dependent oxidation of nicotine- 1.(5.)-iminium ion to

cotinine by rabbit liver microsomes. Biochem Pharmacol

39(1):R1-R4, 1990.

Osawa, Y., and Coon, M.J. Selective mechanism-based inactivation of the major

phenobarbital-inducible P-450 cytochrome from rabbit liver by

phencyclidine and its oxidation product, the iminium

compound. Drug Metab Dispos 17(1):7-13, 1989.

Overton, M.; Hickman, J.A.; Threadgill, M.D.; Vaughan, K.; and Gescher, A.

The generation of potentially toxic, reactive iminium ions from

the oxidative metabolism of xenobiotic N-alkyl compounds.

Biochem Pharmacol 34(12):2055-2061, 1985.

Owens, S.M.; Gunnell, M.; Laurenzana, E.M.; and Valentine, J.L. Dose- and

time-dependent changes in phencyclidine metabolite covalent

binding in rats and the possible role of CYP2DI. J Pharmacol

Exp Ther 265(3):1261-1266, 1993.

Sayre, L.M.; Engelhart, D.A.; Venkataraman, B.; Babu, M.K.M.; and McCoy,

G.D. Generation and fate of enamines in the microsomal

metabolism of cyclic tertiary amines. Biochem Biophys Res

Commun 179(3):1368-1376, 1991.

Serabjit-Singh, C.J.; Wolf, C.R.; and Philpot, R.M. The rabbit pulmonary

monoxygenase system: Immunochemical and biochemical

characterization of enzyme components. J Biol Chem

254:9901-9907, 1979.

Shigenaga, M.K.; Trevor, A.J.; and Castagnoli, N., Jr. Metabolism-dependent

covalent binding of (S)-[5-3H]nicotine to liver and lung

microsomal macromolecules. Drug Metab Dispos 16(3):397-

402, 1988.

Wall, G.M., and Baker, J.K. Metabolism of 3-(p-chlorphenyl)pyrrolidine.

Structural effects in conversion of a prototype -aminobutyric

acid prodrug to lactam and -aminobutyric acid type metabolites.

J Med Chem 32(6):1340-1348, 1989.

Ward, D.P.; Trevor, A.J.; Kalir, A.; Adams, J.D.; Baillie, T.A.; and Castagnoli,

N., Jr. Metabolism of phencyclidine. The role of iminium ion
127

formation in covalent binding to rabbit microsomal protein.

Drug Metab Dispos 10(6):690-695, 1982.

Williams, D.E.; Shigenaga, M.K.; and Castagnoli, N., Jr. The role of

cytochromes P-450 and flavin-containing monoxygenase in the

metabolism of (S)-nicotine by rabbit lung. Drug Metab Dispos

18(4):418-428, 1990.

Zhao, A.; Leung, L.Y.; Trevor, A.; and Castagnoli, N., Jr. C-Formylation in the

presence of rat brain mitochondria of the 2,3,4,5-tetrahydropyridinium metabolite derived from the psychotomimetic drug

phencyclidine. Chem Res Toxicol 4:426-429, 1991.

ACKNOWLEDGMENTS

Work in the authors’ laboratories was supported by National Institutes of

Health grant NS 22688.

AUTHORS

Lawrence M. Sayre, Ph.D.

David A. Engelhart, Ph.D.

Durgesh V. Nadkarni, Ph.D.

M.K. Manoj Babu

Department of Chemistry

and

Ann Marie Flammang, Ph.D.

G. David McCoy, Ph.D.

Department of Environmental Health Sciences

Case Western Reserve University

Cleveland, OH 44106
128

Neurotoxicity of Amphetamines and

Their Metabolites

James W. Gibb, Michel Johnson, Ikram Elayan, Heng Keang Lim,

Lisa Matsuda, and Glen R. Hanson

When amphetamine or an analog is administered in repeated high

doses, neurochemical deficits in both the dopaminergic and

serotonergic systems of selected areas of the brain are observed.

Methamphetamine (10 to 15 milligrams per kilogram (mg/kg)),

administered every 6 hours for 5 doses, decreases rat tyrosine

hydroxylase (TH) activity and dopamine (DA) content in the

neostriatum within 18 hours after the first dose (Koda and Gibb

1973). Interestingly, these neurochemical deficits persist in rats and

nonhuman primates for extended periods of time after the drug is

discontinued (Ellison et al. 1978; Bakhit et al. 1981; Woolverton et

al. 1989).

Buening and Gibb (1974) demonstrated that DA plays a critical role in

the neurochemical deficits observed after methamphetamine

administration. The DA antagonists chlorpromazine and haloperidol,

when administered concurrently with methamphetamine, completely

blocked the metham-phetamine-induced alterations in neostriatal TH

activity and DA content. Subsequent studies (Gibb and Kogan 1979)

demonstrated that when the rate-limiting enzyme TH was inhibited by

administering -methyltyrosine (MT), the neurochemical deficits

normally observed with methamphet- amine were prevented.

Consistent with the concept that DA is necessary for the

methamphetamine-induced neurochemical deficits, the neurochemical deficits were again observed when DA synthesis was restored

by administering L-dopa.

Not only is methamphetamine administration toxic to the

dopaminergic system, but the serotonergic system in the various brain

areas is also altered. Hotchkiss and Gibb (1980) reported that

methamphetamine, administered as described above, decreased

tryptophan hydroxylase (TPH) activity in the serotonergic nerve

terminal of rat brain and spinal cord. Similarly, the content of 5-

hydroxytryptamine (5-HT) and its metabolite 5-hydroxyindole acetic

acid (5-HIAA) were also severely depressed. In contrast to the effects

in the dopaminergic system, these serotonergic parameters were

decreased by methamphetamine within 15 minutes after a single dose
129

(Bakhit and Gibb 1981) and were more pronounced than the

dopaminergic deficits. Like the dopaminergic changes, these

serotonergic alterations persisted for extended periods of time (Bakhit

et al. 1981).

Interestingly, the serotonergic changes resulting from toxic doses of

meth- amphetamine are also dependent on DA. When MT was

administered concurrently with methamphetamine, the decreases in

TPH activity and 5-HT and 5-HIAA content were prevented

(Hotchkiss and Gibb 1980; Schmidt et al. 1985). When DA synthesis

was reinstated by adding L-dopa to the dosing regimen, the

neurochemical deficits were again observed. Further evidence for DA

dependence was reported by Johnson and colleagues (1987), who

destroyed the dopaminergic nerve terminals in the neostriatum by

injecting 6-hydroxydopamine (6-OHDA) into the substantia nigra.

Eleven days later the usual neurotoxic dosage regimen of methamphetamine was administered and TPH activity in the neostriatum,

cerebral cortex, and the hippocampus was determined. The usual

decrease in TPH activity was observed in the cerebral cortex and

hippocampus; however, in the neostriatum, which was deprived of

dopaminergic input by prior administration of 6-OHDA, the deficit of

TPH usually observed with methamphetamine was significantly

attenuated.

The pronounced neurochemical deficits in the dopaminergic and

serotonergic systems posed the question of whether

methamphetamine, administered in these large, repeated doses, was a

general neurotoxin affecting all neurotransmitter systems. Hotchkiss

and colleagues (1979) found no effects on the cholinergic system or

on the glutaminergic system as defined by an absence of any

alteration of the respective synthesizing enzymes of these

neurotransmitter systems.

Ricaurte and colleagues (1985) demonstrated that an analog of

amphetamine, 3,4-methylenedioxyamphetamine (MDA), is

neurotoxic to monoamine systems. They observed that neostriatal

and hippocampal 5-HT and 5-HIAA contents were depressed after

MDA administration and that hippocampal norepinephrine (NE)

content was also compromised. In comparing the effects of

methylenedioxymethamphetamine (MDMA) and methamphetamine,

it was observed (Stone et al. 1986, 1987) that while

methamphetamine decreased both TH and TPH activity, MDMA

depressed only TPH activity without altering the DA-synthesizing

enzyme. Although MDMA releases DA (Yamamoto and Spanos
130

1988), this analog is selectively neurotoxic to the serotonergic

system at doses that do not cause any persisting effect on the

dopaminergic system.

Further studies (Stone et al. 1988) provided extensive evidence that

the neurochemical deficits induced by MDMA are DA dependent.

The deficits in TPH activity and content of 5-HT and 5-HIAA

normally seen in MDMA-treated rats were attenuated by concurrent

administration of MT; these deficits returned when L-dopa was

administered concurrently with MT and MDMA. Prior treatment

with 6-OHDA (described above) selectively attenuated the

serotonergic deficits in the neostriatum while no protection by 6-

OHDA occurred in the hippocampus or cerebral cortex. Prior

depletion of DA with reserpine attenuated the neurotoxicity of

MDMA. From these experiments the authors concluded that DA

and/or its metabolites play a key role not only in the neurotoxicity

observed with methamphetamine, but also with its methylenedioxy

analogs.

Because of the apparent role for DA in the toxicity caused by methamphetamine and its analogs, the authors reasoned that DA and/or a reactive

DA metabolite may be oxidizing components of the dopaminergic and

serotonergic nerve terminals to cause the persisting deficits in these two

neuronal systems. To test this hypothesis, TPH activity was measured in

cerebral cortex from rats treated with toxic doses of methamphetamine,

MDMA, or p-chloroamphetamine (Stone et al. 1989). Three hours after

receiving one of the amphetamines, the rats were killed and the supernatant

fraction of the cerebral cortex containing TPH was obtained. An aliquot of

the supertanant was then exposed to various reducing agents in a nitrogen

atmosphere for a 24-hour period. As previously observed, enzyme activity

from rats treated with the amphetamine analog was markedly impaired

compared to that from untreated rats. TPH activity from rats treated with

the amphetamine analog was essentially normal in those samples incubated

with dithiothreitol, a reducing agent, in a nitrogen atmosphere; other

reducing agents were not as effective. A time-response relationship

revealed that the enzyme activity could be restored only during the first 6-

hour period. After 6 hours, oxidation of the enzyme had apparently

progressed to a point where it was irreversible.

METABOLITES OF AMPHETAMINE

Methamphetamine administered directly into the brain is not neurotoxic

(Matsuda 1987; Molliver et al. 1986; Berger et al. 1990; Paris and
131

Cunningham 1990). Furthermore, when methamphetamine or its analogs

are incubated with brain slices or homogenates, no impairment of TH or

TPH activity is observed. This finding led to an investigation of whether

an amphetamine metabolite is responsible for neurotoxicity (Matsuda et al.

1989). Ketamine-anesthetized rats received bilateral injections of either phydroxyamphetamine (pOHA) or p-hydroxynorephedrine (pOHN) at doses

of 0.5, 5, or 50 micrograms (g) administered directly into the

neostriatum. Three hours later, striatal DA and 5-HT contents were

determined. The 50 g dose of pOHA or pOHN decreased the concentration of striatal DA to 27 percent and 18 percent of control,

respectively. The 5-HT content was also decreased by both metabolites,

but not to the same extent as DA. The DA metabolites

dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)

declined in parallel with DA, but 5-HIAA content was not changed by

pOHA and was actually elevated after injection of pOHN. The decline in

DA and its metabolites reached its nadir at 6 hours and returned to control

levels by 48 hours. Surprisingly, TH activity was not altered when either

pOHA or pOHN was administered intrastriatally. TPH activity was actually

elevated when either of the higher doses of the two amphetamine

metabolites was administered.

The effect of systemic administration of these amphetamine metabolites

on the content of DA and its metabolites and on 5-HT was then examined.

When pOHA was administered systemically (5, 15, or 30 mg/kg), the

concentration of DA and its metabolites in the neostriatum was depressed.

The effect on the neostriatal serotonergic system was not as pronounced as

that on the dopaminergic system; hippocampal serotonin content was

altered only at the higher dose of pOHA and hypothalamic 5-HT

concentrations were decreased at both the 15 mg and 30 mg doses. The

authors then determined the effect of the monoaminergic uptake inhibitors

amfonelic acid and cocaine on the response to systemically administered

pOHA. The decrease in DA and its metabolites was attenuated by

amfonelic acid (1 mg/kg, administered 30 minutes before pOHA), but

cocaine failed to alter the effects of systemic pOHA administration.

The metabolites of amphetamine, in the doses administered, decrease the

concentrations of DA and its metabolites as well as 5-HT. The pronounced

decrease in DA and its metabolites returned to normal within 48 hours. No

decrease in either TH or TPH activity was observed concurrent with the

decrease in DA. From these observations it appears that these

amphetamine metabolites transiently decrease the neurotransmitter

concentrations, but are not neurotoxic since the activity of the

synthesizing enzymes TH and TPH are not altered.
132

The effects of cerebral injections of methamphetamine on striatal

TPH activity were also investigated (figure 1). In an anesthetized rat,

systemically administered methamphetamine (10 mg/kg) decreased

striatal TPH activity. However, bilateral injections of

methamphetamine into the neostriatum (100 g) failed to decrease

striatal TPH activity 3 hours after treatment. To determine whether

multiple areas of the brain are involved in methamphetamine-induced

changes in striatal TPH, microinjections of methamphetamine were

administered into the substantia nigra or lateral midbrain and into the

striatum. Three hours after treatment, injections of

methamphetamine into the striatum (100 g) and the substantia

nigra (25 g) or the lateral midbrain (area of B9 serotonergic cell

bodies) (50 g) did not reduce TPH activity in the neostriatum.
133

Similarly, in rats treated with methamphetamine (800 g

intracerebroventricularly (ICV)), TPH activity was unaffected in the

cerebral cortex and hypothalamus (data not shown) as well as in the

neostriatum. These results suggest that direct application of methamphetamine to neural tissues has no effect on TPH activity; however,

the possibility cannot be ruled out that the duration of exposure of

locally injected methamphetamine, in any region, was too brief to be

effective.

METABOLITES OF MDMA

As indicated above, large doses of MDMA cause profound and

persisting neurochemical deficits in the serotonergic nerve terminals

without any lasting effects on the dopaminergic nerve terminals.

Since the parent compound is not toxic when injected directly into

the brain (Molliver et al. 1986; Paris and Cunningham 1990), the

possibility that one or more MDMA metabolites cause the

neurotoxicity observed after administration of large doses of the drug

was considered.

2,4,5-Trihydroxyamphetamine

While investigating the metabolism of MDMA, two of the authors’

colleagues identified eight metabolites formed in vivo (Lim and Foltz

1991a, 1991b). The authors injected synthesized metabolites into

rats to determine their possible neurotoxicity (figure 2). Rats

received a single injection of either 0.25 or 0.5 micromoles (mole)

of 2,4,5-trihydroxy-amphetamine (THA) ICV and were killed 7 days

later (figure 3).

TPH activity was dramatically decreased in the hippocampus to 5 or

1 percent of control, respectively, with 0.25 or 0.5 moles of the

metabolite. The content of 5-HT and 5-HIAA in the hippocampus

was also decreased. The striatal serotonergic system was more

resistant to the effects of THA. In this structure, TPH activity was

decreased to 74 percent and 81 percent of control 1 week after a

single injection of 0.25 or 0.5 mol of THA, while the concentration

of 5-HT and 5-HIAA remained unaltered. THA administration

dramatically decreased all dopaminergic parameters in the striatum.
134

In response to the administration of 0.25 or 0.5 mol of THA,

striatal TH was reduced to 67 and 10 percent of control, respectively

(figure 4). A similar decrease in content of DA and its metabolites

was observed in the striatum contralateral to the site of injection.

When enzyme activity in the dopaminergic and serotonergic cell

bodies from the THA-treated rats was examined, no change was

observed in the medial or dorsal raphe TPH activity. However, TH

activity in the substantia nigra was decreased to 59 and 20 percent of

control in animals treated with 0.25 or 0.5 mol of THA,

respectively. To assess the response of the noradrenergic system,

hippocampal NE content was measured in the THA-treated animals.

NE content was lowered to 10 and 18 percent of control after 0.25

and 0.5 mol of THA, respectively.

2,4,5-Trihydroxymethamphetamine

Corresponding experiments were conducted with another analog of

MDMA, 2,4,5-trihydroxymethamphetamine (THM). A single dose

of 50, 100, or 200 g was administered ICV 5 days before sacrificing

the rats and TPH activity was determined in various areas of the brain

(figure 5).
135
136

Of the three serotonergic terminal areas examined, TPH activity in

the hippocampus was most impaired to 58 and 10 percent of control

in rats treated with 50 or 200 g of THM. Enzyme activity in the

frontal cortex was decreased less than that observed in the

hippocampus, while the striatum was least affected. TPH activity in

the cell bodies did not decrease; in fact, enzyme activity was elevated

in both the medial and dorsal raphe, depending on the dose of THM.

The effect of THM on 5-HT content followed a pattern similar to

TPH activity in the corres-ponding brain areas, while 5-HIAA

concentrations were not altered by THM in these structures.
137

The effects of THM on the central dopaminergic system were then

examined (figure 6). TH activity was markedly depressed to 76, 56,

and 21 percent of control in the striatum of rats administered a single

ICV injection of 50, 100, or 200 g, respectively, of THM 5 days

previously. A decrease in the content of DA and its metabolites in

these structures was also observed. TH activity was not altered in the

substantia nigra. Another metabolite of MDMA, 3,4-

dihydroxyamphetamine (DHM), was administered ICV (135 g) and

the enzymatic response was examined.
138

The activity of TPH was unaltered by DHM, but TH activity was

significantly elevated by this metabolite of MDMA.

SHORT-TERM EFFECTS OF MDMA METABOLITES

In the studies cited above, the authors determined the effects of

MDMA metabolites 5 to 7 days after administering the drug. In other

experiments (described below), rats were administered 1 mol of

THA, THM, or DHM ICV and the response was determined 3 hours

later. THA decreased TPH activity to 79 percent of control in the

neostriatum, but in the hippocampus enzyme activity was markedly

decreased to 8 percent of control. THM decreased striatal TPH

activity to 87 and 54 percent of control in the striatum and

hippocampus, respectively. The only effect of DHM was an

elevation of enzyme activity in the striatum.
139

When the response of the dopaminergic system was investigated, TH

activity had decreased to 75 percent of control in the striatum

following THA administration, but was unaffected by THM. THA

elevated neostriatal content of DA (135 percent), DOPAC (287

percent), HVA (237 percent), and 5-HT (115 percent). THM

elevated DOPAC (512 percent), HVA (366 percent), and 5-HIAA

(149 percent), but decreased 5-HT (74 percent) striatal content.

The authors had previously demonstrated that the decrease in enzyme

activity observed in rats treated for 3 hours with MDMA was reversed

by exposing the supernatant fraction containing TPH to reducing

conditions (dithiothreitol in a nitrogen atmosphere) for 24 hours.

Incubating hippo- campal TPH from animals treated with THM failed

to prevent the decrease in enzyme activity caused by the metabolite;

this finding suggests that the enzyme changes produced by the

metabolite occur by a different mechanism than those caused by the

parent compound.

Because THA and THM are structurally similar to 6-OHDA, it was

important to ascertain whether 6-OHDA administered in a similar

fashion would produce the same response. Since TPH activity from

the striatum and the hippocampus was not altered by 6-OHDA after 3

hours, the DA analog is not likely to be responsible for the toxicity

associated with administering THA or THM.

It is well established that 5,7-dihydroxytryptamine (DHT) is toxic to

serotonergic neurons. Lim and Foltz (1991b) reported that THA and

THM cyclize, thus forming an indole molecule that is similar in

structure to DHT. The effect of DHT on hippocampal TPH activity

was therefore examined (figure 7). This serotonergic neurotoxin

decreased enzyme activity to 18 percent of control. When TPH was

incubated under the reducing conditions described above, there was no

reversal of the enzyme activity. This observation would argue against

the possibility that DHT is the neurotoxin responsible for MDMAinduced neurotoxicity.

The authors then investigated the effect on hippocampal TPH

activity when incubated in vitro with THA (figure 8). The

hippocampus or striatum of rats was excised and prepared slices were

incubated for 1 hour in buffer containing 0.001, 0.01, 0.1, 0.5, or 5.0

millimoles (mM) THA.
140

As depicted in figure 8, TPH activity was markedly inhibited when the

enzyme was incubated in vitro with THA. When the enzyme was

incubated under reducing conditions, the enzyme activity was not

restored to normal. As was observed in vivo, hippocampal TPH was

more sensitive than striatal TPH since it was inhibited at a lower

concentration of THA. The mechanism responsible for this

interesting response is under investigation.
141
142

CONCLUSIONS

Methamphetamine, when administered in large doses, causes neurochemical deficits in both the dopaminergic and serotonergic nerve

terminals of the brain that persist for extended periods of time.

When the methamphetamine analog MDMA is administered,

selective and persistent neurochemical deficits are observed in the

serotonergic terminal regions. DA is essential for these

neurochemical deficits. Reducing conditions reverse these

amphetamine-induced changes, providing evidence that oxidative

stress is an important component in causing the neurochemical

deficits.

The role of metabolites of amphetamine or MDMA has been

extensively explored. Although some of the responses to these

metabolites are similar to those of the parent compounds, the

neurochemical profile observed with the metabolites is distinctly

different from the deficits induced by parent compounds. From these

observations it appears that there are additional factors responsible

for the neurochemical deficits caused by amphetamine and its

congeners.

REFERENCES

Bakhit, C., and Gibb, J.W. Methamphetamine-induced depression of

tryptophan hydroxylase: Recovery following acute treatment.

Eur J Pharmacol 76:229-233, 1981.

Bakhit, C.; Morgan, M.E.; Peat, M.A.; and Gibb, J.W. Long-term

effects of methamphetamine on synthesis and metabolism of 5-

hydroxy-tryptamine in various regions of the rat brain.

Neuropharmacology 20:1135-1140, 1981.

Berger, U.V.; Molliver, M.E.; and Grzanna, R. Unlike systemic

administration of p-chloroamphetamine, direct intracerebral

injection does not produce degeneration of 5-HT axons. Exp

Neurol 109(3):257-268, 1990.

Buening, M.K., and Gibb, J.W. Influence of methamphetamine and

neuroleptic drugs on tyrosine hydroxylase activity. Eur J

Pharmacol 26:30-34, 1974.

Ellison, G.; Eison, M.S.; Huberman, H.S.; and Daniel, F. Long-term

changes in dopaminergic innervation of caudate nucleus after

continuous amphetamine administration. Science 201:276-278,

1978.
143

Elayan, I.; Gibb, J.W.; Hanson, G.R.; Foltz, R.L.; Lim, H.K.; and

Johnson, M. Long-term alteration in the central monoaminergic

systems of the rat by 2,4,5-trihydroxyamphetamine but not by 2-

hydroxy-4,5-methyl-enedioxymethamphetamine or 2-hydroxy4,5-methylenedio-xyamphetamine. Eur J Pharmacol 221:281-

288, 1992.

Elayan, I.; Gibb, J.W.; Hanson, G.R.; Lim, H.K.; Foltz, R.L.; and

Johnson, M. Short-term effects of 2,4,5-trihydroxyamphetamine,

2,4,5- tri-hydroxymethamphetamine and 3,4-

dihydroxymethamphetamine on central tryptophan hydroxylase

activity. J Pharmacol Exp Ther 265:813-818, 1993.

Gibb, J.W., and Kogan, F.J. Influence of dopamine synthesis on

methamphetamine-induced changes in striatal and adrenal

tyrosine hydroxylase activity. Naunyn-Schmiedeberg Arch

Pharmacol 310:185-187, 1979.

Hotchkiss, A.J., and Gibb, J.W. Long-term effects of multiple doses of

methamphetamine on tryptophan hydroxylase and tyrosine

hydroxylase in rat brain. J Pharmacol Exp Ther 214:257-262,

1980.

Hotchkiss, A.; Morgan, M.E.; and Gibb, J.W. The long-term effects of

multiple doses of methamphetamine on neostriatal tryptophan

hydroxylase, tyrosine hydroxylase activity, choline

acetyltransferase and glutamate decarboxylase activities. Life Sci

25:1373-1378, 1979.

Johnson, M.; Elayan, I.; Hanson, G.R.; Foltz, R.L.; Gibb, J.W.; and

Lim, H.K. Effects of 3,4-dihydroxymethamphetamine and 2,4,5-

trihydroxy-methamphetamine, two metabolites of 3,4-

methylene-dioxymethamphetamine, on central serotonergic and

dopaminergic systems. J Pharmacol Exp Ther 261:447-453,

1992.

Johnson, M.; Stone, D.M.; Hanson, G.R.; and Gibb, J.W. Role of the

dopaminergic nigrostriatal pathway in methamphetamine-induced

depression of the neostriatal serotonergic system. Eur J

Pharmacol 135:231-234, 1987.

Koda, L.Y., and Gibb, J.W. Adrenal and neostriatal tyrosine

hydroxylase activity after methamphetamine. J Pharmacol Exp

Ther 185:42-48, 1973.

Lim, H.K., and Foltz, R.L. In vivo formation of aromatic

hydroxylated metabolites of 3,4-

(methylenedioxy)methamphetamine in the rat: Identification by

ion trap ms/ms and ms/ms/ms techniques. Biol Mass Spectrom

20:677-686, 1991a.

Lim, H.K., and Foltz, R.L. Ion trap tandem mass spectrometric

evidence for the metabolism of 3,4-
144

(methylenedioxy)methamphetamine to the potent neurotoxins

2,4,5-trihydroxymethamphetamine and 2,4,5-

trihydroxyamphetamine. Chem Res Toxicol 4:626-632, 1991b.

Matsuda, L.A. "Effects of Local Application of Methamphetamine

and its Para-hydroxylated Metabolites on Central Monoaminergic

Systems." Ph.D. dissertation, University of Utah, 1987.

Matsuda, L.A.; Hanson, G.R.; and Gibb, J.W. Neurochemical effects of

amphetamine metabolites on central dopaminergic and

serotonergic systems. J Pharmacol Exp Ther 251:901-908, 1989.

Molliver, M.E.; O’Hearn, E.; Battaglia, G.; and DeSouza, E.B. Direct

intracerebral administration of MDA and MDMA does not

produce serotonin neurotoxicity. Soc Neurosci Abstr 12:1234,

1986.

Paris, J.M., and Cunningham, K.A. Lack of neurotoxicity after intraraphe microinjection of MDMA ("Ecstacy"). In: Harris, L., ed.

Problems of Drug Dependence, 1990. National Institute on Drug

Abuse Research Monograph 105. DHHS Pub. No. (ADM) 91-

1754. Washington, DC: Supt. of Docs., U.S. Govt. Print. Off.,

1990.

Ricaurte, G.; Bryan, G.; Strauss, L.; Seiden, L.; and Schuster, C.

Hallucinogenic amphetamine selectively destroys brain serotonin

nerve terminals. Science 229:486-488, 1985.

Schmidt, C.; Ritter, J.K.; Sonsalla, P.K.; Hanson, G.R.; and Gibb, J.W.

Role of dopamine in the neurotoxic effects of methamphetamine.

J Pharmacol Exp Ther 233:539-544, 1985.

Stone, D.M.; Stahl, D.C.; Hanson, G.R.; and Gibb, J.W. The effects of

3,4-methylenedioxymethamphetamine (MDMA) and 3,4-

methylene-dioxyamphetamine (MDA) on monoaminergic

systems in the rat brain. Eur J Pharmacol 128:41-48, 1986.

Stone, D.M.; Johnson, M.; Hanson, G.R.; and Gibb, J.W. A

comparison of the neurotoxic potential of

methylenedioxyamphetamine (MDA) and its N-methylated and

N-ethylated derivatives. Eur J Pharmacol 134:245-248, 1987.

Stone, D.M.; Johnson, M.; Hanson, G.R.; and Gibb, J.W. Role of

endogenous dopamine in the central serotonergic deficits induced

by 3,4-methylenedioxymethamphetamine. J Pharmacol Exp Ther

247:79-87, 1988.

Stone, D.M.; Hanson, G.R.; and Gibb, J.W. In vitro reactivation of rat

cortical tryptophan hydroxylase following in vivo inactivation by

methylenedioxymethamphetamine. J Neurochem 53:572-581,

1989.

Woolverton, W.L.; Ricaurte, G.A.; Forno, L.S.; and Seiden, L.S.

Long-term effects of chronic methamphetamine administration

in rhesus monkeys. Brain Res 486:73-78, 1989.
145

Yamamoto, B.K., and Spanos, L.J. The acute effects of

methylenedioxy-methamphetamine on dopamine release in the

awake behaving rat. Eur J Pharmacol 148:195-203, 1988.

ACKNOWLEDGMENTS

This work was supported by U.S. Public Health Service grants DA

00869 and DA 04222. The parent compounds were kindly

supplied by the National Institute on Drug Abuse.

AUTHORS

James W. Gibb, Ph.D.

Professor and Chair

Michel Johnson, Ph.D.

Research Assistant Professor

Ikram Elayan, Ph.D.

Professor

Glen R. Hanson, Ph.D., DDS

Professor

Department of Pharmacology and Toxicology

University of Utah

Salt Lake City, UT 86112

Heng Keang Lim, Ph.D.

Research Scientist

Wyeth Ayerst Research

CN 8000

Princeton, NJ 08543-8000

Lisa Matsuda, Ph.D.

Assistant Professor

Department of Psychiatry

Medical University of South Carolina

Charleston, SC 29425
146

Potential New Insights Into the

Molecular Mechanisms of

Methamphetamine-Induced

Neurodegeneration

Monika Z. Wrona, Zhaoliang Yang, Fa Zhang, and Glenn

Dryhurst

INTRODUCTION

In humans, methamphetamine evokes alertness, a decreased sense of

fatigue, increased ability to concentrate or perform physical tasks,

euphoria, and increased initiative and confidence (McGeer et al.

1987). Probably because of its mood-elevating and antifatigue

effects, methamphetamine is self-administered by humans and a

number of animal species in experimental models (Schuster 1981).

Methamphet-amine is recognized as a positively reinforcing drug, and

it continues to be extensively abused in many countries. However,

high doses or continuous use of methamphetamine can lead to a

variety of undesirable side effects. These include paranoid delusions,

disordered thought, aggression, and hallucinations (Seiden et al. 1988).

Neurotoxicological studies have established that certain dose regimens

of methamphetamine evoke the degeneration of dopaminergic and

serotonergic fibers in the brains of many animal species including the

monkey (Seiden et al. 1975/1976), rats, mice, and cats (Ricaurte et al.

1980; Wagner et al. 1980; Levine et al. 1980; Seiden and Ricaurte

1987). These observations suggests that methamphetamine might

also evoke similar neurodegenerative effects in the human brain.

For a variety of reasons, most studies aimed at understanding the

neurodegenerative effects of methamphetamine have employed the

rat. Repeated low doses or a single large dose of methamphetamine to

this animal results in the degeneration of serotonergic (Ricaurte et al.

1980; Seiden et al. 1988; Bakhit and Gibb 1981; Hotchkiss and Gibb

1980a, 1980b; Commins and Seiden 1986) and dopaminergic (Seiden

et al. 1988; Ricaurte et al. 1982; Wagner et al. 1980; Fibiger and

McGeer 1971; Buening and Gibb 1974) nerve terminals in several

areas of the brain and of a subpopulation of cell bodies in the

somatosensory cortex (Commins and Seiden 1986). The latter region

of the rat cortex contains no serotonergic or catecholaminergic

perikarya and hence these cell bodies must be associated with another,
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as yet unknown, neurotransmitter system. Based on measurements of

residual levels of 5-hydroxytryptamine (5-HT) and dopamine (DA) in

many brain areas following methamphet-amine administration, it

appears that the most profound degeneration of serotonergic

terminals occurs in the frontal cortex, hippocampus, and amygdala,

whereas dopaminergic terminals are most severely degenerated in the

caudate nucleus, amygdala, and nucleus accumbens (Seiden et al.

1988). Such studies have also suggested that serotonergic terminal

fields are more susceptible than dopaminergic terminal fields to the

neurotoxic effects of methamphetamine. Furthermore, for the

various brain areas examined, it appears that depletions of DA and 5-

HT are proportional. For example, if DA is not depleted then 5-HT

is depleted by only a small amount; when significant dopaminergic

degeneration occurs, serotonergic degeneration is even more

pronounced (Seiden et al. 1988). These observations could imply that

the methamphetamine-induced degeneration of dopaminergic and

serotonergic terminals are not entirely independent processes, but are

in some way related or connected phenomena.

The fundamental molecular mechanisms that underlie the

neurodegenera- tive properties of methamphetamine are unknown,

although they are probably not directly caused by the drug or its

normal metabolites (Gibb et al. 1994). However, several lines of

evidence suggest that oxygen radical species contribute directly or

indirectly to methamphetamine-induced neuronal damage (Cadet et al.

1994). For example, pretreatment of rats with antioxidants

attenuates the neurodegenerative effects of methamphetamine

(DeVito and Wagner 1989). It seems to be widely accepted that

oxygen radical species limit their damage in biological systems to

lipids, proteins, and deoxyribonucleic acid (DNA) (Halliwell 1992).

However, the two neuronal systems severely damaged as a result of

methamphetamine administration utilize neurotransmitters that are

very easily oxidized (i.e., 5-HT and DA). Thus, it appears unlikely

that these neurotransmitters would be spared from oxygen radicalmediated oxidation reactions unless there are very sophisticated

mechanisms available for their protection. Support for this view can

be drawn from the observation that, following the administration of a

single large dose of methamphetamine to the rat, the

catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) has

been detected in the caudate nucleus (Seiden and Vosmer 1984) and

the serotonergic neurotoxin 5,6-dihy-droxytryptamine (5,6-DHT)

has been detected in the cortex and hippocampus (Commins et al.

1987).
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The only known direct chemical pathway leading to 6-OHDA and

5,6-DHT is attack of the hydroxyl radical (HO·) on DA (Slivka and

Cohen 1987) and 5-HT (Wrona et al. 1995), respectively. The

detection of 6-OHDA (Seiden and Vosmer 1984) and 5,6-DHT

(Commins et al. 1987) in rat brain following methamphetamine

administration has been reported to be somewhat sporadic and the

concentrations measured extremely low. Indeed, several investigators

have been unable to detect 6-OHDA in rat brain following

methamphetamine administration (Rollema et al. 1986; Evans and

Cohen 1989; Karoum et al. 1993). Nevertheless, a hypothesis has

been advanced that under conditions of massive methamphetamineinduced release of DA and 5-HT and monoamine oxidase (MAO)-A

and MAO-B inhibition (Suzuki et al. 1980), these neurotransmitters

are non-enzymatically oxidized in the synaptic cleft to give 6-OHDA

and 5,6-DHT, respectively (Seiden et al. 1988; Seiden and Vosmer

1984; Commins et al. 1987). Subsequent carrier-mediated uptake of

6-OHDA and 5,6-DHT into dopaminergic and serotonergic terminals,

respectively, have been proposed to lead to the degeneration of these

neurons. Some support for this hypothesis is provided by the fact

that selective DA and 5-HT uptake inhibitors protect dopaminergic

and serotonergic terminals, respectively, against metham-phetamineinduced damage (Marek et al. 1990a; Ricaurte et al. 1983).

On the assumption that methamphetamine in some way evokes

aberrant oxidation of DA to 6-OHDA and 5-HT to 5,6-DHT, it is of

interest to consider whether such reactions would occur within the

cytoplasm of nerve terminals rather than in the synaptic cleft. In the

dopaminergic system methamphetamine participates in an exchangediffusion process (Raitiri et al. 1979) and displaces DA from

cytoplasmic storage sites, presumably resulting in elevated levels of

free or unbound neurotrans-mitter prior to its massive efflux through

the uptake carrier site (Marek et al. 1990a; Liang and Rutledge 1982).

Elevated cytoplasmic concen- trations of DA appear to expose this

neurotransmitter to oxidation. To illustrate, pretreatment of rats

with pargyline (which increases the cytoplasmic pool of DA) followed

by methamphetamine results in increased levels of 6-OHDA in the

striatum compared to those observed in animals not treated with this

MAO-A and MAO-B inhibitor (Marek et al. 1990c). Similarly

reserpine, which disrupts catecholamine storage vesicles and increases

cytoplasmic levels of DA, potentiates metham-phetamine-induced

damage to dopaminergic terminals (Wagner et al. 1983). Indeed,

treatment of guinea pigs with reserpine alone results in a marked

elevation of striatal levels of 5-S-cysteinyldopamine, indicative of

increased oxidation of cytoplasmic DA (Fornstedt and Carlsson
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1989). In contrast, the catecholamine synthesis inhibitor -methylp-tyrosine (MpT), which depletes the cytoplasmic pool of DA,

attenuates methamphetamine-induced damage to dopaminergic

terminals (Wagner et al. 1983) and decreases 6-OHDA formation

(Axt et al. 1990). Reinstating DA synthesis by administration of Ldopa reverses the protective effects of MpT against

methamphetamine-induced damage to dopaminergic terminals

(Schmidt et al. 1985). However, administration of L-dopa without

methamphetamine does not appear to cause any neurodegenerative

effects, indicating that DA is not a neurotoxic agent (Schmidt et al.

1985).

Taken together, these lines of evidence tend to support the idea that

elevated cytoplasmic concentrations of DA might favor the

intraneuronal oxidation of this neurotransmitter and that such

reactions are linked to the degeneration of dopaminergic terminals.

The influence of manipulations of the cytoplasmic pool of 5-HT on

the methamphetamine-induced oxidation of 5-HT to 5,6-DHT have

not been studied. However, in view of the many apparent similarities

between the neurodegenerative and other biochemical effects of

methamphetamine on dopaminergic and serotonergic terminals, it

might also be concluded that oxidation of 5-HT to 5,6-DHT occurs

intraneuronally.

The hypothesis that 6-OHDA and 5,6-DHT are responsible for

mediating the methamphetamine-induced degeneration of

dopaminergic and serotonergic terminals, respectively, has been

challenged on the basis of the sporadic detection and exceedingly low

levels of these neurotoxins as measured in the brain (Evans and Cohen

1989). However, in the event that these neurotoxins are formed only

at, or in, axon terminals, it is not inconceivable that they might

transiently reach lethal localized concen-trations. Furthermore, 6-

OHDA and 5,6-DHT are believed to be neuro-toxic because of their

facile intraneuronal autoxidation to cytotoxic products and

byproducts. Thus, the highly localized sites of formation of 6-OHDA

and 5,6-DHT and their chemical instability would be expected to

make the detection of these neurotoxins in the brain a significant

analytical challenge.

Inhibition of DA synthesis by the tyrosine hydroxylase inhibitor

MpT attenuates not only 6-OHDA formation and degeneration of

striatal dopaminergic terminals, but also 5,6-DHT formation and the

degeneration of serotonergic terminals and cell bodies of unknown

neurotransmitter content in the somatosensory cortex (Commins and
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Seiden 1986; Axt et al. 1990). These observations suggest that either

DA and/or an aberrant metabolite of this neurotransmitter might play

a role in the processes that result in the degeneration of both

dopaminergic and serotonergic terminals and certain cell bodies in the

somatosensory cortex. Selective DA uptake inhibitors such as

amfolenic acid (AFA) protect striatal dopaminergic terminals against

methamphetamine-induced damage (Marek et al. 1990a) but do not

block the release of DA or 6-OHDA formation (Marek et al. 1990b).

However, AFA does not protect striatal serotonergic terminals against

methamphetamine-induced damage (Marek et al. 1990a).

Significantly, AFA prevents the degeneration of striatal dopaminergic

terminals when administered as late as 8 hours after

methamphetamine (Marek et al. 1990a). Selective 5-HT uptake

inhibitors such as fluoxetine protect serotonergic terminals against

methamphetamine-induced damage but exacerbate damage to striatal

dopaminergic terminals (Ricaurte et al. 1983). Taken together, these

results suggest a number of possible conclusions:

1. Methamphetamine evokes an initial increase in the cytoplasmic

concentrations of free or unbound DA and 5-HT prior to their

massive release;

2. Increased cytoplasmic levels of DA and 5-HT in some way permit

oxidation of these neurotransmitters as evidenced by the

formation of 6-OHDA, 5-S-cysteinyldopamine, and 5,6-DHT;

3. The toxic effects of methamphetamine toward dopaminergic and

serotonergic terminals require intact and functioning

neurotransmitter uptake systems;

4. DA or a toxic DA metabolite might contribute to the

degeneration of both dopaminergic and serotonergic nerve

terminals; and,

5. A toxic metabolite of 5-HT might contribute to the degeneration

of serotonergic and dopaminergic terminals and certain cell bodies

in the somatosensory cortex (there is virtually no dopaminergic

input into this region of the cortex) (Lindvall and Björklund

1978).

The latter two conclusions imply that toxic aberrant metabolites of

DA and 5-HT are transferred between connected dopaminergic and

seroto-nergic terminals and between serotonergic terminals and
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connected cell bodies in the somatosensory cortex. However, while

6-OHDA and 5,6-DHT might in principal contribute to the

degeneration of dopa-minergic and serotonergic terminals,

respectively, it is not likely that 6-OHDA contributes to the

degeneration of serotonergic terminals or that 5,6-DHT contributes

to the degeneration of dopaminergic terminals or neuronal perikarya

in the somatosensory cortex. This is so because 6-OHDA (Johnsson

et al. 1975) and 5,6-DHT (Baumgarten and Lachenmeyer 1972) are

selective catecholaminergic and serotonergic neurotoxins,

respectively, except when present in the brain in very high

concentrations. Indeed, it might be argued that 6-OHDA may not be

responsible for the degeneration of dopaminergic terminals because

this neurotoxin cannot be detected in rat brain after

methamphetamine administration at times when AFA is able to block

the neurodegenerative process (Marek et al. 1990b).

It is possible that toxic metabolites of DA and 5-HT other than, or in

addition to, 6-OHDA and 5,6-DHT might contribute to the neuronal

degeneration evoked by methamphetamine. Several lines of

experimental evidence provide some support for this possibility. For

example, the methamphetamine-induced degeneration of

dopaminergic and serotonergic terminals is significantly attenuated by

N-methyl-D-aspartate (NMDA) receptor antagonists (Sonsalla et al.

1991; Farfel et al. 1992). However, there is no evidence available to

suggest that 6-OHDA, 5,6-DHT, or methamphetamine are NMDA

receptor agonists. Some evidence has been presented that

methamphetamine evokes elevated release of glutamate in the

striatum of rats (Sonsalla et al. 1991; Nash and Yamamoto 1992) that

might result in enhanced activation of NMDA receptors located on

dopaminergic and serotonergic terminals and resultant excitotoxic

damage (Choi 1987; Lafon-Cazal et al. 1993). However, there are

relatively few NMDA receptors located on these terminals in the

striatum, whereas gamma aminobutyric acid (GABA), cholinergic, and

substance P cell bodies in this structure have high densities of these

receptors (Coyle and Schwarz 1976; Sonsalla et al. 1991). Thus, if

methamphetamine evokes widespread release of glutamate from

glutamatergic terminals throughout the striatum, activation of NMDA

receptors and excitotoxic damage would be expected to cause serious

damage to GABAergic, cholinergic, and substance P cell bodies.

However, these neurons suffer no long-lasting damage following

methamphetamine administration (Hotchkiss et al. 1979; Sonsalla et

al. 1986).
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As noted by Sonsalla and colleagues (1991), if dopaminergic and

serotonergic terminal damage induced by methamphetamine is due to

the direct effects of glutamate on NMDA receptors, it would clearly

have to be a very discrete and highly localized action. In principle,

such a localized action could be caused by aberrant metabolites of DA

and/or 5-HT formed at dopaminergic or serotonergic terminals,

respectively, that are either potent NMDA receptor agonists or in

some way potentiate the release of glutamate from connected

glutamatergic terminals. A possible clue to the mechanism that might

evoke the latter process may be drawn from the observation that the

methamphetamine-induced damage to dopaminergic terminals is

blocked by coadministration of pharmacologic agents that elevate

extraneuronal levels of GABA (Hotchkiss and Gibb 1980a).

This finding suggests that interactions of GABA with GABA receptors

can block the neurotoxic effects of methamphetamine. However,

there are no known interactions between 6-OHDA, 5,6-DHT, or

methamphet-amine and GABA receptors. Interestingly, GABAB

receptors are located both on nerve terminals and at postsynaptic

sites in many brain regions (Bowery 1989). Activation of

presynaptic GABAB receptors by GABA reduces the evoked release of

biogenic amine, excitatory amino acid (including glutamate), and

neuropeptide neurotransmitters by blockade of calcium channels

(Bowery 1989; Lev-Tov et al. 1988). Conversely, therefore, a

GABAB inverse receptor agonist would be expected to potentiate the

opening of the calcium channel and evoke elevated release of

neurotransmitters. Following methamphetamine administration,

formation of an aberrant metabolite of DA and/or 5-HT that was a

potent GABAB receptor inverse agonist at dopaminergic and/or

serotonergic terminals could, in principal, account for the release of

glutamate from anatomically connected glutamatergic terminals

leading to very localized NMDA receptor-mediated excitotoxicity and

neuronal damage.

Available evidence thus suggests that methamphetamine evokes

aberrant oxidations of DA to 6-OHDA and 5-HT to 5,6-DHT,

probably in the cytoplasm of dopaminergic and serotonergic

terminals, respectively. While these two neurotoxins might

contribute to the degeneration of the nerve terminals where they are

formed, it is not likely that they are involved with neurodegenerative

mechanisms mediated by NMDA or GABA receptors. The influence

of MpT and selective DA uptake inhibitors on the

neurodegenerative effects of methamphetamine tend to support the

notion that aberrant metabolites derived from this neurotrans-mitter
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(other than or in addition to 6-OHDA) not only contribute to the

degeneration of dopaminergic terminals, but also play a role in the

degeneration of connected serotonergic terminals. Similarly, the

effects of selective 5-HT uptake inhibitors suggest that aberrant

metabolites of 5-HT (other than or in addition to 5,6-DHT) might

contribute to the degeneration of serotonergic terminals, connected

dopaminergic terminals, and certain cell bodies in the somatosensory

cortex. These putative aberrant metabolites of DA and/or 5-HT

might include compounds that either activate NMDA receptors and

evoke excitotoxic damage or facilitate the release of glutamate, but

only in the immediate vicinity of their sites of formation at

dopaminergic and/or serotonergic terminals.

In the event that aberrant metabolites of DA and 5-HT do play roles in

mediating the neurodegenerative effects of methamphetamine, it becomes

of key interest to know the identities of the compounds and mechanisms

that might be responsible for their formation. Extensive studies (Seiden et

al. 1988; Seiden and Vosmer 1984; Commins et al. 1987; Marek et al.

1990a, 1990b; Axt et al. 1990) indicate that methamphetamine evokes

oxidation of DA to 6-OHDA and 5-HT to 5,6-DHT. However, the

autoxidation (i.e., oxidation by molecular oxygen in the absence of enzyme

catalysis) of DA (Graham 1978) and the electrochemically driven oxidation

(Zhang and Dryhurst 1993) of this neurotransmitter in aqueous solution at

physiological pH do not give 6-OHDA as a detectable product. Similarly,

the autoxidation (Wrona et al. 1992) and enzyme-mediated (Wrona and

Dryhurst 1991) and electrochemical (Wrona and Dryhurst 1990) oxidations

of 5-HT at physiological pH do not give 5,6-DHT. However, HO· attack

on DA does give 6-OHDA, although in much lower yields than 2-OHDA

and 5-OHDA (Slivka and Cohen 1987). The latter observation, therefore,

might indicate that 6-OHDA and 5,6-DHT are marker molecules for the

methamphetamine-induced HO· oxidation of DA and 5-HT, respectively.

Indeed, there is good—albeit indirect—evidence that methamphetamine

evokes formation of oxygen radicals in the brain (DeVito and Wagner

1989; Cadet et al. 1994). However, remarkably little is known about the

HO·-mediated oxidations of DA and 5-HT, the products (i.e., putative

aberrant oxidative metabo-lites) that are likely to be formed in vivo, and

the neurobiological properties of these products. Accordingly, the authors

have recently initiated studies aimed at elucidating the oxidation chemistry

of 5-HT and DA, including that mediated by HO·, and assessing the

neurobiological activities of the resulting putative metabolites and their

possible roles in methamphetamine-evoked neurodegenerative processes

and other neurodegenerative brain disorders.
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OXIDATION CHEMISTRY OF 5-HT AND DA

Location is a central question relevant to studies of the oxidation

reactions of 5-HT and DA and the role of such reactions (and

resulting products) in mediating methamphetamine’s

neurodegenerative properties. This question is important because the

course of these reactions and the putative aberrant oxidative

metabolites formed are likely to be strongly dependent on whether

they occur in the synaptic cleft (Seiden et al. 1988; Seiden and

Vosmer 1984; Commins et al. 1987; Marek et al. 1990c) or

intraneuronally. For example, the cytoplasm of dopaminergic and

serotonergic nerve terminals contain relatively high concentrations

of ascorbic acid (AA) (Spector and Eells 1984), glutathione (GSH), Lcysteine (CySH) (Slivka et al. 1987), hydrogen peroxide (H2O2

, a

byproduct of many intraneuronal metabolic processes), molecular

oxygen, and traces of low molecular weight Fe2+ species (Halliwell

1992). By contrast, extraneuronal levels of AA (Spector and Eells

1984), GSH, and CySH (Slivka et al. 1987), for example, are probably

appreciably lower than cytoplasmic concentrations.

A widely used HO·-generating system consists of AA, traces of Fe2+-

ethylenediaminetetraacetic acid (EDTA), H2O2, and molecular

oxygen (Udenfriend et al. 1954). In this medium, HO· is formed by

decomposition of H2O2 by the Fenton reaction (Walling 1975) and

the resulting Fe3+ is reduced back to Fe2+ by AA (equation 1). Thus, as

long as AA and H2O2 are available, HO· is formed by this cyclic

reaction. When incubated with this HO·-generating system, 5-HT is

oxidized extremely rapidly (Wrona et al. 1995). The initial step in

the reaction involves HO· attack on 5-HT to give the 4,5-(1), 2,5-(2)

(1) and 5,6-(3)-dihydroxytryptamine radicals, which are then oxidized

by a second molecule of HO· to give 2,5-DHT, 4,5-DHT, and 5,6-

DHT in approximate yields of 60 percent, 24 percent, and 11

percent, respectively (figure 1). However, 4,5-DHT is not an

isolatable product owing to its very facile oxidation by molecular

oxygen to give tryptamine-4,5-dione (T-4,5-D) in a reaction that

forms H2O2 as a byproduct (Wong and Dryhurst 1990). Furthermore,

2,5-DHT exists predominantly in solution at pH 7.4 as its 2-keto

tautomer, 5-hydroxy-3-ethylamino-2-oxindole (5-HEO). The latter

compound readily deprotonates to give the C(3)-centered carbanion 4

which attacks T-4,5-D to give dimer 5. Autoxidation of the 4,5-

DHT residue of 5 then yields 6, forming H2O2 as a byproduct. Dimer

6 undergoes a slow intramolecular cyclization reaction to give the

pyrrolo[2,3-f]quinoline 7, in which the 4,5-DHT residue is autoxidized
155

to o-quinone 8, again forming H2O2 as a byproduct. A relatively

minor amount of T-4,5-D also dimerizes to give the 7,7.-linked dimer

9, which is immediately autoxidized to 7,7.-D forming H2O2 as a

byproduct. Because of its reactions with 5-HEO and its dimerization

to 7,7.-D, T-4,5-D is not observed as a product of the HO·-mediated

oxidation of 5-HT. 5,6-DHT is clearly formed as a result of

oxidation of 5-HT by HO·, but it represents only a rather minor

product of this reaction. Although 5,6-DHT can be oxidized by

molecular oxygen to give o-quinone 10, which subsequently

polymerizes to indolic melanin, this is a relatively slow reaction in

vitro (Singh and Dryhurst 1990). Furthermore, detection of this

neurotoxin as a product of the HO·-mediated oxidation of 5-HT

presumably reflects the reduction of o-quinone 10 by AA.

The rate of the reaction shown in figure 1 is dependent on the

concen-tration of Fe2+ in the reaction medium. However, even in the

absence of added Fe2+, 5-HEO 6, and 5,6-DHT are observed as

products after several

minutes, indicating that submicromolar concentrations of iron and/or

other transition metal ions that always contaminate buffer

constituents are sufficient to catalyze the decomposition of H2O2 to

HO·. Similarly, in the presence of Fe2+ but without added H2O2, 5-HT

is oxidized to 5-HEO, 6 (subsequently 8 forms), and 5,6-DHT. This is

because autoxidation of AA to dehydroascorbic acid provides the

initial source of H2O2 and hence HO·. When 5-HT is oxidized in the

same HO·-generating system in the presence of GSH, the yields of

5,6-DHT are not significantly altered. However, yields of 5-HEO

increase, those of 6 (and 8) decrease, and a new product, 7-Sglutathionyltryptamine-4,5- dione (7-S-Glu-T-4,5-D), appears. In

the presence of sufficiently high concentrations of GSH,
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formation of 6 (and 8) is almost completely blocked and yields of 5-

HEO and 7-S-Glu-T-4,5-D reach maximal levels. These observations

are explained by the fact that GSH efficiently scavenges T-4,5-D to

give
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7-S-glutathionyl-4,5-dihydroxytryptamine (11, figure 2), which is

then autoxidized to 7-S-Glu-T-4,5-D and forms H2O2 as a byproduct

(Wong et al. 1993).

Quantitative measurements of the yields of products formed when 5-

HT is oxidized with the AA/Fe2+-EDTA/H2O2/O2 system reveal that

the initial yields of 5-HEO, 5,6-DHT, and 6 (and 7-S-Glu-T-4,5-D

when GSH is present) are larger than expected based on the H2O2 (and

hence HO·) concentrations employed (Wrona et al. 1995). This

additional HO· probably results from autoxidations of 4,5-DHT

(Wong and Dryhurst 1990), 5,6-DHT (Singh and Dryhurst 1990), 5

(and 8) (Wrona et al. 1995), 11 (Wong et al. 1993), and redox

cycling reactions of the T-4,5-D/4,5-DHT (formal potential (E%.) =

-240 millivolts (mV) versus the saturated calomel reference electrode

(SCE) at pH 7.4), 6/5 (E%. = -495 mV), 8/7 (E%. = -495 mV), 7-SGlu-T-4,5-D/11 (E%. = -248 mV), 10/5,6-DHT, and 7,7./9 (Singh et

al. 1992) couples which form H2O2 as a byproduct.

These autoxidation-redox cycling reactions and resultant H2O2 and

HO· formation apparently continue the oxidation of 5-HT until AA

is exhausted. Although 5,6-DHT is an initial but minor product of the

HO·-mediated oxidation of 5-HT, this neurotoxin almost completely

disappears within 2 to 3 hours (Wrona et al. 1995) because of its

autoxidation to an insoluble brown-black indolic melanin polymer

(Singh and Dryhurst 1990; Singh et al. 1990). By contrast, the major

reaction product, 5-HEO, is an appreciably more stable compound.
158

The oxidation of DA at physiological pH by HO·-generating systems

consisting of Fe2+/H2O2/O2 or Fe2+/AA/O2 (H2O2 and hence HO· being

formed as a result of AA autoxidation) has been reported to give a

mixture of 2-OHDA, 5-OHDA, and 6-OHDA in relative yields of

3:2:1 (Slivka and Cohen 1987). However, the yields of all of these

products were low based upon the HO· concentrations generated. The

mechanism of these hydroxylation reactions is not completely clear.

However, Richter and Waddell (1983) have reported that addition of

HO·, generated by pulse radiolysis, to each of the available ring

positions of DA is equally probable. Under the conditions employed

by these investigators, which were carried out in the absence of

molecular oxygen and iron salts, the intermediate radical species (12-

14, figure 3) eliminate the elements of water to give semiquinones of

DA. The dehydration of the 6-hydroxy- lated radical (14) was faster

than for 12 and 13. Slivka and Cohen (1987) have suggested that

radicals 12-14 are oxidized by molecular oxygen or Fe3+ to give 2-

OHDA, 5-OHDA, and 6-OHDA (figure 3). The lower yield of 6-

OHDA was attributed to the more rapid water elimination reaction of

14. In the event that methamphetamine evokes oxidation of DA by

HO· in the cytoplasm of dopaminergic nerve terminals, then such

reactions must occur in the presence of CySH and GSH (Slivka et al.

1987) and it is probable that these sulfhydryl compounds would

influence the products formed.

Information bearing on such reactions in vivo can be drawn from

reports that the 5-S-cysteinyl conjugates of DA and other endogenous

catechols are present in human and other mammalian brains

(Rosengren et al. 1985; Fornstedt et al. 1986). These conjugates are

probably formed either by nucleophilic addition of CySH to the oquinones formed by oxidation of these catechols or by addition of

GSH to yield the 5-S-glutathionyl conjugates which are then

hydrolyzed by peptidase enzymes (Rosengren et al. 1985).

Several lines of evidence suggest that the oxidation of DA and other

catechols and formation of their 5-S-cysteinyl conjugates occurs in

the cytoplasm of dopaminergic neurons. To illustrate, both GSH and

CySH, which are clearly required to form these cysteinyl conjugates,

are located in the cytoplasm of dopaminergic terminals and axons

(Slivka et al. 1987). Furthermore, treatment of guinea pigs with

reserpine, which disrupts DA storage vesicles and elevates cytoplasmic

levels of this neurotransmitter, evokes a marked elevation of striatal

levels of
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5-S-cysteinyldopamine (5-S-CyS-DA) (Fornstedt and Carlsson 1989).

The mechanism of the HO·-mediated oxidation of DA in the presence

of molecular oxygen and Fe2+/Fe3+ does not appear to involve an

electro- philic o-quinone intermediate (figure 3), the apparent

precursor of 5-S-CyS-DA (Rosengren et al. 1985; Fornstedt et al.

1986, 1989). Autoxidation of DA apparently does proceed via such

an intermediate, but is a relatively slow reaction at physiological pH

and hence unsuitable for isolation and identification of reaction

products. However, the electrochemically driven oxidation of DA is

not only very facile but undoubtedly proceeds by initial formation of

an o-quinone intermediate (Zhang and Dryhurst 1993). Accordingly,

the authors have recently studied the influence of CySH on the

electrochemical oxidation of DA at physiological pH (Zhang and

Dryhurst 1994). This reaction results in formation of a very

complex mixture of products. However, among the major products

are the dihydrobenzothiazines (DHBTs) 17 to 19 and 22 (figure 4).

In this reaction, DA-o-quinone is initially attacked by CySH to give

5-S-CyS-DA, which is appreciably more easily oxidized than DA to

give o-quinone 15. This intermediate very rapidly cyclizes to give

the bicyclic o-quinone imine 16. In the presence of free CySH 16 can

either be reduced to DHBT 17 or nucleophilic addition of a second

CySH molecule occurs to give DHBTs 18 and 19. The latter

compounds are even more easily oxidized than DA and 5-S-CyS-DA

to o-quinone imines 20 and 21, respectively, which can be either

reduced by CySH or further
160

attacked to give DHBT 22. DHBTs 17 to 19 and 22 survive in the

oxidation reaction simply because their oxidized forms are readily

reduced by free CySH. DA-o-quinone is also scavenged by GSH to

give a number of glutathionyl conjugates that include 5-Sglutathionyl- dopamine (5-S-Glu-DA) (Zhang and Dryhurst, in press).

As suggested by Rosengren and colleagues (1985), if formed in the

cytoplasm of dopaminergic terminals, 5-S-Glu-DA might be

hydrolyzed by peptidase enzymes to 5-S-CyS-DA (figure 5). Under

conditions when DA is oxidized, the latter conjugate must also be
161

oxidized because it is a significantly more easily oxidized compound

(Zhang and Dryhurst 1994). In the event that this reaction is

mediated by molecular oxygen, the expected byproducts would be

superoxide anion radical (O2-·) and/or H2O2, both precursors of HO·.

The resulting products would include DHBTs 17 to 19 and 22 as

conceptualized in figure 5. Again, the facile autoxidation of these

DHBTs and their ability to redox cycle would be expected to

potentiate additional formation of HO· precursors. The oxidation of

DA in the presence of CySH in an HO·-generating system consisting

of AA/Fe2+/H2O2/O2 also forms 5-S-CyS-DA in high yield; this is

further oxidatively cyclized to DHBT 17 (Zhang and Dryhurst,

unpublished results). Preliminary results do not indicate that 6-OHDA

is a significant reaction product.

Studies in this laboratory aimed at understanding the in vitro oxygen

radical-mediated oxidations of 5-HT and DA under experimental

conditions that might mimic those in the cytoplasm of serotonergic

and dopaminergic nerve terminals in vivo are at an early stage.

Nevertheless, these investigations have established that 5,6-DHT is

formed as a result of HO· attack on 5-HT. However, this neurotoxin

is the most minor of the identified products of the reaction and is also

a rather unstable species owing to its oxidative polymerization to

indolic melanin. Furthermore, intraneuronal formation of 5,6-DHT

as a result of methamphetamine administration would necessarily

expose this substance to mitochondria which are known to catalyze

its oxidation by molecular oxygen (Klemm et al. 1980; Singh and

Dryhurst 1990). Taken together, these facts might account for the

sporadic and low levels of 5,6-DHT detected in rat brain following an

acute dose of methamphetamine (Commins et al. 1987).

These observations led Evans and Cohen (1989) to question the

hypothesis that this neurotoxin mediates the methamphetamineinduced degeneration of serotonergic terminals. Assuming that

formation of 5,6-DHT in rat brain following methamphetamine

administration reflects intraneuronal oxidation of 5-HT by HO· (this

is the only known chemical reaction that directly converts 5-HT and

5,6-DHT), the reaction pathways shown in figures 1 and 2 (Wrona et

al. 1995) predict that 5-HEO in particular (and perhaps 6 and 8)

should be formed as major and more stable aberrant metabolites.

However, intraneuronal GSH (Slivka et al. 1987) would be expected to

scavenge T-4,5-D to give 7-S-Glu-T-4,5-D.
162

Thus 5-HEO, 6, 8, or 7-S-Glu-T-4,5-D might represent valuable

analytical marker molecules in brain tissue to provide evidence in

support of the idea that methamphetamine does indeed evoke

intraneuronal formation of HO·. Furthermore, the fact that the in

vitro HO·-mediated oxidation of 5-HT gives additional products in

higher yields than
163

5,6-DHT raises the possibility that one or more of these other

putative aberrant oxidative metabolites might play roles in the

neurodegenerative effects evoked by methamphetamine. The

influence of CySH on the HO·-mediated oxidation of 5-HT remains

to be studied. However, recent studies into the electrochemically

driven oxidation of 5-HT in the presence of CySH suggest that many

additional products are likely to be formed (Wrona et al. 1994).

In vitro studies reveal that the HO·-mediated oxidation of DA at

physiological pH gives 6-OHDA (Slivka and Cohen 1987) (figure 3).

Accordingly, detection of 6-OHDA in the rat striatum shortly

following methamphetamine administration (Seiden and Vosmer

1984) suggests that this drug also evokes formation of HO·.

Nevertheless, this conclusion must be tempered by the fact that

several investigators have been unable to detect even extremely low

levels of 6-OHDA in rat brain following administration of

methamphetamine or other drugs that release DA into the synaptic

cleft (Rollema et al. 1986; Evans and Cohen 1989; Karoum et al.

1993). However, using gas chromatography-mass spectrometry,

Karoum and colleagues (1993) have observed a fluoro-propionylated

derivative of an unknown compound having a retention time very

similar to pentafluoropropionylated 6-OHDA in the brains of

methamphetamine treated rats. In the event that methamphetamine

evokes the intraneuronal oxidation of DA, it seems plausible to

suggest that CySH and or GSH might intervene in the reaction as

shown in figures 4 and 5 to give 5-S-CyS-DA and 5-S-Glu-DA,

precursors of DHBTs such as 17 to 19 and 22. This suggestion raises

the possibility that these putative aberrant oxidative metabolites of

DA, formed only from endogenous substances, might contribute to

the neurodegenerative effects evoked by methamphetamine.

IN VIVO PROPERTIES OF PUTATIVE ABERRANT OXIDATIVE

METABOLITES OF 5-HT AND DA

Investigations into the neurotoxicology and neuropharmacology of

putative aberrant oxidative metabolites of 5-HT and DA are at a very

preliminary stage. 5-HEO, the major product of the in vitro HO·-

mediated oxidation of 5-HT, is not toxic (lethal) when administered

into the brains of mice weighing ~ 30 grams (g) at doses as high as

100-200 micrograms (g) (Dryhurst et al., unpublished results).

However, several other intermediates/products of this reaction are

active in the brain. For example, T-4,5-D is lethal when injected into

the brains of mice (Wong et al. 1993). Furthermore,
164

intracerebroventricular injections of T-4,5-D into rat brain have been

claimed to evoke long-lasting decreases in 5-HT levels in the

hippocampus, striatum, and cortex; reduced activity of tryptophan

hydroxylase (Chen et al. 1992); and degeneration of nerve terminals

(Crino et al. 1989). 7-S-Glu-T-4,5-D is also lethal (median lethal

dose (LD50 ) = 21 g) when administered into mouse brain, evoking

extreme excitation and hyperactivity for about 30 minutes (Wong et

al. 1993). During this period, 7-S-Glu-T-4,5-D evokes statistically

significant declines of whole brain levels of norepinephrine, DA, and

5-HT and elevation of the metabolites of these neurotransmitters

(Wong et al. 1993). This might suggest that 7-S-Glu-T-4,5-D

potentiates the release and elevated turnover of the monoamines

although its influence on the release of other neurotransmitters,

especially glutamate, remains to be determined. 7-S-Glu-T-4,5-D also

redox cycles in the presence of cellular antioxidants/reductants and

molecular oxygen and when incubated with brain homogenates

forming H2O2 as a byproduct (Wong et al. 1993). Dimer 6 is also

lethal (LD50 = 19 g) when administered into mouse brain (Dryhurst

et al., unpublished results). Furthermore, the E%. values of 6 and 8

(both -495 mV at pH 7.4) are such that these compounds might be

expected to redox cycle under intraneuronal conditions (Yamazaki

and Piette 1963) forming O2-· and/or H2O2 as byproducts, that is,

precursors of cytotoxic HO·.

5-S-CyS-DA, a putative aberrant oxidative metabolite of DA in dopaminergic terminals, is not lethal when administered into the brains of

laboratory mice. DHBTs 17 (LD50 = 14 g), 18 (LD50 = 17 g), and

19 (LD50 = 70 g) are lethal (Zhang and Dryhurst 1994).

SUMMARY

In the event that methamphetamine evokes HO· formation within

serotonergic axon terminals, the resultant oxidation of 5-HT would be

expected to generate not only 5,6-DHT but also T-4,5-D, 7-S-Glu-T4,5-D, 6, 8, and 7,7T-D (figure 1), at least three of which (T-4,5-D,

7-S-Glu-T- 4,5-D, and 6) are lethal in mouse brain. Furthermore,

several intermed- iates/products formed in the in vitro oxidation of 5-

HT by HO· are readily autoxidized (4,5-DHT, 5,6-DHT, 5, 7, and 9)

or redox cycled (T-4,5-D, 6, 8, 7,7T-D, 7-S-Glu-T-4,5-D) in

reactions that would be expected to yield O2-· and/or H2O2 as

byproducts. These byproducts, in the presence of trace levels of

transition metal ion catalysts, would be readily converted into HO·

(Walling 1975; Halliwell and Gutteridge 1984). Together these
165

putative aberrant oxidative metabolites of 5-HT and HO·-forming

reactions might contribute to the degeneration of serotonergic nerve

terminals. Similarly, the methamphetamine-induced intraneuronal

formation of HO· in dopaminergic terminals might be expected to

generate not only 6-OHDA (and 2-OHDA and 5-OHDA, figure 3) but

also 5,-S-CyS-DA and 5-S-Glu-DA, precursors of DHBT 17 and other

more complex dihydrobenzothiazines (figure 4).

DHBTs 17 to 19 are lethal in mouse brain, although at this time the

biochemical/chemical mechanisms underlying this toxicity and

specific neuronal systems affected are unknown. However, 5-S-CySDA and 17 to 19 are much more easily oxidized than DA, and the

latter DHBTs appear to be capable of redox cycling reactions (Zhang

and Dryhurst 1994). Thus, the HO·-mediated oxidation of DA in

dopaminergic nerve terminals induced by methamphetamine might be

expected to generate aberrant oxidative metabolites that (as a result

of autoxidation and redox cycling reactions) potentiate formation of

O2-· and/or H2O2, and then HO· and neuronal damage.

A number of lines of evidence, discussed previously, suggest that

aberrant metabolite(s) of DA (other than or in addition to 6-OHDA)

might contribute to the methamphetamine-induced degeneration of

not only dopaminergic terminals but also serotonergic terminals.

Similarly, aberrant metabolite(s) of 5-HT (other than or in addition

to 5,6-DHT) might be involved in the degeneration of serotonergic

and dopaminergic terminals and a subpopulation of cell bodies in the

somatosensory cortex. Experimental evidence indicates that some of

the neurodegenerative effects evoked by methamphetamine are

mediated by NMDA and GABA receptors. Thus, it will be of

considerable interest to investigate the neuro- toxicity of putative

aberrant oxidative metabolites of 5-HT (figures 1 and 2) and DA

(figures 4 and 5) towards serotonergic, dopaminergic, and other

neuronal systems and their interactions with NMDA, GABA, and

other brain receptors.

A central question relates to mechanisms by which methamphetamine

might evoke the intraneuronal formation of oxygen radicals that

appear to play important roles in the overall neurodegenerative

processes evoked by this drug (DeVito and Wagner 1989; Cadet et al.

1994). Once putative oxidative metabolites of 5-HT such as T-4,5-

D, 7-S-Glu-T-4,5-D, 5,6-DHT, 6, 8, and 7,7T-D (figure 1) are formed

intraneuronally, autoxidation/redox cycling reactions should, in

principle, be capable of generating O2-· and/or H2O2, the precursors of

HO·. Similarly, intraneuronal formation of 6-OHDA, 5-S-CyS-DA,
166

and DHBTs 17 to 19 and 22 would also be expected to potentiate

elevated fluxes of O2-·, H2O2, and HO· as a result of the facile

autoxidation/redox cycling reactions of these putative aberrant

metabolites. The presence of very low concentrations of 5-S-CySDA in DA-rich regions of human and other mammalian brains

suggests that autoxidation (Rosengren et al. 1985; Fornstedt et al.

1986, 1989, 1990) or perhaps some other form of DA oxidation is a

normal reaction in vivo. Furthermore, available evidence suggests

that it is cytoplasmic DA that is oxidized to give 5-S-CyS-DA

(Fornstedt et al. 1989; Fornstedt and Carlsson 1989). Presumably,

only free or unbound cytoplasmic DA is available for such oxidation

reactions.

The ability of methamphetamine to displace DA from cytoplasmic

storage sites prior to efflux through the uptake carrier site (Raitiri et

al. 1979; Marek et al. 1990a; Liang and Rutledge 1982) would be

expected to elevate the levels of this neurotransmitter available for

autoxidation. Furthermore, reuptake of released DA through the

uptake carrier site might continue to provide elevated cytoplasmic

levels of the neurotrans-mitter for autoxidation and oxidation by

HO·, generated as a result of autoxidation/redox cycling reactions of

aberrant oxidative metabolites of DA. Such a sequence of events

might result in the gradual increase in the levels of toxic aberrant

oxidative metabolites of DA and account for the observation that

administration of the uptake inhibitor AFA up to 8 hours after

methamphetamine is capable of protecting dopaminergic terminals

(Marek et al. 1990b).

There is presently no experimental information concerning the

normal autoxidation of cytoplasmic 5-HT. However, 5-HT

autoxidizes at about the same rate as DA at physiological pH

(Creveling et al. 1975; Rotman et al. 1976). Methamphetamine

displacement of 5-HT from its cytoplasmic storage sites and reuptake

of the released neurotransmitter might lead to 5-HT autoxidation to

aberrant metabolites; as a result of autoxidation/redox cycling

reactions, these metabolites form elevated fluxes of O2-·, H2O2, and

HO· and thereby potentiate their own synthesis and neuronal damage.

Until recently it was believed that intraneuronal proteins known as

serotonin binding proteins (SBP) were involved in the storage,

protection, and/or transport of 5-HT (Tamir et al. 1976; Gershon and

Tamir 1984) and catecholamines (Jimenez Del Rio et al. 1992,

1993a, 1993b; Pinxteren et al. 1993). The binding of 5-HT and DA

to SBP is increased by Fe2+ but not Fe3+. Thus, it was generally
167

believed that Fe2+ initially binds to sulfhydryl groups of SBP and that

the monoamine neurotrans- mitters form reversible coordinate bonds

with the trapped iron. However, more recent results (Jimenez Del Rio

et al. 1993a) have revealed that in the presence of Fe2+ and molecular

oxygen, SBP covalently (irreversibly) binds the oxidation products of

5-HT and DA. The latter reactions are apparently mediated by

oxygen radicals formed by reaction between Fe2+ and molecular

oxygen. These observations might imply that the physiological role

for SBP is to act as a scavenger of electrophilic oxidation products of

5-HT (e.g., T-4,5-D, 10 (figure 1)) and DA (e.g., DA-o-quinone,

figure 4), which are themselves cytotoxins or precursors of other

toxic metabolites. Interestingly, reserpine inhibits the binding of 5-

HT (or its oxidation products) to SBP in the presence of Fe2+ and

molecular oxygen (Tamir et al. 1976) and hence might account, in

part, for increased formation of 5-S-CyS-DA in the striatum of reserpinized rats (Fornstedt and Carlsson 1989). Thus, reserpine might

not only evoke increased cytoplasmic levels of DA, but also impair

the ability of SBP to bind electrophilic oxidation products. Similarly,

elevated cytoplasmic levels of free DA and 5-HT caused by

methamphetamine, followed by intraneuronal oxidation of these

neurotransmitters, might result in formation of electrophilic

intermediates at concentrations that exceed the scavenging capacity

of SBP with resultant neurodegenerative consequences.

REFERENCES

Axt, K.; Commins, D.L.; Vosmer, G.; and Seiden, L-S. -Methyl- ptyrosine pretreatment partially prevents methamphetamineinduced endogenous neurotoxin formation. Brain Res 515:269-

276, 1990.

Bakhit, C., and Gibb, J.W. Methamphetamine-induced depression of

tryptophan hydroxylase: Recovery following acute treatment.

Eur J Pharmacol 76:229-233, 1981.

Baumgarten, H.G., and Lachenmeyer, L. Chemical lesioning of central

monoamine axons by 5,6-dihydroxytryptamine and 5,7-

dihydroxy-tryptamine. Adv Biochem Pharmacol 10:13-33, 1972.

Bowery, N. GABAB receptors and their significance in mammalian

pharmacology. Trend Pharmacol Sci 10:401-407, 1989.

Buening, M.K., and Gibb, J.W. Influence of methamphetamine and

neuroleptic drugs on tyrosine hydroxylase activity. Eur J

Pharmacol 26:30-34, 1974.

Cadet, J.L.; Sheng, P.; Ali, S.; Rothman, R.; Carlson, E.; and Epstein,

C. Attenuation of methamphetamine-induced neurotoxicity in
168

copper/zinc superoxide dismutase transgenic mice. J Neurochem

62:380-383, 1994.

Chen, J-C.; Schnepper, P.W.; To, A.; and Volicer, L. Neuronal

changes in the rat brain after intraventricular administration of

tryptamine-4,5- dione. Neuropharmacology 31:215-219, 1992.

Choi, D.W. Ionic dependence of glutamate neurotoxicity. J Neurosci

7:369-379, 1987.

Commins, D.L., and Seiden, L. -Methyltyrosine blocks methylamphetamine-induced degeneration in rat somatosensory cortex.

Brain Res 365:15-20, 1986.

Commins, D.L.; Axt, K.J.; Vosmer, G.; and Seiden, L.S. 5,6-

Dihydroxy-tryptamine, a serotonergic neurotoxin, is formed

endogenously in the rat brain. Brain Res 403:7-14, 1987.

Coyle, J.T., and Schwarz, R. Lesion of striatal neurons with kainic

acid provides a model for Huntington's Chorea. Nature 263:244-

246, 1976.

Creveling, C.R.; Lundstrom, J.; McNeal, E.T.; Tice, L.; and Daly,

J.W. Dihydroxytryptamines: Effects on noradrenergic function in

mouse heart in vivo. Mol Pharmacol 11:211-222, 1975.

Crino, P.B.; Vogt, B.A.; Chen, J-C.; and Volicer, L. Neurotoxic effects

of partially oxidized serotonin: Tryptamine-4,5-dione. Brain Res

504:247-257, 1989.

DeVito, M.J., and Wagner, G.C. Methamphetamine-induced neuronal

damage: A possible role for free radicals. Neuropharmacology

28:1145-1150, 1989.

Evans, J.M., and Cohen, G. Can trace amounts of neurotoxins destroy

dopamine neurons? Neurochem Int 15:127-129, 1989.

Farfel, G.M.; Vosmer, G.L.; and Seiden, L.S. The N-methyl-Daspartate antagonist MK-801 protects against serotonin

depletions induced by methamphetamine, 3,4-methylenedioxymethamphetamine and p-chloroamphetamine. Brain Res

595:121-127, 1992.

Fibiger, H.C., and McGeer, E.G. Effect of acute and chronic methamphetamine treatment on tyrosine hydroxylase activity in brain

and adrenal medulla. Eur J Pharmacol 16:176-180, 1971.

Fornstedt, B.; Rosengren, E.; and Carlsson, A. Occurrence and

distribution of 5-S-cysteinyl derivatives of dopamine, DOPA and

DOPAC in the brains of eight mammalian species.

Neuropharmacology 25:451-454, 1986.

Fornstedt, B.; Brun, A.; Rosengren, E.; and Carlsson, A. The apparent

autoxidation rate of catechols in dopamine-rich regions of human

brains increases with the degree of depigmentation of substantia

nigra. J Neural Trans 1:279-295, 1989.
169

Fornstedt, B., and Carlsson, A. A marked rise in 5-Scysteinyldopamine levels in guinea-pig striatum following

reserpine treatment. J Neural Transm 76:155-161, 1989.

Fornstedt, B.; Pilebad, E.; and Carlsson, A. In vivo autoxidation of

dopamine in guinea-pig striatum increases with age. J Neurochem

55:655-659, 1990.

Gershon, M.D., and Tamir, H. Serotonectin and the family of

proteins that bind serotonin. Biochem Pharmacol 33:3115-3118,

1984.

Gibb, J.W.; Hanson, G.R.; and Johnson, M. Neurochemical

mechanisms of toxicity. In: Cho, A.K., and Segal, D.S., eds.

Amphetamine and Its Analogs. Psychopharmacology,

Toxicology, and Abuse. New York: Academic Press, 1994. pp.

269-295.

Graham, D.C. Oxidation pathways for catecholamines in the genesis

of neuromelanin and cytotoxic quinones. Mol Pharmacol 14:633-

643, 1978.

Halliwell, B., and Gutteridge, M.C. Oxygen toxicity, oxygen radicals,

transition metals and disease. Biochem J 219:1-14, 1984.

Halliwell, B. Reactive oxygen species and the central nervous system.

J Neurochem 59:1609-1623, 1992.

Hotchkiss, A.J.; Morgan, M.E.; and Gibb, J.W. The long-term effects

of multiple doses of methamphetamine on neostriatal tryptophan

hydroxylase, tyrosine hydroxylase, choline acetyltransferase and

glutamate decarboxylase activities. Life Sci 25:1373-1378, 1979.

Hotchkiss, A., and Gibb, J.W. Blockade of methamphetamine-induced

depression of tyrosine hydroxylase by GABA transaminase

inhibitors. Eur J Pharmacol 66:201-206, 1980a.

Hotchkiss, A.J., and Gibb, J.W. Long-term effects of multiple doses of

methamphetamine on tryptophan hydroxylase and tyrosine

hydroxylase activity in rat brain. J Pharmacol Exp Ther 214:257-

262, 1980b.

Jimenez Del Rio, M.J.; Pinxteren, J.; De Potter, W.P.; Ebinger, G.;

and Vauquelin, G. Serotonin-binding proteins in the bovine

cerebral cortex: Interactions with serotonin and catecholamines.

Eur J Pharmacol 225:225-234, 1992.

Jimenez Del Rio, M.J.; Velez Pardo, C.; Pinxteren, J.; De Potter, W.;

Ebinger, G.; and Vauquelin, G. Binding of serotonin and dopamine

to ‘serotonin binding proteins’ in bovine frontal cortex: Evidence

for iron-induced oxidative mechanisms. Eur J Pharmacol 247:11-

21, 1993a.

Jimenez Del Rio, M.; Pinxteren, J.; De Potter, W.; Ebinger, G.; and

Vauquelin, G. Serotonin binding proteins in bovine retina: Binding
170

of serotonin and catecholamines. Neurochem Int 22:111-119,

1993b.

Johnsson, G.; Malfors, T.; and Sachs, C. 6-Hydroxydopamine as a

Denervation Tool in Catecholamine Research. Amsterdam: North

Holland, 1975.

Karoum, F.; Chrapusta, S.J.; Egan, M.F.; and Wyatt, R.J. Absence of

6-hydroxydopamine in rat brain after treatment with stimulants

and other dopaminergic agents: A mass fragmentographic study. J

Neurochem 61:1369-1375, 1993.

Klemm, H.P.; Baumgarten, H.G.; and Schlossberger, H.G.

Polarographic measurements of spontaneous and mitochondriapromoted oxidation of 5,6- and 5,7-dihydroxytryptamine. J

Neurochem 35:1400-1408, 1980.

Lafon-Cazal, M.; Pietri, S.; Culcasi, M.; and Bockaert, J. NMDAdependent superoxide production and neurotoxicity. Nature

364:535-537, 1993.

Levine, M.S.; Hull, C.D.; Garcia-Rill, E.; Erinoff, L.; Buchwald, N.A.;

and Heller, A. Long-term decrease in spontaneous firing of

caudate neurons induced by amphetamine in cats. Brain Res

194:263-268, 1980.

Lev-Tov, A.; Meyers, D.E.R.; and Burke, R.E. Activation of type B -

aminobutyric acid receptors in the intact mammalian spinal cord

mimics the effects of reduced presynaptic Ca2+ influx. Proc Nat

Acad Sci U S A 85:5330-5334, 1988.

Liang, N.Y., and Rutledge, C.O. Evidence for carrier-mediated efflux

of dopamine from corpus striatum. Biochem Pharmacol 30:2479-

2484, 1982.

Lindvall, O., and Björklund, A. Organization of catecholamine

neurons in the rat central nervous system. In: Iversen, L.L.;

Iversen, S.D.; and Snyder, S.H., eds. Handbook of

Psychopharmacology. Vol. 9. New York: Plenum Press, 1978.

pp. 139-231.

Marek, G.J.; Vosmer, G.; and Seiden, L.S. Dopamine uptake inhibitors

block long-term neurotoxic effects of methamphetamine upon

dopaminergic neurons. Brain Res 513:274-279, 1990a.

Marek, G.J.; Vosmer, G.; and Seiden, L.S. The effects of monoamine

uptake inhibitors and methamphetamine on neostriatal 6-

hydroxy-dopamine (6-OHDA) formation, short-term monoamine

depletions and locomotor activity in the rat. Brain Res 516:1-7,

1990b.

Marek, G.J.; Vosmer, G.; and Seiden, L.S. Pargyline increases 6-

hydroxydopamine levels in the neostriatum of

methamphetamine-treated rats. Pharmacol Biochem Behav

36:187-190, 1990c.
171

McGeer, P.L.; Eccles, J.C.; and McGeer, E.G. Molecular Neurobiology

of the Mammalian Brain. New York: Plenum Press, 1987.

Nash, J.F., and Yamamoto, B.K. Methamphetamine neurotoxicity

and striatal glutamate release: Comparison to 3,4-

methylenedioxy-methamphetamine. Brain Res 581:237-243,

1992.

Pinxteren, J.; Jimenez Del Rio, M.; Valez-Pardo, C.; Ebinger, G.;

Vauquelin, G.; and De Potter, W. Soluble serotonin and

catecholamine binding proteins in the bovine adrenal medulla.

Neurochem Int 23:343-350, 1993.

Raitiri, M.; Cerrito, F.; Cervoni, A.M.; and Levi, G. Dopamine can be

released by two mechanisms differentially affected by the

dopamine transport inhibitor nomifensine. J Pharmacol Exp Ther

208:195-202, 1979.

Ricaurte, G.A.; Schuster, C.R.; and Seiden, L.S. Long-lasting effects of

repeated methamphetamine administration on dopamine and

serotonin neurons in the rat brain: A regional study. Brain Res

193:153-160, 1980.

Ricaurte, G.A.; Guillery, R.W.; Seiden, L.S.; Schuster, C.R.; and

Moore, R.Y. Dopamine nerve terminal degeneration produced by

high doses of methylamphetamine in the rat brain. Brain Res

235:93-103, 1982.

Ricaurte, G.A.; Fuller, R.W.; Perry, K.W.; Seiden, L.S.; and Schuster,

C.R. Fluoxetine increases long-lasting neostriatal dopamine

depletion after administration of d-methamphetamine and damphetamine. Neuropharmacology 22:1165-1169, 1983.

Richter, H.W., and Waddell, W.H. Oxidation of dopamine by

hydroxyl radical in aqueous solution. J Am Chem Soc 105:5435-

5440, 1983.

Rollema, H.; DeVries, J.B.; Westerink, B.H.C.; VanPutten, F.M.S.; and

Horn, A.S. Failure to detect 6-hydroxydopamine in rat striatum

after the dopamine releasing drugs dexamphetamine,

methamphetamine, and MPTP. Eur J Pharmacol 132:65-69,

1986.

Rosengren, E.; Linder-Eliasson; and Carlsson, A. Detection of 5-Scysteinyldopamine in human brain. J Neural Transm 63:247- 261,

1985.

Rotman, A.; Daly, J.W.; and Creveling, C.R. Oxygen-dependent

reaction of 6-hydroxydopamine, 5,6-dihydroxytryptamine, and

related compounds with proteins in vitro: A model for

cytotoxicity. Mol Pharmacol 12:887-899, 1976.

Schmidt, C.J.; Ritter, J.K.; Sonsalla, P.K.; Hanson, G.R.; and Gibb,

J.W. Role of dopamine in the neurotoxic effects of

methamphetamine. J Pharmacol Exp Ther 233:539-544, 1985.
172

Schuster, C.R. The behavioral pharmacology of psychomotor

stimulant drugs. In: Hoffmeister, F., and Stille, G., eds. Handbook

of Experimental Pharmacology. Berlin/Heidelberg: SpringerVerlag, 1981. pp. 587-605.

Seiden, L.S., and Ricaurte, G.A. Neurotoxicity of methamphetamine

and related drugs. In: Meltzer, H.Y., ed. Psychopharmacology: A

Third Generation of Progress. New York: Raven Press, 1987. pp.

359-366.

Seiden, L.S., and Vosmer, G. Formation of 6-hydroxydopamine in

caudate nucleus of the rat after a single large dose of

methylamphetamine. Pharmacol Biochem Behav 21:29-31,

1984.

Seiden, L.S.; Fischman, M.W.; and Schuster, C.R. Long-term methamphetamine changes in brain catecholamines in tolerant rhesus

monkeys. Drug Alcohol Depend 1:215-219, 1975-1976.

Seiden, L.S.; Commins, D.L.; Vosmer, G.; Axt, K.; and Marek, G.

Neurotoxicity in dopamine and 5-hydroxytryptamine terminal

fields: A regional analysis in nigrostriatal and mesolimbic

projections. Ann NY Acad Sci 537:161-172, 1988.

Singh, S., and Dryhurst, G. Further insights into the oxidation

chemistry and biochemistry of the serotonergic neurotoxin 5,6-

dihydroxy-tryptamine. J Med Chem 33:3035-3044, 1990.

Singh, S.; Jen, J-F.; and Dryhurst, G. Autoxidation of the indolic

neurotoxin 5,6-dihydroxytryptamine. J Org Chem 55:1484-

1489, 1990.

Singh, S.; Wrona, M.Z.; and Dryhurst, G. Synthesis and reactivity of

the putative neurotoxin tryptamine-4,5-dione. Bioorg Chem

20:189-203, 1992.

Slivka, A., and Cohen, G. Hydroxyl radical attack on dopamine. J Biol

Chem 260:15466-15472, 1987.

Slivka, A.; Mytilineou, C.; and Cohen, G. Histochemical evaluation of

glutathione in brain. Brain Res 409:275-284, 1987.

Sonsalla, P.K.; Gibb, J.W.; and Hanson, G.R. Nigrostriatal dopamine

actions on the D2 receptors mediate methamphetamine effects on

the striatonigral Substance P system. Neuropharmacology

25:1221-1230, 1986.

Sonsalla, P.K.; Riordan, D.E.; and Heikkila, R.E. Competitive and

noncompetitive antagonists of N-methyl-D-aspartate receptors

protect against methamphetamine-induced dopaminergic damage

in mice. J Pharmacol Exp Ther 256:506-512, 1991.

Spector, R., and Eells, J. Deoxynucleotide and vitamin transport into

the central nervous system. Fed Proc 43:196-200, 1984.
173

Suzuki, O.; Haltori, H.; Asano, M.; and Katsumata, Y. Inhibition of

monoamine oxidase by D-methamphetamine. Biochem

Pharmacol 29:2071-2073, 1980.

Tamir, H.; Klein, A.; and Rapport, M.M. Serotonin binding protein:

Enhancement of binding by Fe2+ and inhibition of binding by

drugs. J Neurochem 26:871-878, 1976.

Udenfriend, S.; Clark, C.T.; Axelrod, J.; and Brodie, B.B. Ascorbic acid

in aromatic hydroxylation. J Biol Chem 208:731-738, 1954.

Wagner, G.C.; Ricaurte, G.A.; Seiden, L.S.; Schuster, C.R.; Miller, R.J.;

and Westley, J. Long-lasting depletions of striatal dopamine and

loss of dopamine uptake sites following repeated administration of

methamphetamine. Brain Res 181:151-160, 1980.

Wagner, G.; Lucot, J.B.; Schuster, C.R.; and Seiden, L.S. Alphamethyltyrosine attenuates and reserpine increases

methamphetamine-induced neuronal changes. Brain Res 270:285-

288, 1983.

Walling, C. Fenton's reagent revisited. Acc Chem Res 8:125-131,

1975.

Wong, K-S., and Dryhurst, G. Tryptamine-4,5-dione: Properties and

reaction with glutathione. Bioorg Chem 18:253-264, 1990.

Wong, K-S.; Goyal, R.N.; Wrona, M.Z.; and Dryhurst, G. 7-SGlutathio- nyltryptamine-4,5-dione: A possible aberrant

metabolite of serotonin. Biochem Pharmacol 46:1637-1652,

1993.

Wrona, M.Z., and Dryhurst, G. Electrochemical oxidation of 5-

hydroxytryp- tamine in aqueous solution at physiological pH.

Bioorg Chem 18:291-317, 1990.

Wrona, M.Z., and Dryhurst, G. Interactions of 5-hydroxytryptamine

with oxidative enzymes. Biochem Pharmacol 41:1145-1162,

1991.

Wrona, M.Z.; Goyal, R.N.; Turk, D.; Blank, C.L.; and Dryhurst, G.

5,5T-Dihydroxy-4,4T-bitryptamine: A potentially aberrant

neurotoxic metabolite of serotonin. J Neurochem 59:1392-1398,

1992.

Wrona, M.Z.; Singh, S.; and Dryhurst, G. Influence of L-cysteine on

the oxidation chemistry of serotonin. Bioorg Chem 22:421-445,

1994.

Wrona, M.Z.; Yang, Z.; McAdams, M.; O'Connor-Coates, S.; and

Dryhurst, G. Hydroxyl radical-mediated oxidation of serotonin:

Potential insights into the neurotoxicity of methamphetamine. J

Neurochem 64:1390-1400, 1995.

Yamazaki, I., and Piette, L.H. Mechanism of aerobic oxidase reaction

catalyzed by peroxidase. Biochem Biophys Acta 77:47-64, 1963.
174

Zhang, F., and Dryhurst, G. Oxidation chemistry of dopamine:

Possible insights into the age-dependent loss of dopaminergic

nigrostriatal neurons. Bioorg Chem 21:392-410, 1993.

Zhang, F., and Dryhurst, G. Effects of L-cysteine on the oxidation

chemistry of dopamine: New reaction pathways of potential

relevance to idiopathic Parkinson's Disease. J Med Chem

37:1084-1908, 1994.

Zhang, F., and Dryhurst, G. Influence of glutathione on the oxidation

chemistry of the catecholaminergic neurotransmitter dopamine. J

Electroanal Chem Interfacial Electrochem, in press.

ACKNOWLEDGMENTS

This chapter was prepared with support from National Institutes of

Health grants GM-32367 and NS-29886.

AUTHORS

Monika Z. Wrona, Ph.D.

Adjunct Associate Professor

Zhaoliang Yang, Ph.D.

Research Associate

Glenn Dryhurst, Ph.D.

Chairman

George Lynn Cross Research Professor

Department of Chemistry and Biochemistry

University of Oklahoma

Norman, OK 73019

Fa Zhang, Ph.D.

Agricultural Research Division

American Cyanamid Co.

P.O. Box 400

Princeton, NJ 08543
175

Immunotoxicology of Opioids,

Inhalants, and Other Drugs of Abuse

Robert V. House, Peter T. Thomas, and Hemendra N. Bhargava

INTRODUCTION

Drug abuse is a problem of increasing worldwide significance. In

addition to the obvious socioeconomic problems associated with the

use of so-called street drugs, the abuse of ethical pharmaceuticals may

also result in serious untoward health effects depending upon a wide

range of variables (Chiang and Goldfrank 1990). One possible medical

complication of drug abuse is modulation of the immune system, or

immunotoxicity (Pillai and Watson 1990). The immune system is a

highly regulated organ system that presents a variety of potential

targets for modulation by drugs. This modulation may take the form

of immunosuppression, leading to an enhanced susceptibility to

infection or neoplasia; conversely, it may take the form of

immunostimulation, resulting in hypersensitivity (allergy) or

autoimmunity (Luster and Rosenthal 1993). Closely associated with

drug abuse in recent years has been the emergence of the acquired

immunodeficiency syndrome (AIDS), a retroviral infection spread by

sexual contact or hematogenously by the sharing of needles among

intravenous (IV) drug abusers. One of the hallmarks of AIDS is a

profound and irreversible suppression of immune competence, usually

resulting in death of the host from opportunistic infections. Thus,

the combination of drug abuse and human immunodeficiency virus

(HIV) infection represents a formidable challenge to the immune

system. Although the scientific literature is replete with studies

describing drug-related immunosuppression, the sheer number of

abused drugs precludes detailed examination using traditional

methodology. Moreover, as new therapeutics are developed for the

treatment of drug abuse, an efficient screening approach will be

required to assess their immunomodulatory potential.

The studies described herein were performed under the aegis of

National Institute on Drug Abuse (NIDA) contract 271-91-9201,

entitled "Immunomodulatory Effects of Drugs of Abuse and Potential

Medications." The purpose of the these studies was the evaluation of

the potential for a number of drugs of abuse, opioid peptides, and

established and experimental therapeutic agents to alter immune

function associated with host defense. This was approached in a
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twofold manner. First, test materials were evaluated in vitro using

murine splenic lymphocytes from B6C3F1 (C57BL/6 X C3H) hybrid

mice. This hybrid strain was chosen because of the large database that

exists of drug and chemical effects on immunity. The panel of

immune function assays was carefully chosen to represent relevant

host defense mechanisms and be adaptable to drug screening. Second,

standard in vivo pharmacological models of tolerance/abstinence were

validated in this strain and then used for in vitro multidrug exposure

studies as above.

RATIONALE AND METHODOLOGY

For the screening portion, the data were obtained following in vitro

exposure of drugs to isolated splenic lymphocytes and macrophages.

This experimental paradigm allows for a high degree of precision in

drug concentration delivered to target cells, utilizes only small

amounts of test material and a limited number of animals, targets only

the cells of primary interest, and facilitates a high-output screening

approach. In spite of these important advantages, this in vitro

exposure system does not account for drug metabolism (and the

attendant question of metabolite-associated immune effects) or

potential secondary effects on other target cells or tissues. For

example, a drug might exert immunotoxicity toward the bone

marrow, thus affecting the precursors of all immune cells.

Alternatively, a drug that targets the thymus (for example) might

have selective effects on cell-mediated immunity, but only limited

effects on humoral immunity. Additionally, studies have

demonstrated that the metabolites of some drugs exert profound

effects on immune function (Thomas et al. 1995a). Thus, abused

drugs that do not exhibit in vitro immunomodulatory activity may

still display such activity following in vivo exposure. For these

reasons, drugs testing positive in the in vitro screen would require

further testing following in vivo exposure to more fully evaluate the

range and nature of their immunomodulatory potential.

Figure 1 illustrates the general experimental approach. Spleens

harvested from naive female B6C3F1 mice served as a convenient

source of lymphocytes for study. The initial consideration in the in

vitro screen was the physical characteristics of the test compounds

themselves. For the most part, at the in vitro concentrations tested,

the compounds have
177

largely been soluble in aqueous solution. However, drugs that are

primarily or exclusively lipid-soluble require the use of an appropriate,

nontoxic carrier material. The second consideration was the

cytotoxic potential following in vitro exposure to any of these drugs.

This was evaluated by exposing isolated splenocytes to the anticipated

range of drug concentrations in vitro. At appropriate time points,

the cells were evaluated for toxicity by vital dye exclusion. Any drug

found to be cytotoxic would have been excluded from study; however,

no drug yet tested has produced overt toxicity as evaluated by this

approach.

Two ranges of in vitro concentrations were utilized in these studies.

Most of the abused drugs (e.g., amphetamine, phencyclidine (PCP))

were examined at in vitro concentrations between 0.0001 and 100
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micromolars (M). Endogenous and exogenous peptide-type

compounds (e.g., [D-penicillamine2, D-penicillamine5-]-enkephalin

(DPDPE), -endorphin) were evaluated at concentrations between

0.00001 and 10 M. These ranges were chosen to include realistic

pharmacologically achievable concentrations in humans following in

vivo drug exposure.

After establishment of the drug exposure paradigm, the potential of

these compounds to produce immunomodulation was examined in a

panel of in vitro assays. These assays were carefully selected to yield

the maximal amount of information on various immune regulatory

and effector mechanisms. The rationale for the use of each assay and

its execution are described below.

One of the two principal arms of the immune system in vertebrates,

namely humoral immunity, is mediated by soluble factors (antibodies)

produced by B-cells. This form of immunity is vitally important for

efficient host defense. B-cells are primarily responsible for the

production of specific antibody although they also exhibit other

functions, including antigen presentation and cytokine production

(Brown 1992). For the purposes of this work, a rapid screening type

assay of B-cell function was required. B-cell proliferation was chosen

as the endpoint for two principal reasons. First, proliferation of

lymphocytes is an early sequela of activation, and as such represents a

relevant measurement of immune competence. Second, cellular

proliferation is an easily quantitated function, whereas other assays of

B-cell function (e.g., antibody production) are laborious and time

consuming, precluding their efficient use in a large-scale screening

study.

Isolated murine splenocytes were cultured for 3 days in the presence

of various concentrations of drugs and a combination of antiimmuno-globulin M (IgM) antibody/recombinant murine interleukin-4

(IL-4) as adapted from Abbas and colleagues (1990). The activational

signal is mediated via anti-Ig binding with the surface immunoglobulins

present on B-cells, with IL-4 providing the proliferative signal. This

system may represent a more physiologically relevant model than

more routinely used mitogens such as lipopolysaccharide (LPS). Since

this was a screening study requiring a high throughput of test

materials, a more rapid indicator system was needed to replace the

traditional thymidine incorporation method. To this end, a

colorimetric assay based on mitochondrial reduction of a tetrazolium

salt to a colored formazan end product (Roehm et al. 1991) was

employed. This system allowed assay of a large number of samples in
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a greatly reduced time, and will be used in all of the authors’ future

screening studies.

The action of the second principal arm of the immune system is

mediated primarily by cellular mechanisms, and consequently is

referred to as cell-mediated immunity. Cell-mediated immunity is

determined largely by the function of T-cells. For this study, both the

regulatory and the effector functions of T-cells were evaluated. The

regulatory capacity of T-helper cells was evaluated by measurement

of cytokine production. T-helper cells are a subset of T-cells

displaying the CD3/CD4 surface antigens, and are primarily

responsible for producing regulatory cytokines. T-helper cells exhibit

functional differences based on their pattern of cytokine secretion;

these differences are thought to function as mechanisms for

controlling the immune reaction and directing its ultimate expression

as either humoral- or cell-mediated immunity (Mosmann et al. 1991).

In simplistic terms, T-helper cells may be categorized as TH1, which

secrete IL-2 and interferon-gamma (IFN) but not IL-4 or IL-10, and

TH2, which secrete IL-4 and IL-10 but not IL-2 or IFN. Examining

representa- tive cytokines from each of these groups provides

mechanistic information on the potential differential effects of test

materials on T-cell subset functions. In this work, the production of

IL-2 and IL-4, the prototype representatives for TH1 and TH2

respectively, was evaluated. For the in vitro studies, naive murine

splenocytes were cultured in the presence of various concentrations of

test drugs, and were stimulated with a mono- clonal antibody directed

against the T-cell antigen receptor (i.e., anti-CD3). This system

polyclonally simulates the interaction of T-cells with their cognate

antigen, and represents a physiologically relevant activational signal.

Following culture, the supernatant fluids were collected and analyzed

for IL-2 bioactivity by a modification of the method of Gillis and

coworkers (1978), and for IL-4 bioactivity by a modification of the

method of Hu-Li and coworkers (1989). As with the B-cell

proliferation assay, a colorimetric endpoint was utilized to facilitate

evaluation of a large number of samples.

The impact of drugs on effector T-cell function is determined by

evaluating the in vitro induction of cytotoxic T-lymphocytes

(CTLs), a population of T-cells bearing the CD3/CD8 surface

antigens. These cells are capable of exhibiting cytotoxicity toward

specific target cells after prior exposure to antigen, and therefore

represent a central effector mechanism of cell-mediated immunity

and host resistance (Berke 1989). The crucial role that CTLs are
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thought to serve in host defense makes them a prime measure of cellmediated immunity in both normal and immunocompromised states.

In the course of the in vitro studies in this work, the laboratory of

one author has developed a serum-free modification of the standard

CTL induction method (House et al. 1994a). This serum- free

approach results in greater reproducibility of the assay and facilitates

recovery of viable cells, an important consideration when examining

cells with potential immunomodulatory effects. In these studies,

isolated murine splenocytes were bulk cultured for 5 days in the

presence of various concentrations of drugs and mitomycin Cinactivated P815 mastocytoma cells (which also serve as the target

cells). The effector cells were subsequently collected, washed, and

cocultured for 4 hours with radiolabeled P815 target cells. Released

radiolabel was measured in a gamma counter, and the specific lysis of

the target cells was determined as a percentage of total releasable

counts.

An important immune function to evaluate in the context of

immuno-deficiency, and one that represents a form of nonspecific

immunity/host resistance, is the function of natural killer (NK) cells.

NK cells are lymphocytes distinct from either B-cells or T-cells,

which contribute to immunocompetence by mediating major

histocompatibility complex-independent cytotoxicity (Lotzová

1993). For the purposes of these studies, a combined measurement of

both basal and augmented NK cell function was used. In this assay,

murine splenocytes were exposed for 24 hours to various

concentrations of drugs in the presence or absence of an optimum

concentration of recombinant IL-2 (Thomas et al. 1993). The cells

were then washed and cocultured for 4 hours with radiolabeled YAC-1

tumor cells (a murine NK-sensitive cell line). Tumor cell lysis was

quantitated as described above for the CTL procedure.

The approach described above had a dual purpose. First, although NK

cells are generally nonspecific in their cytotoxic capacity, their

action is sensitive to modulation by immune mechanisms, particularly

by the action of cytokines like IL-2 and interferon-gamma (Talmadge

1985). This situation would probably occur in vivo during an

infection or other immune reaction. Basal NK levels are often of low

activity, which would render any decrease in activity difficult to

measure. Thus, measurement of enhanced (augmented) activity may

reveal differential modulation of NK cell function in a physiological

situation. The second reason for this 24-hour culture was the

uncertainty that drug exposure would produce significant effects only

during the 4-hour lytic phase of the assay. This combination assay
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provided mechanistic information on NK cell functional alteration

with a minimum of extra labor and materials.

Finally, the effect of drug exposure on macrophage function was

evaluated. Macrophages subserve both specific and nonspecific host

resistance mechanisms and, as such, are important cells in the

induction and maintenance of various immune and nonimmune

responses to infection. As with the T-cells, macrophages accomplish

this regulatory activity largely through the action of cytokines. In

these studies, the production of IL-6 and tumor necrosis factor (TNF)

by macrophages exposed in vitro to drugs was implemented. IL-6 and

TNF are pivotal cytokines with a multitude of effector and regulatory

functions (Akira et al. 1990) and, as such, represent rational targets

for determining macrophage function. For these studies, naive mice

were given an intraperitoneal (IP) injection of thioglycolate to elicit

peritoneal exudate cells, which were washed from the animal by

peritoneal lavage following sacrifice. Peritoneal macrophages from

these preparations were enriched by plastic adherence. Monolayers of

enriched macrophages were exposed in vitro for 48 hours to various

concentrations of drugs in the presence of an optimum stimulatory

concentration of bacterial LPS. The culture supernatant fluids were

harvested, and IL-6 and TNF were quantitated by specific bioassays.

IL-6 was quantitated by a modification of the method of Van Snick

and colleagues (1986) using the 7TD1 cell line, and TNF was

quantitated by a modification of methods described by Meager and

colleagues (1989) using the L929 fibroblast cell line. Both bioassays

utilized a colorimetric endpoint, as described above.

Although drug action on elicited macrophages may not necessarily

reflect the situation in normal animals, the elicitation procedure was

necessitated by the extremely low recovery of peritoneal cells in the

absence of such treatment; this low recovery would have necessitated

the use of very large numbers of mice to accomplish these screening

studies.

RESULTS FROM IN VITRO SCREENING STUDIES

In general, drugs selected for testing in this study may be grouped into

three general categories: abused drugs previously demonstrated to

suppress the immune response (e.g., heroin, cocaine); abused drugs

that have not previously been associated with immune function

alterations (e.g., lysergic acid diethylamide (LSD),
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methylenedioxymethamphetaine (MDMA)); and drugs with

therapeutic potential for treating drug abuse (e.g., ibogaine).

For the purpose of summarizing the findings of the in vitro screening

assays, these drugs are further classified according to either their

action or chemical description. The sections below describe the

results of these in vitro assays. Representative data are presented for

illustrative purposes.

Hallucinogens

In general, the hallucinogenic drugs have received scant investigation

for potential immunotoxic potential. The reason for this is unclear,

although the limited duration of exposure characteristic of these drugs

may be an important factor. That is, although hallucinogen abuse

may be a long-term habit, actual exposure is usually not chronic. This

pattern of abuse may result in acute alterations of selected immune

functions, but the resiliency of the immune response may compensate

for any drug-induced defects.

One of the first drugs examined in this project was the dissociative

hallucinogen PCP, also known as angel dust. In the absence of effects

on cellular viability, PCP exposure significantly suppressed B-cell

prolif-eration, cytokine production by T-cells, and the generation of

specifically sensitized cytotoxic T-cells. In addition, PCP

significantly suppressed IL-2-augmented NK function. By

comparison, macrophage IL-6 production was not affected by any

concentration of PCP examined (Thomas et al. 1993). Significantly,

these alterations in immune function were observed at

pharmacologically relevant concentrations. These results are in basic

agreement with, and expand upon, the work of Khansari and

colleagues (1984) and Dornand and colleagues (1987), who both

reported immunosuppression after PCP exposure. These data suggest

that chronic PCP abuse may adversely affect immune mechanisms

associated with host defense.

Another hallucinogen examined in this project was LSD, a commonly

abused drug in the 1960s that appears to be enjoying a renaissance. A

careful review of the literature indicated that LSD had not heretofore

been evaluated for immune effects, prompting its investigation in this

work. It was found that in vitro exposure to LSD resulted in

suppressed proliferation of B-cells, production of the cytokines IL-2,

IL-4, and IL-6, and the induction of cytotoxic T-lymphocytes. In

vitro exposure to LSD had differential effects on NK cell activity,
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with significant enhancement of both basal and IL-2-augmented NK

cell function occurring at concen-trations that may be reached upon

human exposure (House et al. 1994b). Potential in vivo

immunological effects of LSD exposure remain to be characterized.

Two additional hallucinogens examined in this project were the indole

alkaloids ibogaine and harmaline. Ibogaine enjoyed a brief career as a

street drug in the 1960s, and was classified by the U.S. Food and Drug

Administration as a Schedule I drug in 1970 (Glick et al. 1991).

Ibogaine was of special interest for study since it is the subject of two

U.S. patents for the treatment of addiction (U.S. patents 4,499,096

and 4,587,243). Another indole alkaloid with hallucinogenic

properties is harmaline, a -carboline compound related to ibogaine.

The -carbolines are of particular interest in that, in addition to their

presence in plant materials, they may be present in mammals as

metabolic byproducts. Although harmaline and its congeners produce

physiological reactions similar to those seen following ibogaine

exposure, the two classes of compounds apparently function by

different mechanisms (Deecher et al. 1992). Harmaline was evaluated

along with ibogaine to determine whether any observed effects might

be related to the chemical structure of these hallucinogens. With the

exception of NK cell function with ibogaine, both drugs produced a

biphasic effect on both natural and antigen-specific immune

responses. By contrast, TH1- and TH2-derived cytokine production

was unaffected by exposure to either ibogaine or harmaline.

Suppression of macrophage function was noted for the different drugs,

but was generally associated only with high concentrations (House et

al. 1995b).

Stimulants

Amphetamine [(phenylisopropyl)amine] is a powerful central nervous

system stimulant that also affects the cardiovascular and peripheral

nervous systems, producing a decreased sense of fatigue, a mood

elevation, an increase in motor activity, and often euphoria

(Hoffman and Lefkowitz 1990). These properties have contributed

to its continuing popularity as a recreational drug. Although

amphetamine abuse represented an important problem in the United

States between the 1940s and the 1960s, the Controlled Substance Act

of 1970 limited the practice (Derlet and Heischober 1990). The Nmethylated homolog methamphetamine is produced in numerous

clandestine laboratories, and its abuse appears to be on the rise,

especially in Hawaii and the U.S. West Coast (Heischober and Miller

1991). A confounding factor in methamphetamine/amphetamine
184

abuse is that these drugs are often self-injected intravenously. This

practice poses the dual concern of an increased probability of

spreading HIV infection during the process of drug abuse, as well as the

potential of methamphetamine-mediated suppression of an already

damaged immune system in normal or HIV-infected drug abusers (Klee

1992). Studies on the immunomodulatory effects of amphetamines

(House et al. 1994a) indicated that in vitro exposure to amphetamine

resulted in a significant suppression of IL-2, but not IL-4, production

by T-cells, as well as a suppression of B-cell proliferation only at the

highest amphetamine concentration examined. An interesting finding

of these studies was that NK cell function was slightly suppressed by

amphetamine exposure, but was enhanced by methamphetamine

exposure (figure 2). The nature of this differential

immunomodulatory activity is currently unknown.

Methamphetamine often serves as the parent compound for synthesis

of new designer drugs (Bost 1988; Buchanan and Brown 1988).

Currently, one of the more popular designer drugs is MDMA, also

known as ecstasy. MDMA is the methylated derivative of the

amphetamine analog methylenedioxyamphetamine (MDA), a

prototype of the hallucinogenic amphetamine drugs (Bost 1988).

MDMA is relatively easy to synthesize
185

in clandestine drug laboratories, and thus will probably remain a

potential drug of abuse for the foreseeable future. Although MDMA

has been used both legally and illegally for a number of years, little is

known concerning its potential health effects. Studies with MDMA

demonstrated that in vitro exposure resulted in a pattern of

immunomodulation similar in most respects to that observed with the

parent drug methamphetamine (House et al. 1995a). No effect was

observed on B-cell proliferation at any concentration tested. In

comparison, production of IL-2 was enhanced at concentrations as

low as 0.0001 M and IL-4 production was unaffected. Basal and

augmented NK cell function were enhanced at concentrations between

0.0001 and 1.0 M, and CTL induction was significantly suppressed

at a concentration of 100 M. Finally, macrophage production of

TNF was slightly suppressed at 10 and 100 M MDMA, although this

inhibition was not statistically significant. The similarity in results

from methamphetamine and MDMA suggests that a common

mechanism of immunomodulation may be, in part, due to chemical

similarities between the two drugs, although elucidation of this

mechanism awaits further investigation.

It is important to remember that drug abuse is not an exclusively

American problem, and increasing population mobility can only serve

to increase exposure to previously unknown types of abused drugs.

Therefore, the immunotoxic potential of exposure to the natural

amphetamine cathinone was investigated. Cathinone is a

phenylalkylamine present in the khat shrub, the leaves of which are

chewed on a regular basis by residents of East Africa and the Arabian

peninsula (Kalix 1992). Consumption of khat is widespread in certain

cultures, but its use in countries outside its growth area has been

limited. Studies with cathinone, performed in tandem with studies of

the synthetic amphetamines, demonstrated that in vitro exposure to

either the (S) or (R) isomers of cathinone resulted in stimulation of

IL-2 production, B-cell proliferation, and CTL induction in murine

cells (House et al. 1994a). No significant effect of cathinone was

noted on NK cell function. These previously unknown findings

indicate that use of this mild stimulant may significantly alter the

immune response.

Opiates

Abuse of morphine and related opiates is well known to alter a number

of immune response parameters, including suppression of cellmediated, humoral-mediated, and natural (nonspecific) immunity

(Bhargava 1990). Unlike the clear demonstration of morphine's

effects on immune responses following in vivo exposure, in vitro

studies have been limited. Although morphine's ultimate deleterious
186

effects on human health are the subject of greatest concern, the use of

in vitro models of immune function offer the possibility of

investigating, at a cellular and molecular level, the mechanisms of the

immunomodulation produced by morphine, its metabolites, and

related opiates.

Following in vivo exposure, morphine is eliminated in a biphasic

manner. In the first phase, morphine is rapidly distributed to all

tissues. In the second phase, morphine is quickly converted to its

principal metabolite, morphine-3-glucuronide, and somewhat more

slowly to a secondary metabolite, morphine-6-glucuronide (Karch

1993). Another metabolite, normorphine, is produced in smaller

amounts than the glucuronides. Normorphine and morphine-6-

glucuronide exhibit the greatest pharmacological activity (Glare and

Walsh 1991).

The immunomodulatory potential of morphine sulfate and its

principal metabolites normorphine, morphine-3-glucuronide, and

morphine-6-glucuronide were explored. B-cell proliferation was

significantly suppressed following exposure to all drugs. Production of

cytokines was affected only moderately by all drugs except morphine6-glucuronide, which produced a marked suppression at 100 M. NK

cell function was unaffected by any drug except morphine-6-

glucuronide, which enhanced NK cell activity at concentrations

between 0.0001 and 1.0 M (figure 3). In contrast, both morphine3-glucuronide and morphine-6-glucuronide significantly inhibited CTL

induction at concentrations between 0.0001 and 100 M, whereas

morphine and normorphine were inactive in this assay. These results

suggest that, in general, immunomodulation produced by morphine

may be at least partially independent from its pharmacological

characteristics (Thomas et al. 1995a).

Another opiate with significant in vivo data, but only limited in vitro

data, is heroin (diacetylmorphine). Heroin has been extensively

associated with suppression of immunity and enhanced susceptibility

to infection (Novick et al. 1991). Methadone, the treatment of

choice for heroin addiction, has also been associated with

immunosuppression, although limited in vitro studies have explored

the nature of this immunosuppression (Novick et al. 1991). Both

drugs were evaluated simultaneously in this laboratory. In general, a

differential modulation of immune function was observed between

these drugs. In vitro exposure to
187

heroin or methadone resulted in decreased B-cell proliferation and

production of IL-2 and IL-4. Cytokine production by macrophages

was differentially affected, with a significant suppression of IL-6

production observed, whereas TNF production was markedly enhanced

in the same cells. Induction of CTL was suppressed by exposure to

heroin, whereas NK cell activity was suppressed by both drugs

(Thomas et al. 1995b).

Finally, the potential immunotoxicity of the opioid analgesics

fentanyl and meperidine was evaluated. Exposure to fentanyl and

meperidine was associated with a differential suppression of IL-4

production by T-cells, as well as a more generalized suppression of

cytokine production by macrophages. In addition, T-cell cytolytic

activity was suppressed at high drug concentrations. B-cell

proliferation and NK cell activity were also inhibited, but to a lesser

degree than noted with T-cell function. Interestingly, the addition of

naltrexone to the cultures did not reverse these alterations in immune

function, suggesting that these changes are not mediated via opioid

receptors (House et al. 1995c).
188

Mu Opioid Agonists

The mu opioid receptor agonists examined in this study have included

[D-Ala2-MePhe4,Gly-ol5]-enkephalin (DAMGO), metkephamide, and

PL017. In general, this laboratory has shown that in vitro exposure

to mu opioid receptor agonists results in immunosuppression (data not

shown). More extensive studies on this group of drugs are currently in

progress.

Endogenous Opioid Peptides

A number of endogenous opioid peptides have been evaluated in these

studies, including met-5-enkephalin, leu-5-enkephalin, -endorphin,

met- enkephalinamide, and dynorphin B. The enkephalins and

endorphins are known to be secreted by cells of the immune system,

and have previously been demonstrated to produce

immunomodulation. This activity may be either suppressive or

stimulatory, depending upon the nature of the target cells and the

conditions of exposure. In the present studies, none of the peptides

examined exhibited any significant effect on B-cell proliferation or

production of IL-2. In comparison, production of IL-4 and IL-6 were

modulated by endorphin. Although the results were statistically

signifi-cant, the limited degree of bioactivity noted was of doubtful

biological significance. A more consistent pattern of activity was

observed with NK cell activity, which was significantly suppressed by

leu-enkephalin, met-enkephalin, and met-enkephalinamide. Neither

-endorphin nor dynorphin had any significant effect on NK cell

activity.

Delta Opioid Receptor Agonists

Recent studies have suggested that compounds acting as delta opioid

receptor agonists may exhibit immunostimulatory activity. For

example, both IL-2 and IL-4 production were markedly enhanced by

DPDPE at concentrations between 0.00001 and 0.1 M. Exposure

to H-Tyr-D-Thr-Gly-Phe-Leu-Thr-OH (DTLET) and DPDPE was

associated with a significant enhancement in NK cell activity. More

extensive mechanistic studies are in progress to define the nature of

this immunomodulatory activity.

Delta Opioid Receptor Antagonists—Peptidic

Representative peptides acting as delta opioid receptor antagonists,

including Tyr-Tic-Phe-Phe-OH (TIPP), Tyr-D-Tic-Phe-Phe-NH2
189

(d-TIPP), and ICI 174864, have been selected for testing. To date,

B-cell proliferation and production of T-cell-derived cytokines

following in vitro exposure to these compounds have been examined.

Neither TIPP nor d-TIPP produced any consistent

immunomodulation following in vitro exposure. Exposure to ICI

174864 resulted in suppression of both B-cell proliferation and

cytokine production only at the 10 M concen-tration; this

suppression is of doubtful biological significance due to the lack of any

obvious dose relationship.

Delta Opioid Receptor Antagonists—Nonpeptidic

Several nonpeptide delta opioid receptor antagonists are currently

under investigation in this project, including 7-benzylidene-7-

dehydronaltrexone HCl (BNTX), naltrindole (NTI), and naltriben

(NTB). In vitro exposure to BNTX or NTI results in

immunosuppression at concentrations between 0.1 and 10 M,

consonant with the hypothesis that delta opioid receptor agonists

enhance, but delta receptor antagonists suppress, immune functional

parameters. In comparison, in vitro exposure to NTB did not result

in any detectable alteration in immune function.

Kappa Opioid Receptor Agonists

A number of kappa opioid receptor agonists have been examined so

far, including dynorphin A (1-9), dynorphin A (1-11), dynorphin A

(1-13), and dynorphin A (1-17). In general, these compounds do not

exhibit a great degree of immunomodulatory activity, with the

exception of B-cell proliferation. Exposure to dynorphins at

concentrations between 0.1 and 10 M was associated with a

significant enhancement in B-cell prolif-eration. However, due to the

high (i.e., nonphysiological) concentrations, the biological relevance

of these findings is uncertain.

Inhalants

A form of substance abuse of increasing interest is the recreational use

of inhalants. Taken literally, this classification is useless, as it may be

construed to include use of tobacco, crack cocaine, or marijuana

(Sharp 1992). A better designation may be "volatile substance" abuse,

a more accurate description of an important drug abuse pattern.

Volatile substances that are commonly abused include glues, aerosols,

anesthetics, cleaning agents, and solvents (Sharp 1992). Abuse of

inhalants has been associated with a variety of toxicologies
190

(Tenenbein 1992). More important from the perspective of

immunomodulation is the abuse of so-called room odorizers or

volatile nitrites. These compounds—such as amyl nitrite, butyl

nitrite, and isobutyl nitrite (IBN)—produce vasodi-lation of the

cerebral vessels, resulting in euphoria (Haverkos and Dougherty 1988)

and a reported enhancement in sexual function. Initially used

clinically in the treatment of angina pectoris, their use appears to be

prevalent in the homosexual community and may be associated with

Kaposi's sarcoma (Newell et al. 1984). Use of these compounds has

been demonstrated to result in decreased immune function,

particularly T-cell-mediated immunity (Dax et al. 1991; Lotzová et

al. 1984; Soderberg and Barnett 1991; Ratajczak et al. 1995). The

association between inhalants and Kaposi's sarcoma is currently the

subject of increased research interest.

EFFECT OF TOLERANCE TO AND ABSTINENCE FROM MORPHINE

In vivo exposure to morphine via implantable pellets was evaluated in

female B6C3F1 mice using the model illustrated in figure 4. This

system, originally developed in the rat to investigate pharmacological

parameters (Bhargava and Matwyshyn 1985), was modified to accommodate immunological assessment in the B6C3F1 mouse (Bhargava et

al. 1994). The model indicated a reproducible induction of tolerance,

and was subsequently used to evaluate the effect of

tolerance/abstinence on immune function. Tolerance to morphine

was associated with a significant suppression of host immunity.

Abstinence from morphine after removal of the pellets resulted in a

differential effect on the immune system, with some parameters

exacerbated and other slightly ameliorated (Bhargava et al. 1994).

As a followup on these studies, additional experimental groups were

included in which naltrexone pellets were coimplanted with either the

morphine pellets or placebo pellets (Bhargava et al. 1995).

Implantation of naltrexone pellets reversed the morphine-induced

analgesic response, as was anticipated. Once this model was in place,

the same exposure regimen was utilized to examine the effects on

immune function. As demonstrated previously, implantation of

morphine pellets produced a significant suppression in B-cell

proliferation. Coimplantation of naltrexone pellets did not reverse

this suppression, but rather appeared to slightly exacerbate

suppression of B-cell function (figure 5). Also consistent with earlier

findings, morphine exposure resulted in suppres-ion of IL-2

production by T-cells. Unlike the B-cell response, the
191

suppression of IL-2 production was completely reversed in animals

coimplanted with naltrexone pellets (figure 6a). In contrast, IL-4

production was unaffected by morphine or naltrexone (figure 6b).

EFFECT OF TOLERANCE TO AND ABSTINENCE FROM L-TRANS- 9-TETRAHYDROCANNABINOL

An experimental model of tolerance/abstinence for

tetrahydrocannabinol (THC) exposure was also developed as

illustrated in figure 7, and was used to evaluate the effect of THC

exposure on immune function

 (Bhargava et al., submitted). Mice were injected subcutaneously (SC)

with THC (10 milligrams per kilogram (mg/kg)) twice daily for 4

days. On day 5, analgesic and hypothermic responses to THC were

determined. Multiple injections of THC resulted in the development

of tolerance to both the analgesic and hypothermic effects of THC.
192

Immune function studies were subsequently performed on mice

rendered tolerant or abstinent by this procedure. Neither tolerance

nor abstinence to the 4-day THC administration had any effect on

bodyweight or thymus weight and cellularity, although spleen weight

and cellularity were both decreased in THC-abstinent animals.

Likewise, there were no significant effects on B-cell proliferation

observed in either group. Production of IL-2 by cells was suppressed

in both tolerant and abstinent mice, whereas production of IL-4 was

significantly suppressed only in THC-abstinent mice. Significant

suppression of CTL and NK cells activity was only observed in THCabstinent mice. These results suggest that THC-mediated modulation

of the immune response may result from a differential effect on

cellular populations.
193
194

RECOMMENDATIONS FOR THE FUTURE

Continuation of In Vitro Screening Program

This program has resulted in the development of a rapid and costeffective screening protocol that has produced a substantial database

of information on the in vitro and in vivo effects of abused drugs on

immune function. The advantages of such a program include

examination of the effects of drugs of abuse on individual cells and

components of the immune system, evaluation of the

immunomodulatory effects of both parent compounds and

metabolites, and generation of dose-response curves for immune

function at pharmacologically relevant drug concentrations.

Delta Opioid Receptor Agonist Compounds as Immunomodulators

As mentioned previously, a potentially significant observation made

in these studies is that the delta opioid receptor agonist compounds
195

tend to exhibit distinct immunostimulatory activity at picomolar to

nanomolar concentrations. In addition, both peptide and nonpeptide

delta receptor antagonists tend to suppress both B-cell and T-cell

function. This trend is consonant with the authors' hypothesis that,

in general, delta receptor agonists and antagonists are

immunostimulatory and immunosuppressive, respectively. Data

obtained in these various studies will be invaluable in designing novel

immunostimulant or immunorestorative drugs based on this class of

compounds.

Potential of Kappa Opioid Receptor Agonists To Produce

Immunomodulation

A considerable body of data has been published documenting the

immunosuppressive nature of compounds active at the mu opioid

receptors (e.g., heroin, morphine). As described above, the delta

opioid receptor-specific compounds appear to be immunostimulatory.

However, the role of the third major type of opioid receptor, the

kappa receptor, in immune function has not been described in detail.

Defining the role of this receptor in immunomodulation would be

vital in understanding the nature of neuroimmune interactions. To

date, the kappa opioid receptor agonists dynorphin A (1-9),

dynorphin A (1-11), dynorphin A (1-13), dynorphin A (1-17),

dynorphin B, and U-50488H have been evaluated for

immunomodulatory activity in the B-cell proliferation and T-cell

cytokine production assays only. This group of drugs produces only

marginal effects on B-cell proliferation, cytokine production, or NK

cell activity. These drugs have not yet been evaluated for effects on

CTL induction or macrophage cytokine production. These assays will

be necessary to more fully examine any possible immunomodulation

by kappa opioid receptor specific compounds.

Development of Animal Models of Polydrug Abuse

It is well established that drug abuse increasingly involves the use of

multiple agents (e.g., ethanol, cocaine, heroin, THC). As mentioned

previously, the authors' laboratories have developed in vivo models

for tolerance to and abstinence from morphine and 9-THC in

B6C3F1 mice. This particular mouse strain has been used extensively

by the National Toxicology Program, resulting in a sizable database of

background toxicology and immunology data. In addition, a similar

model for in vivo exposure to cocaine is in the preliminary stages of

development. These models reveal that in vivo exposure to these

drug types results in a significant suppression of a variety of immune
196

effector and regulatory mechanisms; this suppression is at least

partially reversible with antagonists. These models will be used for

determining the immuno-modulatory effect of polydrug exposure. In

these studies, mice tolerant to morphine, THC, or cocaine will also

receive various other drugs in combination. These studies are

expected to provide vital information on the nature of immune

dysfunction following drug exposure relevant to human usage.

Abused Drugs as Cofactors in AIDS-Related Infections

Another area of interest relevant to this work is the role of drug abuse

in the pathology of AIDS (Pillai et al. 1991). Two particular research

areas that would generate valuable information are drug exposure in

animals infected with opportunistic infections and the potential

reconstitution of suppressed immunity by drug treatment. In the first

instance, opportunistic infections are normally benign organisms that

often lead to fulminant infections in immunocompromised hosts.

Examples include tuberculosis, toxoplasmosis, and pneumocystosis.

An animal model of some interest is murine acquired

immunodeficiency syndrome (MAIDS), a murine analog of the human

disease. There is a need for studies to evaluate the potential of drugs

of abuse or potential medications to either exacerbate or ameliorate

this condition.

REFERENCES

Abbas, A.K.; Urioste, S.; Collins, T.L.; and Boom, W.H. Heterogeneity of

helper/inducer T lymphocytes: IV. Stimulation of resting and

activated B cells by Th1 and Th2 clones. J Immunol 144:2031-

2037, 1990.

Akira, S.; Hirano, T.; Taga, T.; and Kishimoto, T. Biology of multifunctional

cytokines: IL-6 and related molecules (IL-1 and TNF). FASEB J

4:2860-2867, 1990.

Berke, G. The cytolytic T lymphocyte and its mode of action. Immunol Lett

20:169-178, 1989.

Bhargava, H.N. Opioid peptides, receptors, and immune function. In: Pham,

P.T.K., and Rice, K., eds. Drugs of Abuse: Chemistry,

Pharmacology, Immunology, and AIDS. National Institute on

Drug Abuse Research Monograph 96. DHHS Pub. No.

(ADM)90-1676. Washington, DC: Supt. of Docs., U.S. Govt.

Print. Off., 1990.
197

Bhargava, H.N., and Matwyshyn, G.A. Comparative effect of thyrotropin

releasing hormone, MK-771 and DN-1417 on morphine

abstinence syndrome. Psychopharmacology 97:141-146, 1985.

Bhargava, H.N.; Thomas, P.T.; Thorat, S.N.; and House, R.V. Effects of

morphine tolerance and abstinence on cellular immune function.

Brain Res 642:1-10, 1994.

Bhargava, H.N.; House, R.V.; Thorat, S.N.; Pong, M.S.; and Thomas, P.T.

Effect of l-trans9-tetrahydrocannabinol induced

tolerance and abstinence on cellular immune function.

Pharmacol Toxiol, submitted.

Bhargava, H.N.; House, R.V.; Thorat, S.N.; and Thomas, P.T. Effects of

naltrexone on morphine-induced tolerance and physical

dependence and changes in cellular immune function. Brain Res

690:121-126, 1995.

Bost, R.O. 3,4-Methylenedioxymethamphetamine (MDMA) and other

amphetamine derivatives. J Forensic Sci 33:576-587, 1988.

Brown, A.R. Immunological functions of splenic B-lymphocytes. Crit Rev

Immunol 11:395-417, 1992.

Buchanan, J.F., and Brown, C.R. ‘Designer drugs’: A problem in clinical

toxicology. Med Toxicol 3:1-17, 1988.

Chiang, W., and Goldfrank, L. The medical complications of drug abuse. Med J

Australia 152:83-88, 1990.

Dax, E.; Adler, W.; Nagel, J.; Lange, W.; and Jaffe, J. Amylnitrite alters human

in vitro immune function. Immunopharmacol Immunotoxicol

13:577-587, 1991.

Deecher, D.C.; Teitler, M.; Soderlund, D.M.; Bornmann, W.G.; Kuehne, M.E.;

and Glick, S.D. Mechanisms of action of ibogaine and harmaline

congeners based on radioligand binding studies. Brain Res

571:242-247, 1992.

Derlet, R.W., and Heischober, B. Methamphetamine: Stimulant of the 1990s?

West J Med 153:625-628, 1990.

Dornand, J.; Kamenka, J.-M.; Bartegi, A.; and Mani, J.-C. PCP and analogs

prevent the proliferative response of T lymphocytes by

lowering IL2 production. Biochem Pharmacol 36:3929-3936,

1987.

Gillis, S.; Ferm, M.M.; Ou, W.; and Smith, K.A. T cell growth factor: Parameters

of production and a quantitative microassay for activity. J

Immunol 120:2027-2032, 1978.

Glare, P.A., and Walsh, T.D. Clinical pharmacokinetics of morphine. Ther Drug

Monitor 13:1-23, 1991.

Glick, S.D.; Rossman, K.; Steindorf, S.; Maisonneuve, I.M.; and Carlson, J.N.

Effects and aftereffects of ibogaine on morphine selfadministration in rats. Eur J Pharmacol 195:341-345, 1991.
198

Haverkos, H., and Dougherty, J. Health hazards of nitrite inhalants. Am J Med

84:479-482, 1988.

Heischober, B., and Miller, M.A. Methamphetamine abuse in California. In:

Miller, M.A., and Kozel, N.J., eds. Methamphetamine Abuse:

Epidemiologic Issues and Implications. National Institute on

Drug Abuse Research Monograph 115. DHHS Pub. No.

(ADM)91-1836. Washington, DC: Supt. of Docs., U.S. Govt.

Print. Off., 1991.

Hoffman, B.B., and Lefkowitz, R.J. Catecholamines and sympathomimetic

drugs. In: Gilman, A.G.; Rall, T.W.; Nies, A.S.; and Taylor, P.,

eds. The Pharmacological Basis of Therapeutics. New York:

Pergamon Press, 1990. pp. 187-220.

House, R.V.; Thomas, P.T.; and Bhargava, H.N. Comparison of immune

functional parameters following in vitro exposure to natural and

synthetic amphetamines. Immunopharmacol Immunotoxicol

16:1-21, 1994a.

House, R.V.; Thomas, P.T.; and Bhargava, H.N. Immunological consequences of

in vitro exposure to lysergic acid diethylamide.

Immunopharmacol Immunotoxicol 16:23-40, 1994b.

House, R.V.; Thomas, P.T.; and Bhargava, H.N. Selective modulation of immune

function resulting from in vitro exposure to methylenedioxymethamphetamine (Ecstasy). Toxicology 96:59-69, 1995a.

House, R.V.; Thomas, P.T.; and Bhargava, H.N. Comparison of the

hallucinogenic indole alkaloids ibogaine and harmaline for

potential immunomodulatory activity. Pharmacology 51:56-

65, 1995b.

House, R.V.; Thomas, P.T.; and Bhargava, H.N. In vitro evaluation of fentanyl

and meperidine for immunomodulatory activity. Immunol Lett

46:117-124, 1995c.

Hu-Li, J.; Ohara, J.; Watson, C.; Tsang, W.; and Paul, W.E. Derivation of a T

cell line that is highly responsive to IL-4 and IL-2 (CT.4R) and

an IL-2 hyporesponsive mutant of that line (CT.4S). J

Immunol 142:800-807, 1989.

Kalix, P. Cathinone, a natural amphetamine. Pharmacol Toxicol 70:77-86,

1992.

Karch, S.B. Individual narcotic agents: Morphine. In: The Pathology of Drug

Abuse. Boca Raton, FL: CRC Press, 1993. pp. 251-259.

Khansari, N.; Whitten, H.D.; and Fudenberg, H.H. Phencyclidine-induced

immunodepression. Science 225:76-78, 1984.

Klee, H. A new target for behavioural research—amphetamine misuse. Br J

Addict 87:439-446, 1992.

Lotzová, E.; Savary, C.A.; Hersh, E.M.; Khan, A.A.; and Rosenblum, M.

Depression of murine natural killer cell cytotoxicity by isobutyl

nitrite. Cancer Immunol Immunother 17:130-134, 1984.
199

Lotzová, E. Definition and functions of natural killer cells. Nat Immun 12:169-

176, 1993.

Luster, M.I., and Rosenthal, G.J. Chemical agents and the immune response.

Environ Health Perspect 100:219-236, 1993.

Meager, A.; Leung, H.; and Woolley, J. Assays for tumour necrosis factor and

related cytokines. J Immunol Meth 116:1-17, 1989.

Mosmann, T.R.; Schumacher, J.H.; Street, N.F.; Budd, R.; O'Garra, A.; Fong,

T.A.T.; Bond, M.W.; Moore, K.W.M.; Sher, A.; and Fiorentino,

D.F. Diversity of cytokine synthesis and function of mouse

CD4+ T cell. Immunol Rev 123:209-229, 1991.

Newell, G.R.; Adams, S.C.; Mansell, P.W.A.; and Hersh, E.M. Toxicity,

immunosuppressive effects and carcinogenic potential of

volatile nitrites: Possible relationship to Kaposi's sarcoma.

Pharmacotherapy 4:284-291, 1984.

Novick, D.M.; Ochshorn, M.; and Kreek, M.J. In vivo and in vitro studies of

opiates and cellular immunity in narcotic addicts. Adv Exp Med

Biol 288:159-170, 1991.

Pillai, R.M., and Watson, R.R. In vitro immunotoxicology and

immunopharmacology: Studies on drugs of abuse. Toxicol Lett

53:269-283, 1990.

Pillai, R.; Nair, B.S.; and Watson, R.R. AIDS, drugs of abuse and the immune

system: A complex immunotoxicological network. Arch Toxicol

65:609-617, 1991.

Ratajczak, H.V.; Thomas, P.T.; House, R.V.; Gaworski, C.L.; Sherwood, R.L.;

Luster, M.I.; Hagen, K.L.; Abdo, K.; Jackson, C.D.; Roycroft, J.;

and Aranyi, C. Local versus systemic immunotoxicity of

isobutyl nitrite following subchronic inhalation exposure of

female B6C3F1 mice. Fundam Appl Toxicol 27:177-184, 1995.

Roehm, N.W.; Rodgers, G.H.; Hatfield, S.M.; and Glasebrook, A.L. An improved

colorimetric assay for cell proliferation and viability utilizing

the tetrazolium salt XTT. J Immunol Meth 142:257-265, 1991.

Sharp, C.W. Introduction to inhalant abuse. In: Sharp, C.W.; Beauvais, F.; and

Spence, R., eds. Inhalant Abuse: A Volatile Research Agenda.

National Institute on Drug Abuse Research Monograph 129.

NIH Pub. No. 93-3495. Washington, DC: Supt. of Docs., U.S.

Govt. Print. Off., 1992.

Soderberg, L.S.F., and Barnett, J.B. Exposure to inhaled isobutyl nitrite reduces T

cell blastogenesis and antibody responsiveness. Fundam Appl

Toxicol 17:821-824, 1991.

Talmadge, J.E. Immunoregulation and immunostimulation of murine

lymphocytes by recombinant human interleukin-2. J Biol Res

Modif 4:18-34, 1985.

Tenenbein, M. Clinical/biophysiologic aspects of inhalant abuse. In: Sharp,

C.W.; Beauvais, F.; and Spence, R., eds. Inhalant Abuse: A
200

Volatile Research Agenda. National Institute on Drug Abuse

Research Monograph 129. NIH Pub. No. 93-3495. Washington,

DC: Supt. of Docs., U.S. Govt. Print. Off., 1992.

Thomas, P.T.; House, R.V.; and Bhargava, H.N. Phencyclidine exposure alters in

vitro cellular immune response parameters associated with host

defense. Life Sci 53:1417-1427, 1993.

Thomas, P.T.; Bhargava, H.N.; and House, R.V. Immunomodulatory effects of

in vitro exposure to morphine and its metabolites.

Pharmacology 50:51-62, 1995a.

Thomas, P.T.; House, R.V.; and Bhargava, H.N. Direct cellular

immunomodulation produced by diacetylmorphine (heroin) or

methadone. Gen Pharmacol 26:123-130, 1995b.

Van Snick, J.; Caypahs, S.; Vink, A.; Uyttenhove, C.; Coulie, P.G.; Rubira, M.R.;

and Simpson, R.J. Purification and NH2-terminal amino acid

sequence of a T-cell-derived lymphokine with growth factor

activity for B-cell hybridomas. Proc Natl Acad Sci U S A

83:9679-9683, 1986.

AUTHORS

Robert V. House, Ph.D.

Senior Immunologist, Life Sciences Department

Peter T. Thomas, Ph.D.

Manager, Microbiology/Immunology

Life Sciences Department

IIT Research Institute

10 West 35th Street

Chicago, IL 60616

Hemendra N. Bhargava, Ph.D.

Professor of Pharmacology

Department of Pharmaceutics and Pharmacodynamics (M/C 865)

University of Illinois at Chicago, Health Sciences Center

833 South Wood Street

Chicago, IL 60612
201

Inhalation Studies With Drugs of

Abuse

Yun Meng, Aron H. Lichtman, D. Troy Bridgen, and Billy R.

Martin

INTRODUCTION

In recent years, smoking or inhalation of drugs has become a popular route of

administration among drug users. Various drugs of different classes have been

abused by inhalation or smoking, including phencyclidine (PCP), cocaine,

heroin, methamphetamine, and marijuana. This increased popu-larity of

smoking drugs has resulted from the fast onset of drug action and fears related

to contracting acquired immunodeficiency syndrome (AIDS) or other

infectious diseases from intravenous (IV) injections. In particular, heroin

smoking has increased dramatically; approximately 74 percent of the heroin

addicts in India use this method of administration, and it is also popular in the

United States (Griffiths et al. 1994). The shift toward smoking heroin has

also been associated with an increased availability of illicit heroin on the

streets, which has declined in price and increased in purity (Huizer 1987).

Consequently, there is an increased risk of overdose.

Inhalation is a very potent route of drug administration, and is characterized

by fast absorption from the nasal mucosa and the extensive lung capillaries.

Inhalation results in an immediate elevation of arterial blood drug concentration and a higher bioavailability due to avoiding drug metabolism by the

liver. The fact that smoking or inhalation provides rapid delivery of drugs to

the brain may result in an immediate reinforcing effect of the drug and further

contribute to its abuse liability or risk of dependency. Smoking or inhalation

of drugs may also lead to other adverse effects, such as pulmonary diseases or

deleterious cardiovascular consequences. Moreover, the parent drugs may be

degraded, leading to inhalation of toxic pyrolytic products (Benowitz 1990;

Wesson and Washburn 1990). For example, there have been reports of

heroin leucoencephalopathy, a life-threatening condition that has occurred in

some individuals after smoking heroin (Wolters et al. 1982). This condition

was not associated with IV administration and no impurities present in the

drug were identified as the cause, but may have been caused by pyrolytic

products of either heroin or impurities present in the samples (Wolters et al.

1982).

The rapid increase in smoking drugs of abuse raised concerns about drugs that

are currently abused using other routes of administration: Can they also be
202

smoked? Furthermore, it is unknown whether their abuse liability, side

effects, and toxicity would be increased by smoking. Thus the major goal of

this research has been to develop guidelines for predicting which drugs can

potentially be abused by smoking or inhalation.

To reach this goal, the first step was to establish a predictive parameter for

the volatility of drugs. Underscoring the importance of volatility is the

practice of smoking various drugs of abuse in combination or with other

agents in an effort to enhance their volatility and thereby increase their

pharmacological effects. For example, the addition of either caffeine or

barbiturates has been shown to improve the volatilization of heroin (Huzier

1987).

One physiochemical parameter that might play a critical role in the volatility

of a compound is vapor pressure. Accordingly, the authors hypothesized that

vapor pressure could be positively correlated with both volatility and

pharmacological potency after smoking. Thus, the vapor pressures of a

variety of drugs were determined, and then the volatili-zation of selected

compounds was studied.

A second goal of this work was to investigate the volatilization of these drugs

and identify their major pyrolysis products. Analytical methods employing

gas chromatography/mass spectrometry (GC/MS) and high performance liquid

chromatography (HPLC) were developed to identify and quantify drugs and

their pyrolytic products after volatilization.

Despite the increase in smoking and inhalation of drugs of abuse, relatively

little is known about the consequences of this route of administration.

Therefore, the third research goal has been to develop a reliable animal model

to evaluate the pharmacology and biodisposition of drugs after inhalation. In

these studies, the pharmacological effects of volatilized drugs were assessed in

mice. In addition, the brain, plasma, and whole-body levels of drugs occurring

after inhalation were quantified in order to obtain biodisposition information.

These studies provided the strategy and the tools for prediction of drugs that

can be abused by smoking or inhalation.

VAPOR PRESSURE AND VOLATILITY

In general, absorption of inhaled drug is dependent upon the physical

characteristics of the drug, including particle size, lipid solubility, and

volatility. Clearly, a drug’s volatility would play an important role in

determining its inhalation potential. Drug volatility is determined by many

factors, including boiling point, melting point, and vapor pressure. Since

many abused substances are less volatile than organic solvents and are smoked
203

at very high

temperatures,

effects of

melting and

boiling points

on the

volatilization

would be negligible. On the other hand, vapor pressure may serve as a good

indication of volatility.

Based on the ideal gas law,

equation 1

where p is the partial pressure of the gas, R is the gas constant, n is the

number of molecules, V is the volume of the gas phase of the compound, and

T is the temperature in kelvin, the partial pressure of the gas becomes the

vapor pressure (Pv) when the gas and liquid phases of the compound reach

equilibrium at a particular temperature. Then, equation 1 can be expressed as

equation 2

where C = n/V and is the concentration of the compound in the gas phase and

is directly proportional to the vapor pressure. This relationship suggests that

the vapor

pressure of a

compound is

positively

correlated with

volatility; the

higher concentration of the gas, the more volatile it is. Therefore, the

volatility of a drug at a certain temperature is determined by its vapor

pressure and the volatilization temperature.

Due to a lack of information on vapor pressure for a variety of drugs of abuse

and related compounds, the authors determined this parameter by an indirect

method based on a system using gas chromatography and relative retention

times. This approach is a modification those described by others (Hamilton

1980; Westcott and Bidleman 1981; Bidleman 1984). The original method is

based on in the relationship between solid vapor pressure and GC column

retention time (or volume retention time, VR), and has been used in

determination of vapor pressures for herbicides, pesticides, and a variety of

nonpolar organic compounds. The vapor pressure (Pv) of two substances at

the same temperature (as well as their latencies of vaporization, LV) are

related by

pV = nRT

Pv = CRT
204

equation 3

and the fact

that vapor

pressure has

been shown to

be related to

column

retention

volumes by

equation 4

The

relationship

between vapor

pressure and

column

retention times

can be determined from the combination of equations 3 and 4:

equation 5

Therefore, a plot of ln [(VR)1/(VR)2] versus ln P2 should yield a straight line

with either a positive or negative correlation coefficient depending on the

ratio of (VR)1/(VR)2 . The value of (L1/L2) can be calculated from the slope

and thus P1 determined from equation 3. If substances 1 and 2 are the

unknown and standard compounds, respectively, then the vapor pressure of

the unknown at a given temperature can be determined. The relationship

between vapor pressure and temperature can be simply described by the

Clausius-Clapeyron equation:

equation 6

Therefore, the vapor pressure at any temperature can be extrapolated by the

linear regression between ln P and 1/T.

Since this technique has primarily been used for estimating vapor pressures of

pesticides, these chemicals were utilized to establish a working model to

determine the vapor pressures of drugs of abuse. The authors’ strategy was to

determine the vapor pressure of drugs of abuse by employing a pesticide with

known Pv values as a standard. A GC/MS was equipped with a 4-meter

capillary column. The helium carrier gas was adjusted to a 1.11 milliliter per

minute (mL/min) flow rate and a 78.26 mL/min split for a split ratio of 79:1.

The injector port was kept at 200%C, detector port at 225%C, and the

source of the MS at 200%C. The oven temperature was kept constant during

any given analysis. All test compounds were dissolved in hexane or

ln P1= (L1/L2) ln P2 - C

(VR)1/(VR)2 = P2/P1

ln [(VR)1/(VR)2] = [1-(L1/L2)] ln P2 -

C

ln P = A + B/T
205

chloroform at 0.5 to 4.0 milligrams per mL (mg/mL) in order to obtain a

substantial peak from a 1 microliter (L) injection. Since eicosane and

octadecane are commonly used as standard compounds in determining the

vapor pressure of other pesticides, both compounds were used to standardize

the GC column. Various pesticides (nonpolar organic compounds), such as

naphthalene, phenanthrene, pyrene, and benzo[a]pyrene were then injected

and retention times obtained for 5 to 8 temperatures at 10%C increments,

ranging from 40%C to 190%C. The natural logs of the ratios of the

retention times of the standard and test compounds were then plotted against

the natural log of the vapor pressure of the standard at each temperature.

Both octadecane and eicosane standardization yielded vapor pressures of the

pesticides very close to published values. Of these two compounds, the values

obtained with eicosane exhibited a higher correlation coefficient. Thus,

eicosane appeared to be the better standard for approximating the values of

various drugs of abuse. However, the plot of relative retention time ratios of

several of the drugs of abuse to the published vapor pressure of eicosane

(equation 5) correlated poorly (table 1). This result eliminated eicosane as a

standard for measuring the vapor pressures of drugs of abuse. In the search

for another standard, dibutyl phthalate proved to be a good candidate. Using

eicosane as a standard, the vapor pressure of dibutyl phthalate, at 25%C, was

determined as 6.89 x 10-5 torr, which fell within the published vapor pressure

range of 1.2 x 10-6 to 4.4 x 10-5 torr (Small et al. 1948). By the same

method, the vapor pressures of dibutyl phthalate at different temperatures

were then determined against eicosane. Equation 6 was then solved for

dibutyl phthalate: ln P = A + B/T where A = 25.179, B = -10,364, P is in torr

and T is in kelvins. Using dibutyl phthalate as a standard, the natural log of

the relative retention time ratios plotted against the natural log of its vapor

pressures at the respective temperatures yielded a high correlation for various

drugs of abuse (figure 1). Thus, vapor pressures at 25%C could be

approximated for drugs representing a variety of classes (table 1).

Vapor pressures of selected compounds are listed in table 1. In comparing

different classes of drugs, the opioids appear to have relatively low vapor

pressure, suggesting that they are less volatile than other drugs. As can also

be seen in table 1, nicotine exhibited relatively high volatility, which is

consistent with the fact that cigarette smoking is the most popular method of

nicotine administration. Vapor pressures for methamphetamine and

amphetamine appeared to be higher than that of nicotine at 25%C, but their

vapor pressures could not be determined by the present method since their

volatility exceeded the range of the standard at the temperatures that were
206

TABLE 1. Determining the vapor pressures of drugs of abuse using dibutyl

phthalate as the standard.

Correlation coefficient of

In (VR)1/(VR)2 to In P

Drugs

(Free base) Eicosane Dibutyl

phthalate

Vapor pressures

(Torr, at

25%C)

Nicotine A 0.871 2.61x10-2

MDMA — 0.997 4.47x10-3

Caffeine — 0.997 8.56x10-4

PCP — 0.984 1.49x10-4

Secobarbital — 0.996 5.72x10-5

Pentalbarbital — 0.948 4.16x10-5

Methaqualone 0.751 0.896 1.60x10-5

Cocaine 0.748 0.996 9.79x10-6

Morphine — 0.943 9.49x10-7

 9-THC — 0.986 1.01x10-7

Heroin 0.981 0.983 5.71x10-8

Fentanyl — 0.982 2.41x10-8

KEY: A = Values were not determined.

studied. Compounds with high vapor pressures are predicted to be

much more volatile and consequently more likely to be smoked than

those possessing low vapor pressures.
207
208

INHALATION EXPOSURE OF DRUGS OF ABUSE IN THE MOUSE

INHALATION MODEL: PHARMACOLOGICAL ASSESSMENT

Although drug volatility can provide fundamental information about

the inhalation potential for drugs of abuse, it is only one of many

factors necessary for producing a pharmacological effect.

Pharmacokinetic and pharmacodynamic considerations have

considerable bearing on a drug's ability to produce an effect.

Presently, relatively little is known about the potency of drugs of

abuse after inhalation or smoking. In order to deter-mine the

relationship between volatility and pharmacological potency by the

inhalation route, the authors developed an animal model to approximate the conditions of human inhalation. The approach involved a

volatilization-inhalation drug delivery system developed over the past

10 years in this laboratory. The design of this inhalation apparatus is

illustrated in figure 2.

The apparatus consisted of a U-shape glass volatilization pipe

preheated to a designed temperature, a nose-only exposure unit

containing six mice, a glass wool trap (packed with 0.5 g of glass wool

fiber) that sequestered the vaporized test compound, and a vacuum

system that created negative pressure and pulled the air through the
209

entire apparatus. The airflow was regulated at a rate of 400 mL/min

by a flow meter placed after the glass wool trap. The entire system

was contained within a hood. A known amount of test compound

(either in dry powder or liquid form), packed in between two pieces of

glass wool in a glass plunger, was injected into the preheated pipe and

volatilized. Animals were exposed to the vapor for 5 minutes, and

then the appropriate pharmacological effects for each respective drug

were measured.

These studies allowed the authors to optimize the volatilization

conditions by using a pharmacological endpoint. Once conditions for

volatilizing a drug had been established, then pharmacological potency

was determined by exposing mice to different quantities of volatilized

drug. This strategy represents a deviation from traditional inhalation

approaches involving optimization of volatilization using analytical

methods. The rationale for using a pharmacological endpoint is that

failure of an agent to produce behavioral effects via inhalation renders

the analytical considerations moot.

To determine the feasibility of evaluating the volatilization of drugs

with different pharmacological properties, several compounds that are

abused by smoking or inhalation were selected. It is well known that

changing the route of administration has important effects on the

development of drug dependency (Griffiths 1994); thus the authors

elected to compare pharma- cological effects after inhalation and IV

administration. Heroin was chosen because of the shift toward

smoking among heroin users and the lack of systematic studies on the

pharmacological potency, onset, and duration of action of smoked

heroin. Other drugs of abuse such as PCP and meth-amphetamine are

also commonly smoked (Wesson and Washburn 1990). PCP was

initially abused by oral and IV administration routes associated with

many adverse effects.

The discovery that smoking PCP-laced cigarettes allowed for a better

titration of doses and fewer side effects propelled it to the forefront

of drug abuse. Previously, the authors studied the pyrolysis of PCP in

parsley cigarettes, which employed a much higher temperature than

the currently used system (Freeman and Martin 1981; Lue et al. 1986,

1988; Martin and Boni 1990). Those studies demonstrated that more

than 50 percent of the drug was delivered intact. The pharmacology

of smoked PCP-laced cigarettes in mice and rats has also been

characterized (Freeman and Martin 1982; Martin and Freeman 1983;

Wessinger et al. 1985).
210

The incidence of inhalation or smoking of methamphetamine has

risen very recently in the United States. Several investigators have

examined the volatilization and pyrolysis of this compound (Cook et

al. 1991, 1993; Miller and Kozel 1991). Sekine and Nahahara (1987)

studied the volatilization of methamphetamine applied to tobacco

cigarettes and found that about 15 percent of the drug was delivered in

the main stream of the smoke and several pyrolysis products were

formed. Studies of the volatilization, biodisposition, and

pharmacokinetics of smoked metham-phetamine hydrocloride (Cook

et al. 1991, 1993; Perez-Reyes et al. 1991) demonstrated that this

compound can be easily volatilized in the temperature range of 200 to

400%C, while 90 percent of the parent drug is delivered intact. The

pharmacological effects of methamphetamine administered by

inhalation or IV injection appeared to be similar.

Using the rodent inhalation model depicted in figure 2, mice were

assessed for locomotor activity after exposure to methamphetamine

vapor, antinociception after exposure to heroin, and motor

coordination after exposure to PCP. Temperatures used for

volatilization of these drugs are listed in table 2. These temperatures

were empirically derived

TABLE 2. Relative potencies of drugs by inhalation exposure and

IV administration.

Drugs Inhalation ED50 (mg)AIV ED50 (mg/kg)

Heroin B1.1 B 0.28

PCP HCl C2.8 C0.1

Methamphetamine-HCl

D

3.9 D0.9

KEY: A = Based upon the amount of drug added to the volatilization

chamber. B = Free base volatilized at 250%C. C = Volatilized at

275%C. D = Volatilized at 200 %C. ED50 = Median effective

dose.
211

based upon their pharmacological effectiveness in mice in inhalation

studies. In order to measure the antinociceptive effects of heroin, the

tail-flick apparatus described by Dewey and colleagues (1970) was

used. Fifteen minutes after the inhalation exposure or IV

administration of heroin, the mouse's tail was placed under a radiant

heat lamp and the amount of time required for the animal to flick its

tail from under the heat source was recorded. Baseline reaction

latencies ranged from 2 to 4 seconds, and the maximal allowable

reaction time was 10 seconds. The percent of maximal percent effect

(percent MPE) was calculated for each animal. Volatilization of

heroin free base resulted in a dose-related antinociception with

maximal effects occurring in mice exposed to the vapor from 3 mg of

heroin free base. Dose-response curves generated for heroin are

illustrated in figure 3 and the ED50 values are listed in table 2. These

data clearly show that heroin-induced antinociception is qualitatively

similar after inhalation and IV administration. Similar to the results

in the present study, smoking heroin has been reported to be as

potent as IV heroin in humans (Jenkings et al. 1994). In a controlled

clinical study, the pharmacokinetic and pharmacodynamic profile of

smoked heroin was evaluated in human subjects. It was demonstrated

that the behavioral effects of smoked heroin were as potent and rapid

in onset as IV administration.
212

Stimulant effects are readily quantified by measuring spontaneous

activity. For these studies mice were placed in individual photocell

activity cages (6.5 x 11 in) with 16 photocell beams per chamber.

Individual mice were placed into one of six chambers and allowed to

acclimate for 10 minutes. They were removed from the activity

chambers and either injected IV with saline or drug or exposed to

volatilized methamphetamine. Immediately after the injection or

inhalation exposure, the mice were returned to the chambers, and

interruptions of the photocell beams were recorded for the next 40

minutes using an animal activity monitor. Activity in the chamber

was then expressed as the total number of beam interruptions for the

total 40 minutes. Maximal possible stimulation was determined from

double reciprocal plots of photocell interruptions versus dose so that

the data could be expressed as percentage of maximal stimulation.

Methamphetamine produced a dose-related stimulation of

spontaneous activity with maximal stimulation occurring with

volatilization of 25 mg of drug. This stimulation was comparable to

that produced by IV administration of methamphetamine in the dose

range of 0.5 to 4.0 mg/kg (figure 4). The ED50 of methamphetamine by both routes of administration are summarized in table 2.

The final drug to be evaluated for pharmacological effects following

inhalation was PCP hydrochloride (HCl). Motor incoordination

produced by PCP HCl was evaluated by the inverted-screen test

(Coughenour et al. 1977). Fifteen minutes after drug exposure, the

mice were placed on a wire screen that was immediately inverted.

The percentage of animals that climbed onto the top within 60

seconds was recorded. Only mice successfully completing the task in a

pre-experimental test were used. Mice (in groups of six) exposed to

the volatilization of PCP HCl in the range of 2 to 6 mg exhibited a

dose-related inhibition of motor function as depicted in figure 5.

Mice exposed to the volatilization of 1 mg of PCP displayed altered

behavior to a greater degree than those exposed to 2 mg; this most

likely represents an aberration. The disruption of motor function

following inhalation is comparable to that produced by IV

administration of PCP in the dose range of 0.03 to 1.0 mg/kg.

These inhalation studies demonstrate the feasibility of evaluating the

potency of drugs with different pharmacological actions following

inhalation exposure. The comparison of pharmacological effects

after both inhalation and IV administration revealed very similar

dose-response relationships for each of these three drugs. However,

valid potency comparisons can be made between inhalation and IV

administration only
213
214

if the biological concentrations of the parent drug are known. In

addition to differences in the pharmacokinetics of drugs administered

by these two routes, the dose of the drug administered by inhalation

must be deter- mined. In the studies described above, the potencies

are expressed in terms of amount of drug added to the volatilization

apparatus. In order to establish dosimetry, the animals are exposed to

the volatilization of the radiolabeled drug under the same conditions

used for the potency measurement. Whole-body determination of

total radioactivity provides the dose of the volatilized drug.

An additional possible confound is the formation of pyrolysis

products during volatilization, which could contribute pharmacological

effects. The authors’ strategy is to utilize volatilization conditions in

such a way that minimal pyrolysis occurs, thus eliminating the

possible contribution of pharmacological effects and simplifying

determination of dosimetry.

To demonstrate the feasibility of this approach, the authors have

chosen to determine the volatilization and dosimetry of heroin for

the purpose of making valid potency comparisons between IV and

inhalation exposure.

VOLATILIZATION OF HEROIN

Heroin represents a logical choice for establishing inhalation

procedures. Smoking and inhalation of heroin, known as "chasing the

dragon," have largely replaced opium smoking for almost a century.

It has become the most popular method of heroin use in recent years

due to searches for alternatives to IV injections and the drug's

increased availability. The most common method of smoking heroin

involves heating the drug on a piece of aluminum foil and inhaling the

vapor. An often reported obser-vation is that the aluminum foil

contains black residues of decomposed heroin after smoking. In

pyrolysis studies, Huzier (1987) and Cook and Jeffcoat (1990)

reported that heroin undergoes extensive decomposition at

temperatures that are presumably required for volatilization.

Studies of the volatilization of the drugs of abuse together with the

assessment of pharmacological effects in laboratory animals after

inhalation exposure can provide important information for drug

inhalation potential in humans. Using the same volatilizationinhalation apparatus as shown in figure 2, the volatilization of heroin

was investigated. Heroin free base, 1 and 5 mg doses, was heated for 5
215

minutes at 250%C. The glass wool trap and the pipe were then

flushed with ethanol. Concentrations of the heroin and its pyrolytic

products were analyzed by GC/MS and HPLC.

Results from volatilization of heroin free base indicated high

efficiency at the employed temperature. The extent of volatilization

after 5 minutes of heating appeared to be independent of the drug

quantity. At 250%C, heroin was found to be volatilized over 75

percent. More than 90 percent of the initial amount was recovered as

unchanged heroin after volatilization. Monoacetylmorphine, the

only degradation product that resulted from the volatilization of

heroin, accounted for less than 5 percent of the total drug. In

contrast to the authors’ results, Cook (1991) found that heroin was

extensively degradated to a variety of products, including 6-acetyl

morphine, N,6-diacetylnormorphine, and N-acetylnormorphine, after

heating in a quartz furnace tube at 250%C. On the other hand, Huizer

(1987) studied the pyrolysis of both heroin free base and its

hydrochloride salt by using a TAS oven (a thermomicro separation,

transfer, and application procedure) or heating on a piece of

aluminum foil. They found that heroin HCl required a higher

volatilizing temperature than the free base to completely
216

volatilized (275 versus 225%C for the salt and free base,

respectively). Heating heroin HCl resulted in the parent compound

and 6-monoacetyl-morphine, along with small amounts of N,6-

diacetylnormorphine and N,3,6-triacetylnormorphine; the amounts

of the later two pyrolysis products increased when the temperature

was increased from 275 to 325%C. Pyrolysis of heroin free base

produced mainly heroin and 6-monoacetyl-morphine; as the

temperature was increased from 225 to 325%C, trace amounts of

morphine were detected. Analyses of the results from heating heroin

on aluminum foil indicated that 17 and 62 percent of the heroin salt

and free base, respectively, were recovered in the condensate. This

later value is similar to the percentage of heroin recovered in the

present study. Huizer's method was similar to the system described in

this chapter in terms of employing a steady airflow through the

system. Variations in the composition and nature of the heroin

samples being smoked by users may be expected to influence

volatilization efficiency. Huzier (1987) also demonstrated that the

presence of caffeine and barbiturate increased the volatilization of

heroin free base and the hydrochloride salt, respectively. Taken

together, these results demonstrate that, in addition to volatility,

temperature and airflow are important determinants of the

volatilizing efficiency of a drug.

BIODISPOSITION OF HEROIN AFTER INHALATION EXPOSURE

The ED50 values obtained from inhalation studies (table 2) were based

on the amounts of drug that were added into the pipe prior to

volatilization, which precluded direct comparison of potencies to

those obtained by IV administration. To evaluate the relative

pharmacological potency of the inhaled drugs, the actual tissue

concentrations of the drugs resulting from inhalation are required.

Therefore, the biodisposition of heroin was investigated by exposing

mice to volatilized [3H.]-heroin.

Results of the biodisposition analysis of [3H]-heroin are summarized

in table 3. The whole-body concentrations of heroin equivalents that

produced a 50 percent MPE for inhalation exposure and IV injection

were 0.60 and 0.28 mg/kg, respectively. The concentrations of

heroin equivalents in brain and plasma resulting from inhalation and

IV administration were also very close. Doses of heroin that produced

100 percent MPE through inhalation exposure or IV injection showed

a similar pattern of results. These data suggest that heroin is
217

equipotent when administered by inhalation exposure or IV injection.

It is known that the acetyl groups on the heroin molecule enable it to

enter the brain easily, suggesting that the equilibrium of heroin

between the brain and plasma can be reached rather quickly regardless

of the route of administration. Furthermore, since heroin is

extensively metabolized by the liver and other tissues, the immediate

metabolite of heroin, 6-monoacetylmorphine, has been reported to be

both as potent and lipophilic as heroin (Way et al. 1959). Thus, the

concentrations of drug equivalents found in brain in the present study

undoubtedly reflect heroin and its metabolites.

It has also been demonstrated that, in humans, the time course of

heroin in plasma and the appearance and disappearance of heroin

metabolites after inhalation and IV administration are similar

(Jenkings et al. 1994). The half-lives of heroin after smoking and IV

administration were 3.3 and 3.6 minutes, respectively.

TABLE 3. Recovery of heroin free base after volatilization. A

Heroin recovered after volatilization (mg)

Amount (mg) Glass wool Pipe Total

1.10 Å 0.01 0.77 Å 0.03 0.16 Å 0.03 0.92 Å 0.06

5.11 Å 0.03 3.36 Å 0.22 1.42 Å 0.13 4.73 Å 0.18

KEY: A = Volatilization was carried out at 250%C. Values represent

means Å SE.

This rodent inhalation model has proven to be reliable in studying the

pharmacological effects of a variety of opioids, stimulants, and other

drugs of abuse. In addition to predicting the inhalation potential of

drugs of abuse by the comparison of inhalation and IV routes of

administration, actual tissue concentrations of drug can be

approximated with the use of radiolabeled compounds.

CONCLUSION

Inhalation has become known to drug abusers as a rapid and potent

route of administration. This route has also increased in popularity

because of
218

TABLE 4. Biodisposition of [H]- heroin free base.

Drug equivalentsA Drug equivalents ratioA

Route Dose % MPE

Brain

(&g/g)

Plasma

(&g/ml)

Body

(&g/g) Brain/body Brain/plasma

IV

N = 6 0.28 mg/kg 50 0.04Å0.01 0.20Å0.05 0.19Å0.0

1

0.20Å0.02 0.15Å0.03

1.0 mg/kg 100 0.24Å0.02 0.82Å0.14 0.82Å0.0

4

0.30Å0.01 0.34Å0.07

Inhalation

N = 3 1.0 mg 50 0.11Å0.02 0.38Å0.08 0.06Å0.1

4

0.31Å0.01 0.18Å0.01

3.0 mg 100 0.24Å0.01 0.86Å0.10 1.20Å0.2

5

0.29Å0.04 0.22Å0.05

KEY: A = Values represent means Å SE.
219

fears of contracting diseases such as AIDS from IV injection.

However, smoking may increase the risk of other hazardous effects

caused by pyrolytic products that are not associated with other modes

of administration. Studies of the volatility and inhalation of drugs of

abuse can provide important information for developing guidelines to

predict their abuse potential upon inhalation.

One of the goals of this investigation was to use vapor pressure to

predict drug volatility and thus the abuse potential through inhalation.

Preliminary evidence from studies on the vapor pressure and pharmacology of the drugs of abuse is consistent with this notion. In table 5,

the estimated vapor pressures of several drugs were compared with the

temperature required to produce optimum pharmacological effects

following inhalation. These results suggest that as vapor pressure

increases (i.e., a more volatile drug), lower volatilization temperatures

are required to produce a pharmacological effects.

Drugs in the salt form are much less volatile than their free bases and

would be expected to require high temperatures for volatilization. It

should be noted that vapor pressure information on the salts not

currently available; however, the relative order of their vapor

pressures is assumed to be the same as their free bases (table 5). In

addition, the present results suggest that methamphetamine and

amphetamine are more volatile than most of the other drugs tested.

This is consistent with the popularity of methamphetamine smoking

among drug users. Conversely, a drug that has extremely low vapor

pressure would not be expected to be readily used by inhalation.

In order to study the pharmacological effects of a drug when smoked,

and presumably its potential for abuse by inhalation, efforts were

made to examine the relationship between a drug’s volatility and its

pharmacological potency upon inhalation. A rodent inhalation model

was developed that enabled systematic investigation of the inhalation

route of drug admini-stration. Using this model of inhalation, the

pharmacological effects of a variety of drugs, including opioids,

stimulants, and PCP, were evaluated in mice. It was demonstrated

that inhalation produced pharmacological effects similar to those

obtained from IV administration of heroin, PCP, and

methamphetamine. Biodisposition studies with radiolabeled heroin

enabled the authors to evaluate the relative potency of a drug

following inhalation and IV injection. The tissue concentrations of

heroin equivalents obtained after these two routes of administration

revealed that smoking is equipotent to IV injection.
220

TABLE 5. Comparison of vapor pressures and volatilizing

temperatures.

Vapor pressures at Volatilizing temperature (%C)

Drug 25%C (Torr) Free base Salt

Methamphetamine > 10-2 200

Amphetamine > 10-2 200

PCP 8.56 x 10-4 275

Cocaine 9.79 x 10-6 220

Morphine 9.49 x 10-7 250

Heroin 5.71 x 10-8 250

Fentanyl 2.41 x 10-8 300

Although the authors propose that vapor pressure can be used to

predict whether a drug of abuse may be smoked, other physiochemical

parameters, including particle size (Snyder et al. 1988) and lipid

solubility (McQuay et al. 1989), also influence drug potency. The

determination of these physiochemical parameters used concurrently

with a reliable inhalation animal model will serve as useful tools for

identifying which drugs may potentially be abused by inhalation.

REFERENCES

Benowitz, N.L. Clinical pharmacology of inhaled drugs of abuse:

Implications in understanding nicotine dependence. In:

Chiang, C.N., and Hawks, R.L., eds. Research Findings on

Smoking of Abused Substances. National Institute on

Drug Abuse Research Monograph Series 99. DHHS Pub.

No.(ADM)90-1690. Washington, DC: Supt. of Docs., U.S.

Govt. Print. Off., 1990. pp. 12-29.

Bidleman, T.F. Estimation of vapor pressures for nonpolar organic

compounds by capillary gas chromatography. Anal Chem

56:2490-2496, 1984.

Cook, C.E. Pyrolytic characteristics, pharmacokinetics, and bioavailability

of smoked heroin, cocaine, phencyclidine, and

methamphetamine. In: Miller, M.A., and Kozel, N.J., eds.

Methamphetamine Abuse: Epidemiologic Issues and

Implications. National Institute on Drug Abuse Research

Monograph 115. DHHS Pub. No. (ADM) 91-1896.

Washington, DC: Supt. of Docs., U.S. Govt. Print. Off.,

1991. pp. 6-23.
221

Cook, C.E., and Jeffcoat, A.R. Pyrolytic degradation of heroin, and

cocaine: Identification of products and some observations

on their metabolism. In: Chiang, C.N., and Hawks, R.L.,

eds. Research Findings on Smoking of Abused

Substances. National Institute on Drug Abuse Research

Monograph 99. DHHS Pub. No.(ADM)90-1690.

Washington, DC: Supt. of Docs., U.S. Govt. Print. Off.,

1990. pp. 97-120.

Cook, C.E.; Jeffcoat, A.R.; and Perez-Reyes, M. Pharmacokinetic studies

of cocaine and phencyclidine in man. In: Barnett, G., and

Chiang, C.N., eds. Pharmacokinetics and

Pharmacodynamics of Psychoactive Drugs. Foster City,

CA: Biomedical Publications, 1985. pp. 48-74.

Cook, C.E.; Jeffcoat, A.R.; Perez-Reyes, M.; Sadler, B.M.; Hill, J.M.;

White, W.R.; and McDonald, S. Plasma levels of

methamphetamine after smoking of methamphetamine

hydrocloride. In: Harris, L.S., and Kozel, J., ed. Problems

of Drug Dependence, 1990. National Institute on Drug

Abuse Research Monograph 105. DHHS Pub.

No.(ADM)91-1753. Washington, DC: Supt. of Docs., U.S.

Govt. Print. Off., 1991. pp. 575-577.

Cook, C.E.; Jeffcoat, A.R.; Hill, J.M.; Pugh, D.E.; Patetta, P.K.; Sadler,

B.M.; White, W.R.; and Perez-Reyes, M.

Pharmacokinetics of methamphetamine self-administered

to human subjects by smoking S-(+)-methamphetamine

hydrocloride. Drug Metab Disp 21:717-723, 1993.

Coughenour, L.L.; McClean, J.R.; and Parker, R.B. A device for rapid

measurement of impaired motor function in mice.

Pharmacol Biochem Behav 6:351-353, 1977.

Dewey, W.L.; Harris, L.S.; Howes, J.F.; and Nuite, J.A. The effect of

various neurohumoral modulators on the activity of

morphine and the narcotic antagonists in the tail-flick and

phenylquinone tests. J Pharmacol Exp Ther 175:435-442,

1970.

Freeman, A.S., and Martin, B.R. Quantitation of phencyclidine in main

stream smoke and identification of 1-phenylcyclohexene

as pyrolysis product. J Pharmaceut Sci 70:1002-1004,

1981.

Freeman, A.S., and Martin, B.R. Disposition of phencyclidine in mice after

smoking exposure. Drug Metab Disp 10:680-684, 1982.

Griffiths, P.; Gossop, M.; Powis, B.; and Strang, J. Transitions in patterns

of heroin administration: A study of heroin chasers and

heroin injectors. Addiction 89:301-309, 1994.
222

Hamilton, D.J. Gas chromatographic measurement of volatility of

herbicides esters. J Chromatogr 195:75-83, 1980.

Huizer, H. Analytical studies on illicit heroin V. Efficacy of volatilization

during heroin smoking. Pharmaceutish Weekblad

Scientific Edition 9:203-211, 1987.

Jenkings, A.J.; Keenan, R.M.; Henningfield, J.E.; and Cone, E.J.

Pharmacokinetics and pharmacodynamics of smoked

heroin. J Anal Toxicol 18:317-330, 1994.

Lue, L.P.; Scimeca, J.A.; Thomas, B.F.; and Martin, B.R. Identification

and quantification of phencyclidine pyrolysis products

formed during smoking. J Anal Toxicol 10:81-86, 1986.

Lue, L.P.; Scimeca, J.A.; Thomas, B.F.; and Martin, B.R. Pyrolytic fate of

piperidinenocyclohexanecarbonitrile, a contamination of

phencyclidine, during smoking. J Anal Toxicol 12:57-61,

1988.

Martin, B.R., and Freeman, A.S. Disposition of phencyclidine and its

pyrolytic products in mice exposed to smoke. Fed Proc

42:2561-2565, 1983.

McQuay, H.J.; Sullivan, A.F.; Smallman, K.; and Dickenson, A.H.

Intrathecal opioids, potency and lipophilicity. Pain

36:111-115, 1989.

Miller, M.A., and Kozel, N.J. Introduction and overview. In: Miller, M.A.,

and Kozel, J., eds. Methamphetamine Abuse:

Epidemiologic Issues and Implications. National Institute

on Drug Abuse Research Monograph 115. DHHS Pub.

No.(ADM)91-1836. Washington, DC: Supt. of Docs., U.S.

Govt. Print. Off., 1991. pp. 1-5.

Perez-Reyes, M.; White, W.R.; McDonald, S.; Hill, J.M.; Jeffcoat, A.R.;

and Cook, C.E. Clinical effects of methamphetamine

vapor inhalation. Life Sci 49:953-959, 1991.

Martin, B.R., and Boni, J. Pyrolysis and inhalation studies with

phencyclidine and cocaine. In: Chiang, C.N., and Hawks,

R.L., eds. Research Findings on Smoking of Abused

Substances. National Institute on Drug Abuse Research

Monograph 99. DHHS Pub. No.(ADM)90-1690.

Washington, DC: Supt. of Docs., U.S. Govt. Print. Off.,

1990. pp. 141-158.

Sekine, H., and Nahahara, Y. Abuse of smoking methamphetamine mixed

with tobacco: I. Inhalation efficiency and pyrolysis

products of methamphetamine. J Forensic Sci 32:1271-

80, 1987.

Small, P.A.; Small, K.W.; and Cowley, P. The vapor pressures of some

high boiling esters. Trans Faraday Soc 44:810-816, 1948.
223

Snyder, C.A.; Wood, R.W.; Graefe, J.F.; Bowers, A.; and Magar, K. "Crack

smoke" is a respirable aerosol of cocaine base. Pharmacol

Biochem Behav 29:93-95, 1988.

Way, E.L.; Kemp, J.W.; Young, J.M.; and Grassetti, D.R. The

pharmacologic effects of heroin in relationship to its rate

of biotransformation. J Pharmacol Exp Ther 129:144-

154, 1959.

Wessinger, W.D.; Martin, B.R.; and Balster, R.L. Discriminative stimulus

properties and brain distribution of phencyclidine in rats

following administration by injection and smoke

inhalation. Pharmacol Biochem Behav 23:607-612,

1985.

Wesson, D.R., and Washburn, P. Current patterns of drug abuse that

involve smoking. In: Chiang, C.N., and Hawks, R.L., eds.

Research Findings on Smoking of Abused Substances.

National Institute on Drug Abuse Research Monograph

99. DHHS Pub. No.(ADM)90-1690. Washington, DC:

Supt. of Docs., U.S. Govt. Print. Off., 1990. pp. 5-11.

Westcott, J.W., and Bidleman, T.F. Determination of polychlorinated

biphenyl vapor pressures by capillary gas

chromatography. J Chromatogr 210:331-336, 1981.

Wolters, E.C.; Stam, F.C.; Lousberg, R.J.; van Wyngaarden, G.K.;

Rengelink, H.; Schipper, M.E.I.; and Verbeeten, B.

Leucoencephalopathy after inhaling "heroin" pyrolysate.

Lancet 8310:1233-1237, 1982.

ACKNOWLEDGMENTS

The author would like to thank Ms. Carlota Jackson and Ms. Yulin Chen

for their technical assistance and Dr. William Glassco for advice and

assistance in purifying free bases and chemical preparations. This research

was supported by National Institute on Drug Abuse grant DA 082396.
224

AUTHORS

Yun Meng, M.D., Ph.D.

Aron H. Lichtman, Ph.D.

D. Troy Bridgen, M.S.

Billy R. Martin, Ph.D.

Department of Pharmacology and Toxicology

Medical College of Virginia

Virginia Commonwealth University

Richmond, VA 23298
225

Biocatalysts in Detoxication of Drugs

of Abuse

John R. Cashman

INTRODUCTION

Detoxication of drugs of abuse has generally followed a traditional

approach involving pharmacological intervention in the biochemical

action of the abused substance. This intervention has usually taken

the form of the development of inhibitors or antagonists of biological

macromolecules such as central nervous system (CNS) receptors.

Some of the shortcomings of this approach include the fundamental

biological activity of the antagonist, the possible occurrence of side

effects or pharmacological liabilities of the antagonist itself, and the

stoichiometric nature of the antagonism.

The idea that biocatalysts (e.g., enzymes, catalytic antibodies,

ribozymes) could play a role in human therapy is not a new one.

However, few examples of the application of biocatalysts to the

detoxication of drugs of abuse have been reported. The use of

biocatalysts in the detoxication of drugs of abuse has tremendous

potential. First, the catalyst would likely have no intrinsic

pharmacological activity. That is, the biomacro- or small-molecule

catalyst would probably have minimal direct effect on CNS receptors.

As such, the biocatalyst itself should have a markedly reduced

potential for pharmacological side effects. Of course, numerous

advances in "humanizing" biocatalysts must be undertaken before they

are useful in the clinical setting, but it is likely that those advances

will come to fruition in the near future based on the recent rapid

progress of recombinant biological approaches to developing human

therapeutics. Finally, biocatalysts work in an exponential catalytic

fashion; it is possible that less material will be needed to be employed

therapeutically, thus decreasing the possible side effects and increasing

the therapeutic efficacy as a detoxication agent.

In principle, there are two fundamentally different ways to develop

biocatalysts useful in detoxication of drugs of abuse. The first

approach involves the procurement or design of a small molecule or

macro-molecule that will selectively catalyze the detoxication

reaction of interest. The second approach relies on selection of a

catalyst from a large pool of candidates. However, several important
226

but difficult questions associated with such an exercise need to be

addressed. For example, what is the critical rate-limiting step in the

chemical detox-ication process? How will the catalyst be synthesized

or otherwise obtained? Will the detoxication catalyst be of clinical

relevance?

Selection of a catalyst for specific chemical detoxication reactions

from a large biologically or chemically generated library also poses

several important questions. How does one create a large biologically

or chemically generated library of putative detoxication catalysts?

How will the library be screened for catalytic detoxication activity?

Are dependable methods available to amplify the catalyst to

reasonable amounts for further study? Is it sufficient to simply screen

existing stockpiles of manmade chemicals or natural products for

detoxication catalytic activity, or is it imperative to develop

fundamentally new technology so that novel approaches and novel

agents are introduced for evaluation? It is likely that the time to

develop new technology to address important questions of drug abuse

is now; traditional approaches are limited, and have not appeared to

solve the clinical problems thus far.

There are a number of secondary questions that arise from a

consideration of the above-mentioned approaches to procuring

detoxication catalysts or developing new detoxication strategies. Of

course, one of the first such questions is: Will the catalysts obtained

by design or selection possess any clinical relevance or be useful in

human therapy? If so, will the clinical use be limited to acute toxic

overdose situations or will the catalyst be useful only in drug cessation

paradigms? Will the catalyst play any role in decreasing or reversing

addiction liability of drugs of abuse? To a certain extent these latter

questions are somewhat premature (like the field of catalyst design

and selection itself). At present, a larger question should be: What is

the fundamental technology useful in discovering catalysts or

underlying principles of catalysis that might later support rational

design? What can the technology reveal about the way detoxication

catalysts (such as they are) naturally evolved? How should a drug of

abuse detox-ication catalyst be evaluated in the clinic?

Several fundamentally distinct approaches to the procurement and

evalu-ation of biocatalysts useful in the detoxication of drugs or

chemicals of abuse are outlined below. The author has arbitrarily

limited the presen-tation to a few select detoxication catalysts in the

field of drug abuse. In some cases, other nondetoxicating biochemical

routes of transformation are included as well.
227

The first section focuses on naturally occurring adult human

biocatalysts that transform (S)-nicotine. (S)-Nicotine is discussed

because it is one of the most well-studied CNS-active compounds and

because it constitutes perhaps the most widely used pharmacologically

active agent in humans. In the second section, discussion of the

metabolism of cocaine focuses on cocaine hydrolysis as a major route

of detoxication. Cocaine has been chosen because it illustrates some

of the advantages and pitfalls of the use of cocaine esterolytic

catalytic antibodies as a new potential therapeutic approach. Finally,

a brief discussion of catalytic ribonucleic acid (RNA) (i.e., ribozymes)

is presented. The use of catalytic RNAs as therapeutic agents is in its

infancy but some of the relevant concepts discussed for enzymes or

catalytic antibodies are also relevant to ribozymes. In addition, other

principles observed in RNA catalysis have found considerable

application in the use of oligonucleotides in antisense therapy, and

there may be some useful extensions of antisense drug design that

could be useful in the field of detoxication catalysts.

ENZYMES IN (S)-NICOTINE METABOLISM AND DETOXICATION

(S)-Nicotine1 is one of the most widely used psychoactive drugs in the

world. Almost 25 percent of adult Americans smoke despite

convincing evidence of the health hazards of smoking (Surgeon

General 1988). Nicotine causes complex CNS, behavioral,

cardiovascular, and endocrine neuromuscular effects in humans

(Benowitz 1988). Most notably, nicotine is the primary cause of

tobacco addiction in humans (Surgeon General 1988). The molecular

basis for tobacco addiction is unknown but it is possible that a

biologically active metabolite of nicotine plays a role in nicotine

tolerance and dependence, and individual differences in the

metabolism and disposition of nicotine may explain why some

humans become highly dependent on nicotine and others do not.

Clearly, a full appreciation of the role of human enzymes in nicotine

metabolism and detoxication could provide much insight into the

interindividual variation in the biological responses to smoking and

the variation in the pharmacological effects of nicotine.

The untoward biological properties of nicotine and nicotine

metabolites are controversial. Extensive epidemiological studies have

supported a role of smoking in several types of cancer. However,

although nicotine is a prominent component of cigarette smoke,

there are thousands of other chemicals present in tobacco smoke,

including most classes of known chemical carcinogens (Hecht and
228

Hoffmann 1989). The determination of the agent(s) in tobacco

smoke responsible for causing cancer has been and continues to be an

extremely challenging problem. Most notable for this discussion,

however, is that several nicotine-related metabolites are formed in

humans in apparent detoxication processes. Study of these enzymatic

processes could reveal why certain people are less susceptible to the

harmful effects of nicotine. That is, certain humans may be endowed

with the ability to decrease the potential untoward effects of nicotine

by an abundance of detoxication processes. Careful study of the

enzymes involved in nicotine detoxication could provide valuable

clues for designing new catalysts useful in decreasing the

pharmacological effects of nicotine. For example, nicotine-,

cotinine-, and 3-hydroxy-cotinine glucuronides are all formed in

apparent enzymatic detoxication processes. The apparent

detoxication of nicotine by glucuronidation metabolic pathways in

humans is quite variable and it is possible that an undiscovered

relationship exists between glucuronidation and susceptibility to the

untoward effects of nicotine ingestion. While nicotine addiction is a

complex phenomenon involving many biological, behavioral, and

other parameters, it is unknown what relationship, if any, exists

between metabolic biotransformation of nicotine and nicotine

addiction.

Even though the pharmacokinetics and metabolism of nicotine have

been extensively studied (Beckett et al. 1971; Benowitz and Jacob

1994; Booth and Boyland 1970; Byrd et al. 1992; Caldwell et al.

1992; Jacob et al. 1988; Kyerematen et al. 1990; Neurath et al. 1987;

Schepers et al. 1992; Scherer et al. 1988), the molecular basis for

addiction to nicotine remains unclear. Following smoking or

intravenous (IV) infusion, the terminal half-life of nicotine is about 2

hours (and is urine pH-dependent) (Benowitz et al. 1982). Total

clearance averages 1300 milliliters per minute (mL/min) (and is

highly variable between individuals) and the relatively large volume of

distribution (183 liters (L)) explains the relatively long half-life in the

presence of rapid clearance. Plasma protein binding of nicotine is

very low (Duan et al. 1991). In humans, nicotine is rapidly and

extensively metabolized. Liver monooxygenases and, to a lesser

extent, lung monooxygenases have been implicated, and at present

about 90 percent of ingested nicotine can be accounted for as urinary

metabolites (Benowitz and Jacob 1994). The major excreted urinary

metabolites of nicotine in humans are cotinine (10 to 15 percent),
229

nicotine N-1'-oxide (4 percent), trans-3-hydroxycotinine (30 to 35

percent) and cotinine-, 3-hydroxycotinine-, and nicotine-glucuronides

(together, approximately 24 percent) (figure 1) (Benowitz and Jacob

1994). Cotinine and nicotine N-1'-oxide have been used as markers

of nicotine exposure. Cotinine has a long half-life (16 hr) and blood

levels are stable (Benowitz et al. 1983). However, it may be the

glucuronide metabolites of nicotine or cotinine that are the most

useful as bioindicators of nicotine exposure. Regardless, cotinine and

nicotine N-1'-oxide have historically been used to verify human

nicotine exposure.

In 26 smokers, mean cotinine excretion was 1.39 mg/24 hr, while

nicotine N-1'-oxide was excreted at a rate of 0.56 mg/24 hr in the

same smokers (Jacob et al. 1986). The disposition of nicotine N-1'-

oxide in humans has been characterized (Park et al. 1993).

Administration of deuterated nicotine N-1'-oxide (figure 2) to humans

results in the rapid excretion of the N-1'-oxide largely unchanged in

the urine. Examination of the stereochemistry of the nicotine N-1'-

oxide before and after administration showed that the stereochemistry

of the excreted material was essentially identical to that of the

administered compound. That no
230

change in stereochemistry and no reduction of IV-administered N-1'-

oxide to nicotine was observed in humans (Beckett et al. 1971; Park

et al. 1993) suggests that N-1'-oxygenation of nicotine is a

detoxication process. In animals, considerable reduction of oral or

intraperitoneally (IP)-administered nicotine N-1'-oxide was observed

(Dajani et al. 1975). In rabbits, less than 3 percent is reduced

following IV dosing, but after oral administration 45 percent of

nicotine N-1'-oxide is reduced. It is likely that gut bacteria or

intestinal reductases are responsible for the reduction of nicotine N1'-oxide after oral administration (Duan et al. 1991). In rabbits,

nicotine N-1'-oxide clearance was 7.5 mL/min per kilogram (kg), had

a half-life of 42.6 min, a volume of distribution of 340 mL/kg, and

was not plasma protein bound (Duan et al. 1991). The idea that

nicotine N-1'-oxide serves as a reservoir for nicotine (i.e., by

reduction back to nicotine) and participates in maintaining nicotine

levels required for addiction and nicotine-related toxicity is

apparently not true in humans (Park et al. 1993). This is not to

imply that nicotine N-1'-oxide does not have pharmacological

activity; it causes vasoconstrictive effects on isolated rabbit-ear

vessels and increases the tonus of isolated rabbit intestine, and in mice

causes qualitatively similar tremor, dyspnea, and spasms that are

similar (IV administration) in effects to nicotine, but the dose required

is much larger (Barass et al. 1969). More important, the N-1'-oxide

administered before challenge with nicotine conferred protection

against a subsequent lethal dose of nicotine (Barass et al. 1969). The

conclusion is that formation of nicotine N-1'-oxide constitutes a true

detoxication route of disposition, but in most adult humans, this

pathway represents only a small percentage of the overall metabolism

of nicotine. In principle, development of catalysts that could form
231

nicotine N-1'-oxide may be a useful adjuvant in smoking cessation

therapy.

Cytochromes P-450

Human cytochrome P-450 is a ubiquitous heme-containing enzyme

that participates in the oxidation of a wide variety of chemicals,

drugs, and even some endogenous materials (Guengerich and Shimada

1991). Although the cytochrome P-450 transformation of nicotine

has been extensively described for animals, nicotine has not been

extensively used as a probe substrate for adult human metabolism.

The molecular basis for metabolism of nicotine in humans is

considerably different from that observed in animals (Cashman et al.

1992). In humans, nicotine is primarily eliminated by metabolism,

with total clearance varying by about fourfold among individuals.

Men metabolize nicotine more rapidly than women (Benowitz and

Jacob 1984). Age also influences urinary nicotine metabolite levels in

humans. The pathway leading to cotinine is the major route of

nicotine metabolism, and the initial metabolic step is nicotine 1',5'-

iminium ion formation. In previous studies, the author and colleagues

identified human liver cytochrome P-450 2A6 as the principal

enzyme responsible for nicotine 1',5'-iminium ion formation

(Cashman et al. 1992). This result is in contrast to the situation in

animals where cytochrome P-450 2B (identical to the phenobarbitalinducible form) has most often been implicated as the putative

nicotine oxidase (McCoy et al. 1989; Williams et al. 1990). In rats

and rabbits, phenobarbital pretreatment increased cotinine formation

in liver microsomes supplemented with aldehyde oxidase that was

isolated from the animals. In the absence of aldehyde oxidase,

cytochrome P-450 is responsible for the metabolism-dependent

covalent binding of nicotine to tissue macromolecules, implicating

nicotine 1',5'-iminium ion as an electrophilic metabolite (Williams

et al. 1990; Shigenaga et al. 1988). In rabbit lung microsomes,

cytochrome P-450 2 (or LM2), which is similar to the phenobarbitalinducible hepatic form, is primarily responsible for nicotine 1',5'

-

iminium ion formation that can be trapped by cyanide or converted

to cotinine by aldehyde oxidase (Williams et al. 1990). It is

important to note that the cytochrome P-450 corresponding to the

rat or rabbit liver phenobarbital-inducible form (i.e., adult human liver

cytochrome P-450 2B6) is highly variable and is detectable in less

than 3 percent of adult human livers examined (Mimura et al. 1993).

Because there is strong evidence that P-450 2A6 is the primary

enzyme forming nicotine 1',5'-iminium ion in adult human liver
232

microsomes, it is likely that in human tissues about 90 percent of the

nicotine iminium ion formation is mediated by cytochrome P-450

2A6 (Berkman et al. 1995; Cashman et al. 1992). Interestingly,

increased hepatic cytochrome P-450 2A6 levels have been observed

in humans with a history of barbiturate administration (Cashman et al.

1992). Production of nicotine 1',5'-iminium ion showed some

dependence on the previous drug administration history of the subject

from which the hepatic microsomes were isolated (table 1). Two of

the highest rates of iminium ion formation occurred in the presence

of human liver microsomes from barbiturate-pretreated subjects (i.e.,

E and I) (table 1). Of the seven major human cytochrome P-450

enzymes examined (i.e., 1A2, 2A1, 2C8, 2C9, 2D6, 2E1, 3A5, and

3A total), the greatest linkage between immunoreactivity and

nicotine iminium ion formation (i.e., at 10 micromolar (M)

substrate concentration) was observed for cytochrome P-450 2A6 (r

= 0.9) (Berkman et al. 1995). Neither gender, age, nor smoking

history provided a direct relation between nicotine 1'

,5'-iminium

formation and cytochrome P-450 2A6 levels. Others have suggested

that cytochrome P-450 2D6 or 2B6 plays a role in adult human liver

nicotine oxidase activity. However, these studies employed

extremely high nonphysiological concentrations of nicotine

(Flammang et al. 1992). Based on their studies, the author and

colleagues anticipate that non-cytochrome P-450 2A6 enzymes

contribute less than 10 percent to the oxidation of nicotine (Berkman

et al. 1995).

In summary, in adult human liver, cytochrome P-450 2A6 is

responsible for formation of nicotine 1',5'-iminium ion that is

subsequently converted by aldehyde oxidase to cotinine. Because

cotinine is extensively glucuronidated or metabolized to 3-hydroxycotinine (which is in turn glucuronidated), detoxication of nicotine via

the cotinine glucuronidation pathway must pass through the relatively

electrophilic iminium ion pathway. However, with the exception of a

few studies (most notably by Castagnoli and coworkers) (Peterson and

Castagnoli 1988; Peterson et al. 1987), the pharmacological or

toxicological effects of nicotine 1',5'-iminium ion have not been

reported. It is interesting to note that human liver cytochrome P450 2A6 and 2E1 catalyze metabolic activation of the tobacco

smoke-related nitrosoamines 4-(methylnitrosoamino)-1-(3-pyridyl)-

1-butanone (NNK), 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol

(NNAL), and N-nitrosonornicotine (NNN). Thus, cytochrome P-450

2A6 and 2E1 could catalyze the conversion of several tobacco

smoke-related nitrosoamines to genotoxic products (Yamazaki et al.

1992).
233

Metabolite formed [pmol/(min mg of protein)]

patient

code gender age

drug

history

FMO

immunoreactivity

b

nicotine

N-11-

oxide cotinine nornicotine

A M 25 none 3 226 294 ND

B M 50 none 2 88 238 ND

C M 22 ethanol 2 181 187 ND

D M 31 MPME 1 54 568 ND

E M 14 pentobarb 2 180 376 ND

F F 50 ethanol 2 55 353 ND

G F 48 teldrin 5 225 299 ND

H F 28 none 2 34 284 ND

I N 43 phenobarb 3 45 750 ND

J F 55 none 4 133 377 ND

K M 23 ethanol 4 192 431 ND

L F 58 dopamine 4 141 208 ND

M M 18 ethanol 3 67 234 ND

N M 21 ethanol 2 127 311 ND

KEY: a = Incubations were performed with 0.4-2.0 mg of microsomal protein, 4.3

mg of rat liver microsome supernatant (as a source of aldehyde oxidase), 0.5

mM NADPH at pH 8.4 and 37%C for 10-20 min, and products were

quantified by HPLC. For cotinine, nornicotine, and 7-OH-coumarin

formation, incubations were at pH 7.4. Values are the mean of 2-4

determinations 10%. b = Relative immunoquantitation; 5 = greatest, 1 =

weakest, ND = not detected.

SOURCE: From Cashman et al. 1992, used with permission.
234

The conclusion of the study was that interindividual variations in the

amount of human cytochrome 2A6 and 2E1 could contribute to the

susceptibility of environmental procarcinogens including those

derived from tobacco smoke. Cytochromes P-450 largely bioactivate

nicotine and nicotine-related nitrosoamines to electrophilic materials.

Presumably, the balance between bioactivating enzyme activity and

detoxicating enzyme activity is important in determining the overall

susceptibilities of humans to exposure to chemicals. Below, the

properties of several additional enzymes in nicotine detoxication are

described.

Aldehyde Oxidase

Aldehyde oxidase is a cytosolic, iron-containing molybdoflavoprotein

that mediates the oxidation of aldehydes and nitrogen-containing

heterocyclic drugs, chemicals, and endogenous substrates (Beedham

1985). The structural biology of the various forms of the enzyme are

just beginning to be described, but animal and human aldehyde oxidase

apparently consists of homogenous homodimers of subunit molecular

size (approximately 150,000 daltons (Da)). The substrate specificity

of human liver aldehyde oxidase has not been as exhaustively

examined as animal liver aldehyde oxidase but some data point to a

similar spectrum of substrate activity (Beedham 1985; Krenitsky et

al. 1974). Thus, prototypic substrates such benzaldehyde, 6-

methylpurine, and N-1-methylnicotinamide appear to be good

substrates for both animal and human forms of aldehyde oxidase

(Rodrigues 1994).

Electronic factors and the relative lipophilicity of the molecule

probably help to determine the affinity of the substrate for the

enzyme as well as turnover properties. It is likely that the

fundamental instability of the enzyme has hampered progress in the

characterization of human liver aldehyde oxidase. At least in animals,

the specific activity of the enzyme is quite dependent on the way the

tissue is procured, processed, and stored; this may lead to considerable

intersample variability. Enzyme instability may at least in part

explain why aldehyde oxidase activity from different species is so

variable (Duley et al. 1985). However, it is likely that in addition to

intrinsic differences in stability, the determination of aldehyde

oxidase activity for a given substrate in various tissue preparations is

dependent on the analytical methodology employed to assay the

enzyme and the likelihood of the presence of different forms of the

enzyme that possess distinct substrate specificity and kinetic

properties (Johns 1967; Beedham 1985). For example, in the
235

presence of six different adult human liver preparations (i.e., the

9000 g supernatant or S-9 fraction), the oxidation of benzaldehyde,

6-methylpurine, and N-1-methylnicotinamide by aldehyde oxidase

varied by 3.6-, 2.3-, and > 40-fold, respectively (Rodrigues 1994)

(table 2).

TABLE 2. Aldehyde oxidase activity in human liver 9000 g

supernatant (S-9) fractions.

Activity* (nmol/

min/mg S-9 protein

Subject code Age

(years)

Sex BA 6-MP NMN

FGL 26 F 11.4 17.5 1.4

GDD 25 F 14.5 14.7 <0.5

GEQ 20 M 19.5 14.1 <0.5

GFE 50 F 40.7 32.8 20.4

GC4 29 M 26.3 23.1 <0.5

FRX 58 M 22.2 15.5 1.5

Mean Å SD 22.4 Å 10.4 19.6 Å 7.2 7.78 Å 10.9

Fold

variation**

 3.6 2.3 >40

KEY: * = Data for each subject represent the mean of duplicate

determinations. BA = benzaldehyde; 6-MP = 6-methylpurine;

NMN = N-1'-methylnicotinamide. ** = Ratio of the

highest/lowest activity.

SOURCE: From Rodrigues 1994, used with permission.

In agreement with what has been observed in animals, the level of

aldehyde oxidase activity present in human liver is also markedly

dependent on the substrate employed, the analytical method used to

evaluate the substrate, and most importantly, the intrinsic activity of

the preparation (Beedham et al. 1992). For N-1-methylnicotinamide

oxidase activity, the rank order was cynomolgus monkey > rat >

beagle dog > human liver (Rodrigues 1994). The relative levels of

aldehyde oxidase activity may help determine the relative extent of

lactim formation from N-heterocyclic compounds such as N-1-

methylnicotinamide. Of course, formation of lactim metabolites

from iminium ions can also be coordinately regulated by other

enzyme activities including cytochromes P-450. Thus, formation of
236

iminium ions by cytochrome P-450 and conversion to lactams by

aldehyde oxidase can be thought of as an important detoxication

mechanism whereby N-heterocyclic compounds are converted into

more polar materials that are excreted (Ohkubo and Fujimura 1982).

In addition to its role as an oxidative enzyme, aldehyde oxidase has

been reported to reduce a number of tertiary amine N-oxides back to

the parent tertiary amine (Kitamura and Tatsumi 1984a, 1984b).

Because a number of heterocyclic tertiary amines can undergo

sequential 1-electron oxidation (i.e., via the iminium ion by

cytochrome P-450) or 2-electron oxygenation (i.e., to the tertiary

amine N-oxide by the flavin-containing monooxygenase), the

participation of aldehyde oxidase activity in the disposition of

tertiary amines may represent an important aspect of drug of abuse

detoxication.

Flavin-Containing Monooxygenase

The mammalian flavin-containing monooxygenase (FMO) is a widely

distributed membrane-associated family of enzymes that catalyze the

oxygenation of nucleophilic nitrogen-, sulfur-, and phosphorouscontaining xenobiotics, drugs, and endogenous substances (Ziegler

1993). Nucleophilic tertiary amines such as nicotine are readily

converted to relatively stable tertiary amine N-oxides that are more

polar and readily excreted unchanged (Park et al. 1993). As described

above, nicotine N-1'-oxygenation is a route of detoxication for

nicotine in humans. In contrast to cytochromes P-450, the

mechanism of human FMO form 3 (FMO3)-catalyzed N-1'-

oxygenation of nicotine involves 2-electron oxygenation to provide

exclusively the trans nicotine N-1'-oxide (Cashman et al. 1992).

Formation of nicotine N-1'-oxide diastereomers is highly FMO

enzyme form-dependent. Thus, pig FMO1 forms approximately a

40:60 mixture of cistrans nicotine N-1'-oxides (Damani et al. 1988;

Park et al. 1993). In contrast, human stereoselective formation of

trans nicotine N-1'-oxide may serve as a highly sensitive

stereochemical probe of adult human FMO3 activity in vitro and in

vivo.

In the presence of liver microsomes, human FMO3 nicotine N-1'-

oxygenase activity is not strongly dependent on the age, gender, or

drug administration history of the subject from whom the liver was

obtained (Cashman et al. 1992). In good agreement with in vitro

studies, in vivo metabolism of nicotine in humans by three routes of

administration including ad libitum smoking, intravenous infusion of

nicotine-d2, and dermal patch administration of nicotine all produced
237

only the trans diastereomer of nicotine N-1'-oxide in the urine (Park

et al. 1993). In addition, trans nicotine N-1'-oxide is not appreciably

reduced or oxidized further because infusion of nicotine-d2 N-1'-oxide

in humans with a known ratio of cis to trans N-1'- oxide

diastereomers gave recovered urinary metabolite N-1'-oxides in high

yield and with metabolite diastereoisomer ratios essentially identical

to that of the infusate. In addition, human administration of highly

purified preparations of stereochemically characterized nicotine-N-1'-

oxide resulted in the material being rapidly excreted unchanged. This

data supports the idea that formation of nicotine N-1'-oxide is a

detoxication process.

Although a slight difference between the average amount of nicotineN-1'- oxide formed and excreted in the urine of smokers versus the

transdermal route of administration (i.e., 3.7 versus 2.7 percent of

total urinary metabolites) could possibly reflect a minor contribution

from pulmonary human FMO2 (Benowitz and Peyton 1994), the

preferential N-1'-oxygen-ation of nicotine by hepatic FMO as

opposed to lung FMO observed in animals (Williams et al. 1990)

suggests that human liver FMO3 is the major contributing metabolic

pathway. Formation of trans nicotine N-1'-oxide has been proposed

as a selective functional marker for adult human FMO3 activity. The

fact that nicotine N-1'-oxide formation possesses significantly less

pharmacological activity than nicotine suggests that catalysts

designed to convert nicotine to nicotine N-1'-oxide might be useful in

smoking cessation treatment.

As stated above, it is likely that human FMO3-catalyzed formation of

trans nicotine N-1'-oxide constitutes a detoxication process whereby

the pharmacologically active nicotine alkaloid is converted to a polar,

readily excreted tertiary amine N-1'-oxide. Previously, it has been

suggested that the role of FMO in human metabolism is to metabolize

xenobiotics from plant sources to benign materials that do not pose a

pharmacological or toxicological challenge to the organism ingesting

the chemical (Ziegler 1993). Because many CNS drugs commonly

abused by humans contain a tertiary amine center, elaboration of

catalysts to form tertiary amine N-oxides could constitute the basis

for a new class of detoxication catalysts.
238

URIDINE DIPHOSPHOSPHATE GLUCURONOSYL TRANSFERASE

Glucuronosyl transferases mediate the transfer of uridine-5'-

diphospho-- D-glucuronic acid to acceptor molecules in metabolic

transformations that are generally considered detoxication reactions.

While acyl glucu-ronides in some cases have been associated with

potentially toxic conse-quences, in most instances, formation of

glucuronide metabolites is associated with detoxication reactions. In

humans, there have been numerous reports of the urinary excretion of

quaternary N-linked glucu-ronides (Chaudhary et al. 1988; Lehman et

al. 1983; Macrae et al. 1990). Recently, studies have confirmed that

major metabolites of nicotine in humans are in fact glucuronide

conjugates (Caldwell et al. 1992). Thus, humans administered

nicotine by smoking or by a dermal patch route of administration

excrete the glucuronide conjugates of (S)-nicotine, cotinine, and 3-

hydroxy cotinine (i.e., on average, 30 percent, 49 percent, and 16

percent of a dose is conjugated, respectively) (Benowitz and Jacob

1994). While there is significant interindividual variability in the

excretion of glucuronide conjugates, the pattern of metabolism is

generally similar when nicotine is inhaled or absorbed transdermally.

However, formation of nicotine and cotinine glucuronides does not

exactly parallel the formation of 3-hydroxy cotinine glucuronide.

Apparently, conjugation of 3-hydroxy cotinine is done by a different

glucuronosyl transferase than the enzyme responsible for nicotine and

cotinine glucuronidation (Benowitz and Jacob 1994). This may

reflect the fact that one enzyme form is a nicotine Nglucuronosyltransferase while the other enzyme form is a nicotine Oglucuronosyltransferase.

Conjugation is a quantitatively important route of nicotine

detoxication in humans. The large amount of nicotine glucuronosyl

conjugates observed in human urine following nicotine administration

and the significant degree of interindividual variability in the amount

of glucuronide formed have confounded accurate assessment of

nicotine metabolic disposition. By taking into account the

glucuronidation pathway, a more complete quantification of nicotine

metabolism and disposition could enhance the accuracy of

bioindicators of nicotine exposure. In the gut, glucuronosyl

conjugates of nicotine may become substrates for microflora -

glucuro- nidase. As such, it is possible that enterohepatic recirculation

of nicotine could arise via this pathway. A cycle of conjugation and

hydrolysis could represent a mechanism to prolong the bioavailability

of nicotine or its metabolites. While glucuronidation of nicotine or

one of its metabolites represents a detoxication reaction, considering
239

other possible metabolic pathways makes the analysis of the

contribution of conjugation to detoxication less straightforward.

GLUTATHIONE TRANSFERASE

While no published studies of the conjugation of electrophilic nicotine

metabolites with glutathione have been reported, in principle,

enzyme-catalyzed glutathione addition could produce metabolites that

are more polar and more readily excreted. Nucleophilic addition of

low molecular weight thiols to nicotine 1',5'-iminium ions has been

shown to occur under pseudo first-order reaction conditions (Brandage

and Lindblom 1979). In the liver where glutathione reaches

millimolar concentrations, it is possible that glutathione conjugation

contributes to the disposition of the nicotine iminium ion, especially

in animals with low amounts of aldehyde oxidase. Presumably

glutathione conjugates, if formed, could also be transformed into

mercapturates that should be readily excreted. While investigations

of a role for glutathione conjugates or mercapturates of nicotine have

not been reported in the literature, the feasibility of trapping

electrophilic iminium ion metabolites of nicotine with thiol

nucleophiles has ample precedence (Brandage and Lindblom 1979).

At this point it is only speculation that addition of biologically

relevant sulfur-containing nucleophiles constitutes a detoxication

reaction for metabolites of nicotine. Finally, because nucleophilic

addition to the nicotine iminium ion is reversible, from a practical

standpoint, an addition compound will probably only be formed and

detected under conditions where the nucleophile is present in large

excess.

Tobacco Addiction

Tobacco addiction is complex, and involves behavioral as well as

pharmacologic factors. The importance of nicotine in tobacco

dependence has been demonstrated in many studies (Benowitz and

Jacob 1990; Surgeon General 1988). However, the neurochemical

mechanisms of nicotine tolerance and dependence are not known, and

the possibility exists that a reactive metabolite may be involved.

Thus, identification of nicotine-derived electrophilic metabolites that

covalently modify human proteins may provide insight into the

fundamental mechanisms underlying tobacco addiction. This is a

largely unexplored area of research. Knowledge of the bioactivation

as well as detoxication steps in the metabolism of nicotine may help
240

identify the reasons for the interindividual variation and susceptibility

to the injurious effects of this widely used chemical.

Cocaine Use and Related Toxicity in the United States

During recent years, cocaine has been most commonly ingested in the

United States by smoking cocaine base (in chunk or crack form)

(NIDA 1990). Smoking crack cocaine is preferred by abusers because

the concentration of cocaine in venous blood peaks sooner after

smoking crack (or after IV administration of cocaine) than by other

routes of administration. Cocaine is generally not abused by the oral

route of administration, but oral absorption is efficient and is a

significant route of dosing as a cause of toxicity (Jones 1990). Thus,

cocaine toxicity could be greater for children who have swallowed

cocaine in mother’s milk, for example.

Cocaine is hydrolyzed by esterases of the blood, liver, and other

organs (Benowitz 1992). The major (inactive) metabolite of cocaine

is benzoylecgonine, but ecgonine and ecgonine methyl ester are also

formed (Benowitz 1992) (figure 3). Hepatic metabolism of cocaine

to norcocaine (pharmacologically active) accounts for only 2 to 6

percent of the total amount eliminated (Benowitz 1992). After IV

administration, cocaine has a plasma half-life of 70 mins. The

transient nature of the CNS effects of cocaine is due to the relatively

rapid redistribution of cocaine into and out of the brain and into other

tissues. This type of rapid influx-efflux brain distribution is typical of

lipophilic agents such as cocaine. In contrast to IV or smoking routes

of administration, systemic effects and plasma levels may be sustained

for longer periods because of continuous absorption after oral

administration and selective binding to CNS monoamine reuptake

transporters.

The primary effects of cocaine in humans are CNS stimulation

resulting in euphoria and activation of the peripheral sympathetic

nervous system with tachycardia and blood pressure elevation.

Tolerance to the euphoric effects of cocaine develops quickly (Foltin

and Fischman 1991), but is incomplete for the cardiovascular effects

of cocaine (Ambre et al. 1988). It is possible that repeated use of

cocaine to seek the high may lead to progressive cardiovascular

toxicity. Medical problems arising from cocaine abuse are among the

most common causes for emergency room visits in U.S. hospitals,

especially in the inner cities (Brody et al. 1990). Three types of lifethreatening medical toxicities reported include
241

cocaine-related sudden death, cardiovascular disease, and reproductive

disturbances (Benowitz 1992). Sudden death is believed to result from

cardiac arrhythmias due to massive catecholamine release with or

without acute myocardial ischemia. However, a review of 935

cocaine-related deaths reported in 1986 in New York City showed

that the top causes of death were homicide (37.5 percent), acute

narcotic exposure (12 percent), other natural causes (11.1 percent),

and suicide (6.6 percent) (Litovitz et al. 1991). Another serious type

of injury from cocaine use with consequences for long-term harm

involves brain damage. Episodes of angina and myocardial infarction

due to cocaine abuse have also become commonplace in reports from

major U.S. metropolitan medical centers.

In inner-city areas of the United States, surveys of pregnant women

reported rates of maternal cocaine use as high as 30 percent.

Maternal cocaine use can affect reproduction by adversely affecting

the pregnancy. In addition, cocaine use may directly injure the fetus

and/or produce behavioral and developmental abnormalities in the

neonate. Indirect toxicities of cocaine abuse may result in severe

health consequences including child neglect and abuse, loss of family

structure, an increased risk of AIDS, and congenitally acquired

sexually transmitted diseases (Benowitz 1992). A study of data from

72 U.S. Poison Control Centers in 1990 that analyzed 1.7 million
242

human drug exposure cases (Litovitz et al. 1991) showed that cocaine

was implicated in more major medical complications and deaths than

any other chemical agent. Cocaine use among polydrug abusers,

presumably occurring at increasingly higher doses via IV injection or

by smoking, has become more toxic to humans. In summary, the

current picture of cocaine abuse is associated with more emergency

room visits and more deaths than any other illicit drug of abuse.

Completely new strategies for preventing the toxicity and abuse of

cocaine are needed and novel methods may be best developed by

employing modern biological and biochemical approaches (Jones

1992). It is unlikely that antagonists of neurotransmitter

transporters or other pharmacological intervention (i.e., desipramine

or other antidepressants) will be efficacious because the blood levels

required to saturate the cocaine receptor will be too toxic in humans

(Ritz et al. 1987) or have an induction period of several weeks

(Fischman et al. 1990). The reasons for cocaine deaths probably

stem partly from a contribution of the intrinsic toxicity and

mechanism of action of cocaine and partly from the current lack of

appropriate treatment for cocaine overdose. With heroin, the opiate

receptor antagonist naloxone is likely to save someone brought to the

emergency room with a heroin overdose. A comparable antagonist,

such as the opiate antagonist naloxone, is currently not available to

treat cocaine overdose. Antibodies against opiates have been reported

to antagonize the reinforcing effect of low doses of heroin (Bonese et

al. 1974) but because the antibodies were removed from circulation

presumably by clearance processes, the therapeutic paradigm failed at

high doses of heroin. Only symptomatic treatment of cocaine

overdose is available today.

Powerful selective antidotes or antagonists to treat cocaine abuse are

necessary to offset the increased cocaine toxicity currently observed in the

United States. The use of catalytic antibodies in the creation of selective

binding agents and detoxication catalysts of cocaine could represent a novel

approach that may result in significant advances in the field of detoxication

of drugs of abuse. An anti-cocaine catalytic antibody directed to hydrolyze

the benzoyl ester could in principle catalyze the formation of ecgonine and

benzoic acid, hydrolysis products of cocaine that do not possess the

reinforcing or CNS stimulation properties of cocaine (Spealman et al. 1989).

Catalytic Antibodies

Antibodies are proteins that can recognize a wide array of chemicals,

drugs, and other biological materials with great specificity and efficacy
243

(Harlow and Lane 1988). An antibody may bind to its ligand with an

equilibrium dissociation constant (Kd) equal to 10-12 molar (M). In

contrast, an enzyme typically devotes only a fraction of its overall

free energy to binding phenomena and generally reserves most of its

expendable free energy on catalytic events. In principle, an antibody

can afford to bind a ligand with less avidity and channel the difference

in free energy into catalytic activities. The fundamental similarity

between antibodies and enzymes has important implications for

combining the catalytic action of enzymes with the selective

recognition function of antibodies.

The general approach to producing catalytic antibodies is based on the

principle of reaction intermediate-enzyme transition state

complementarity (Pauling 1946; Wolfenden and Kati 1991). Small

molecule analogs of putative enzyme reaction transition states have

been used as haptens to induce antibodies with complementary binding

sites (Janda et al. 1989; Lerner et al. 1991; Pollock et al. 1986;

Tramontano et al. 1986a, 1986b). As in the case of enzymes that

have evolved to preferentially stabilize the transition state, antibodies

induced to recognize the transition state have been shown to stabilize

the putative transition state of the reaction and afford catalysis

(Lerner et al. 1991; Tramontano et al. 1986a, 1986b). One of the

first examples of catalytic antibody activity led to the development

of hydrolytic or acyl transfer catalytic antibodies (Janda et al. 1988,

1989). Thus, simple tetrahedral carbon mimics representing the

transition state for ester hydrolysis (i.e., phosphonates) were

synthesized and antibodies directed against these materials (i.e.,

antibodies directed against small molecules covalently attached to a

large carrier molecule such as a protein) possessed esterolytic activity

against the corresponding ester substrate.

In principle, catalytic antibodies can be developed to facilitate

selective chemical reactions that do not have biological counterparts

(Benkovic et al. 1988). For example, toxins could be selectively

detoxicated, novel chemical reactions could be developed, or

extremely sensitive bioprobes of antibody ligands could be readily

developed.

Esterolytic Catalytic Antibodies

Simple structures such as phenyl phosphonates have been synthesized

and used to generate monoclonal antihapten antibodies (Tramontano

et al. 1988). Antibody-producing hybridoma cells can be cloned to

obtain colonies, each producing a single type of antibody
244

(Tramontano and Schloeder 1989). Because a large number of

hybridomas can be generated, it was thought to be impractical to

screen all cultures for catalytic activity. Today, with advances in

rapid screening techniques, it is now practical to screen hundreds if

not thousands of hybridomas for antibodies with catalytic activity.

Typically, an antibody is produced in microgram quantities in cell

culture and a subset of the population that recognizes the original

hapten itself is chosen. One of the first catalytic antibodies studied

(i.e., esterase 50D8) (Tramontano et at. 1986a, 1986b) has been well

characterized. Esterase 50D8 shows a high degree of substrate

specificity; the rate advantage of 6 x 106 over the background (i.e.,

uncatalyzed rate) and the catalytic efficiency (i.e., kcat 29 sec-1) both

illustrate the catalytic properties of the antibodies; and the antigen or

hapten inhibits the substrate (i.e., Ki=60-80 nM). However, the rate

factor (i.e., kcat/Km of 104 M-1 sec-1) is still several orders of magnitude

less than those of a good enzyme that catalyzes an analogous reaction

(i.e., values of 107 or 108 M-1 sec-1) and the activity increases sharply

above neutral pH and becomes pH-independent above pH 10. This

latter observation has been found with most esterolytic catalytic

antibodies, and probably reflects the fact that deacylation of the

antibody by hydroxide ion is rate limiting. The design of future

esterolytic catalytic antibodies must address this apparent

shortcoming.

Cocaine Catalytic Antibodies

Typical antibody-catalyzed reaction rates are several hundredfold to

100,000-fold faster than the uncatalyzed reaction of the substrate.

Several fundamental postulates have been proposed to explain the

rate enhancements that nevertheless fall short of the enormous rate

accelerations of enzymes. Is activity truly due solely to transition

state stabilization by antibody-binding interactions? Can additional

binding interactions be built into the combining site or into the

substrate molecule itself to increase the overall rate of the reaction?

Can new screening methods and immunological methods be developed

to uncover novel catalysts with diverse activities? Most important,

can novel esterolytic catalysts be developed based on currently

available catalytic antibody technology to efficiently hydrolyze and

detoxicate cocaine?

The answer is unquestionably yes. In fact, cocaine is quite

immunogenic; a catalytic antibody was obtained that catalyzed the

hydrolysis of cocaine, but the rate enhancement was considerably less

than desirable (Landry et al. 1993). The ratio of the catalytic rate

constant (kc) to the spontaneous or water-catalyzed rate constant (ko)
245

was 540 and 440 for two catalytic antibodies, respectively, which is

considerably less than the rate of hydrolysis of other esters by

catalytic antibodies reported in the literature. Thus, immunization of

mice with a bovine serum albumin conjugate linked to a phosphonate

analog of cocaine (figure 4) afforded antisera that provided

monoclonal antibodies after using standard purification procedures.

The primary screening for anti-cocaine antibodies was done with an

enzyme-linked immunosorbent assay (ELISA) against a phospho-nate

analog of cocaine (figure 4). Hybridoma cells immunopositive for

anti-phosphonate analog antibodies were next screened for cocaine

benzoyl ester hydrolytic activity. The selectivity of the most active

anti-cocaine catalytic antibody was shown by the inhibition of the

hydrolysis reaction in the presence of the phosphonate transition

state analog (figure 4). The Km values for the two most active anticocaine catalytic antibodies were 490 and 1020 M, with kcat values of

0.11 and 0.07 min-1, respectively. These values were significantly less

impressive than the values obtained for butyryl cholinesterase, the

principal cocaine esterase in human serum (Gatley 1991; Stewart et al.

1978), that provided Km and kcat values of 38 M and 1.2 min -1,

respectively. As stated by Landry and colleagues, a catalytic antibody

against cocaine should ideally have a turnover number of at least 2

sec-1 and a Km of approximately 30 M to deactivate cocaine before

the molecule partitions into the CNS (Landry et al. 1993). Clearly,

room for significant improvement in the catalytic and binding

properties of antibodies against cocaine is required before such

biocatalysts become part of a human therapeutic regime. In addition,

advances in humanizing catalytic antibodies are also needed before

artificial esterases will find clinical usefulness in decreasing the serum

levels of cocaine during overdose or intoxication in humans.

Structure and Modification of Antibodies

The attachment of drugs and enzymes to antibodies is a common

practice in commercial diagnostics, experimental immunoassays, and

drug-delivery therapeutic approaches (Chaudhary et al. 1989;

Pinchera et al. 1985). Antibody-enzyme conjugates are now being

used for targeting cytotoxic pro-drugs to cancerous tissue (Chaudhary

et al. 1989). In principle, antibody-enzyme conjugates could offer

another possible way
246

to pharmacologically interdict cocaine or other drugs of abuse. For

example, a biocatalyst with cocaine hydrolytic activity could be

coupled to an antibody to produce a conjugate with enhanced catalytic

and pharmacokinetic properties.

Coupling methods between antibodies and ligands generally rely on

chemical modification to selectively introduce the enzyme onto the

antibody. Antibodies offer a range of sites for chemical modification.

As shown in figure 5, the combining site where antibody-hapten

recognition occurs is contained in the Fab fragment. The Fab

fragment can be produced by selective proteolytic digestion of the

antibody. Thus, a minimal high-affinity binding protein can be

obtained by papain digestion of an antibody prepared against the

hapten. The variable domains of the light chains (VL) and heavy

chains (VH) have been frequently documented as having the same

antigen-binding activities as the whole antibody (Weir 1986). VL and

VH fragments can be engineered by molecular biological means to have

reduced background (i.e., nonspecific) binding sometimes associated

with intact antibodies. In addition, use of the light and heavy chain

fragments may be clinically advantageous in that it is possible that

they are less immunogenic than the intact antibody but still possess

high affinity (Condra et al. 1990). In principle, VL and VH fragments

are also easier to construct by recombinant methods than intact

antibodies because they possess fewer disulfide bonds (Better et al.

1989). However, the stoichiometric expression of VL and VH

fragments must be linked (Pluckthun 1991). The Fab fragments are
247

coupled through cysteine residues at the C-terminal end of the heavy

chains. Additional cysteines, which are normally involved in

interchain disulfide linkages in the hinge region, are retained in a

pepsin-generated F(ab')2 fragment (figure 5). Selective coupling of

small or large biomacromolecules to F(ab')2 fragments is simple and

straightforward. The potential for antibody-enzyme or antibody

fragment-enzyme conjugates for use in detoxication of drugs of abuse

is essentially unexplored. It is possible that combining the favorable

properties of antibody-mediated localization with enzymes or other

biocatalysts could provide a new class of detoxication agents.

Anti-Idiotype Antibodies

The immune system discriminates between self and nonself (i.e.,

foreign) antigen. The foreign antigen can stimulate an immune

response, and the immune system sets in motion a steady-state

memory lymphocyte which, upon reinfection with the same or

closely related antigen, is able to respond with a vigorous immune

response. If the foreign antigen is a complementary structure, then

one might influence the immune system to recognize the mirror

image of the complementary structure. Thus, immunization of an

animal with a monoclonal antibody that recognizes the transition

state for detoxication of a drug of abuse should elicit an immune

response and produce antibodies that resemble the structure of the

drug of abuse. The second antibody, the anti-idiotype antibody, thus

mimics the transition state for detoxication. Injection of this second

antibody into an animal could create a relatively long-term memory

effect to give protection against the drug of abuse; the third antibody

produced could act as a catalyst to detoxicate the drug (Landry et al.

1993). For example, passive immunization with an anti-cocaine

catalytic antibody "could provide a window for appropriate

psychosocial and relapse-prevention interventions" (Landry et al.

1993). Of course, researchers are a long way away from passively

immunizing humans with catalytic antibodies. However, future use of

antibodies in a therapeutic paradigm may provide novel approaches to

promoting cessation of use of drugs of abuse as well as in the

maintenance of abstinence (Rocio et al. 1995).

Catalytic Antibodies and Catalytic RNA

As discussed above, in vitro selection techniques have been used to

isolate biocatalysts useful in the development of catalytic antibodies

(Tawfik et al. 1990). While the cocaine catalytic antibodies reported

thus
248

far do not possess impressive hydrolytic catalytic rate accelerations,

the selection technology has nevertheless allowed the discovery of

some fundamentals of catalysts that might later support rational

design of detoxication catalysts and answer fundamental questions in

catalysis (Benner 1993). Catalytic antibody technology relies on the

selection of biocatalysts using the enormous diversity of the immune

system. Procurement of catalytic species depends on the ability of

substrates to chemically bind or transform a target molecule. The key

feature of the catalytic antibody is the specificity of the reaction

catalyzed. It is interesting to note that the relatively recent advances

in catalytic RNA technology have revealed considerable parallels

between the concepts of catalytic antibodies and catalytic RNA.

Catalytic RNA

Several structurally and mechanistically distinct classes of catalytic

RNAs have been discovered (Altman 1990; Cech 1990; Pace and

Smith 1990). Generally, for catalytic RNA reactions reported thus

far, RNA and deoxyribonucleic acid (DNA) are substrates and

transesterification or hydrolysis of phosphate esters are the reactions

catalyzed (Piccirilli et al. 1992). However, binding of amino acids,

organic dyes, and other small molecules to RNA suggest that catalytic

RNAs may catalyze other reactions (Prudent et al. 1994). Some

catalytic RNA molecules are metalloenzymes and require magnesium

for their three-dimensional structure and catalytic activity. Like
249

catalytic antibodies, catalytic RNAs (i.e., ribozymes) catalyze very

specific reactions. For example, DNA-cleaving ribozymes with

relatively high catalytic efficiency that undergo relatively rapid

turnover and operate in a highly specific manner have been

discovered (Tsang and Joyce 1994). Future studies may show that

catalytic RNA has utility as sequence-specific DNA endonucleases.

The specificity stems from the first step in the ribozyme reaction

which is due to specific Watson-Crick base-pairing interactions

(Mueller et al. 1993). In every ribozyme thus far examined, a

template region (named the internal guide sequence) (Seiwert and

Stuart 1994) has been observed near the 5'-end of the molecule that

forms Watson-Crick base pairs with the target RNA substrate. The

3'-hydroxy of a base at the other end of the ribozyme attacks the

phosphodiester bond within the ribozyme-bound substrate complex.

A transesterication reaction occurs that results in cleavage of the

substrate and ligation of the 3'-portion of the substrate (Tsang and

Joyce 1994). Just like in the case of enzyme- or catalytic antibodycatalyzed reactions, the rate depends upon substrate binding affinity

and the intrinsic catalytic rate parameters. For example, in ester

hydrolysis there is a hyperbolic dependence on the concentration of

the ribozyme: at low concentration of catalyst the rate of hydrolysis

is first order, while at high concentration of catalyst the reaction rate

is indepen-dent of ribozyme concentration (Piccirilli et al. 1992).

This type of saturation or Michaelis-Menten kinetic behavior is

typical of ribozymes and is completely analogous to the enzymesubstrate complex observed for enzymes and catalytic antibodies.

Like other biocatalysts, RNA-cleaving ribozymes are highly specific

for RNA and catalyze hydrolysis with high catalytic efficiency,

undergo rapid turnover, and operate in a highly selective manner. In

addition, inhibition of ribozyme catalysis has been observed for

substrate analogs; like enzymes and other biomacro-molecules,

changes in ribozyme solvation and conformation have been observed

when the inhibitor binds (Piccirilli et al. 1992).

Ribozymes may be developed in the future to act as aminoacyl tRNA

synthetases. Ribozymes might be developed to selectively break acyl

bonds to oligonucleotides or might also be engineered to selectively

charge oligonucleotides with amino acids. Again, due to the

specificity of Watson-Crick base-pair interactions, oligonucleotide

catalysts may find use in the therapeutics of human disease. Highly

selective ribozyme endonucleases may be created to perform

sequence-specific cleavages of potential therapeutic value. The ratio

of a toxic dose to a therapeutic dose (i.e., [toxic dose]/[therapeutic

dose]) presumably will be very high for an effective ribozyme
250

primarily due to the high specificity of oligonucleo-tides (i.e.,

containing 15 to 17 nucleotides) that should have a unique sequence

relative to the entire human genome (Stein and Cheng 1993). In

principle, suitable oligonucleotides should be able to interfere in a

sequence-specific manner with processes such as translation of

messenger RNA (mRNA) into a specific receptor or other protein. If

the synthesis of the target protein encoded by the mRNA is required

for susceptibility to drug addiction, it may be possible to develop new

therapeutic strategies based on elaboration of ribozymes or

oligonucleotides targeted to inhibit or inactivate key biological

processes.

SUMMARY

Currently there is a significant amount of information about the way

biocatalysts from animals detoxicate and bioactivate drugs of abuse.

In some cases, biotransformation data concerning drugs of abuse

obtained from animal systems are analogous to the human situation,

but in many cases the data are not. Clearly, significant work needs to

be done with human biocatalysts to define a role in the

biotransformation of drugs of abuse and to relate the work that has

already been done in animals. New metabolic pathways will likely be

discovered that may link drug metabolism to addiction liability or drug

susceptibility in humans.

New design and selection technologies are providing the basis to allow

the discovery of new biocatalysts that may be useful in the

detoxication of drugs of abuse in humans. Fundamentally new

approaches using biocatalysts including rationally engineered

enzymes, catalytic antibodies, catalytic antibody fragments,

ribozymes, oligonucleotides, and other biomacromolecules may

provide basic information that may later support the rational design

of biocatalysts, which may in turn provide the basis for designing

detoxication catalysts for drugs of abuse.

NOTES

1. In this chapter, nicotine or nicotine metabolites shall all be

assumed to have the (S) stereochemistry at the benzylic center.

REFERENCES

Altman, S. Ribonuclease P. J Biol Chem 265:20053-20056, 1990.
251

Ambre, J.J.; Belknap, S.M.; Nelson, J.; Ruo, T.I.; Shin, S.; and Atkinson,

A.A., Jr. Acute tolerance for cocaine in humans. Clin

Pharmacol Ther 44:1-8, 1988.

Barass, B.C.; Blackburn, J.W.; Brimblecombe, R.W.; and Rich, P.

Modification of nicotine toxicity by pretreatment with

different drugs. Biochem Pharmacol 18:2145, 1969.

Beckett, A.H.; Gorrod, J.W.; and Jenner, P. The analysis of nicotine-1'-

oxide in urine, in the presence of nicotine and cotinine,

and its application to the study of in vivo nicotine

metabolism in man. Supplement. J Pharm Pharmacol

23:55S-61S, 1971.

Beedham, C. Molybdenum hydroxylases as drug-metabolizing enzymes.

Drug Metab Rev 16:119-156, 1985.

Beedham, C.; al-Tayib, Y.; and Smith, J.A. Role of guinea pig and rabbit

hepatic aldehyde oxidase in oxidative in vitro metabolism

of cinchona antimalarials. Drug Metab Dispos 20:889,

1992.

Benkovic, S.J.; Napper, A.D.; and Lerner, R.A. Catalysis of stereospecific

biomolecular amide synthesis by an antibody. Proc Natl

Acad Sci U S A 85:5355-5358, 1988.

Benner, S.A. Catalysis: Design versus selection. Science 261:1402-1403,

1993.

Benowitz, N.L. Pharmacological aspects of cigarette smoking and nicotine

addiction. N Engl J Med 319:1318, 1988.

Benowitz, N.L. How toxic is cocaine? In: 1992 Cocaine: Scientific and

Social Dimensions. Ciba Foundation Symposium 166.

Chichester, U.K.: Wiley, 1992. pp. 125-148.

Benowitz, N.L., and Jacob, P., III. Daily intake of nicotine during cigarette

smoking. Clin Pharmacol Ther 35:499, 1984.

Benowitz, N.L., and Jacob, P., III. Intravenous nicotine replacement

suppresses nicotine intake from cigarette smoking. J

Pharmacol Exp Ther 254:1000, 1990.

Benowitz, N.L., and Jacob, P., III. Nicotine metabolic profile in man:

Comparison of cigarette smoking and transdermal

nicotine. J Pharmacol Exp Ther 268:296, 1994.

Benowitz, N.L.; Jacobs, P., III; Jones, R.T.; and Rosenberg, T.

Interindividual variability in the metabolism and

cardiovascular effects of (S)-nicotine in man. J

Pharmacol Exp Ther 221:368, 1982.

Benowitz, N.L.; Kuyt, F.; Jacob, P., III; Jones, R.T.; and Osman, A.L.

Cotinine disposition and effects. Clin Pharmacol Ther

34:604, 1983.
252

Berkman, C.E.; Park, S.B.; Wrighton, S.A.; and Cashman, J.R. In vitro-in

vivo correlations of human (S)-nicotine metabolism.

Biochem Pharmacol 50:565-570, 1995.

Better, M.; Chang, C.P.; Robinson, R.R.; and Horwitz, A.H. E. Coli

secretion of active chimeric antibody fragment. Science

246:1275-1281, 1989.

Bonese, K.F.; Wainer, B.H.; Fitch, F.W.; Rothberg, R.M.; and Schuster,

C.R. Changes in heroin self-administration by a rhesus

monkey after morphine immunization. Nature 252:708-

710, 1974.

Booth, J., and Boyland, E. The metabolism of (S)-nicotine into two

optically active stereoisomers of (S)-nicotine-1'-N-oxide

by animal tissues in vitro and by cigarette smokers.

Biochem Pharmacol 19:733, 1970.

Brandage, S., and Lindblom, L. Synthesis, structure and stability of nicotine

1',5'-iminium ion, an intermediary metabolite of

nicotine. Acta Chemica Scandinavica B 33:187-191,

1979.

Brody, S.L.; Slovis, C.M.; and Wrenn, K.D. Cocaine-related medical

problems: Consecutive studies of 233 patients. Am J Med

88:325-331, 1990.

Byrd, G.D.; Chang, K.; Greene, J.M.; and deBithzy, J.D. Evidence for

urinary excretion of glucuronide conjugates of nicotine,

cotinine, and trans-3'-hydroxycotinine in smokers. Drug

Metab Dispos 20:192, 1992.

Caldwell, W.S.; Greene, J.M.; Byrd, G.D.; Chang, K.M.; Uhrig, M.;

deBithizy, J.D.; Crooks, P.A.; Bhatti, B.S.; and Riggs,

R.M. Characterization of the glucuronide conjugate of

cotinine: A previously unidentified major metabolite of

nicotine in smokers' urine. Chem Res Toxicol 5:280,

1992.

Cashman, J.R.; Park, S.B.; Yang, Z.C.; Wrighton, S.A.; Jacob, P., III; and

Benowitz, N.L. Metabolism of nicotine by human liver

microsomes: Stereoselective formation of trans-nicotine

N'-oxide. Chem Res Toxicol 5:639, 1992.

Cech, T.R. Self-splicing of Group I introns. Ann Rev Biochem 59:543-568,

1990.

Chaudhary, A.K.; Hubbard, J.W.; McKay, G.; and Midha, K.K.

Identification of a quaternary ammonium-linked

glucuronide of chlorpromazine in the urine of a

schizophrenic patient treated with chlorpromazine. Drug

Metab Dispos 16:506-508, 1988.

Chaudhary, V.K.; Queen, C.; Junghans, R.P.; Waldmann, T.A.; Fitzgerald,

D.J.; and Pastan, I. A recombinant immunotoxin
253

consisting of two antibody variable domains if used to

Pseudomonas exotoxin. Nature 339:294-397, 1989.

Condra, J.H.; Sardana, V.V.; Tomassini, J.E.; Schlaback, A.J.; Davies, M.E.;

Lineberger, D.W.; Gragam, D.J.; Gotlieb, L.; and Colonno,

R.J. Bacterial expression of antibody fragments that block

human rhinovirus infection of cultured cells. J Biol Chem

265:2292-2295, 1990.

Dajani, R.M.; Gorrod, J.W.; and Beckett, A.H. Hepatic and extrahepatic

reduction of nicotine-1'-N'-oxide in rats. Biochem

Pharmacol 24:648, 1975.

Damani, L.A.; Pool, W.F.; Crooks, P.A.; Kaderlik, R.K.; and Ziegler, D.M.

Stereoselectivity in the N'-oxidation of nicotine isomers

by FMO. Mol Pharmacol 33:702-705, 1988.

Duan, M.J.; Yu, L.; Savanapridi, C.; Jacob, P., III; and Benowitz, N.L.

Dispositional kinetics and metabolism of (S)-nicotine N1'-oxide in rabbits. Drug Metab Dispos 19:667, 1991.

Duley, J.A.; Harris, O.; and Holmes, R.S. Analysis of human alcohol- and

aldehyde-metabolizing isozymes by electrophoresis and

isoelectric focusing. Alcohol Clin Exp Res 9:263-271,

1985.

Fischman, M.W.; Foltin, R.W.; Nestadt, G.; and Perlson, G.D. Effects of

desipramine maintenance on cocaine self-administration

by humans. J Pharmacol Exp Ther 253:760-770, 1990.

Flammang, A.M.; Gelboin, H.V.; Aoyama, T.; Gonzalez, F.J.; and McCoy,

G.D. Nicotine metabolism by cDNA-expressed human

cytochrome P-450's. Biochem Arch 8:1-8, 1992.

Foltin, R.W., and Fischman, M.W. Smoked and i.v. cocaine in humans:

Acute tolerance, cardiovascular and subjective effects. J

Pharmacol Exp Ther 257:247-261, 1991.

Gatley, S.J. Activities of the enantiomers of cocaine and some related

compounds as substrates and inhibitors of plasma

butyrylcholin-esterase. Biochem Pharmacol 41:1249-

1254, 1991.

Guengerich, F.P., and Shimada, T. Oxidation of toxic and carcinogenic

chemicals by human cytochrome P-450 enzymes. Chem

Res Toxicol 4:391-407, 1991.

Harlow, E., and Lane, D. Antibodies: A Laboratory Manual. Cold Spring

Harbor, NY: Cold Spring Harbor Laboratory, 1988.

Hecht, S.S., and Hoffman, D. The relevance of tobacco-specific

nitrosoamines to human cancer. Cancer Surveys 8:273-

294, 1989.

Jacob, P., III; Benowitz, N.L.; and Shulgin, A.J. Recent studies of (S)-

nicotine metabolism in humans. Pharmacol Biochem

Behav 30:249, 1988.
254

Jacob, P., III; Benowitz, N.L.; Yu, L.; and Shulgin, A.T. Determination of

(S)-nicotine N-1'oxide by gas chromatography following

thermal conversion to 2-methyl-6-(3-pyridyl)-

tetrahydro-1,2-oxazine. Analyt Chem 58:2218, 1986.

Janda, K.D.; Benkovic, S.J.; and Lerner, R.A. Catalytic antibodies with

lipase activity. Science 244:437-440, 1989.

Janda, K.D.; Schloeder, D.; Benkovic, S.J.; and Lerner, R.A. Induction of

an antibody that catalyzes the hydrolysis of an amide

bond. Science 241:1188-1191, 1988.

Johns, D.G. Human liver aldehyde oxidase: Differential inhibition of

oxidation of charged and uncharged substrates. J Clin

Invest 46:1492-1505, 1967.

Jones, R.T. Alternative strategies. In: 1992 Cocaine: Scientific and Social

Dimensions. Ciba Foundation Symposium 166. Chichester,

UK: Wiley, 1992. pp. 224-241.

Jones, R.T. The pharmacology of cocaine smoking in humans. In: Chiang,

C.N., and Hawks, R.L., eds. Research Findings on

Smoking of Abused Substances. National Institute on

Drug Abuse Research Monograph 99. DHHS Pub. No.

(ADM)90-1690. Washington, DC: Supt. of Docs., U.S.

Govt. Print. Off., 1990. pp. 30-41.

Kitamura, S., and Tatsumi, K. Reduction of tertiary amine N-oxides by

liver preparations: Function of aldehyde oxidase as a

major N-oxide reductase. Biochem Biophys Res Commun

121:749-754, 1984a.

Kitamura, S., and Tatsumi, K. Involvement of liver aldehyde oxidase in

the reduction of nicotinamide N-oxide. Biochem Biophys

Res Commun 120:692-703, 1984b.

Krenitsky, T.A.; Tuttle, J.V.; Cattan, E.L.; and Wang, P. A comparison of

the distribution and the electron acceptor specificities of

xanthine oxidase and aldehyde oxidase. Comp Biochem

Physiol 49B:687-703, 1974.

Kyerematen, G.A.; Morgan, M.L.; Chattopadhyay, B.; deBithizy, J.D.; and

Vessel, E.S. Disposition of nicotine and eight metabolites

in smokers and nonsmokers: Identification in smokers of

two metabolites that are longer lived than cotinine. Clin

Pharmacol Ther 48:641, 1990.

Landry, D.W.; Zhao, K.; Yang, G.X.-Q.; Glickman, M.; and Georgiadis,

T.M. Antibody-catalyzed degradation of cocaine. Science

259:1899-1901, 1993.

Lehman, J.P.; Fenselau, C.; and Depaulo, J.R. Quaternary ammoniumlinked glucuronides of amitriptyline, imipramine, and

chlorpromazine. Drug Metab Dispos 11:221-225, 1983.
255

Lerner, R.A.; Benkovic, S.J.; and Schultz, P.G. At the crossroad of

chemistry and immunology: Catalytic antibodies. Science

252:659-667, 1991.

Litovitz, T.L.; Bailey, K.M.; Schmitz, B.F.; Holm, K.C.; and KleinSchwartz, W. 1990 Annual Report of the American

Association of Poison Control Centers National Data

Collection System. Am J Emerg Med 9:461-509, 1991.

Macrae, P.V.; Kinns, M.; Pullen, F.S.; and Tarbit, M.H. Characterization

of a quaternary N-glucuronide metabolite of the imidazole

antifungal, tioconazole. Drug Metab Dispos 16:1100-

1102, 1990.

McCoy, G.D.; DeMarco, G.J.; and Koop, D.R. Microsomal nicotine

metabolism: A comparison of relative activities of six

purified rabbit cytochrome P-450 isozymes. Biochem

Pharmacol 38:1185, 1989.

Mimura, M.; Baba, T.; Yamazaki, H.; Ohmori, S.; Inui, Y.; Gonzalez, F.J.;

Guengerich, F.P.; and Shimada, T. Characterization of

cytochrome P-450 2B6 in human liver microsomes. Drug

Metab Dispos 21:1048, 1993.

Mueller, M.W.; Hetzer, M.; and Schweyen, R.J. Group II intron RNA

catalysis of progressive nucleotide insertion: A model for

RNA editing. Science 261:1035-1038, 1993.

National Institute on Drug Abuse. Annual Data 1989: Data from the Drug

Abuse Warning Network. Series 1, No. 9. DHHS Pub. No.

(ADM)90-1717. Alcohol, Drug Abuse, and Mental Health

Administration. Washington, DC: Supt. of Docs., U.S.

Govt. Print. Off., 1990.

Neurath, G.B.; Dungen, M.; Orth, D.; and Pein, F.G. Trans-3'-

hydroxycotinine as a main metabolite in urine of smokers.

Int Arch Occup Environ Health 59:199, 1987.

Ohkubo, M., and Fujimura, S. Rat liver N-methyl-nicotinamide oxidase I

and II: Substrate non-specific enzyme "aldehyde oxidase"

and specific enzyme. Biochem Int 4:353-358, 1982.

Pace, N.R., and Smith, D. Ribonuclease P: Function and variation. J Biol

Chem 265:3587-3590, 1990.

Park, S.B.; Jacob, P., III; Benowitz, N.L.; and Cashman, J.R.

Stereoselective metabolism of (S)-(-)-nicotine in humans:

Formation of trans-(S)-nicotine N-1'-oxide. Chem Res

Toxicol 6:880, 1993.

Pauling, L. Comments. Chem Eng News 24:1375, 1946.

Peterson, L.A., and Castagnoli, N., Jr. Regio- and stereo-chemical studies

on the -carbon oxidation of (S)-nicotine by cytochrome

P-450 model systems. J Med Chem 31:637, 1988.
256

Peterson, L.A.; Trevor, A.; and Castagnoli, N., Jr. Stereochemical studies

on the cytochrome P-450 catalyzed oxidation of (S)-

nicotine to the (S)-nicotine 1',5'-iminium species. J Med

Chem 30:249, 1987.

Piccirilli, J.A.; McConnell, T.S.; Zang, A.J.; Noller, H.F.; and Cech, T.R.

Aminoacyl esterase activity of the Tetrahymena

ribozyme. Science 256:1420-1424, 1992.

Pinchera, A.; Doris, G.; Damacco, F.; and Baargellesi, A. Monoclonal

Antibodies: Biological and Clinical Applications. Milan:

E. Kurtis, 1985.

Pluckthun, A. Antibody engineering: Advances from the use of

Escherichia Coli expression systems. Biotechnology

9:545-551, 1991.

Pollock, S.J.; Jacobs, J.W.; and Schultz, P.G. Selective chemical catalysis

by an antibody. Science 234:1570-1573, 1986.

Prudent, J.R.; Uno, T.; and Schultze, P.G. Expanding the scope of RNA

catalysis. Science 264:1924-1927, 1994.

Ritz, M.C.; Lamb, R.J.; Goldberg, S.R.; and Kuhar, M.J. Cocaine receptors

on dopamine transporters are related to selfadministration of cocaine. Science 237:1219-1223, 1987.

Rocio, M.; Carrera, A.; Ashley, J.A.; Parsons, L.H.; Wirsching, P.; Koob,

G.F.; and Jamda, K.D. Suppression of psychoactive effects

of cocaine by active immunization. Nature 378:727-729,

1995.

Rodrigues, A.D. Comparison of levels of aldehyde oxidase with

cytochrome P-450 activities in human liver in vitro.

Biochem Pharmacol 48:197-200, 1994.

Schepers, G.; Demetriou, D.; Rustenmeier, K.; Voncken, P.; and Diehl, B.

Nicotine phase 2 metabolites in human urine—structure of

metabolically formed trans-3'-hydroxycotinine

glucuronide. Med Sci Res 20:863, 1992.

Scherer, G.; Jarczyk, L.; Heller, W.D.; Biber, A.; Neurath, G.B.; and

Adlkofer, F. Pharmacokinetics of nicotine, cotinine, and

3'-hydroxy-cotinine in cigarette smokers. Supplement.

Klin Wochenschr 66:5, 1988.

Seiwert, S.D., and Stuart, K. RNA editing: Transfer of genetic information

from gRNA to precursor mRNA in vitro. Science

266:114-117, 1994.

Shigenaga, M.K.; Trevor, A.J.; and Castagnoli, N., Jr. Metabolismdependent covalent binding of (S)-(5-3H) nicotine to liver

and lung microsomal macromolecules. Drug Metab Dispos

16:397, 1988.

Spealman, R.D.; Madras, B.K.; and Bergman, J. Effects of cocaine and

related drugs in nonhuman primates. II. Stimulant effects
257

on schedule-controlled behavior. J Pharmacol Exp Ther

251:142-149, 1989.

Stein, C.A., and Cheng, Y.-C. Antisense oligonucleotides as therapeutic

agents—Is the bullet really magical? Science 261:1004-

1012, 1993.

Stewart, D.J.; Inaba, T.; Lucassen, M.; and Kalow, W. Cocaine metabolism:

Cocaine and norcocaine hydrolysis by liver and serum

esterases. Clin Pharmacol Ther 25:464-468, 1978.

Surgeon General. The Health Consequences of Involuntary Smoking. A

report of the Surgeon General. DHHS Pub. No. (PHS)87-

8398. Washington, DC: Supt. of Docs., U.S. Govt. Print.

Off., 1986.

Surgeon General. The Health Consequences of Smoking: Nicotine

Addiction. A report of the Surgeon General. DHHS Pub.

No. (CDC)88-8406. Washington, DC: Supt. of Docs., U.S.

Govt. Print. Off., 1988.

Tawfik, D.S.; Zemel, R.R.; Yellin, R.A.; Green, B.S.; and Eshhaz, A. Simple

method for selecting catalytic monoclonal antibodies that

exhibit turnover and specificity. Biochemistry 29:9916-

9921, 1990.

Tramontano, A.; Amman, A.A.; and Lerner, R.A. Antibody catalysis

approaching the activity of enzymes. J Am Chem Soc

110:2282-2286, 1988.

Tramontano, A., and Schloeder, D. Production of antibodies that mimic

enzyme catalytic activity. Methods Enzymol 178:531-

550, 1989.

Tramontano, A.; Janda, K.D.; and Lerner, R.A. Catalytic antibodies.

Science 234:1566-1573, 1986a.

Tramontano, A.; Janda, K.D.; and Lerner, R.A. Chemical reactivity at an

antibody biding site elicited by mechanistic design of a

synthetic antigen. Proc Natl Acad Sci U S A 83:6736-

6740, 1986b.

Tsang, J., and Joyce, G.F. Evolutionary optimization of the catalytic

properties of a DNA-cleaving ribozyme. Biochemistry

33:5966-5973, 1994.

Weir, D.M., ed. Immunochemistry Handbook of Experimental

Immunology. 4th ed., Vol. 1. Oxford: Blackwell Scientific

Publications, 1986.

Williams, D.E.; Shigenaga, M.K.; and Castagnoli, N., Jr. The role of

cytochrome P-450 and flavin-containing monooxygenase

in the metabolism of (S)-nicotine by rabbit lung. Drug

Metab Dispos 18:418, 1990.
258

Wolfenden, R., and Kati, W.M. Testing the limits of protein-ligand

binding discrimination with transition-state analogue

inhibitors. Acc Chem Res 24:209-215, 1991.

Yamazaki, H.; Inui, Y.; Yun, C-H.; Guengerich, F.P.; and Shimada, T.

Cytochrome P-450 2F1 and 2A6 enzymes as major

catalysts for metabolic activation of Nnitrosodialkylamines and tobacco-related nitrosoamines in

human liver 'somes Carcinogenesis 13:1789-1794, 1992.

Ziegler, D.M. Recent studies on the structure and function of multisubstrate flavin-containing monooxygenases. Ann Rev

Pharmacol Toxicol 33:179-199, 1993.

ACKNOWLEDGMENTS

I thank Drs. Neal Benowitz, Neal Castagnoli, Jr., and Peyton Jacob III

for many stimulating discussions regarding nicotine metabolism and

disposition and my many collaborators on the projects published that

are listed in the references (especially Drs. Sang Park and Clifford

Berkman). This work was financially supported in part by grants

from National Institutes of Health (GM 36426, ES 06973, and

DA08531) and the University of California Tobacco-Related Disease

Research Program (1IT0071).

AUTHOR

John R. Cashman, Ph.D.

Senior Scientist

Seattle Biomedical Research Institute

4 Nickerson Street, Suite 200

Seattle, WA 98109
259

Antibodies as Pharmacokinetic and

Metabolic Modifiers of Neurotoxicity

S. Michael Owens

INTRODUCTION

Abuse of psychoactive chemicals can result in neurotoxic effects that

are difficult to treat medically. Successful therapy is often hindered

by the lack of useful antagonists for many of these chemicals and by

the extensive distribution of these chemicals out of the bloodstream.

Although there are treatments for opiate addiction and an antagonist

for opiate overdose, there are no such medical treatments for most

drugs of abuse such as phencyclidine (PCP) and cocaine. Therefore,

this chapter focuses on recent advances in immunotherapy which

suggest this novel approach could be beneficial in the treatment of

drug abuse.

PROBLEMS ASSOCIATED WITH TREATMENT OF PCP TOXICITY

PCP use by drug abusers sometimes results in behavioral toxicity that

manifests as a wide variety of frightening effects that include extreme

violence; self-destructive and psychotic behavior; and, at very high

doses, even death (Peterson and Stillman 1978). Often the acute

behavioral toxicity is not realized or even felt by the individual

because of the anesthetic effects of the drug. Indeed, the lack of a

pain sensation may contribute to PCP's ability to make users become

violent and self-destructive. The acute toxicity of PCP is often

misdiagnosed because of its similarity to schizophrenic episodes.

Arylcyclohexylamines like PCP are considered to produce one of the

best drug-induced models of schizophrenia (Luby et al. 1962; Itil et al.

1967). These clinical manifestations are even more frightening when

misinterpreted by the public. In addition, chronic PCP use in some

individuals can produce a long-lasting schizophrenic episode, which in

some cases may not be reversible. These effects can be produced in

humans at doses ranging from 5 milligrams (mg) to greater than 10

mg. Consequently, treatment for PCP abuse is needed and, in fact,

may be most necessary to offset the behavioral toxicity that occurs at

only moderate doses.
260

The pharmacological effects of PCP and related

arylcyclohexylamines are produced through interaction with several

neurotransmitter systems, ion channels, and catecholamine uptake

systems. These sites include the so-called PCP receptor that is

associated with the N-methyl-D-aspartate (NMDA) receptor complex

(Lodge et al. 1983; Vincent et al. 1979; Zukin and Zukin 1979) and

the dopamine reuptake site that may also significantly contribute to

PCP abuse and psychosis (Chaudieu et al. 1989; Vignon et al. 1988).

NMDA receptor antagonists such as PCP can be protective against

brain damage in neurological disorders such as stroke (Olney et al.

1989; Rothman 1984), but can also cause dose-dependent

morphological damage to neurons in the cerebral cortex of rats

(Olney et al. 1991). Although these effects appear to be reversible

after a single dose, effects after chronic doses have not been studied

and it is conceivable that chronic use of the drug could be associated

with more permanent neurological damage. In addition, female rats

are twice as sensitive to these effects as male rats (Olney et al. 1989).

This increased potency of the drug in female rats is also found with

other pharmacological effects of PCP. The mechanisms underlying

these sex differences in the pharmacological effects in rats are poorly

understood, but they are dose dependent and are found after single and

chronic doses of the drug (Olney et al. 1989; Wessinger 1995). In

addition, females are much less efficient at metabolizing PCP than

males. Although sex differences of this type are not found in humans,

this sexual dimorphism in the rat could be a useful model for

mimicking the wide range of differences in effects and metabolism of

the drug in humans.

IMMUNOTHERAPY FOR TREATMENT OF DRUG OVERDOSE

Treatment of the adverse effects of PCP is difficult for several

reasons. PCP has a very high volume of distribution (6.2 liters per

kilogram (L/kg) in humans) and its clearance is primarily by

metabolism (Cook et al. 1982) with only a small contribution from

renal excretion. Its major sites of action in the central nervous

system (CNS) are far removed from the beneficial effects of most

traditional treatment methods such as dialysis. In addition, there is no

specific antagonist for PCP’s adverse effects. These pharmacokinetic

and receptor-medicated characteristics make it very difficult to

develop effective treatment strategies. Some of the current methods

for treatment of overdose are urine acidification, diazepam

administration to control convulsions (Aronow and Done 1978;
261

Peterson and Stillman 1978), and simply waiting for the patient to

get better. Nevertheless, Mayersohn (1985) predicts that even under

ideal conditions, urine acidification would only increase systemic

clearance (Cls) by about 28 percent and would only decrease the halflife (t_) by about 23 percent (t_ in humans averages about 17 hrs, but

can range from 7 to 58 hrs; Cook et al. 1985). This small change in

kinetic parameters is not surprising due to PCP's very high volume of

distribution and low renal clearance (Mayersohn 1985).

Consequently, treatments that can only reduce the amount of drug in

the bloodstream will not be effective.

One approach to treating drug overdose resulting from CNS-acting,

high- distribution volume drugs is the use of high-affinity, drugspecific antibodies (Colburn 1980; Owens and Mayersohn 1986; Smith

et al. 1979). Currently, the only drug toxicity routinely treated by

immunotherapy is the cardiac toxicity due to digitalis (Smith et al.

1982). The use of anti-drug antibodies for treating toxicity due to

CNS-acting agents like PCP is currently being tested in a rat model

(McClurkan et al. 1993; Valentine et al. 1994). These studies show

that the anti-PCP Fab can dramatically change PCP

pharmacokinetics (table 1).

Earlier pharmacokinetic studies with a tracer dose of [3H]PCP (4

micrograms (g)) and very low doses of goat anti-PCP Fab (0.5 to 1

mg) in dogs suggested the therapy could be beneficial (Owens and

Mayersohn 1986). However, the major limitation at the time of

these earlier studies was the inability to produce large quantities of

drug-specific antibody. The development of large-scale techniques for

the production of monoclonal antibodies of desired specificity has

made immunotherapy a more attractive possibility for treating

toxicity associated with drugs of abuse. These large-scale techniques

for monoclonal antibody production include ascites production and

hollow fiber bioreactors. Hollow fiber bioreactor techniques for largescale production of a high-affinity murine monoclonal antibody are

capable of producing gram quantities of monoclonal antibodies on a

daily basis for extended periods of time. This technique is the

preferred method since it does not require the use of live animals, and

the production scale can be significantly increased over other

methods. These are essential features since treatment of PCP

overdose in humans will probably require several grams of Fab.
262

TABLE 1. Comparison of the pharmacokinetics of PCP, anti-PCP

Fab, and PCP after treatment with anti-PCP Fab in Sprague-Dawley

rats.

Pharmacokineti

c parameter

Anti-PCP FabaPCPbPCP after

treatment with

anti-PCP Fabb

Vss*0.55 L/kg 12.6 L/kg 0.6 L/kg

Cls 2.7 mL/min/kg 66.3

mL/min/kg

6.8 mL/min/kg

t_ 7.5 hrs 3.9 hrs 4.9 hrs

KEY: * = Vss = volume of distribution at steady state.

SOURCES: a = data from McClurkan et al. 1993; b = data from

Valentine et al. 1994.

From a pharmacokinetic standpoint, the potential for this new class

of biologically based drugs (designer antibodies) is very exciting.

Antibodies would be expected to have a totally different set of

disposition parameters (volume of distribution, clearance, metabolism,

and t_) from the drugs against which they are used as antagonists.

Table 1 shows a comparison of the pharmacokinetic parameters for

monoclonal anti-PCP Fab, PCP, and PCP after administration of

anti-PCP Fab. In addition, the antigenicity can be significantly

decreased or completely eliminated simply by digesting the

immunoglobulin G (IgG) with papain and removing the antigenic Fc

(crystalline fragment) portion by chromatographic procedures (Porter

1959; Smith et al. 1979). This yields two Fab fragments (antigenbinding fragments) each with the same affinity as the parent IgG

(figure 1). The Fab fragments also have a ninefold greater apparent

volume of distribution compared to intact IgG (Smith et al. 1979).

Furthermore, the use of monoclonal antibodies allows greater

refinement of both antibody specificity and affinity as well as the

possibility for the production of large quantities of a homogeneous

protein. When the therapeutic use of human monoclonal antibodies

becomes more practical, the potential for protein antigenicity should

be reduced even further when used in humans.

From a basic science point of view, it is very interesting to study the

effects of a high-affinity antibody on the disposition of a drug such as

PCP. This is because PCP has some unique characteristics in terms of

its metabolism and pharmacokinetics. First, in the rat and other

animal
263

species including humans, PCP has an extremely high volume of

distribution (12.6 L/kg in the rat (table 1; Valentine et al. 1994); 6.2

L/kg in man (Cook et al. 1982; see Owens et al. 1987 for a review of

PCP pharmacokinetics across species). In contrast, monoclonal antiPCP Fab appears to have a much smaller volume of distribution (0.55

L/kg or about twice the value of extracellular fluid volume (table 1)

(Valentine et al. 1994)). Second, the systemic clearance of PCP in

the male rat is over 95 percent due to metabolism, with renal

clearance composing only about 1 to 5 percent of total body

clearance (Valentine et al. 1994). For comparison, the renal

clearance of Fab in the rat comprises about 17 percent of total body

clearance. Also, the kidney is an important site for metabolism of

Fab (Arend and Silverblatt 1975; Wochner et al. 1967).

Consequently, it is essential to determine how the high-affinity

monoclonal Fab changes PCP effects and disposition at toxic doses of

PCP (Valentine et al. 1994).

A possible drawback to the use of antibodies for the treatment of

toxicity is the possibility of anti-idiotypes being produced against the

monoclonal antibody binding site. These antibodies could potentially

mimic drug-like structural features necessary for binding to a receptor.

However, antibodies do not cross the blood-brain barrier in significant

amounts and studies to date have not found this to be a problem,

especially when Fab fragments are used.
264

With careful consideration for hapten design, anti-drug antibodies can

be used in the treatment of toxicity due to other structurally related

compounds. These compounds are usually Schedule I drugs because of

their potential for abuse (e.g., 1-[1-(2-thienyl) cyclohexyl] piperdine

(TCP) and N-ethyl-1-phencyclohexylamine (PCE). If these other

drugs were to suddenly become available to drug abusers, as have a wide

variety of fentanyl- and amphetamine-like derivatives, the medical

community would not be prepared to treat the toxic effects of these

other arylcyclohexylamines. Therefore, it would be most prudent if

strategies could be developed for the treatment of a whole class of

drugs rather than individual drugs. Indeed, several

arylcyclohexylamines appear to have similar effects to PCP, except

that they are even more potent. TCP and PCE are about 1.3 to 6

times more potent (respectively) than PCP in drug discrimination

assays (Shannon 1981, 1983) and PCP receptor binding assays (see

Owens et al. 1988 for a review).

After careful consideration for the structure-activity relationship of

arylcyclohexylamines, monoclonal antibodies against a PCP-like

hapten (5-[N-(1'-phenylcyclohexyl)amino]pentanoic acid) were

generated (McClurkan et al. 1993). These antibodies bind to PCP,

TCP, and PCE with a greater affinity than the binding of these ligands

to the PCP receptor (Owens, unpublished observation). The

development of antibody-based approaches for treating drug classes,

rather than just one specific drug, is an exciting possibility and could

provide a prototypic model for designing immunotherapeutic

approaches for other classes of drugs of abuse.

CATALYTIC ANTIBODIES FOR TREATMENT OF DRUG ABUSE

Another experimental approach for the treatment of drug abuse is the

use of catalytic antibodies. The idea behind this treatment is to create

an artificial, antibody-based enzyme that can convert the parent drug

to an inactive metabolite. This catalytic antibody would then be

administered in medical emergencies for overdose or perhaps

administered on a chronic basis to persons trying to withdraw from

addiction to the drug. In the field of drug abuse, the possibility of

using cocaine catalytic antibodies as a therapeutic aid to the treatment

of toxicity and addiction has generated the most clinical interest.

Cocaine is a good prototypic ligand to study for this purpose because

the drug is naturally metabolized by in vivo estearases, and catalytic

monoclonal antibodies with esterase activity are among the few
265

metabolic reactions that have been successfully mimicked. Landry

and colleagues (1993) have generated a monoclonal antibody that can

metabolize cocaine to ecgonine methyl ester and benzoic acid. While

this approach may have some theoretical advantages over the use of

high-affinity anti-drug antibodies, it also has some serious and perhaps

insurmountable disadvantages.

The major theoretical advantage to catalytic antibodies for therapy is

that, unlike most anti-drug antibodies, they release the breakdown

product (metabolite) and are ready to metabolize more drug. Since

they continuously regenerate their capacity (like a natural enzyme),

they could reduce the body burden of drug with perhaps lower molar

ratios of antibody to drug than would be needed for an anti-cocaine

antibody. It has been suggested that they could even be used for

passive immunization of cocaine addicts during periods of

psychological and social rehabilitation to help prevent or blunt drug

effects if the patient self-administered cocaine. While these suggested

uses could be important from a pharmacokinetic, pharmacodynamic,

and immunological point of view, these uses may prove difficult to

achieve.

The use of catalytic antibodies for acute toxicity in humans would

most likely be only marginally helpful. It is difficult to conceive that

this medical approach would have significant advantages over highaffinity anti-cocaine antibodies such as those currently in use for

treating digoxin toxicity. It is true that catalytic antibodies could

potentially regenerate their capacity after enzymatic turnover of

cocaine, theoretically allowing a higher overall capacity for the

antibody antagonist. However, in terms of therapeutic effect, rapid

reduction of tissue concentrations may be the critical factor in saving

lives.

Sudden death is a major factor in cocaine-related fatalities. The

mechanism for the cardiotoxicity is not fully understood, but it

appears to be poorly predicted from patient to patient. Nevertheless,

a rapid immuno-therapeutic response would be essential in treating

cocaine-related toxicity, and even catalytic antibodies with low

micromolar (M) Michaelis-Menton constant (K m) values may not

be fast-acting enough. By comparison, high-affinity anti-drug

antibodies typically have dissociation constant (Kd) values in the low

nanomolar (nM) to high picomolar (pM) range. Therefore, to test

the usefulness of catalytic antibodies, it will be important to conduct

full dose-response curves with the cocaine catalytic antibodies and to
266

compare these results with the therapeutic effects of similar doses of

high-affinity anti-cocaine antibodies as the control.

Currently the immunotherapy literature is not clear about the dose of

antibody that would be needed to rapidly reduce the body burden, and

thereby the toxicity, of drugs. Nevertheless, some studies suggest it is

necessary to use approximately 0.3 to > 1.0 mole equivalent doses of

anti-drug antibodies. Since the Km values for cocaine catalytic

antibodies are likely to be relatively high compared to the Kd values

for anti-drug antibodies, it is especially difficult to estimate the

amount of catalytic antibodies that would be necessary to be effective

in various medical situations. Nevertheless, based on the expected

high Km values for cocaine metabolism, the amount of antibody

needed for effective treatment will likely be significantly greater than

the 0.3 to 1.0 mole equivalents of a high-affinity (low Kd value) anticocaine antibody.

Therefore, the greater potential capacity for enzymatic inactivation

by catalytic antibodies might be more than offset by their lower

effective affinities (i.e., higher Km values). The higher Km values

would result in very slow metabolic inactivation. The metabolic

capacity of endogenous enzyme systems that metabolize cocaine are

very high and very efficient relative to the currently available

catalytic antibodies. In addition, high- affinity anti-cocaine

antibodies are easier to produce than cocaine catalytic antibodies.

Since the terminal elimination t_ of cocaine is less than 2 hours

(Cook et al. 1985) and the terminal elimination t_ for Fab fragments

and IgG are on the order of several hours to a day or more, the

normal in vivo metabolic pathways for cocaine could also serve to

metabolize unbound drug and thereby regenerate these cocainespecific antibodies. This would assume that the high-affinity anticocaine antibodies would not significantly cross-react with cocaine

metabolites.

As another minor point, the use of catalytic antibodies with esterase

activity could theoretically lead to autoimmunity against endogenous

enzymes. This problem could be partially overcome if Fab fragments

were used, or perhaps completely overcome if humanized antibodies

or their fragments were used. Also, it is not currently known if

endogenous ligands will be substrates for these catalytic enzymes; this

could potentially decrease the effective rate for cocaine metabolism

or lead to other medical problems.
267

It has also been suggested that cocaine catalytic antibodies could be

used for chronic treatment (for months) of cocaine addicts to

augment behavioral modification therapy while patients are

withdrawing from the drug. The theory is that if high doses of

cocaine catalytic antibodies were present in a recovering addict who

then used cocaine, the drug would be efficiently removed or the

effects would at least be blunted by the catalytic antibodies. Although

this treatment would presumably prevent addicts from adding to their

dependence on the drug, it could possibly lead to a dangerous clinical

situation. It is difficult to imagine that enough cocaine catalytic

antibody could be continuously admini-stered to blunt the effects of

cocaine, since enterprising patients would simply take increasing

amounts of drug until they obtained the desired effect. In addition,

the presence of cocaine catalytic antibodies could actually be a

problem because the patient’s attempts to titrate effects would be

very unpredictable. For instance, if the patient surmounted the

effects of the catalytic antibody by self-administering high doses soon

after immunotherapy, this dose of cocaine would be too high even a

day later when the concentration of the catalytic antibodies had

significantly deceased. This could lead to an unexpected overdose.

For now, the major strength in the use of catalytic antibodies is the

basic science underlying generation of this unique pharmacological

tool. However, it is proving extremely challenging to develop a

catalytic antibody with a turnover rate that even approaches the

turnover rates of natural cocaine-metabolizing enzymes such as

butyrylcholinesterase. Therefore, researchers should first determine if

similar approaches, which are currently more technically feasible,

might serve the same purpose. For instance, butyrylcholinesterase

purified from human blood or high- affinity monoclonal antibodies

might be more useful. Nevertheless, basic research on cocaine

catalytic antibodies should be pursued since the development of

pharmacokinetic and metabolic modifiers of abused drugs is an

underexplored area of medications development.

ANIMAL MODELS FOR TESTING IMMUNOTHERAPY

As discussed earlier, high-affinity anti-drug antibodies produce effects

on the pharmacokinetics and pharmacodynamics of drugs in animals

and humans. For new therapeutic applications, these effects need to

be fully tested in animals before administration to humans. In

addition, from a basic science viewpoint, it will be necessary to

develop relevant pharmacokinetic and pharmacodynamic models of
268

the effects of immunotherapy on drug abuse. Examples of the

scientific questions that need to be addressed are as follows.

The relationships between antibody dose, antibody affinity (or

catalytic antibody Km), and their effects on the drug’s

pharmacokinetics and pharmacodynamics are poorly understood. For

instance, if the drug effect compartment is associated with the

pharmacokinetic peripheral compartment, the time needed to reverse

drug effects with antibodies would be predicted to be slower than if the

effect compartment is associated with the pharmacokinetic central

compartment. In addition, it appears that high-affinity antibodies

block the metabolism and/or change the metabolic profile of drugs

(Owens and Mayersohn 1986; Valentine et al. 1994). These are

complex changes that need to be studied in detail, and

pharmacokinetic and pharmacodynamic models of these effects need

to be developed in animal models before use in humans.

One way to model the genetic polymorphism of metabolism and drug

effects in humans is to use the genetic diversity in different strains

and sexes of animals such as rats. Although there are not significant

sex-related differences in metabolism in humans, sex-related

differences in rat liver function have been identified in the last few

decades (see Zaphiropoulos et al. 1989 for a review). For example,

sexual dimorphism in metabolism with cytochrome P-450 (CYP) rat

isozymes occurs with CYP2C13, CYP2C11, CYP2C12, CYP3A2,

CYP2A2, and CYP2E1 (Zaphiropoulos et al. 1989). In particular,

there are major differences in either the amounts or the expression of

the CYP family of enzymes. These enzymes catalyze the oxidation

of many steroids, xenobiotics, and drugs (Waxman et al. 1985).

These sexual differences are most extensively characterized in rats; in

general, the CYP-catalyzed reactions are more efficient in male rats

than in female rats. Studying these differences in the pharmacological

response resulting from sex-related differences in rat metabolism

should provide a useful model for predicting differences in the

pharmacological response resulting from genetic polymorphism in

human metabolism.

Although these sex-related differences in metabolism have been

known for a long time, there is very little data available concerning

the role of gender in the metabolism and pharmacokinetics of drugs of

abuse. For some drugs of abuse, sexual dimorphism could have

important implications and adverse consequences. For PCP, a subject

of this discussion, metabolism is the major mechanism for

inactivation of pharmacological effects (Mayersohn 1985). In
269

normal rats, recent data implicate the constitutive CYP2D1 and

CYP2C11 isozymes in the formation of PCP metabolites (Hiratsuka

et al. 1995; Shelnutt et al., in press).

CONCLUSIONS

Immunotherapy for treating drug abuse appears to be a viable

therapeutic approach. To make this experimental treatment a

reality, some major hurdles must be overcome. These include the

production and purification of very large quantities of antibody at a

reasonable cost and increasing the safety of using antibodies in

humans. Increased production is currently possible through the use of

bioreactors for large-scale production of monoclonal antibodies.

Nevertheless, the cost of the antibody is still too high for the day-today use in most emergency rooms and clinics. As the technology

improves, the cost should be dramatically reduced. To make

antibodies safer for use in humans it should be possible to engineer

human monoclonal antibodies or to make very small, high-affinity

binding proteins based on the knowledge gained from antibody binding

site templates.

REFERENCES

Arend, W.P., and Silverblatt, F.J. Serum disappearance and catabolism of

homologous immunoglobulin fragments in rats. Clin Exp

Immunol 22:502-513, 1975.

Aronow, R., and Done, A.K. Phencyclidine overdose: An emerging concept of

management. J Am Coll Emerg Phys 7:56-59, 1978.

Chaudieu, I.; Vignon, J.; Chicheportiche, M.; Kamenka, J.; Trouiller, G.; and

Chicheportiche, R. Role of an aromatic group in the inhibition

of phencyclidine binding and dopamine uptake by PCP analogs.

Pharm Biochem Behav 32:699-705, 1989.

Colburn, W.A. Specific antibodies and Fab fragments to alter the

pharmacokinetics and reverse the pharmacologic/toxicologic

effects of drugs. Drug Metab Rev 11:223-262, 1980.

Cook, C.E.; Brine, D.R.; Jeffcoat, A.R.; Hill, J.M.; Wall, M.E.; Perez- Reyes, M.;

and DiGuiseppi, S.R. Phencyclidine disposition after intravenous

and oral doses. Clin Pharmacol Ther 31:625-634, 1982.

Cook, C.E.; Jeffcoat, A.R.; and Perez-Reyes, M. Pharmacokinetic studies of

cocaine and phencyclidine in man. In: Barnett, G., and Chiang,

C.N., ed. Pharmacokinetics and Pharmacodynamics of
270

Psychoactive Drugs: A Research Monograph. Foster City, CA:

Biomedical Publications, 1985. pp. 48-74.

Hiratsuka, A.; Chu, T.Y.Y.; DiStefano, E.W.; Lin, L.Y.; Schmitz, D.A.; and Cho,

A.K. Inactivation of constitutive hepatic cytochromes P450 by

phencyclidine in the rat. Drug Metab Dispos 23:201-206, 1995.

Itil, T.; Keskiner, A.; Kiremitci, N.; and Holden, J.M.C. Effects of phencyclidine

in chronic schizophrenics. Can Psychiatr Assoc J 12:209-212,

1967.

Landry, D.W.; Zhoa, K.; Yang, G.X.-Q.; Glickman, M.; and Georgiadis, T.M.

Antibody-catalyzed degradation of cocaine. Science 259:1899-

1900, 1993.

Luby, E.D.; Gottlieb, J.S.; Cohen, B.D.; Rosenbaum, G.; and Domino, E.F. Model

psychoses and schizophrenia. Am J Psychiatr 119:61-67, 1962.

Lodge, D.; Anis, N.A.; Berry, S.C.; and Burton, N.R. Arylcyclo-hexylamines

selectively reduce excitation of mammalian neurons by

aspartate-like amino acids. In: Kamenka, J.-M.; Domino, E.F.;

and Geneste, P., eds. Phencyclidine and Related

Arylcyclohexylamines: Present and Future Applications. Ann

Arbor, MI: NPP Books, 1983. pp. 595-616.

Mayersohn, M. Rational approaches to treatment of drug toxicity: Recent

considerations and application of pharmacokinetic principles.

In: Barnett, G., and Chiang, C.N., eds. Pharmacokinetics and

Pharmaco- dynamics of Psychoactive Drugs: A Research

Monograph. Foster City, CA: Biomedical Publications, 1985.

pp. 120-142.

McClurkan, M.B.; Valentine, J.L.; Arnold, L.; and Owens, S.M. Disposition of a

monoclonal anti-phencyclidine Fab fragment in rats. J

Pharmacol Exp Ther 266:1439-1445, 1993.

Olney, J.W.; Labruyere, J.; and Price, M.T. Pathological changes induced in

cerebrocortical neurons by phencyclidine and related drugs.

Science 244:1360-1362, 1989.

Olney, J.W.; Labruyere, J.; Wang, G.; Wozniak, D.F.; Price, M.T.; and Sesma,

M.A. NMDA antagonist neurotoxicity: Mechanism and

prevention. Science 254:1515-1518, 1991.

Owens, S.M., and Mayersohn, M. Phencyclidine-specific Fab fragments alter

phencyclidine disposition in dogs. Drug Metab Disp 14:52-58,

1986.

Owens, S.M.; Hardwick, W.C.; and Blackall, D. Phencyclidine pharmacokinetic

scaling among species. J Pharmacol Exp Ther 242:96-101,

1987.

Owens, S.M.; Zorbas, M.; Lattin, D.L.; Gunnell, M.; and Polk, M. Antibodies

against arylcyclohexylamines and their similarities in binding

specificity with the phencyclidine receptor. J Pharmacol Exp

Ther 246:472-478, 1988.
271

Peterson, R.C., and Stillman, R.C. Phencyclidine: An overview. In: Peterson,

R.C., and Stillman, R.C., eds. Phencyclidine (PCP) Abuse: An

Appraisal. National Institute on Drug Abuse Research

Monograph 21. DHHS Pub. No. (ADM)78-728. Washington,

DC: Supt. of. Docs., U.S. Govt. Print. Off., 1978. pp. 1-17.

Porter, R.R. The hydrolysis of rabbit gamma-globulin and antibodies with

crystalline papain. Biochemistry 73:119-126, 1959.

Rothman, S. Synaptic release of excitatory amino acid neurotransmitter

mediates anoxic neuronal death. J Neurosci 4:1884-1891, 1984.

Shannon, H.E. Evaluation of phencyclidine analogs on the basis of their

discriminative stimulus properties in the rat. J Pharmacol Exp

Ther 216:543-551, 1981.

Shannon, H. Discriminative stimulus effects of phencyclidine: Structure-activity

relationships. In: Kamenka, J.-M.; Domino, E.F.; and Geneste,

P., eds. Phencyclidine and Related Arylcyclohexylamines:

Present and Future Applications. Ann Arbor, MI: NPP Books,

1983. pp. 311-335.

Shelnutt, S.R.; Badger, T.M.; and Owens, S.M. Phencyclidine metabolite

irreversible binding in the rat: Gonadal steroid regulation and

CYP2C11. J Pharmacol Exp Ther, in press.

Smith, T.W.; Butler, V.P.; Haber, E.; Fozzard, H.; Marcus, F.I.; Brenner, W.F.;

Schulaman, I.C.; and Phillips, A. Treatment of life-threatening

digitalis intoxication with digoxin-specific Fab antibody

fragments. N Engl J Med 307:1357-1362, 1982.

Smith, T.W.; Lloyd, B.L.; Spicer, N.; and Haber, E. Immunogenicity and kinetics

of distribution and elimination of sheep digoxin-specific IgG and

Fab fragments in the rabbit and baboon. Clin Exp Immunol

36:384-396, 1979.

Valentine, J.L.; Arnold, L.W.; and Owens, S.M. Anti-phencyclidine Fab

fragments markedly alter phencyclidine pharmacokinetics in

rats. J Pharmacol Exp Ther 269:1079-1085, 1994.

Vignon, J.; Pinet, V.; Cerruti, C.; Kamenka, J.; and Chicheportiche, R. [3H]N-[1-

(2-Bento (b) thiophenylcyclohexyl] piperidine ([3H]BTCP): A

new phencyclidine analog selective for the dopamine uptake

complex. Eur J Pharmacol 148:427-436, 1988.

Vincent, J.P.; Kartalorski, B.; Geneste, P.; Kamenka, J.-M.; and Lazdunski, M.

Interaction of phencyclidine ("angel dust") with a specific

receptor in rat brain membranes. Proc Nat Acad Sci U S A

76:4678-4682, 1979.

Waxman, D.J.; Dannan, G.A.; and Guengerich, F.P. Regulation of rat hepatic

cytochrome P-450: Age-dependent expression, hormonal

imprinting, and xenobiotic inducibility of sex-specific isozymes.

Biochemistry 24:4409-4417, 1985.
272

Wessinger, W.D. Sexual dimorphic effects of chronic phencyclidine in rats. Eur

J Pharmacol 277:107-112, 1995.

Wochner, R.D.; Strober, W.; and Waldman, T.A. The role of the kidney in the

catabolism of Bence-Jones proteins and immunoglobulin

fragments. J Exp Med 126:207-221, 1967.

Zaphiropoulos, P.G.; Mode, A.; Norstedt, G.; and Gustafsson, J. Regulation of

sexual differentiation in drug and steroid metabolism. Trends

Pharm Sci 10:149-153, 1989.

Zukin, S.R., and Zukin, R.S. Specific 3H phencyclidine binding in rat central

nervous system. Proc Nat Acad Sci U S A 76:5372-5376, 1979.

ACKNOWLEDGMENTS

This chapter was prepared with the support of NIDA grants DA 04136 and

DA 07610, and a Research Scientist Development Award (K02 DA 00110)

to S.M. Owens.

AUTHOR

S. Michael Owens, Ph.D.

Professor

Department of Pharmacology and Toxicology

University of Arkansas for Medical Sciences

Slot 611

4301 West Markham Street

Little Rock, AR 72205
273

Toxicokinetics in the National

Toxicology Program

Thomas J. Goehl

BACKGROUND

The National Toxicology Program (NTP) was established by the

Department of Health and Human Services (DHHS) in 1978 to

coordinate and manage the department’s toxicology studies. The

objective of DHHS’ toxicology efforts is to develop scientific

information necessary to protect the health of the public from

exposure to hazardous chemicals. The NTP consists of the National

Institutes of Health's (NIH) National Institute of Environmental

Health Sciences (NIEHS), the Centers for Disease Control and

Prevention's (CDC) National Institute of Occupational Safety and

Health (NIOSH), and the Food and Drug Administration's (FDA)

National Center for Toxicological Research (NCTR). The NTP is

administered by a director, who is also the director of NIEHS. An

executive committee consisting of heads of Federal health research

and regulatory agencies (Consumer Products Safety Commission

(CPSC), Environmental Protection Agency (EPA), FDA, National

Cancer Institute (NCI), NIEHS, NIOSH, and Occupational Safety and

Health Administration (OSHA)) and a board of scientific counselors

(composed of governmental, industrial, and academic scientists)

provide guidance for the program (figure 1).

The NTP toxicological evaluations are initiated on chemicals that are

nominated for study. These nominations can come from the private

or public sector, and are sent to Chemical Nominations, National

Toxicology Program, NIEHS, PO Box 12233, Maildrop A001,

Research Triangle Park, NC 27709. After an extensive nomination

review process, selections are made and each chemical assigned to an

NTP scientist who serves as the toxicology study project director. A

project team is then formed by the project director to review the

literature and identify data gaps. Basic information on chemical

disposition, chemistry, genetic toxicology, and health and safety are

developed as needed and provided to the project director for

consideration in the design of toxicology protocols. The toxicology

study protocols that are developed may include a general toxicological

evaluation and/or specific target organ studies (figure 2).
274

OVERVIEW

Toxicokinetics is a term used for describing kinetic studies conducted

in conjunction with toxicology evaluations (Di Carlo 1982) that deal

with absorption, distribution, and elimination processes of chemicals

present at concentrations that produce toxic effects. By monitoring

the blood concentrations of the chemical and/or metabolites over

time after administration by different routes, the test chemical's

bioavailability and kinetic characteristics can be readily obtained. The

data also permit the determination of the so-called linear dose range

based on area under the plasma versus time curve and clearance or

other related toxicokinetic parameters, as well as the prediction of

possible bioaccumulation after multiple doses. Changes in kinetic

parameters after multiple exposures
275

are indicative of perturbations in the normal way the animal handles

the chemical (e.g., enzyme induction or inhibition). Techniques such

as physiologically based toxicokinetic (PBTK) modeling have been

used (Bischoff 1967; Ramsey and Andersen 1984; Teorell 1937) to

predict the target organ and blood concentrations at various doses.

Other work has centered on developing interspecies scaling factors

(Mordenti 1986; Travis and White 1988).

During recent years, the importance of toxicokinetic information in

risk evaluation has become more widely recognized (Yacobi et al.

1989), including by the NTP. In fact, as part of the activities prior

to the conduct of an NTP toxicology study, the determination of

basic toxicokinetic information is often conducted to aid in study

design. During the conduct of NTP toxicology studies, evaluation of

internal dose and changes in kinetic parameters are also frequently

conducted to aid in the interpretation of toxicology study outcomes.

Generally the internal dose is based on plasma concentrations, since

the target organ for the toxicologic effect generally has not been

identified as yet. However, after a PBTK model has been developed,
276

the target organ dose can later be estimated using the collected plasma

data. Most of the NTP effort to date has been given to 2-year

toxicology and carcinogenicity studies. The application of NTP's

toxicokinetic studies to toxicology study design and interpretation is

discussed in this chapter.

GOALS

Over the years, NTP, a worldwide leader in toxicology, has conducted

chemical disposition studies. More recently, NTP and others

(Jameson and Goehl 1994; World Health Organization 1986) have

placed even greater emphasis on such studies, especially those that

evaluate the kinetics. The scope of the NTP toxicology effort can be

seen in the listing of projected NTP toxicology studies for Fiscal Year

1996 (figure 3). Many of the studies listed will include toxicokinetic

evaluations. It is anticipated that the toxicokinetic data collected will

ultimately improve the usefulness of the toxicology study for risk

characterization.

Specifically, the goals of the toxicokinetic evaluations conducted

prior to the conduct of toxicology studies are to assist in the selection

of the animal species and strain, dose vehicle, dosing route, and

dosages. The goals of the determination of concentrations of test

chemical or metabolite concentrations in biological samples during

the course of the toxicology study are to estimate the internal dose

and its possible correlation to toxic effects, as well as to evaluate the

effect of age and multiple exposure on the disposition kinetics (figure

4).

APPROACH

If the available toxicokinetic information does not provide enough

information to aid in toxicology study design, NTP designs an

upfront toxicokinetic study. The doses for the toxicokinetic

study are chosen to reflect those anticipated to be used in the

toxicology studies. The most common species used by the NTP

are Fischer 344/N rats and B6C3F1 mice. The approach used by

the NTP involves three steps: preliminary
277
278

studies, definitive studies, and studies conducted during the in life

toxicology evaluations. The first step in producing the needed

toxicokinetic data is to develop an analytical method to quantify the

test chemical or metabolite in a biological sample. Most of the time

blood plasma is used; all subsequent discussions will specifically refer to

the analysis of the analyte in plasma. If the anticipated plasma

concentrations and optimal blood sampling times are not known, it is

often necessary to conduct a preliminary toxicokinetic study. This

type of study involves the administration of the test chemical at the

lowest and highest doses proposed for the toxicology study to a small

number of animals by appropriate routes and then drawing blood

samples at approximately 12 time points. The samples are analyzed

using an unvalidated analytical method. Based on these results, the

analytical method is further developed to accommodate the

determined concentration range and then validated (see next section

on analytical method validation). The stability of the analyte in the

biological material is subsequently determined at 75 percent of the

highest expected concentration for the period of time over which the

samples are to be stored prior to analysis (figure 5).

The second step is to conduct definitive toxicokinetic studies (figure

6). In the initial single-exposure definitive study, the chemical is

intravenously administered to the animals at two doses, using

sufficient animals so that three data points are available at each of 10

sampling times. (The number of sampling times depends on the

kinetic profile suggested by the preliminary toxicokinetic studies.) It

is hoped that the intravenous (IV) single-exposure definitive study

results in plasma concentrations that are directly proportional to dose

and provides basic toxicokinetic parameters. The IV study is

conducted regardless of the exposure route planned for the toxicology

study.

In the case of toxicology studies that will use the gavage or dermal

route of exposure, additional animals are dosed by gavage or dermally

at three doses covering the proposed toxicology study dose range,

again using sufficient animals so that three data points will be

available at each sampling time. Often the doses for the

toxicokinetic evaluation are the minimally toxic dose (MTD) and

fractions of the MTD. This study allows for the determination of the

absorption rate, dose proportionality, and, along with the results of

the IV route study, the bioavailability. It is important to note that

since the interpretation of the results of the toxicokinetic studies

depends on the accuracy of animal dosing, the dose
279

formulations need to be characterized by homogeneity, stability, and

accuracy of dose preparation. A number of the NTP 2-year
280

toxicology and carcinogenicity studies use the feed or drinking water

route for test chemical exposure. How to use the kinetic data from

single bolus dose studies in the design and evaluation of feed or

drinking water studies is a challenge to the toxicokineticist. To help

address this issue, a computer model was developed (Yuan 1993) to

incorporate the animal feed or drinking habits together with the

kinetic behavior of chemicals to predict the blood concentration

profiles during dosed-feed or drinking water studies. The needed

absorption rate and dose proportional range are determined from

kinetic studies using the gavage route. The top dose for the gavage

toxicokinetic study is calculated to be between one-half and onequarter of the total daily exposure at the MTD based on normal feed

or water consumption and bodyweight while the other doses are

fractions of the top dose. Using the model, plasma concentrations

are predicted for the planned feed or drinking water study. A feed or

drinking water toxicokinetic study is then conducted and blood

samples are taken over the course of a 7- to 14-day exposure period.

If the determined plasma concentrations fall in the range of those

achieved after gavage dosing, which themselves resulted in dose

proportional concentrations, then the dosed-feed/water

concentrations are assumed to be in the dose- proportional range as

well.

In support of inhalation studies, blood samples are taken after a 4- to

6-hour whole-body or nose-only exposure period. The steady-state

plasma concentration and the elimination rate are determined. The

steady-state concentrations are plotted versus exposure concentration

to make the determination of dose proportionality. A kinetic model

is developed and used to predict steady-state concentrations and the

time to reach steady state.

Multiple exposure studies may include obtaining information on

possible enzymatic inhibition or induction effects as well as the

possibility of bioaccumulation. In such studies, animals are dosed by

gavage (or other appropriate route) for 14 days with one dose, usually

the highest anticipated dose; sufficient animals are used so that three

data points are available at each blood sampling time. Blood samples

are taken at multiple time points after dosing and analyzed for test

chemical or metabolite. These results are compared to the results of

the single- exposure definitive study to determine possible enzymatic

inhibition or induction effects and bioaccumulation of the test

chemical.
281

The third step is to conduct toxicokinetic studies in conjunction with

the actual toxicology studies to estimate internal dose as well as

possible changes in the kinetics after repeated dosing and aging. The

doses used in the 2-year carcinogenicity study are chosen so that, if

possible, the two lower doses are in the linear kinetic range to

facilitate extrapolation to lower doses and interspecies scaling while

the top dose is chosen to be the MTD.

Another challenge to the toxicokineticist involves relating any

observed changes in kinetics during a long-term toxicology study to

age effects. In an attempt to address this issue, the kinetics of the

chemical in a control group of animals are determined at 18 months

of a 2-year study. Extra animals are added to each dose group in the

2-year study and used at 2 weeks and at 3, 12, and 18 months of

dosing so that it is possible to follow any changes in kinetic behavior

as the dosing progresses.

An appendix is included that provides examples of four current

toxicokinetic study protocols for chemicals scheduled for

toxicological evaluation using oral (gavage and feed), topical, and

inhalation routes.

ANALYTICAL METHOD VALIDATION

Validation of biological sample analysis methods is critical to the

evaluation of the final data. A description of the validation procedure

utilized by the NTP is provided here. The method needs to

demonstrate the appropriate specificity, precision, absolute recovery,

measurement limits, and relative error. An evaluation of the blank

biological sample matrix contribution to responses seen in spiked

samples also needs to be determined.

In conducting the validation, spiked biological sample standards are

prepared at six different concentrations, using two independently

prepared stock standards of different concentrations. Spiked

biological sample standards at the lowest and highest concentrations

are prepared in triplicate. Single spikes are prepared at the four

intermediate concentrations. Biological sample blanks collected from

five animals are prepared in triplicate. The spiked standards are

prepared so that every other standard comes from one stock solution

while the remaining standards are prepared from the second stock

solution. The instrumental response from a single analysis of each

biological standard and blank is then recorded. A series of solvent
282

standard solutions with the same final concentrations (i.e., after any

extraction, dilution, or concentration step) are also prepared. The

same standard stock solutions, prepared as described above, are used

for making these solutions. A reagent blank in the same solvent as

the standards is also prepared. Once all standards are prepared, the

analytical response is determined and calculations made to evaluate

the correlation coefficient for the solvent and biological sample

standard curve data as well as the precision, percent relative standard

deviation (percent RSD), the percent recovery (based on a

comparison of responses of the biological and solvent standards), the

relative error, and the measurement limits.

The limit of detection (LOD) is defined as three times the standard

deviation (SD) of the blank response expressed as concentration. The

limit of quantitation (LOQ) is defined as 10 times the SD of the blank

response expressed as concentration. If there is no blank value, the

SD of the lowest concentration should be used in the calculations.

Often another estimate of the LOQ is used, the experimental limit of

quantitation (ELOQ). This value is defined as the lowest biological

sample standard concentration that has been analyzed and that has a

defined relative error and a defined RSD (e.g., 15 percent relative

error and 15 percent RSD).

During the analysis of the actual study samples, a standard curve using

a series of spiked biological sample standards is generated with each

batch of samples. The standard curve is analyzed prior to any study

samples. Once analysis of study samples has been initiated, quality

control samples are run at fixed intervals throughout the analytical

run. An acceptable frequency depends on the method being used, but a

common approach is to run a quality control sample after every 10

study samples. Quality control samples are spiked biological sample

standards that are prepared at the time the actual study samples are

collected and stored under the same conditions as the study samples.

Quality control samples are usually prepared at two or three

concentrations that cover the expected study sample concentrations.

EXAMPLES

Over the last several years, the NTP has increasingly focused on the

generation of toxicokinetic data and its application in toxicology

study design and interpretation. Examples are presented to illustrate

how NTP has utilized the toxicokinetic data.
283

3'-Azido-3'-deoxythymidine

3'-Azido-3'-deoxythymidine (AZT) is the first FDA-approved drug in

the United States for the treatment of acquired immunodeficiency

syndrome (AIDS). To aid in the design of a planned 2-year

carcinogenicity study, the bioavailability and dose proportionality of

AZT in B6C3F1 mice of both sexes were evaluated (Trang 1993).

The chemical was administered IV or intragastrically at doses of 15,

30, and 60 milligrams per kilogram (mg/kg). There were no

differences in the kinetics of AZT between the sexes. The

elimination half-life was 18 minutes and the bioavailability was 85

percent. Dose proportionality was confirmed over the doses planned

in the chronic study.

Benzyl Acetate

Benzyl acetate is widely used as a flavoring agent in the food industry

and as a fragrant ingredient in a variety of consumer products such as

soaps and lotions. The NTP conducted two 2-year carcinogenicity

studies in F344 rats and B6C3F1 mice, one using the daily gavage

administration and the other using dosed-feed administration.

Although the daily doses of benzyl acetate were comparable between

the two studies, hepatic tumorigenicity was only observed in the

gavage studies of mice. In contrast, necrotic lesions were seen in the

brain in both the feed and gavage studies. Comparative toxicokinetic

studies were conducted in F344 rats and B6C3F1 mice to estimate the

impact of gavage (500 mg/kg in rats and 1000 mg/kg in mice using

corn oil as the vehicle) versus dosed-feed administration (dosed-feed

pentachlorophenol concentrations provide a daily dose of 648 mg/kg

for rats and 900 mg/kg for mice) on the toxicokinetics of benzyl

acetate and to provide further information for interpreting the

toxicity differences observed (Yuan et al. 1995). In an in vitro study,

the half-life of benzyl acetate was found to be less than a minute;

therefore, it was decided to monitor internal dose based on benzyl

alcohol, hippuric acid, and benzoic acid plasma concentrations. In

rats and mice, the benzoic acid plasma concentrations after gavage

dosing were about a hundred times the concentrations found in a study

in which benzyl acetate was administered in the feed. In both studies

the benzyl alcohol plasma concentrations were less than 0.1

micrograms per milliliter (g/mL) while hippuric acid concentrations

were about 20 percent of the benzoic acid concentrations. In the

benzyl acetate study, results of the plasma analyses showed that the

method of dosing had a major impact on the internal dose of the
284

metabolite, benzoic acid, which may explain the route-dependent

differences in toxicity.

Cadmium Oxide

Cadmium oxide has many industrial uses, including anticorrosion

coating for iron, copper, and steel. The lung burden and systemic

exposure in male Fischer 344 rats were evaluated during a 13-week

toxicology study in which the animals were exposed to aerosolized

cadmium oxide at concen-trations of 0.1, 0.25, or 1.0 mg per cubic

meter (mg/m3) for 6 hours a day (Dill et al. 1994). Concentrations of

cadmium were deter-mined in the blood, kidney, and lung during the

course of the toxicology study. Accumulated cadmium in the lungs

was not proportional to exposure concentration. Although the

concentrations of cadmium in the blood were very low, the amount in

the kidneys represented a significant fraction of the lung burden.

Thus there was a significant systemic exposure after inhalation of the

aerosolized cadmium oxide.

p-Chloro-,,-trifluorotoluene

p-Chloro-,,-trifluorotoluene (CTFT) is a widely used chemical

intermediate in the manufacture of dinitroaniline herbicides. Various

routes of administration were considered. The inhalation route was

excluded because of the expense involved in the conduct of inhalation

studies. The chemical's volatility excluded the conduct of a dermal or

dosed-feed study, while its insolubility in water excluded the conduct

of a drinking water study. Therefore a gavage study was selected.

Corn oil was considered as the vehicle but administration of corn oil

has been related to increases in the incidences of pancreatic lesions in

male F344 rats. Therefore a new approach was considered, that is,

molecular encapsulation of CTFT with a-cyclodextrin (CD), which

might then be soluble in water. To evaluate the acceptability of this

approach a toxicokinetic study was conducted in male Fischer 344N

rats (Yuan et al. 1991a). Animals were dosed IV at 4.7 mg/kg using a

10 percent Tween 80 aqueous solution, and intragastrically at 10, 50,

and 400 mg/kg doses in either corn oil or CD-complex in water.

Bioavailability was shown to be complete for both vehicles and dose

proportionality was established up to at least 400 mg/kg. It was

concluded that CD could be used in the planned toxicology studies of

CTFT.

Codeine
285

Codeine is an opioid that is an effective analgesic and antitussive

therapeutic agent. NTP conducted a 2-year carcinogenicity study in

Fischer 344 rats of both sexes with codeine administered in the feed

(400, 800, and 1,600 parts per million (ppm)). Toxicokinetic

information from rats in the 2-year study was developed for

comparison to human data to serve as one basis for extrapolating the

results from the rodent study to humans (Yuan et al. 1992, 1994c).

Blood samples were collected from the rats on days 7, 21, and 90 at

7:00 pm, 11:00 pm, 3:00 am, and 7:00 am. Additional collections

were made at 16 and 24 months between 6:00 and 8:00 am. Plasma

concentrations of codeine and its metabolite, morphine, were

determined. Bioavailability of codeine using the dosed feed route

increased with dose (from 10 to 25 percent). The concentrations of

codeine in rats receiving 800 ppm in the feed were comparable with

the mean concentration reported in humans receiving a 60 mg oral

dose, while the determined concentrations of morphine conjugates in

the rat are much higher than reported in humans. The presence of

morphine conjugates might have a significant impact on the

interpretation of the rat codeine 2-year toxicology study.

2’,3’-Dideoxycytosine

2’,3’-Dideoxycytosine (DDC) is a drug approved for the treatment of

AIDS. Plasma concentrations of DDC were determined at the

conclusion of the 180-day short-term toxicology study, and the

kinetic profile of DDC was determined for B6C3F1 and NIH Swiss

mice. The incidence of thymic lymphoma found in this 6-month

study correlated directly to internal dose as measured by area under

the plasma versus time curve, but not the administered dose. This

finding was applied to both strains of mice studied (B. Collins,

personal communication, September 12, 1994).

2'3'-Dideoxyinosine

2'3'-Dideoxyinosine (DDI) is a therapeutic agent used in the

treatment of AIDS. DDI is known to be easily hydrolyzed in the

presence of acid. To aid in the selection of a vehicle for

administration of DDI in planned toxicological evaluations, the

bioavailability of DDI was determined in B6C3F1 mice of both sexes

after intragastric administration in buffered and unbuffered

formulations (R. Handy, personal communication, January 11, 1994).

The significant effect of vehicle pH on the absorption of DDI was

demonstrated by comparison of plasma DDI concentrations from

buffered versus unbuffered oral dose formulations. The bioavailability
286

was 60 percent from buffered aqueous formulations and only 10

percent from unbuffered formulations.

o-Nitroanisole

o-Nitroanisole is widely used as a dye intermediate. A toxicology

study was planned with the chemical administered in feed. In

preliminary work to develop a method for confirmation of the

accuracy of dose formulations, it was found that the complete

recovery of the chemical from feed formulations required more severe

extraction conditions as the feed formulations aged. There was

concern that the increased strong binding of the chemical to feed

constituents might affect the bioavailability in toxicology studies. To

evaluate the bioavailability of o-nitroanisole from feed formulations,

the urinary concentrations of o-nitroanisole's main metabolites, free

and conjugated o-nitrophenol, were determined in 7-day dosed-feed

studies conducted in male F344 rats using freshly prepared and aged

dosed-feed formulations (Yuan et al. 1991b). No differences were

found in the extent of bioavailability from the fresh and aged feed.

Oxazepam

Oxazepam is a widely prescribed benzodiazepine antianxiety agent and

a common metabolite of many other benzodiazepines including

diazepam and chlordiazepoxide. To aid in the assessment of risks

associated with human use of this drug, the comparative toxicokinetic

studies were conducted in F344 rats, B6C3F1 mice, and Swiss-Webster

mice of both sexes after an IV dose of 20 mg/kg and oral gavage doses

of 50, 200, and 400 mg/kg (Yuan et al. 1994b). In addition, since the

NTP 2-year toxicology study used dosed feed, the toxicokinetics of

oxazepam were also investigated in a 3-week dosed-feed study in male

B6C3F1 mice at 125 and 2,500 ppm. Results indicated that the

elimination of oxazepam from plasma after IV injection in both rats

and mice were first-order and could best be described by a twocompartment model with a terminal elimination half-life of 4 to 5

hours for rats and 5 to 7 hours for mice. At all doses studied, the

females had significantly higher plasma concentrations than males.

At 50 mg/kg the bioavailability of oxazepam in rats (< 50 per-cent)

was lower than in Swiss-Webster mice (> 80 percent). Based on the

maximum plasma concentration achieved (Cmax), dose

proportionality was not observed in rats or mice after gavage dosing.

In the B6C3F1 mouse dosed-feed study, plasma concentrations

increased proportionally with concentration of oxazepam in feed.
287

The estimated relative bioavailability from dosed feed (relative to the

gavage study at 50 mg/kg) was about 43 percent.

Pentachloroanisole

Pentachloroanisole (PCA) has been found to be an environmental

pollutant even though it has no major industrial uses. It is postulated

that PCA is derived from methylation of pentachlorophenol, a widely

known environmental pollutant. Since no internal dose

determinations were conducted during the NTP-sponsored 2-year

toxicology studies in B6C3F1 mice and F344 Fischer rats in which

some evidence for carcino-genic potential was found, a retrospective

toxicokinetic study was conducted to evaluate its basic kinetics as well

as its potential for bioac-cumulation (Yuan et al. 1993). It this

toxicokinetic study, B6C3F1 mice and F344 Fischer rats of both sexes

were administered PCA intravenously (10 mg/kg) and intragastrically

(10, 20, and 40 mg/kg). Both PCA and pentachlorophenol plasma

concentrations were determined. PCA was shown to be rapidly

demethylated to pentachlorophenol in both species, and the resulting

pentachlorophenol plasma concentrations were in the range of one

hundred times higher than those found for the parent PCA.

Bioavailability of PCA was only about 36 percent in rats but was

about 74 percent in mice. Plasma concentrations increased with dose,

but were not directly proportional. No sex differences were observed.

Significant potential for bioaccumulation of pentachlorophenol from

exposure to PCA was predicted.

Pentachlorophenol

Pentachlorophenol is an effective broad-spectrum biocide widely used

as a wood preservative. Two-year carcinogenicity studies had been

conducted in B6C3F1 mice and similar studies were planned in Fischer

344 rats. To aid in future comparison of the results of the toxicology

studies in both species and to provide information for dose-response

relationships, toxicokinetic evaluations were conducted. In singleand multiple-exposure studies the toxicokinetics of

pentachlorophenol were studied in the Fischer 344 rat using IV and

oral (gavage and dosed-feed) routes of exposure (Yuan et al. 1994a).

The elimination kinetics of pentachlorophenol after IV

administration of 5 mg/kg were independent of sex. After gavage

administration, dose proportionality was established up to at least 38

mg/kg. To aid in establishing dose-response relationships, the

bioavailability of pentachlorophenol administered in dosed feed was

calculated and compared to that after gavage dosing. The
288

bioavailability of pentachlorophenol administered in dosed feed at

concentrations of 302 and 1,010 ppm ranged from 30 to 52 percent,

which was significantly lower than bioavailability of

pentachlorophenol administered by gavage (86 to 100 percent). The

time course of pentachlorophenol plasma concentrations during the

dosed-feed study was simulated using a computer model based on linear

theory (Yuan 1993). The simulations were comparable with the

experimentally determined concentrations.

SUMMARY

Toxic responses to test chemicals are known to be dependent on the

exposure route, the kinetic behavior of the chemical, and the dose

used in the toxicology study. Therefore, knowledge of internal dose is

indispensable for the interpretation of toxicology study results, for

the facilitation of interspecies scaling, and for risk assessment. By

monitoring the blood and/or tissue concentrations of test chemical

and/or metabolites versus time after administration of study chemicals

by different routes, the bioavailability and kinetic characteristic of

test chemicals can be readily obtained. This data can define the socalled linear dose range using area under the plasma concentration

versus time curve, clearance, or other related toxicokinetic

parameters, and can also be used to predict the possible

bioaccumulation under multiple dose regimes. Changes in kinetic

parameters after multiple exposures indicate alteration in how the

animal handles the chemical (e.g., that there was enzyme induction or

inhibition).

A recommended approach for conducting toxicokinetic studies

generally involves three steps. Step 1 is a preliminary study, which

uses a minimum number of animals to estimate the range of

blood/tissue concentrations, the required quantitation limit for the

analytical method, and the optimal sampling times for the definitive

toxicokinetic studies. Step 2 is the definitive study and generates

blood and/or tissue concentration data for calculating the

toxicokinetic parameters. Step 3 is the toxicokinetic study conducted

in conjunction with the toxicology study to determine the internal

dose and the effects of age and continuous exposure on kinetic

parameters.

Examples of the application of NTP toxicokinetic evaluations were

also presented in this chapter, demonstrating their use in the design

and interpretation of toxicology studies.
289

REFERENCES

Bischoff, K.B. Applications of mathematical model for drug distribution in

mammals. In: Hershey, D., ed. Chemical Engineering in

Medicine and Biology. New York: Plenum Press, 1967.

pp. 417-446.

Di Carlo, F.J. Metabolism, pharmacokinetics, and toxicokinetics defined.

Drug Metab Rev 13:1-4, 1982.

Dill, J.A.; Greenspan, B.J.; Mellinger, K.H.; Roycroft, J.H.; and Dunnick, J.

Disposition of inhaled cadmium oxide aerosol in the rat.

Inhalation Toxicol 6:379-393, 1994.

Jameson, C.W., and Goehl, T.J. Chemistry requirements for toxicological

and carcinogenicity evaluation of chemicals. In: Milman,

H.A., and Weisburger, E.K., eds. Handbook of Carcinogen

Testing. 2d ed. Park Ridge, NJ: Noyes Publications, 1994.

pp. 294-297.

Mordenti, J. Dosage regimen design for pharmaceutical studies conducted

in animals. J Pharmacol Sci 75:852-857, 1986.

Ramsey, J.C., and Andersen, M.E. A physiologically based description of

the inhalation pharmacokinetics of styrene in rats and

humans. Toxicol Appl Pharmacol 73(1):159-175, 1984.

Teorell, T. Kinetics of distribution of substances administered to the body.

Arch Intern Pharmacodynam Ther 57:205-225, 1937.

Trang, J.M.; Prejean, J.D.; James, R.H.; Irwin, R.D.; Goehl, T.J; and Page,

J.G. Zidovudine bioavailability and linear

pharmacokinetics in female B6C3F1 Mice. Drug Metabol

Disposition 21:189-193, 1993.

Travis, C.C., and White, R.K. Interspecies scaling of toxicity data. Risk

Analysis 8:119-125, 1988.

World Health Organization. Principles of Toxicokinetic Studies.

Environmental Health Criteria 57. Geneva: World Health

Organization, 1986.

Yacobi, A.; Skelly, J.P.; and Batra, V.K. Toxicokinetics and New Drug

Development. New York: Pergamon, 1989.

Yuan, J.H. Modeling blood/plasma concentrations in dosed feed and dosed

drinking water toxicology studies. Toxicol Appl

Pharmacol 119:131-141, 1993.

Yuan, J.H.; Jameson, C.W.; Goehl, T.J.; Collins, B.J.; Purdie, W.; and Judd,

L. Application of molecular encapsulation for toxicology

studies: Toxicokinetics of p-chloro-,,-

trifluorotoluene in -cyclodextrin of corn oil vehicles in

male F344 rats. Toxicol Appl Pharmacol 111:107-115,

1991a.
290

Yuan, J.H.; Jameson, C.W.; Goehl, T.J.; Collins, B.J.; Corniffe, G.; Kuhn,

G.; and Castro, C. Effects of physical binding of onitroanisole with feed: Its systemic availability in male

F344 rats. Bull Environ Contam Toxicol 47:152-159,

1991b.

Yuan, J.H.; Jameson, C.W.; Goehl, T.J.; Elwell, M.R.; Leininger, J.R.;

Thompson, M.B.; and Corniffe, G. Application of

molecular encapsulation for toxicology studies:

Comparative toxicity of p-chloro-,,-trifluorotoluene

in a-cyclodextrin vehicle versus corn oil vehicle in male

and female Fischer 344 rats and B6C3F1 mice. Fundam

Appl Toxicol 18:460-470, 1992.

Yuan, J.H.; Goehl, T.J.; Murrill, E.; Moore, R.; Clark, J.; Hong, H.L.; and

Irwin, R.D. Toxicokinetics of pentachloroanisole in rats

and mice. Xenobiotica 23:427, 1993.

Yuan, J.H.; Goehl, T.J.; Murrill, E.; Moore, R.; Clark, J.; Hong, H.L.; and

Irwin, R.D. Toxicokinetics of pentachlorophenol in F344

rat gavage and dosed feed studies. Xenobiotica 24:553-

560, 1994a.

Yuan, J.H.; Goehl, T.J.; Hong, H.L.; Clark, J.; Murrill, E.; and Moore, R.

Toxicokinetics of oxazepam in rats and mice. J

Pharmacol Sci 83:1373-1379, 1994b.

Yuan, J.H.; Dunnick, J.K.; Barnes, E.R.; and Findlay, J.W.A. Codeine

toxicokinetics in rats during a two-year dosed feed study.

Drug Metabol Disposition 22:14-20, 1994c.

Yuan, J.H.; Goehl, T.J.; Abdo, K.; Clark, J.; Espinosa, O.; Bugge, C.; and

Garcia, D. Effects of gavage versus dosed feed

administration upon the toxicokinetics of benzyl acetate

in rats and mice. Food Chem Toxicol 33:151-158, 1995.

AUTHOR

Thomas J. Goehl, Ph.D.

Head, Chemistry and Toxicokinetics

National Institute of Environmental Health Sciences

National Toxicology Program

111 Alexander Drive

Research Triangle Park, NC 27560
291

Appendix:

Examples of NTP Toxicokinetic Protocols

TOXICOKINETICS STUDY PROTOCOL FOR METHYLENE BLUE (AN

ORAL STUDY USING INTRAGASTRIC DOSING)

Rationale

The toxicokinetic studies are designed to: determine the basic kinetics

including dose proportional range and bioavailability, provide an

estimate of the internal dose, and provide information about longterm exposure on the kinetics.

Preliminary Study

Analytical Method. A preliminary analytical method shall be

developed for quantitation of test chemical in plasma over the target

concentration range of 0.1 to 50 g/mL.

Preliminary IV Study. Groups of 12 male Fisher 344N rats and female

B6C3F1 mice (13Å2 weeks old) shall be dosed intravenously with test

chemical (2.5 mg/kg for rats and 2.5 mg/kg for mice). The preferred

vehicle for IV formulation is Emulphor/ethanol/water (1:1:8). The

volumes used for dosing by the IV route will be 2 mL/kg for rats and 4

mL/kg for mice. As much heparinized blood as possible shall be

collected from the vena cava from one dosed animal at each of 12

time points (3, 5, 10, 20, 30, and 40 min; 1, 1.5, 2, 3, 4, and 6

hours). Test chemical concentrations in plasma shall be determined

using the unvalidated analytical method.

Preliminary Gavage Study. Groups of 12 Fisher 344N rats and

B6C3F1 mice of both sexes (13Å2 weeks old) shall be gavaged at one

of two doses of test chemical (2.5 and 50 mg/kg for rats and 2.5 and

25 mg/kg for mice). The vehicle for the gavage formulation will be

0.5 percent aqueous methylcellulose. The volumes used for dosing by

the gavage route will be 5 mL/kg for rats and 10 mL/kg for mice. As

much heparinized blood as possible shall be collected from the vena

cava from one dosed animal at each of 12 time points (5, 10, 20, 30,

and 45 min; 1, 1.5, 2, 3, 4, 6, and 8 hours). Test chemical

concentrations in plasma shall be determined using the unvalidated

analytical method.
292

General Information. Confirmatory dose formulation analyses shall

not be conducted in the preliminary IV and gavage studies, but shall be

conducted for the dosed-feed studies. Pooled blank plasma from rats

shall be used to construct the standard curves for each preliminary

study if no species differences are noted in the method using blank

plasma.

Method Performance Evaluation. If the preliminary study results are

satisfactory and quantitation of test chemical in plasma is in a range

for which quantitation is technically feasible, then the analytical

method shall be validated over the range dictated by the preliminary

study results. Meanwhile, a stability study of the analyte in plasma

shall be conducted over a period of time covering the expected

storage time of the biological samples. The concentration used in the

stability study shall be the midpoint concentration of the standard

curve.

Single Administration Study

Rat IV Route. Groups of 12 Fisher 344N rats of both sexes (13Å2

weeks old) shall be dosed intravenously with a test chemical IV

formulation at one dose (2.5 mg/kg). Blood samples shall be taken at

eight time points (to be determined based on the results of the

preliminary study) after IV administration, with three animals bled at

each time point. Two blood samples at different times (preferably >2

hr apart) shall be collected via alternating orbital sinuses from each

dosed rat. Test chemical concentrations shall be determined in

plasma using a validated analytical method.

Mouse IV Route. Groups of 24 B6C3F1 mice of both sexes (13Å2

weeks) shall be dosed intravenously with a test chemical IV

formulation at one dose (2.5 mg/kg). Blood samples shall be taken at

eight time points (to be determined based on the results of the

preliminary study) after IV administration, with three animals bled at

each time point. Each mouse shall be bled once. Test chemical

concentrations shall be determined in plasma using a validated

method.

Rat Gavage Route. Groups of 12 Fisher 34N rats of both sexes

(13Å2 weeks old) shall be gavaged at each of three doses of test

chemical (2.5, 25, 50 mg/kg). Blood samples shall be taken at eight

time points (to be determined based on the results of the preliminary

study) after gavage administration, with three animals bled at each

time point. Two blood samples at different times (preferably >2 hr
293

apart) shall be collected via alternating orbital sinuses from each dosed

rat. Test chemical concentrations shall be determined in plasma using

a validated analytical method.

Mouse Gavage Route. Groups of 24 B6C3F1 mice of both sexes

(13Å2 weeks old) shall be gavaged at each of three doses of test

chemical (2.5, 12.5, 25 mg/kg). Blood samples shall be taken at eight

time points (to be determined based on the results of the preliminary

study) after gavage administration, with three animals bled at each

time point. Each mouse shall be bled once. Test chemical

concentrations shall be determined in plasma using a validated

analytical method.

General Information. Confirmatory dose formulation analyses shall

be conducted for the single administration studies. Pooled control

plasma from rats shall be used to construct the standard curves if no

species differences are noted in the blank plasma.

Two-Year Studies

Special Study Animals. Special groups of 10 rats and 10 mice of

both sexes shall be added to each dose group of the 2-year study. No

control rats or control mice are needed for this study. For rats, blood

samples shall be collected at 3, 6, 12, and 18 months using orbital

sinus bleeding, with five rats being bled at each of two time points (to

be specified by NTP). Rats shall be returned to their cages after the

3-, 6-, and 12-month bleeding intervals and sacrificed after the 18-

month interval. Test chemical concentrations shall be determined in

plasma using a validated analytical method.

For mice, blood samples shall be collected only at 12 months with

five mice being bled at each of two time points (to be specified by

NTP). After sampling, mice shall be sacrificed. Test chemical

concentrations shall be determined in plasma using a validated

analytical method.

Aged Rat Study. Using the sentinel rats in the 2-year study, 15 male

and 15 female rats (18 months old) shall be gavaged at the middle

dose. Blood samples shall be taken at five time points (to be specified

by NTP), with three animals bled at each time point. Each rat shall be

bled only once. Test chemical concentrations shall be determined in

plasma using a validated analytical method.
294

Aged Mouse Study. Using the sentinel mice in the 2-year study, 15

male and female mice (18 months old) shall be gavaged at the middle

dose. Blood samples shall be taken at five time points (to be specified

by NTP), with three animals bled at each time point. Each mouse

shall be bled only once. Test chemical concentrations shall be

determined in plasma using a validated analytical method.

TOXICOKINETICS STUDY PROTOCOL FOR 2-HYDROXY-4-

METHOXYBENZOPHENONE (AN ORAL STUDY USING DOSED

FEED)

Rationale

The toxicokinetic studies are designed to: determine the basic

kinetics including dose proportional range and bioavailability, provide

an estimate of the internal dose, and provide information about longterm exposure on the kinetics.

Preliminary Study

Analytical Method. A preliminary analytical method shall be

developed for quantitation of test chemical in plasma over the target

concentration range of 0.1 to 50 g/mL.

Preliminary IV Study. Groups of 10 Fisher 344N rats and B6C3F1

mice of both sexes (13Å2 weeks old) shall be dosed intravenously

with test chemical (8 mg/kg for rats and 50 mg/kg for mice). The

preferred vehicle for IV formulation is Emulphor/ethanol/water

(1:1:8). The volumes used for dosing by the IV route will be 2 mL/kg

for rats and 4 mL/kg for mice. As much heparinized blood as possible

shall be collected from the vena cava from one dosed animal at each

of 10 time points (3, 5, 10, 20, and 40 min; 1, 2, 3, 4, and 6 hours).

Test chemical concentrations in plasma shall be determined using the

unvalidated analytical method.

Preliminary Gavage Study. Groups of eight Fisher 344N rats and

B6C3F1 mice of both sexes (13Å2 weeks old) shall be gavaged at one

of two doses of test chemical (8 and 80 mg/kg for rats and 50 and 500

mg/kg for mice). The vehicle for the gavage formulation will be 0.5

percent aqueous methylcellulose. The volumes used for dosing by the

gavage route will be 5 mL/kg for rats and 10 mL/kg for mice. As

much heparinized blood as possible shall be collected from the vena

cava from one dosed animal at each of 8 time points (5, 15, 30, and
295

45 min; 1, 2, 4, and 8 hours). Test chemical concentrations in

plasma shall be determined using the unvalidated analytical method.

Preliminary Feed Study. Groups of eight Fisher 344N rats and

B6C3F1 mice of both sexes (13Å2 weeks old) shall be dosed with the

test chemical in the feed for up to 7 days (time to be dosed will be

based on the preliminary IV study results) at one of two dosed-feed

concentrations (1,000 and 10,000 ppm). As much heparinized blood

as possible shall be collected from the vena cava from one dosed

animal at each of seven time points (4 am, 6 am, 8 am, 10 am, 12

pm, 4 pm, and 6 pm).

General Information. Confirmatory dose formulation analyses shall

not be conducted in the preliminary IV and gavage studies, but shall be

conducted for the dosed feed studies. Pooled blank plasma from rats

shall be used to construct the standard curves for each preliminary

study if no species differences are noted in the method using blank

plasma.

Method Performance Evaluation. If the preliminary study results are

satisfactory and quantitation of test chemical in plasma is in a range

for which quantitation is technically feasible, then the analytical

method shall be validated over the range dictated by the preliminary

study results (determined by NTP) per the current NTP general

specifications requirements. Meanwhile, a stability study of the

analyte in plasma shall be conducted over a period of time covering

the expected storage time of the biological samples. The

concentration used in the stability study shall be the midpoint

concentration of the standard curve.

Single Administration Study

Rat IV Route. Groups of 12 Fisher 344N rats of both sexes (13Å2

weeks old) shall be dosed intravenously with a test chemical IV

formulation at one dose (to be chosen based on results of the

preliminary study). Blood samples shall be taken at eight time points

(to be determined based on the results of the preliminary study) after

IV administration, with three animals bled at each time point. Two

blood samples at different times (preferably > 2 hr apart) shall be

collected via alternating orbital sinuses from each dosed rat. Test

chemical concentrations shall be determined in plasma using a

validated analytical method.
296

Mouse IV Route. Groups of 24 B6C3F1 mice of both sexes (13Å2

weeks) shall be dosed intravenously with a test chemical IV

formulation at one dose (to be chosen based on results of the

preliminary study). Blood samples shall be taken at eight time points

(to be determined based on the results of the preliminary study) after

IV administration, with three animals bled at each time point. Each

mouse shall be bled once. Test chemical concentrations shall be

determined in plasma using a validated method.

Rat Gavage Route. Groups of 12 Fisher 34N rats of both sexes

(13Å2 weeks old) shall be gavaged at each of three doses of test

chemical (to be chosen based on results of the preliminary study).

Blood samples shall be taken at eight time points (to be determined

based on the results of the preliminary study) after gavage

administration, with three animals bled at each time point. Two

blood samples at different times (preferably >2 hr apart) shall be

collected via alternating orbital sinuses from each dosed rat. Test

chemical concentrations shall be determined in plasma using a

validated analytical method.

Mouse Gavage Route. Groups of 24 B6C3F1 mice of both sexes

(13Å2 weeks old) shall be gavaged at each of three doses of test

chemical (to be chosen based on results of the preliminary study).

Blood samples shall be taken at eight time points (to be determined

based on the results of the preliminary study) after gavage

administration, with three animals bled at each time point. Each

mouse shall be bled once. Test chemical concentrations shall be

determined in plasma using a validated analytical method.

General Information. Confirmatory dose formulation analyses shall

be conducted for the single administration studies. Pooled control

plasma from rats shall be used to construct the standard curves if no

species differences are noted in the blank plasma.

Two-Year Studies

Special Study Animals (GLP). Special groups of 10 rats and 10 mice

of both sexes shall be added to each dose group of the 2-year study.

No control rats or control mice are needed for this study. For rats,

blood samples shall be collected at 3, 6, 12, and 18 months using

orbital sinus bleeding, with five rats being bled at each of two time

points (to be specified by NTP). Rats shall be returned to their cages

after the 3-, 6-, and 12-month bleeding intervals and sacrificed after
297

the 18-month interval. Test chemical concentrations shall be

determined in plasma using a validated analytical method.

For mice, blood samples shall be collected only at 12 months, with

five mice being bled at each of two time points (to be specified by

NTP). After sampling, mice shall be sacrificed. Test chemical

concentrations shall be determined in plasma using a validated

analytical method.

Aged Rat Study. Using the sentinel rats in the 2-year study, 15 male

and 15 female rats (18 months old) shall be gavaged at the middle

dose. Blood samples shall be taken at five time points (to be specified

by NTP), with three animals bled at each time point. Each rat shall

be bled only once. Test chemical concentrations shall be determined

in plasma using a validated analytical method.

Aged Mouse Study. Using the sentinel mice in the 2-year study, 15

male and female mice (18 months old) shall be gavaged at the middle

dose. Blood samples shall be taken at five time points (to be specified

by NTP), with three animals bled at each time point. Each mouse

shall be bled only once. Test chemical concentrations shall be

determined in plasma using a validated analytical method.

TOXICOKINETICS STUDIES FOR CAMPHOR (A TOPICAL

EXPOSURE)

Rationale

The camphor toxicokinetic studies using the topical exposure route

are designed to determine: absolute bioavailability and basic kinetics,

percent of internal dose from nontopical exposure, the extent of

bioaccumulation, the internal dose, and the effects of age and longterm exposure on the kinetics.

Preliminary Study

Analytical Method Development. A preliminary analytical method

shall be developed for quantitation of camphor in plasma over the

target concentration range of 0.5 to 50 g/mL.

Preliminary IV Study. Groups of 10 rats and 10 mice of each sex

(13Å2 weeks old purchased by the contractor) shall be dosed

intravenously with camphor at 6 and 50 mg/kg for rats and mice,
298

respectively. Emulphor/ethanol/water (1:1:8) is to be used as the

vehicle. The volumes used for dosing by the IV route will be 2 mL/kg

for rats and 4 mL/kg for mice. As much heparinized blood as possible

shall be collected from the vena cava from one dosed rat and mouse at

each of 10 time points (5, 15, 30, and 45 min; 1, 2, 4, 6, 8, and 10

hrs). Camphor concentrations in plasma shall be determined using the

unvalidated analytical method.

Preliminary Topical Exposure Study. Groups of eight rats and eight

mice of each sex (13Å2 weeks old) shall be topically exposed to

camphor in ethanol/water//1/1 at two dosages (25 and 400 mg/kg for

rats and 200 and 1,000 mg/kg for mice). The volumes used for dosing

by the topical route shall be the same as in chronic study (i.e., 0.6

mL/kg for rats and 2.0 mL/kg for mice). The concentrations of the

dose formulations are to be kept constant. The site of application

shall be protected to avoid grooming by the rodent but the site shall

not be occluded. As much heparinized blood as possible shall be

collected from the vena cava from one dosed rat and mouse at each of

10 time points (15, 30, and 45 min; 1, 2, 4, 8, 12, 16, and 24 hrs).

Camphor concentrations in plasma shall be determined using the

unvalidated analytical method.

General Information. Confirmatory dose formulation analyses shall

not be conducted in the preliminary IV or topical exposure studies.

Pooled control plasma from male and female rats shall be used to

construct the standard curves for each preliminary study.

Method Performance Evaluation. If the preliminary study results are

satisfactory and quantitation of camphor in plasma is in a range for

which quantitation is technically feasible, then the analytical method

shall be validated over the range dictated by the preliminary study

results (determined by NTP). Meanwhile, a stability study of the

analyte in plasma shall be conducted over a period of time covering

the expected storage time of the biological samples. The

concentration in plasma evaluated in the stability study shall be 75

percent of the highest concentration used for the standard curve.

Single Administration Study

Rat IV Route. Groups of 12 male and 12 female rats (13Å2 weeks

old) shall be dosed intravenously with a camphor IV formulation at

one dose (to be chosen based on results of preliminary study). Blood

samples shall be taken at eight time points (to be determined based on

the results of the preliminary study) after IV administration, with
299

three animals bled at each time point. Two blood samples at different

times (preferably >2 hr apart) shall be collected via alternating orbital

sinuses from each dosed rat. Camphor concentrations in plasma shall

be determined using the validated analytical method.

Mouse IV Route. Groups of 24 male and 24 female mice (13Å2

weeks old) shall be dosed intravenously with a camphor IV

formulation at one dose (to be chosen based on results of the

preliminary study). Blood samples shall be taken at eight time points

(to be determined based on the results of the preliminary study) after

IV administration, with three animals bled at each time point.

Camphor concentrations in plasma shall be determined using the

validated analytical method.

Rat Topical Exposure Route. Two groups of 12 male and 12 female

rats (13Å2 weeks old) shall be exposed to camphor at three doses (to

be chosen based on results of the preliminary study). One group will

have the site of application protected to prevent grooming by the

rodents while the second group will not have the site protected.

Blood samples shall be taken at eight time points (to be determined

based on the results of the preliminary study) after topical exposure,

with three animals bled at each time point. Two blood samples at

different times (preferably >2 hr apart) shall be collected via

alternating orbital sinuses from each dosed rat. Camphor

concentrations in plasma shall be determined using the validated

analytical method.

Mice Topical Exposure Route. Two groups of 24 male and 24 female

mice (13Å2 weeks old) shall be exposed to camphor at three dosages

(to be chosen based on results of the preliminary study). One group

will have the site of application protected to prevent grooming by the

rodents while the second group will not have the site protected.

Blood samples shall be taken at eight time points (to be determined

based on the results of the preliminary study) after topical exposure,

with three animals bled at each time point. Camphor concentrations

in plasma shall be determined using the validated analytical method.

General Information. Confirmatory dose formulation analyses shall

be conducted for the single administration studies. Pooled control

plasma from male and female rats shall be used to construct the

standard curves.

Chronic Studies
300

Special Study Animals (GLP). Special groups of 10 rats and 10 mice

of both sexes shall be added to each core dose group of the chronic

study. No control rats or control mice are needed for this study. For

rats, blood samples shall be collected at 3, 6, 12, and 18 months using

orbital sinus bleeding, with two rats being bled at each of five time

points (to be specified by NTP). Rats shall be returned to their cages

after the 3-, 6-, and 12-month bleeding intervals and sacrificed after

the 18-month interval. Plasma concentrations of camphor shall be

determined.

For mice, blood samples shall be collected only at 12 months, with

two mice being bled at each of five time points (to be specified by

NTP). After sampling, mice shall be sacrificed. Plasma

concentrations of camphor shall be determined.

Aged Rat Study. Using the sentinel rats in the chronic study, 15

male and 15 female rats (18 months old) shall be dosed at the middle

dose. Blood samples shall be taken at five time points (to be specified

by NTP) after topical exposure, with three animals bled at each time

point. Each rat shall be bled only once. Plasma concentrations of

camphor shall be determined.

Aged Mouse Study. Using the sentinel mice in the chronic study, 15

male or female mice (18 months old) shall be dosed at the middle

dose. Blood samples shall be taken at five time points (to be specified

by NTP) after topical exposure, with three animals bled at each time

point. Each mouse shall be bled only once. Plasma concentrations of

camphor shall be determined.

TOXICOKINETICS STUDIES PROTOCOL FOR DECALIN/TETRALIN

(AN INHALATION EXPOSURE)

Rationale

The NTP toxicokinetic studies are designed to: determine the

kinetics of decalin/tetralin in blood, determine the internal dose during

inhalation studies as well as any possible changes in kinetics, establish

dose proportional range, provide information on the effect of age on

the kinetic parameters, and determine the concentrations of known

metabolites in kidneys (only for the rat decalin studies).
301

Preliminary Studies

Analytical Method. A preliminary analytical method shall be

developed for quantitation of decalin/tetralin in blood. In the decalin

studies methods are also to be developed for the determination of the

concentration of known metabolites (cis-2-decalone and trans-2-

decalone) in rat kidneys. This range shall be estimated from literature

and previous experience.

Preliminary IV Study. Groups of 10 rats and 10 mice of each sex

(13Å2 weeks old purchased by the contractor) shall be dosed

intravenously with decalin/tetralin at two doses proposed by the

contractor and approved by NTP. Emulphor/ethanol/water (1:1:8)

solutions shall be used for IV administration. The volumes used for

dosing by the IV route will be 2 mL/kg for rats and 4 mL/kg for mice.

As much heparinized blood as possible shall be collected from the

vena cava from one dosed rat and mouse at each of 10 time points (5,

10, 20, and 40 min; 1, 2, 4, 6, 8, and 12 hours). Blood

concentrations of decalin/tetralin shall be determined using the

unvalidated analytical method.

Concentrations of the two decalin ketone metabolites are to be

determined in the kidneys from the 1-, 4-, 8-, and 12-hour time point

in rats of both sexes.

Preliminary Inhalation Study. Groups of five rats and five mice of

each sex (13Å2 weeks old purchased by the contractor) shall be

exposed for 6 hours to decalin/tetralin vapor via inhalation route at

each of two exposure concentrations. The exposure concentrations

shall be set after the completion of the 14-day toxicology study and

shall be the highest and lowest concentrations proposed for the 90-

day study. Blood samples shall be taken from five animals per sex per

specie immediately after shutdown of the exposure (not to exceed 10

minutes). Samples shall be analyzed to estimate the possible steadystate concentration range.

Concentrations of the two decalin ketone metabolites are to be

determined in the rat kidneys of both sexes.

General Requirement. Confirmatory analyses of the dose

formulation shall not be conducted in the preliminary study. Pooled

control blood from male and female rats shall be used to construct the

standard curves for each preliminary study.
302

Validating the Method. If the preliminary study results are

satisfactory and quantitations of decalin/tetralin in blood are in a

technically feasible range, then the analytical methods shall be

validated over the range dictated by the preliminary study results

(determined by NTP). A stability study of the analytes in blood shall

be conducted over a period of time covering the expected storage

time of the biological samples. The samples are to be analyzed as

soon as possible but not to exceed 3 weeks. The concentrations used

in the stability study shall be the midpoint concentration of the

standard curve.

Single Administration Study

Rat IV Route. Groups of three jugular-cannulated rats of each sex

(13Å2 weeks old purchased by the contractor) shall be dosed

intravenously with a decalin/tetralin IV formulation at each of two

doses (to be chosen based on results of preliminary study and the

results of the 90-day inhalation toxicology study). Blood samples

shall be taken at eight time points (to be determined based on the

results of the preliminary study) after IV administration, with three

animals bled at each time point. Blood concentrations of

decalin/tetralin shall be determined using the validated analytical

method.

Concentrations of the two ketone metabolites of decalin are to be

determined in the rat kidneys of both sexes at four time points (to be

determined by NTP).

Mouse IV Route. Groups of 12 mice (13Å2 weeks old purchased by

the contractor) of each sex shall be dosed intravenously with a

decalin/ tetralin IV formulation at each of two doses (to be chosen

based on results of the preliminary study and the 90-day inhalation

toxicology study). Blood samples shall be taken at eight time points

(to be determined based on the results of the preliminary study) after

IV administration, with three animals bled at each time point. Each

mouse shall be sampled twice via alternating orbital sinuses at

different times (preferably > 2 hr apart). Blood concentrations of

decalin/tetralin shall be determined using the validated analytical

method.

Rat Inhalation Route (Single Exposure). Groups of 12 rats (13Å2

weeks old purchased by the contractor) of each sex shall be exposed

to decalin/tetralin vapor for 6 hours at each of three exposure

concentrations via inhalation route. The exposure concentrations
303

shall be based on the results of the 90-day toxicology study.

Immediately after shutdown of the exposure, the decline of blood

concentrations of decalin/tetralin in rats shall be followed at eight

time points (to be determined based on the results of the preliminary

study), with three animals bled at each time point. The first time

point shall be as close as possible to time zero. Time zero is defined

as the time of shutdown of the chamber exposure. Each rat shall be

sampled twice via alternating orbital sinuses at different times

(preferably > 2 hr apart). Blood concentrations of decalin/tetralin

shall be determined using the validated analytical method.

Concentrations of the two ketone metabolites of decalin are to be

determined in the rat kidneys of both sexes at four time points (to be

determined by NTP).

Aged Rat Inhalation Route. Groups of 24 aged rats (18 months old

purchased by the contractor) of each sex shall be exposed to decalin/

tetralin vapor for 6 hours using the proposed middle chronic exposure

concentration via inhalation route. Immediately after shutdown of

the exposure, the decline of blood concentrations of decalin/tetralin

shall be followed at eight time points (to be determined based on the

results of the preliminary study), with three animals bled at each time

point. The first time point shall be as close as possible to time zero.

Time zero is defined as the time of shutdown of the chamber

exposure. Each rat shall be sampled only once. Blood concentrations

of decalin/ tetralin shall be determined using the validated analytical

method.

Mouse Inhalation Route (Single Exposure). Groups of 12 mice

(13Å2 weeks old purchased by the contractor) of each sex shall be

exposed to decalin/tetralin vapor for 6 hours at each of three

exposure concentrations via inhalation route. The exposure

concentrations shall be based on the results of the 90-day toxicology

study. Immediately after shutdown of the exposure, the decline of

blood concentrations of decalin/tetralin in mice shall be followed at

eight time points (to be determined based on the results of the

preliminary study), with three animals bled at each time point. The

first time point shall be as close as possible to time zero. Time zero is

defined as the time of shutdown of the chamber exposure. Each

mouse shall be sampled twice via alternating orbital sinuses at

different times (preferably > 2 hr apart). Blood concentrations of

decalin/tetralin shall be determined using the validated analytical

method.
304

Aged Mouse Inhalation Route. Groups of 24 aged mice (18 months

old purchased by the contractor) of each sex shall be exposed to

decalin/tetralin vapor for 6 hours using the proposed middle chronic

exposure concentration via inhalation route. Immediately after

shutdown of the exposure, the decline of blood concentrations of

decalin/tetralin in mice shall be followed at eight time points (to be

determined based on the results of the preliminary study), with three

animals bled at each time point. The first time point shall be as close

as possible to time zero. Time zero is defined as the time of shutdown

of the chamber exposure. Each mouse shall be sampled only once.

Blood concentrations of decalin/tetralin shall be determined using the

validated analytical method.

General Requirement. Confirmatory dose formulation analyses for

the IV studies shall be conducted for the single administration studies.

Pooled control blood from male and female rats shall be used to

construct the standard curves.

Chronic Studies

Special Study Animals. If the single administration study is

successful, then groups of nine rats of each sex shall be added to each

dose group of the chronic study. No control rats are needed for this

study. Blood samples shall be collected at 2 weeks and 3, 6, 12, and

18 months using orbital sinus bleeding, with three rats being bled at

each of six time points (to be determined based on the single

administration study ). Each rat shall be sampled twice via alternating

orbital sinuses at different times (preferably >2 hr apart). Rats shall

be returned to their cages after being sampled and sacrificed after the

18-month bleedings. Blood concentrations of decalin/tetralin shall be

determined using the validated analytical method.

Groups of nine mice of each sex shall be added to each dose group of

the chronic study. No control mice are needed for this study. Blood

samples shall be collected only at 12 months with three mice being

bled at each of six time points (to be determined based on the singleadministration study). Each mouse shall be sampled twice via

alternating orbital sinuses at different times (preferably >2 hr apart).

After sampling, mice shall be sacrificed. Blood concentrations of

decalin/tetralin shall be determined using the validated analytical

method.
305

While limited

supplies last, single

copies of the following monographs may be obtained free of charge

from the National Clearinghouse for Alcohol and Drug Information

(NCADI). Please also contact NCADI for information about other

publications of the National Institute on Drug Abuse relevant to drug

abuse research.

Additional copies may be purchased from the U.S. Government

Printing Office (GPO) and/or the National Technical Information

Service (NTIS) as indicated. NTIS prices are for paper copy; add

$3.00 handling charge for each order. Microfiche copies also are

available from NTIS. Prices from either source are subject to change.

Addresses are:

NCADI

National Clearinghouse for Alcohol and Drug Information

P.O. Box 2345

Rockville, MD 20852

(301) 468-2600

(800) 729-6686

GPO

Superintendent of Documents

U.S. Government Printing Office

P.O. Box 371954

Pittsburgh, PA 15220-7954

(202) 738-3238

FAX (202) 512-2233

NTIS

National Technical Information Service

U.S. Department of Commerce

Springfield, VA 22161

(703) 487-4650

For information on availability of NIDA Research Monographs from

1975-1996 and those not listed, write to NIDA, Public Information

Branch, Room 10A-39, 5600 Fishers Lane, Rockville, MD 20857.
306

26 THE BEHAVIORAL

ASPECTS OF SMOKING.

Norman A. Krasnegor, Ph.D., ed. (Reprint from 1979 Surgeon

General's Report on Smoking and Health.)

NCADI #M26 NTIS PB #80-118755/AS (A09)

$27.00

42 THE ANALYSIS OF CANNABINOIDS IN BIOLOGICAL

FLUIDS. Richard L. Hawks, Ph.D., ed.

NCADI #M42 NTIS PB #83-136044/AS (A07)

$27.00

50 COCAINE: PHARMACOLOGY, EFFECTS, AND

TREATMENT OF ABUSE. John Grabowski, Ph.D., ed.

NCADI #M50 NTIS PB #85-150381/AS (A07)

$27.00

52 TESTING DRUGS FOR PHYSICAL DEPENDENCE

POTENTIAL AND ABUSE LIABILITY. Joseph V. Brady,

Ph.D., and Scott E. Lukas, Ph.D., eds.

NCADI #M52 NTIS PB #85-150373/AS (A08)

$27.00

53 PHARMACOLOGICAL ADJUNCTS IN SMOKING

CESSATION. John Grabowski, Ph.D., and

Sharon M. Hall, Ph.D., eds.

NCADI #M53 NTIS PB #89-123186/AS (A07)

$27.00

54 MECHANISMS OF TOLERANCE AND DEPENDENCE.

Charles Wm. Sharp, Ph.D., ed.

NCADI #M54 NTIS PB #89-103279/AS (A19)

$52.00

56 ETIOLOGY OF DRUG ABUSE: IMPLICATIONS FOR

PREVENTION. Coryl LaRue Jones, Ph.D., and

Robert J. Battjes, D.S.W., eds.

NCADI #M56 NTIS PB #89-123160/AS (A13)

$36.50

61 COCAINE USE IN AMERICA: EPIDEMIOLOGIC AND

CLINICAL PERSPECTIVES. Nicholas J. Kozel, M.S., and

Edgar H. Adams, M.S., eds.

NCADI #M61 NTIS PB #89-131866/AS (A11)

$36.50

62 NEUROSCIENCE METHODS IN DRUG ABUSE RESEARCH.

Roger M. Brown, Ph.D., and David P. Friedman, Ph.D., eds.

NCADI #M62 NTIS PB #89-130660/AS (A08)

$27.00

63 PREVENTION RESEARCH: DETERRING DRUG ABUSE

AMONG CHILDREN AND ADOLESCENTS. Catherine S. Bell,

M.S., and Robert J. Battjes, D.S.W., eds.

NCADI #M63 NTIS PB #89-103287/AS (A11)

$36.50

64 PHENCYCLIDINE: AN UPDATE. Doris H. Clouet, Ph.D., ed.
307

NCADI #M64 NTIS PB #89-131858/AS (A12)

$36.50

65 WOMEN AND DRUGS: A NEW ERA FOR RESEARCH.

Barbara A. Ray, Ph.D., and Monique C. Braude, Ph.D., eds.

NCADI #M65 NTIS PB #89-130637/AS (A06)

$27.00
308

69 OPIOID PEPTIDES: MEDICINAL CHEMISTRY.

Rao S. Rapaka, Ph.D.; Gene Barnett, Ph.D.; and

Richard L. Hawks, Ph.D., eds.

NCADI #M69 NTIS PB #89-158422/AS (A17)

$44.50

70 OPIOID PEPTIDES: MOLECULAR PHARMACOLOGY,

BIOSYNTHESIS, AND ANALYSIS. Rao S. Rapaka, Ph.D., and

Richard L. Hawks, Ph.D., eds.

NCADI #M70 NTIS PB #89-158430/AS (A18)

$52.00

72 RELAPSE AND RECOVERY IN DRUG ABUSE.

Frank M. Tims, Ph.D., and Carl G. Leukefeld, D.S.W., eds.

NCADI #M72 NTIS PB #89-151963/AS (A09)

$36.50

74 NEUROBIOLOGY OF BEHAVIORAL CONTROL IN DRUG

ABUSE. Stephen I. Szara, M.D., D.Sc., ed.

NCADI #M74 NTIS PB #89-151989/AS (A07)

$27.00

77 ADOLESCENT DRUG ABUSE: ANALYSES OF TREATMENT

RESEARCH. Elizabeth R. Rahdert, Ph.D., and

John Grabowski, Ph.D., eds.

NCADI #M77 NTIS PB #89-125488/AS (A0) $27.00

78 THE ROLE OF NEUROPLASTICITY IN THE RESPONSE TO

DRUGS. David P. Friedman, Ph.D., and

Doris H. Clouet, Ph.D., eds.

NCADI #M78 NTIS PB #88-245683/AS (A10)

$36.50

79 STRUCTURE-ACTIVITY RELATIONSHIPS OF THE

CANNABINOIDS. Rao S. Rapaka, Ph.D., and

Alexandros Makriyannis, Ph.D., eds.

NCADI #M79 NTIS PB #89-109201/AS (A10)

$36.50

80 NEEDLE SHARING AMONG INTRAVENOUS DRUG

ABUSERS: NATIONAL AND INTERNATIONAL

PERSPECTIVES. Robert J. Battjes, D.S.W., and

Roy W. Pickens, Ph.D., eds.

NCADI #M80 NTIS PB #88-236138/AS (A09)

$36.50

82 OPIOIDS IN THE HIPPOCAMPUS. Jacqueline F. McGinty,

Ph.D., and David P. Friedman, Ph.D., eds.

NCADI #M82 NTIS PB #88-245691/AS (A06)

$27.00

83 HEALTH HAZARDS OF NITRITE INHALANTS.

Harry W. Haverkos, M.D., and John A. Dougherty, Ph.D., eds.

NCADI #M83 NTIS PB #89-125496/AS (A06)

$27.00

84 LEARNING FACTORS IN SUBSTANCE ABUSE.

Barbara A. Ray, Ph.D., ed.
309

NCADI #M84 NTIS PB #89-125504/AS (A10)

$36.50

85 EPIDEMIOLOGY OF INHALANT ABUSE: AN UPDATE.

Raquel A. Crider, Ph.D., and Beatrice A. Rouse, Ph.D., eds.

NCADI #M85 NTIS PB #89-123178/AS (A10)

$36.50

86 COMPULSORY TREATMENT OF DRUG ABUSE:

RESEARCH AND CLINICAL PRACTICE. Carl G. Leukefeld,

D.S.W., and Frank M. Tims, Ph.D., eds.

NCADI #M86 NTIS PB #89-151997/AS (A12)

$36.50

87 OPIOID PEPTIDES: AN UPDATE. Rao S. Rapaka, Ph.D.,

and

Bhola N. Dhawan, M.D., eds.

NCADI #M87 NTIS PB #89-158430/AS (A11)

$36.50

88 MECHANISMS OF COCAINE ABUSE AND TOXICITY.

Doris H. Clouet, Ph.D.; Khursheed Asghar, Ph.D.; and

Roger M. Brown, Ph.D., eds.

NCADI #M88 NTIS PB #89-125512/AS (A16)

$44.50

89 BIOLOGICAL VULNERABILITY TO DRUG ABUSE.

Roy W. Pickens, Ph.D., and Dace S. Svikis, B.A., eds.

NCADI #M89 NTIS PB #89-125520/AS (A09)

$27.00

92 TESTING FOR ABUSE LIABILITY OF DRUGS IN HUMANS.

Marian W. Fischman, Ph.D., and Nancy K. Mello, Ph.D., eds.

NCADI #M92 NTIS PB #90-148933/AS (A17)

$44.50

93 AIDS AND INTRAVENOUS DRUG USE: FUTURE

DIRECTIONS FOR COMMUNITY-BASED PREVENTION

RESEARCH. Carl G. Leukefeld, D.S.W.; Robert J. Battjes,

D.S.W.; and Zili Amsel, D.S.C., eds.

NCADI #M93 NTIS PB #90-148933/AS (A14)

$44.50

94 PHARMACOLOGY AND TOXICOLOGY OF

AMPHETAMINE AND RELATED DESIGNER DRUGS.

Khursheed Asghar, Ph.D., and Errol De Souza, Ph.D., eds.

NCADI #M94 NTIS PB #90-148958/AS (A16)

$44.50

95 PROBLEMS OF DRUG DEPENDENCE, 1989.

PROCEEDINGS OF THE 51st ANNUAL SCIENTIFIC

MEETING. THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC.

Louis S. Harris, Ph.D., ed.

NCADI #M95 NTIS PB #90-237660/AS (A99)

$67.00
310

96 DRUGS OF ABUSE: CHEMISTRY, PHARMACOLOGY,

IMMUNOLOGY, AND AIDS. Phuong Thi Kim Pham, Ph.D.,

and Kenner Rice, Ph.D., eds.

NCADI #M96 NTIS PB #90-237678/AS (A11)

$36.50

97 NEUROBIOLOGY OF DRUG ABUSE: LEARNING AND

MEMORY. Lynda Erinoff, Ph.D., ed.

NCADI #M97 NTIS PB #90-237686/AS (A11)

$36.50

98 THE COLLECTION AND INTERPRETATION OF DATA

FROM HIDDEN POPULATIONS.

Elizabeth Y. Lambert, M.S., ed.

NCADI #M98 NTIS PB #90-237694/AS (A08)

$27.00

99 RESEARCH FINDINGS ON SMOKING OF ABUSED

SUBSTANCES. C. Nora Chiang, Ph.D., and

Richard L. Hawks, Ph.D., eds.

NCADI #M99 NTIS PB #91-141119 (A09) $27.00

100 DRUGS IN THE WORKPLACE: RESEARCH AND

EVALUATION DATA. VOL II. Steven W. Gust, Ph.D.; J. Michael

Walsh, Ph.D.; Linda B. Thomas, B.S.; and

Dennis J. Crouch, M.B.A., eds.

NCADI #M100 GPO Stock #017-024-01458-3 $8.00

101 RESIDUAL EFFECTS OF ABUSED DRUGS ON BEHAVIOR.

John W. Spencer, Ph.D., and John J. Boren, Ph.D., eds.

NCADI #M101 NTIS PB #91-172858/AS (A09)

$27.00

102 ANABOLIC STEROID ABUSE. Geraline C. Lin, Ph.D., and

Lynda Erinoff, Ph.D., eds.

NCADI #M102 NTIS PB #91-172866/AS (A11)

$36.50

103 DRUGS AND VIOLENCE: CAUSES, CORRELATES, AND

CONSEQUENCES. Mario De La Rosa, Ph.D.;

Elizabeth Y. Lambert, M.S.; and Bernard Gropper, Ph.D., eds.

NCADI #M103 NTIS PB #91-172874/AS (A13)

$36.50

104 PSYCHOTHERAPY AND COUNSELING IN THE

TREATMENT OF DRUG ABUSE. Lisa Simon Onken, Ph.D., and

Jack D. Blaine, M.D., eds.

NCADI #M104 NTIS PB #91-172874/AS (A07)

$27.00

106 IMPROVING DRUG ABUSE TREATMENT.

Roy W. Pickens, Ph.D.; Carl G. Leukefeld, D.S.W.; and

Charles R. Schuster, Ph.D., eds.

NCADI #M106 NTIS PB #92-105873(A18) $50.00

107 DRUG ABUSE PREVENTION INTERVENTION RESEARCH:

METHODOLOGICAL ISSUES. Carl G. Leukefeld, D.S.W., and

William J. Bukoski, Ph.D., eds.
311

NCADI #M107 NTIS PB #92-160985 (A13) $36.50

108 CARDIOVASCULAR TOXICITY OF COCAINE:

UNDERLYING MECHANISMS. Pushpa V. Thadani, Ph.D., ed.

NCADI #M108 NTIS PB #92-106608 (A11) $36.50

109 LONGITUDINAL STUDIES OF HIV INFECTION IN

INTRAVENOUS DRUG USERS: METHODOLOGICAL ISSUES IN

NATURAL HISTORY RESEARCH. Peter Hartsock, Dr.P.H., and

Sander G. Genser, M.D., M.P.H., eds.

NCADI #M109 NTIS PB #92-106616 (A08) $27.00

111 MOLECULAR APPROACHES TO DRUG ABUSE RESEARCH:

RECEPTOR CLONING, NEUROTRANSMITTER EXPRESSION,

AND MOLECULAR GENETICS: VOLUME I. Theresa N.H. Lee,

Ph.D., ed.

NCADI #M111 NTIS PB #92-135743 (A10) $36.50

112 EMERGING TECHNOLOGIES AND NEW DIRECTIONS IN

DRUG ABUSE RESEARCH. Rao S. Rapaka, Ph.D.;

Alexandros Makriyannis, Ph.D.; and Michael J. Kuhar, Ph.D.,

eds.

NCADI #M112 NTIS PB #92-155449 (A15) $44.50

113 ECONOMIC COSTS, COST EFFECTIVENESS, FINANCING,

AND COMMUNITY-BASED DRUG TREATMENT.

William S. Cartwright, Ph.D., and James M. Kaple, Ph.D., eds.

NCADI #M113 NTIS PB #92-155795 (A10) $36.50

114 METHODOLOGICAL ISSUES IN CONTROLLED STUDIES

ON EFFECTS OF PRENATAL EXPOSURE TO DRUG ABUSE.

M. Marlyne Kilbey, Ph.D., and Khursheed Asghar, Ph.D., eds.

NCADI #M114 NTIS PB #92-146216 (A16) $44.50

115 METHAMPHETAMINE ABUSE: EPIDEMIOLOGIC ISSUES

AND IMPLICATIONS. Marissa A. Miller, D.V.M., M.P.H., and

Nicholas J. Kozel, M.S., eds.

NCADI #M115 NTIS PB #92-146224/ll (AO7)

$27.00

116 DRUG DISCRIMINATION: APPLICATIONS TO DRUG

ABUSE RESEARCH. R.A. Glennon, Ph.D.;

Toubjörn U.C. Järbe, Ph.D.; and J. Frankenheim, Ph.D., eds.

NCADI #M116 NTIS PB #94-169471 (A20) $52.00

117 METHODOLOGICAL ISSUES IN EPIDEMIOLOGY,

PREVENTION, AND TREATMENT RESEARCH ON DRUGEXPOSED WOMEN AND THEIR CHILDREN.

M. Marlyve Kilbey, Ph.D., and Kursheed Asghar, Ph.D., eds.

GPO Stock #O17-024-01472-9

$12.00

NCADI #M117 NTIS PB #93-102101/LL (A18)

$52.00

118 DRUG ABUSE TREATMENT IN PRISONS AND JAILS.

Carl G. Leukefeld, D.S.W., and Frank M. Tims, Ph.D., eds.

GPO Stock #O17-024-01473-7

$16.00
312

NCADI #M118 NTIS PB #93-102143/LL (A14)

$44.50

120 BIOAVAILABILITY OF DRUGS TO THE BRAIN AND THE

BLOOD-BRAIN BARRIER. Jerry Frankenheim, Ph.D., and

Roger M. Brown, Ph.D., eds.

GPO Stock #017-024-01481-8

$10.00

NCADI #M120 NTIS PB #92-214956/LL (A12)

$36.50

121 BUPRENORPHINE: AN ALTERNATIVE TREATMENT

FOR OPIOID DEPENDENCE. Jack D. Blaine, Ph.D., ed.

GPO Stock #017-024-01482-6 $5.00

NCADI #M121 NTIS PB #93-129781/LL (A08)

$27.00

123 ACUTE COCAINE INTOXICATION: CURRENT METHODS

OF TREATMENT. Heinz Sorer, Ph.D., ed.

GPO Stock #017-024-01501-6 $6.50

NCADI #M123 NTIS PB #94-115433/LL (A09)

$27.00

124 NEUROBIOLOGICAL APPROACHES TO BRAIN-BEHAVIOR

INTERACTION. Roger M. Brown, Ph.D., and

Joseph Fracella, Ph.D., eds.

GPO Stock #017-024-01492-3 $9.00

NCADI #M124 NTIS PB #93-203834/LL (A12)

$36.50

125 ACTIVATION OF IMMEDIATE EARLY GENES BY DRUGS

OF ABUSE. Reinhard Grzanna, Ph.D., and

Roger M. Brown, Ph.D., eds.

GPO Stock #017-024-01503-2 $7.50

NCADI #M125 NTIS PB #94-169489 (A12) $36.50

126 MOLECULAR APPROACHES TO DRUG ABUSE RESEARCH

VOLUME II: STRUCTURE, FUNCTION, AND EXPRESSION.

Theresa N.H. Lee, Ph.D., ed.

NCADI #M126 NTIS PB #94-169497 (A08) $27.00

127 PROGRESS AND ISSUES IN CASE MANAGEMENT.

Rebecca S. Ashery, D.S.W., ed.

NCADI #M127 NTIS PB #94-169505 (A18) $52.00

128 STATISTICAL ISSUES IN CLINICAL TRIALS FOR

TREATMENT OF OPIATE DEPENDENCE.

Ram B. Jain, Ph.D., ed.

NCADI #M128 NTIS PB #93-203826/LL (A09)

$27.00

129 INHALANT ABUSE: A VOLATILE RESEARCH AGENDA.

Charles W. Sharp, Ph.D.; Fred Beauvais, Ph.D.; and

Richard Spence, Ph.D., eds.

GPO Stock #017-024-01496-6

$12.00

NCADI #M129 NTIS PB #93-183119/LL (A15)

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313

130 DRUG ABUSE AMONG MINORITY YOUTH: ADVANCES IN

RESEARCH AND METHODOLOGY. Mario De La Rosa, Ph.D.,

and Juan-Luis Recio Adrados, Ph.D., eds.

GPO Stock #017-024-01506-7

$14.00

NCADI #M130 NTIS PB #94-169513 (A15) $44.50

131 IMPACT OF PRESCRIPTION DRUG DIVERSION CONTROL

SYSTEMS ON MEDICAL PRACTICE AND PATIENT CARE.

James R. Cooper, Ph.D.; Dorynne J. Czechowicz, M.D.;

Stephen P. Molinari, J.D., R.Ph.; and

Robert C. Peterson, Ph.D., eds.

GPO Stock #017-024-01505-9

$14.00

NCADI #M131 NTIS PB #94-169521 (A15) $44.50

132 PROBLEMS OF DRUG DEPENDENCE, 1992:

PROCEEDINGS OF THE 54TH ANNUAL SCIENTIFIC MEETING

OF THE COLLEGE ON PROBLEMS OF DRUG DEPENDENCE.

Louis Harris, Ph.D., ed.

GPO Stock #017-024-01502-4

$23.00

NCADI #M132 NTIS PB #94-115508/LL (A99)

133 SIGMA, PCP, AND NMDA RECEPTORS.

Errol B. De Souza, Ph.D.; Doris Clouet, Ph.D., and

Edythe D. London, Ph.D., eds.

NCADI #M133 NTIS PB #94-169539 (A12) $36.50

134 MEDICATIONS DEVELOPMENT: DRUG DISCOVERY,

DATABASES, AND COMPUTER-AIDED DRUG DESIGN.

Rao S. Rapaka, Ph.D., and Richard L. Hawks, Ph.D., eds.

GPO Stock #017-024-01511-3

$11.00

NCADI #M134 NTIS PB #94-169547 (A14) $44.50

135 COCAINE TREATMENT: RESEARCH AND CLINICAL

PERSPECTIVES. Frank M. Tims, Ph.D., and

Carl G. Leukefeld, D.S.W., eds.

GPO Stock #017-024-01520-2

$11.00

NCADI #M135 NTIS PB #94-169554 (A13) $36.50

136 ASSESSING NEUROTOXICITY OF DRUGS OF ABUSE.

Lynda Erinoff, Ph.D., ed.

GPO Stock #017-024-01518-1

$11.00

NCADI #M136 NTIS PB #94-169562 (A13) $36.50

137 BEHAVIORAL TREATMENTS FOR DRUG ABUSE AND

DEPENDENCE. Lisa Simon Onken, Ph.D.; Jack D. Blaine, M.D.;

and John J. Boren, Ph.D., eds.

GPO Stock #017-024-01519-9

$13.00

NCADI #M137 NTIS PB #94-169570 (A15)

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314

138 IMAGING TECHNIQUES IN MEDICATIONS

DEVELOPMENT: CLINICAL AND PRECLINICAL ASPECTS.

Heinz Sorer, Ph.D., and Rao S. Rapaka, Ph.D., eds.

NCADI #M138

139 SCIENTIFIC METHODS FOR PREVENTION

INTERVENTION RESEARCH. Arturo Cazares, M.D., M.P.H., and

Lula A. Beatty, Ph.D., eds.

NCADI #M139

140 PROBLEMS OF DRUG DEPENDENCE, 1993:

PROCEEDINGS OF THE 55TH ANNUAL SCIENTIFIC MEETING,

THE COLLEGE ON PROBLEMS OF DRUG DEPENDENCE, INC.

VOLUME I: PLENARY SESSION SYMPOSIA AND ANNUAL

REPORTS. Louis S. Harris, Ph.D., ed.

NCADI #M140

141 PROBLEMS OF DRUG DEPENDENCE, 1993:

PROCEEDINGS OF THE 55TH ANNUAL SCIENTIFIC MEETING,

THE COLLEGE ON PROBLEMS OF DRUG DEPENDENCE, INC.

VOLUME II: ABSTRACTS. Louis S. Harris, Ph.D., ed.

NCADI #M141

142 ADVANCES IN DATA ANALYSIS FOR PREVENTION

INTERVENTION RESEARCH. Linda M. Collins, Ph.D., and

Larry A. Seitz, Ph.D., eds.

NCADI #M142

143 THE CONTEXT OF HIV RISK AMONG DRUG USERS AND

THEIR SEXUAL PARTNERS. Robert J. Battjes, D.S.W.;

Zili Sloboda, Sc.D.; and William C. Grace, Ph.D., eds.

NCADI #M143

144 THERAPEUTIC COMMUNITY: ADVANCES IN RESEARCH

AND APPLICATION. Frank M. Tims, Ph.D.;

George De Leon, Ph.D.; and Nancy Jainchill, Ph.D., eds.

NCADI #M144

145 NEUROBIOLOGICAL MODELS FOR EVALUATING

MECHANISMS UNDERLYING COCAINE ADDICTION.

Lynda Erinoff, Ph.D., and Roger M. Brown, Ph.D., eds.

NCADI #M145

146 HALLUCINOGENS: AN UPDATE. Geraline C. Lin, Ph.D.,

and Richard A. Glennon, Ph.D., eds.

NCADI #M146

147 DISCOVERY OF NOVEL OPIOID MEDICATIONS.

Rao S. Rapaka, Ph.D., and Heinz Sorer, Ph.D., eds.

NCADI #M147

148 EPIDEMIOLOGY OF INHALANT ABUSE: AN

INTERNATIONAL PERSPECTIVE.

Nicholas J. Kozel, M.S.; Zili Sloboda, Sc.D.;

and Mario R. De La Rosa, Ph.D., eds.

NCADI #M148
315

149 MEDICATIONS DEVELOPMENT FOR THE TREATMENT

OF PREGNANT ADDICTS AND THEIR INFANTS.

C. Nora Chiang, Ph.D., and Loretta P. Finnegan, M.D., eds.

NCADI #M149

150 INTEGRATING BEHAVIORAL THERAPIES WITH

MEDICATIONS IN THE TREATMENT OF DRUG

DEPENDENCE. Lisa Simon Onken, Ph.D.; Jack D. Blaine, M.D.;

and John J. Boren, Ph.D., eds.

NCADI #M150

151 SOCIAL NETWORKS, DRUG ABUSE, AND HIV

TRANSMISSION. Richard H. Needle, Ph.D., M.P.H.;

Susan L. Coyle, Ph.D.; Sander G. Genser, M.D., M.P.H.; and

Robert T. Trotter II, Ph.D., eds.

NCADI #M151

152 PROBLEMS OF DRUG DEPENDENCE 1994: PROCEEDINGS

OF THE 56TH ANNUAL SCIENTIFIC MEETING, THE

COLLEGE ON PROBLEMS OF DRUG DEPENDENCE, INC.

VOLUME I: PLENARY SESSION SYMPOSIA AND ANNUAL

REPORTS. Louis S. Harris, Ph.D., ed.

NCADI #M152

153 PROBLEMS OF DRUG DEPENDENCE 1994: PROCEEDINGS

OF THE 56TH ANNUAL SCIENTIFIC MEETING, THE

COLLEGE ON PROBLEMS OF DRUG DEPENDENCE, INC.

VOLUME II: ABSTRACTS. (1995) Louis S. Harris, Ph.D., ed.

NCADI #M153 GPO Stock #017-024-01564-4

$22.00

154 MEMBRANES AND BARRIERS: TARGETED DRUG

DELIVERY. (1995) Rao S. Rapaka, Ph.D., ed.

NCADI #M154 GPO Stock #017-024-01583-1

$10.00

155 REVIEWING THE BEHAVIORAL SCIENCE KNOWLEDGE

BASE ON TECHNOLOGY TRANSFER. (1995)

Thomas E. Backer, Ph.D.; Susan L. David; and Gerald Soucy,

Ph.D., eds.

NCADI #M155 GPO Stock #017-024-01581-4

$12.00

156 ADOLESCENT DRUG ABUSE: CLINICAL ASSESSMENT

AND THERAPEUTIC INTERVENTIONS. (1995)

Elizabeth Rahdert, Ph.D.; Zili Sloboda, Ph.D.; and Dorynne

Czechowicz, M.D., eds.

NCADI #M156 GPO Stock #017-024-01585-7

$14.00

157 QUALITATIVE METHODS IN DRUG ABUSE AND HIV

RESEARCH. (1995)

Elizabeth Y. Lambert, M.S.; Rebecca S. Ashery, D.S.W.; and

Richard H. Needle, Ph.D., M.P.H., eds.

NCADI #M157 GPO Stock #017-024-01581-4

158 BIOLOGICAL MECHANISMS AND PERINATAL EXPOSURE

TO DRUGS. (1995) Pushpa V. Thadani, Ph.D., ed.
316

NCADI #M158 GPO Stock #017-024-01584-9

159 INDIVIDUAL DIFFERENCES IN THE BIOBEHAVIORAL

ETIOLOGY OF DRUG ABUSE. (1996) Harold W. Gordon, Ph.D.,

and Meyer D. Glantz, Ph.D., eds.

NCADI #M159
317

161 MOLECULAR APPROACHES TO DRUG ABUSE RESEARCH.

VOLUME III: RECENT ADVANCES AND EMERGING

STRATEGIES. (1996) Theresa N.H. Lee, Ph.D., ed.

NCADI #M161

162 PROBLEMS OF DRUG DEPENDENCE, 1995.

PROCEEDINGS FROM THE 57TH ANNUAL SCIENTIFIC

MEETING OF THE COLLEGE ON DRUG DEPENDENCE, INC.

(1996)

Louis Harris, Ph.D., ed.

NCADI #M162

163 NEUROTOXICITY AND NEUROPATHOLOGY

ASSOCIATED WITH COCAINE/STIMULANT ABUSE. (1996)

Dorota Majewska, Ph.D., ed.

NCADI #M163

164 BEHAVIORAL STUDIES OF DRUG-EXPOSED OFFSPRING:

METHODOLOGICAL ISSUES IN HUMAN AND ANIMAL

RESEARCH. (1996) Cora Lee Wetherington, Ph.D.;

Vincent L. Smeriglio, Ph.D.; and Loretta P. Finnegan, Ph.D.,

eds.

NCADI #M164

165 BEYOND THE THERAPEUTIC ALLIANCE: KEEPING THE

DRUG-DEPENDENT INDIVIDUAL IN TREATMENT. (1996)

Lisa Simon Onken, Ph.D.; Jack D. Blaine, M.D.; and

John J. Boren, Ph.D., eds.

NCADI #M165

166 TREATMENT FOR DRUG-EXPOSED WOMEN AND

CHILDREN: ADVANCES IN RESEARCH METHODOLOGY.

(1996) Elizabeth Rahdert, Ph.D., ed.

NCADI #M166

167 THE VALIDITY OF SELF-REPORTED DRUG USE:

IMPROVING THE ACCURACY OF SURVEY ESTIMATES.

(1996) Lana Harrison, Ph.D., and Arthur Hughes, M.D., eds.

NCADI #M167

168 RURAL SUBSTANCE ABUSE: STATE OF KNOWLEDGE

AND ISSUES. (1996) Zili Sloboda, Sc.D.;

Eric Rosenquist; and Jan Howard, Ph.D., eds.

NCADI #M168

169 TREATMENT OF PAIN IN ADDICTS AND OTHERS WHO

MAY HAVE HISTORIES OF DEPENDENCE. (1996)

Alan I. Trachtenberg, M.D., M.P.H., F.A.A.F.P., D.AA.P.M.,

ed.

NCADI #M169

170 META-ANALYSIS OF DRUG ABUSE PREVENTION

PROGRAMS. (1996) William J. Bukoski, Ph.D., ed.

NCADI #M170

171 TREATMENT OF COCAINE DEPENDENCE: OUTCOME

RESEARCH. (1996). Frank M. Tims, Ph.D., ed.

NCADI #M171
318

172 TREATMENT OF DRUG-DEPENDENT INDIVIDUALS

WITH COMORBID MENTAL DISORDERS. (1996). Lisa Simon

Onken, Ph.D.; Jack D. Blaine, M.D.; Sander Genser, M.D.,

M.P.H.; and Arthur MacNeill Horton, Jr., Ed.D., eds.

NCADI #M172