National

Institute on

Research Findings

on Smoking of

Abused Substances

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service Alcohol, Drug Abuse, and Mental Health Administration
Research Findings on

Smoking of Abused

Substances

Editor:

C. Nora Chiang, Ph.D.

Richard L. Hawks, Ph.D.

Division of Preclinical Research

National Institute on Drug Abuse

NIDA Research Monograph 99

1990

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public-Health Service

Alcohol, Drug Abuse, and Mental Health Administration

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, Maryland 20657

For sale by the Superintendent of Documents, U.S. Government Printing Office

Washington, D.C. 20402

Research Findings on Smoking of

Abused Substances
ACKNOWLEDGMENT

This monograph is based on the papers and discussion from a technical

review on “Research Findings on Smoking of Abused Substances” held on

November 10, 1988, in Rockville, MD. The review meeting was sponsored

by the Office of Science and the Division of Preclinical Research, National

Institute on Drug Abuse.

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The National Institute on Drug Abuse has obtained permission from the

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Opinions expressed in this volume are those of the authors and do not

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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

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context of the studies reported herein.

DHHS publication number (ADM)90-1690

Printed 1990

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.

i v
Contents

Page

Introduction and Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

C. Nora Chiang

Current Patterns of Drug Abuse That Involve Smoking . . . . . . . . . 5

Donald R. Wesson and Peter Washburn

Clinical Pharmacology of Inhaled Drugs of Abuse:

Implications in Understanding Nicotine Dependence . . . . . . . . . . . 12

Neal L. Benowitz

The Pharmacology of Cocaine Smoking in Humans . . . . . . . . . . . 30

Reese T. Jones

Marijuana Smoking: Factors That Influence the

Bioavailability of Tetrahydrocannabinol . . . . . . . . . . . . . . . . . . . 42

Mario Perez-Reyes

Effects of Habitual Use of Marijuana and/or Cocaine

on the Lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Donald P. Tashkin, Suzanne Fligiel, Tzu-Chin Wu,

Henry Gong, Jr., Richard G. Barbers, Anne H. Coulson,

Michael S. Simmons, and Theodore F. Beals

Marijuana Effects and Urinalysis After Passive Inhalation

and Oral Ingestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

Edward J. Cone

v
Pyrolytic Degradation of Heroin, Phencyclidine, and

Cocaine: Identification of Products and Some

Observations on Their Metabolism . . . . . . . . . . . . . . . . . . . . . . 97

C. Edgar Cook and A. Robert Jeffcoat

Chemical and Biological Analysis of Marijuana

Smoke Condensate . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . 121

Charles M. Sparacino, Patricia A. Hyldburg,

and Thomas J. Hughes

Pyrolysis and Inhalation Studies With Phencyclidine

and Cocaine . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . 141

Billy R. Martin and Joseph Boni 

Animal Models of Drug Self-Administration by Smoking . . . . . . . . 159

Ronald W. Wood

List of NIDA Research Monographs . . . . . . . . . . . . . . . . . . . . . 172

v i
Introduction and Overview

C. Nora Chiang and Richard L. Hawks

For centuries, smoking has been a popular route for the self-administration

of recreational drugs such as tobacco, marijuana, and opium. Recently,

smoking has also gained popularity for the use of phencyclidine (PCP), cocaine, and methamphetamine. It is likely that smoking as a route of administration is being more frequently chosen by drug abusers as a “safer” route

because of the dangers of intravenous (IV) transmission of the human

immunodeficiency virus. This route of administration is a major contributing factor to the current epidemic of cocaine and methamphetamine abuse.

The toxicity resulting from this route of administration is complex. Not

only the drug itself but also the resulting degradation products contribute to

the pharmacological and toxicological effects. In addition, there are unique

pharmacokinetic and behavioral characteristics associated with smoking as a

route of administration. To understand the particular toxicity associated

with smoking, it is necessary to integrate research findings in several areas.

These include chemistry, pharmacology and toxicology, pharmacokinetics,

and behavioral and clinical observations. A technical review concerning

research finding on smoking of abused substances was held by the National

Institute on Drug Abuse on November 10, 1988.

The first presentation by Dr. Donald Wesson on the pattern as well as the

techniques for the self-administration of abused drugs revealed that cocaine

and methamphetamine smoking is on the rise and that the methods for

smoking abused drugs are quite variable. Often, combinations of drugs are

smoked. This information is important for the interpretation of clinical

observations and for the design of research experiments for the investigation

of various aspects of smoking of abused drugs.

Dr. Neal Benowitz discus& the unique pharmacokinetic and pharmacodynamic aspects of the smoked route of drug administration that may contribute to the abuse of tobacco cigarettes. Using nicotine as an example,

Dr. Benowitz demonstrated that the inhalation route provides a very rapid

delivery of the drug into the brain. The smoker is therefore able to titrate

the level of the drug in the brain to achieve a desired mental state. This

user control would be expected to enhance the reinforcing nature of the

1
drug and thus contribute to the preferred use of inhalation for abused

substances.

The pharmacokinetic and pharmacodynamic data on cocaine presented by

Dr. Reese Jones showed rapid peak effects and peak plasma levels from

smoked and IV doses as compared with those from intranasal and oral

doses. He explained that the rapidly developed tolerance, which occurs

even with the first dose of cocaine, is a contributing factor to the much

less intense drug effects perceived from the oral and intranasal doses. As

the intense peak effect is important to drug abusers, the differences between

addictive potential and lethality of cocaine from different routes of administration undoubtedly could be partly due to the pharmacokinetic differences.

The dynamics of smoking marijuana was presented by Dr. Mario PerezReyes. A great portion of tetrahydrocannabinol (THC) is either degraded

during pyrolysis or lost in sidestream smoke. Therefore, the manner in

which marijuana cigarettes are smoked plays an important role in determining the amount of THC inhaled or the resultant plasma concentrations.

Consequently, great variability in both plasma levels and observed effects

has been reported for the smoked dose. The assumption that smokers have

control over the rate of THC inhaled is also evidenced by the observation

that the smoke volume inhaled per puff during smoking progressively

decreases.

Lungs are the principal organ exposed to the smoke and pyrolysis products

of abused drugs. The long-term effects of such smoking on the lung were

discussed by Dr. Donald Tashkin. Chronic smoking of only a few marijuana cigarettes a day has been shown to have long-term adverse effects on

the lung. A comparison of habitual smoking of marijuana and tobacco indicates that both cause functional alterations in the respiratory tract but on

different sites. The adverse effects on the respiratory tract and the lung

caused by marijuana plus tobacco are additive. Preliminary data from

studies with free-base cocaine smokers revealed a high proportion of subjects with acute cardiopulmonary symptoms temporally related to cocaine

smoking. Further research should be directed at the effect of cocaine smoking on lung function and structure.

Health concerns about passive exposure to tobacco smoke have led to similar concerns about marijuana smoke. Additionally, passively inhaled THC

has been raised as an issue in urine testing. As summarized by Dr. Edward

Cone, the duration of a positive test for urine for cannabinoids and the

intensity of behavioral and pharmacological effects depend upon the amount

of THC that actually gets into the body. Passive inhalation of great

amounts of smoke could no doubt result in significant behavioral responses

as well as positive urine analyses. However, exposures that occur in social

situations are unlikely to result in levels that could produce noticeable

pharmacological effects or positive results for cannabinoids in urinalysis.

2
On the other hand, oral ingestion of marijuana in a brownie containing the

equivalent of one 2.8 percent cigarette was shown to result in measurable

pharmacological effects and positive urines for about a week. It is conceivable that such ingestion could be inadvertent.

Since all the pyrolysis products of abused substances in the mainstream

smoke are inhaled and could contribute to the pharmacological effects and

toxicities associated with smoking, the identification and quantification

of these products are important for understanding the drug’s effects.

Drs. Edgar Cook and Billy Martin presented comprehensive data obtained

from both in vivo and in vitro studies of smoked cocaine, PCP, and heroin.

Some of the pyrolytic products from heroin, identified by Dr. Cook, such as

6-acetylmorphine and N-acetylnormorphine, might be expected to contribute

to pharmacological and toxicological effects observed with smoking. In

addition, the contaminants or adulterants in an abused drug could also pose

health risks when smoked. Quantitative determination of the constituents of

the smoke actually inhaled by a drug abuser on the street is very difficult.

The amounts of the intact drug and its degradation products in mainstream

smoke are quite variable and highly dependent upon the physicochemical

properties of the drug and the smoking conditions employed. These include

temperature, flow rate, and the presence or absence of other materials. The

decomposition of a compound generally is much more extensive if it is in

its acid-salt form rather than its free-base form. This explains why cocaine

free base is the predominant form for smoked cocaine abuse. An exception

to this is methamphetamine for which the hydrochloride is typically the

smoked form (ice), as this form is extensively vaporized on heating.

When plant materials or abused drugs mixed with plant materials are

smoked, the resultant pyrolysis byproducts can be even more complex.

Studies by Dr. Charles Sparacino indicate that marijuana smoke condensate

contains several thousand compounds. So far, several hundred compounds

have been identified, and a significant portion of these are nitrogen

containing aromatic compounds. Dr. Sparacino showed that fractions of

smoke condensate that contain such amine compounds are mutagenic.

The use of proper animal models has made significant contributions to our

understanding of the behavioral pharmacokinetic and pharmacologic effects

of abused drugs. Studies by Dr. Martin in rats have shown that behavioral

effects and biodisposition for PCP were similar following both smoking and

intraperitoneal administration of PCP.

Dr. Ronald Wood explained that animal models for studying self-administration of abused drugs by smoking have been very difficult to devise. In

addition to the fact that animals cannot be trained to inhale like humans, the

pattern of particulate deposition and absorption is dependent on both species

and particle size. Research is currently attempting to develop a special generator to produce reliable exposure concentrations, stable particle size

3
distribution, and less irritation. This system will be used for studies of

behavioral and pharmacological effects from self-administration of smoked

drugs

The conclusion can be easily drawn from this technical review that smoking

of abused drugs is a very complex issue. So far, a limited body of information has accumulated on some of the unique characteristics of the

smoked dose. Much additional research is needed in this area and will

involve the collaboration of both basic and clinical researchers to understand

sufficiently the fundamental mechanisms, toxicities, and behaviors, a collaboration that can lead to the design of better treatment and prevention

strategies.

AUTHORS

C. Nora Chiang, Ph.D.

Research Technology Branch

Richard L. Hawks, Ph.D.

Chief, Research Technology Branch

Division of Preclinical Research

National Institute on Drug Abuse

Rockville, MD 20857

4
Current Patterns of Drug Abuse

That Involve Smoking

Donald R. Wesson and Peter Washburn

INTRODUCTION

Smoking is a method of drug use that is acceptable to many people, including many for whom injection of street drugs would not be acceptable. Familiarity with smoking is, of course, an important factor. Because of extensive tobacco and marijuana smoking in this culture, most people are familiar

with drug use by smoking, and they do not perceive smoking to be as culturally deviant as self-administration of drugs by intravenous injection.

Most people associate intravenous injection of drugs with “hardcore drug

abusers.”

Smoking is perceived as safer than intravenous injection; however, the safety is somewhat illusionary. Although smoking does not produce many of

the medical complications that occur with intravenous drug abuse, e.g.,

human immunedeficiency virus (HIV) infection, infective endocarditis, or

serum hepatitis, the toxicity of some drugs that are smoked is substantial.

Cocaine smokers, for example, can ingest lethal quantities of cocaine by

smoking. Of particular public health concern is cocaine’s toxicity for the

fetuses of pregnant women who smoke crack.

The willingness of many people to smoke drugs played a major role in the

emergence of the crack cocaine epidemic of the eighties. Table 1 shows

the sources of commonly smoked drugs. The introduction of cocaine in

small lots ready for smoking opened a large new marketplace for cocaine

among people whose former drug choices were alcohol, marijuana, and

nicotine.

For drugs that volatilize at low temperature, smoking can be an easy, efficient method of consumption, and large amounts of a drug can be rapidly

delivered to the brain. Most psychoactive drugs exert the maximum subjective effects when blood levels of the drug are rapidly increasing. Decause smokable drugs enter the blood stream rapidly through the lungs, an

5
inhalation can produce a sharp increase in arterial blood concentration of the

drug. The drug is carried by the arterial blood directly to the brain. The

high concentration of drug in the arterial blood can produce an intense psychoactive drug effect, which can be qualitatively different from the drug’s

steady state effect. The intense psychoactive drug effect, often called a

“rush,” is the effect desired by the abuser. This is why, for example, drug

abusers prefer to smoke or inject methamphetamine rather than take it

orally. Methamphetamine is bioavailable orally; however, when absorbed

from the gastrointestinal tract, blood levels rise slowly and do not produce

the desired “rush.”

TABLE 1. Drugs that are smoked and their source

Drug Source

Freebase cocaine

(freebase crystals and crack)

Opium

Freebase heroin

Phencyclidine (PCP)

Methamphetamine

Delta-9-tetrahydrocannabinol (THC)

marijuana (.1-9.5%) -9-THC*

hashish (1040%) -9-THC*

Coca plant

Poppy plant

Opium

Illicit synthesis

Illicit synthesis

Cannabis indica,

Cannabis sativa

*Range of THC values from Mikuriya and Aldrich (1988).

COCAINE

In the United States, cocaine is sold as either the hydrochloride salt or as

free base. Cocaine hydrochloride, which is water soluble, is used by snorting or by intravenous injection. Some cocaine abusers smoke cocaine

hydrochloride; however, cocaine hydrochloride volatilizes at a high temperature and most of it is destroyed by burning. Most cocaine abusers who

smoke cocaine use free-base cocaine. Cocaine free base volatilizes at a

lower temperature, and a greater percentage of it is spared during the smoking

process. The form of free base called crack has become the most popular form of cocaine that is smoked in the United States.

Figure 1 shows the number of emergency room mentions of cocaine in the

Drug Abuse Warning Network (DAWN) between 1982 and 1987. The total

number of cocaine mentions per year have increased for all methods of

amine use; however, the largest increase has ocurred in the mentions of

smoked cocaine-which is now primarily crack.

6
FIGURE 1. DAWN national emergency room mentions of cocaine

Note: Data from the annual publication Data from the Drug Abuse Warning Network, 1982-

1987. Shown are the national total of emergency room visits in which the administration

route of cocaine was specified. The relationship between national prevalence of cocaine use

and mentions in DAWN is complex. DAWN cocaine data are consistent with many other

indices of cocaine use that support a marked increase in prevalence of cocaine smoking.

Free-Base Cocaine

Freebase cocaine can be prepared from cocaine hydrochloride as either

free-base crystals or as crack.

The preparation of free-base crystals involves dissolving the cocaine hydrochloride in water, converting the hydrochloride to the alkaloid form by addition of a strong base (such as sodium or ammonium hydroxide), extraction

of the alkaloid with an organic solvent (such as petroleum ether), and

crystallization of the free base. Before the current crack epidemic in the

United States, most cocaine free-base users made their free base from cocaine hydrochloride. The preparation of free-base crystals was part of the

ritual of use. The free-base conversion also removed sugars and other

water-soluble diluents used to “cut” cocaine. Until most States passed drug

paraphernalia laws that made free-base conversion kits illegal, most users

made their free base with the kits, which contained all the ingredients for

the conversion-except, of course, the cocaine hydrochloride. The kits were

sold in drug paraphernalia shops and by mail order. A history of cocaine

free-base use before 1982 is detailed in a theme issue of the Journal of

Psychoactive Drugs (Siegel 1982).

Crack cocaine is also made from cocaine hydrochloride. The cocaine

hydrochloride is dissolved, mixed with baking soda or sodium bicarbonate,

7
and heated. While the mixture is being heated, the “cooker” gently swirls

or rotates the container. The freebase cocaine precipitates and coalesces

into a soft mass that becomes hard when it is dried. Sugars and other

water-soluble diluents remain in the water, which is then poured off; leaving

the solid mass. Now, most crack users buy crack in the form of pellets or

“rocks” that are already in the free-base form. Some users prefer to make

their own crack from the cocaine hydrochloride because they feel more

confident of its purity.

Several years before the current crack epidemic, smoking cocaine paste, an

extract of coca leaves containing 40 to 85 percent cocaine sulfate, became a

major public health problem in Peru. Subsequently, paste smoking became

common in Bolivia, Columbia, and Ecuador (Jeri 1984). Cocaine paste

abusers there are mostly people in the lower socioeconomic classes. The

paste is generally smoked in marijuana or tobacco cigarettes.

Crack and cocaine free-base crystals are often smoked in glass pipes. The

glass pipe has a bowl fitted on the bottom with one or more fine-mesh

copper screens, which support the cocaine. The user heats the side of the

bowl with a small butane torch or lighter. The free base vaporizes and is

drawn through the pipe. A pipe used in this manner gets hot and users

often bum fingers or lips. Some free-base smokers have burns on their

groins from suddenly hiding hot free-base pipes in their pockets.

Cocaine and Marijuana Combinations

Cocaine free base and occasionally cocaine hydrochloride are mixed with

marijuana and smoked. A marijuana cigarette laced with crack is commonly called a “grimmie.” Crack is crushed between two coins, mixed with

marijuana, and rolled into a “joint.” Although smokers of grimmies are

smoking crack, they seem to view it as a less intense and leas severe form

of use than using crack in a pipe. Many users of grimmies do not realize

that they are free basing as they associate free basing with using a pipe.

Some free-base abusers report using grimmies as a means of controlling

their cocaine use. When cocaine is smoked in marijuana, users report that

there is less compulsion for continued use and less difficulty sleeping after

use. In the San Francisco Bay area, grimmies are particularly popular

among black youth.

Cocaine and Tobacco Combinations

The combination of crack cocaine and a tobacco cigarette is known as caviar (or cavies). Cocaine hydrochloride is also used in this manner. The

user makes the combination by reducing the crack to a powder and using

the cigarette as a straw, sucking the cocaine inside the cigarette. The advantage of this dosage form is that it can be smoked in public without its

being apparent that the person is using cocaine.

8
Phencyclidine and Cocaine

Phencyclidine (PCP) and cocaine are sometimes mixed together. The combination is known as “whack” or “spacebase.” Some users seek the combination, but PCP is more often included because it is a less expensive

substitute for cocaine. Thus, some cocaine users who believe they are

using only cocaine are also using PCP.

Cocaine Substitutes

Cocaine substitutes, most often procaine, are still used either as drugs of

deception or intentionally as a “poor man’s cocaine substitute.” Sold under

names such as Snowcaine or Rock, procaine am be purchased in drug paraphernalia shops or by mail order. Sellers escape drug regulation by selling

the substitute as incense. The label disclaimer gives directions for use:

“Do not inhale vapors, as it may cause stimulation.”

Acquired Immunodeficiency Syndrome and Crack

Although smoking avoids the intravenous drug abuser’s risk of being infected with HIV by contaminated needles, smoking crack cocaine is still

associated with behavior that puts some abusers at risk. Increasingly,

women offer sex in exchange for crack.

PCP

Most users of PCP use it by smoking, usually mixing it with parsley,

tobacco, or marijuana. The PCP may be dissolved and a cigarette dipped in

the solution. A dark-brown colored cigarette is often used for this purpose

as the discoloration produced by PCP is not as obvious as it would be on a

white cigarette. Sherman cigarettes, because they are dark brown, are often

used, thus the street name of this combination is known as “Shermans” or

“shermies.”

METHAMPHETAMlNE (ICE)

Methamphetamine can be smoked in either the hydrochloride salt or free

base form. Smoking of the d-isomer of methamphetamine, called “ice,” has

become popular in Hawaii. Drug Enforcement Agency (DEA) seizures of

ice in Hawaii began in 1985. Ice sold in Hawaii probably originates in

Korea; however, mainland U.S. manufacture is beginning. In 1990, the

DEA seized ice crystals manufactured in California.

Methamphetamine can be smoked in one of several ways:

1. Methamphetamine is placed on aluminum foil, the aluminum foil heated

from below, and the methamphetamine vapors inhaled. (As with heroin,

this mode of use is sometimes called “chasing the dragon.“)

9
2. Methamphetamine is smoked in small, straight glass pipes. (Unlike

free-base cocaine, which is not water soluble, methamphetamine cannot

be smoked in water pipes, as the methamphetamine is water soluble and

would be trapped in the water.)

3. Methamphetamine is smoked in combination with tobacco or marijuana.

HEROIN AND OPIUM SMOKING

Smoking heroin is a dominant form of opiate use in Asia, where the form

of freebase heroin prepared for smoking is designated by the U.S. Drug

Enforcement Agency as heroin #3. Smoking free-base heroin also occurs in

the United States and England.

In the early seventies, Persian heroin, a freebase form of heroin, was

favored by cocaine free-base users on the West Coast, who used it to attenuate cocaine-induced anxiety, agitation, or paranoia. With continued, frequent use, some cocaine users who used Persian heroin as a secondary drug

became physically dependent on the heroin, much to their surprise, as they

thought that physical dependency could occur only with intravenous

injection.

Smoking opium is a primary and stable mode of use among some youthful

heroin abusers in England (Gossop et al. 1988). Opium smoking also

occurs in the United States, particularly among immigrants from Southeast

Asia.

STIMULANT AND OPIATE COMBINATIONS

Drug abusers have long used combinations of stimulants and opiates intravenously. The combination is commonly called a “speedball.” Cocaine

freebase abusers also smoke cocaine free base or methamphetamine mixed

with freebase heroin. The street term for smoking a mixture of cocaine

and heroin is “chasing and basing.”

RECOMMENDATIONS FOR FUTURE RESEARCH

Future research in pyrolytic chemistry should examine both street drugs and

drug combinations, emulating as closely as possible the way abusers actually

smoke the drugs. Much pyrolytic chemistry research on drugs of abuse

involves the use of pure drugs. This is important in delineating the toxicity

of specific drugs, but does not completely address clinicians’ need to understand the toxicity of abused drugs combined with street impurities, adulterants, and other drugs. Much of the toxicity information currently available

to clinicians is based on case reports that lacked adequate analysis of the

drug.

10
The role of cigarette smoking in drug abuse relapse needs systematic study.

Some patterns of drug abuse involve combining drugs with cigarettes. In

addition, many patients report that they smoke more cigarettes when using

cocaine. The role of continued cigarette smoking in inducing cravings for

other drug of abuse and relapse to other drug abuse deserves careful clinical study. If researchers should find that smoking cigarettes contributes to

relapse, treatment programs should incorporate smoking cessation as part of

drug abuse treatment.

REFERENCES

Gossop, M.; Griffiths, P.; and Strang, J. Chasing the dragon: Characteristics of heroin chasers. Br J Addict 83:1159-1162, 1988.

Jeri, F.R. Coca-paste smoking in some Latin American countries: A

severe and unabated form of addiction. Bull Narc 36(2):15-31, 1984.

Mikuriya, T.H., and Aldrich, M.R. Cannabis 1988: Old drug, new

dangers: The potency question. J Psychoactive Drugs 20(1):47-55, 1988.

Siegel, R.K. Cocaine smoking. J Psychoactive Drugs 14(4):271-358, 1982.

AUTHORS

Donald R. Wesson, M.D.

Associate Clinical Professor of Psychiatry

University of California, San Francisco

and

Staff Psychiatrist

San Francisco Veterans Administration Medical Center

4150 Clement Street, 116F

San Francisco, CA 94121

Peter Washburn, M.D.

Medical Director

Merritt Peralta Institute

Oakland, CA

11
Clinical Pharmacology of Inhaled

Drugs of Abuse: Implications in

Understanding Nicotine

Dependence

Neal L. Benowitz

INTRODUCTION

Why do people abuse drugs by the smoked or inhaled route? How does

the pharmacology of inhaled drugs differ from that of drug administered by

other routes? This chapter will discuss these and other aspects of the clinical pharmacology of inhaled drugs of abuse using nicotine as an example.

Pharmacokinetic and pharmacodynamic characteristics of a drug are important determinants of dependence liability, the temporal patterns of drug use,

and the level of drug use. ultimately, an understanding of pharmacokinetics

and pharmacodynamics may be useful in developing effective treatment

strategies. Several pharmacokinetic and pharmacodynamic characteristics

appear to be necessary or optimal for a drug to produce dependence:

(1) the drug must be effectively absorbed into the blood stream; (2) the

drug must rapidly enter into the brain; and (3) the drug must be psychoactive and that psychoactivity related to levels of the drug in the brain.

These characteristics allow for the drug abuser to manipulate the dose of his

or her drug to optimize mood and psychological functioning and are most

likely to result in the behavior described as criteria for drug dependence,

Inhalation of a drug provides a unique delivery system that is conducive to

producing drug dependence. Inhalation of a drug facilitates rapid absorption

into the circulation which, in turn, results in rapid delivery to the brain.

Studies on the clinical pharmacology of inhaled drugs of abuse present

some particular research problems. For example, the bioavailability of inhaled drugs is highly variable from person to person and difficult to quantitate. Inhaled drugs are delivered and distributed to target organs rapidly.

This means that pharmacokinetic and pharmacodynamic behaviors are in

12
disequilibrium conditions. This makes it difficult to study actions of a drug

by the usual techniques of comparing venous concentrations with effects.

Finally, tolerance develops rapidly to many or all effects of drug of abuse.

Thus, even if the concentration in the brain could be known, the effect will

vary over time and as a function of previous drug exposure history.

Approaches to studying these and other research problems concerning the

pharmacology of inhaled drugs will be presented.

THE LUNGS AND DRUG ACTION

Inhaled drugs are taken into the lungs, from which they are absorbed into

the systemic circulation (Bend et al. 1985) (table 1). Before discussing the

pharmacology of inhaled drugs, it is useful to consider how the anatomy

and physiology of the lung may influence drug action.

TABLE 1. Lungs and drug action

Pharmacologic Function Action

Absorption of Inhaled Drugs Rapid transfer of unionized

lipophilic drugs from alveolar

spaces to circulation

High pulmonary blood flow

Accumulation (Uptake) of Drugs Retention of drugs after inhalation

First pass uptake after IV dosing

Local toxicity

Metabolism Clearance of drugs and endogenous

chemicals

Oxidation, reduction, hydrolysis,

biosynthesis

The lung may be conceived as having three major pharmacologic actions.

The first is the absorption of inhaled drugs. Absorption of drugs from the

lungs is facilitated by a huge alveolar surface area, thin alveolar epithelial

and endothelial layers, and an extensive capillary bed. As a result of these

anatomical factors, many drugs, particularly un-ionized lipophilic drugs

(which includes all of the common drugs of abuse), move rapidly from the

alveolar spaces into the systemic circulation. Also of importance is that

pulmonary capillary blood flow is high, representing passage of the entire

blood volume through the lung every minute. Thus, drugs that are absorbed

are carried quickly to various parts of the body and to target organs.

13
A second function of the lung is the uptake of drugs by the lung parenchyma. Such uptake may involve extracellular binding, intracellular binding, or both to the various proteins, including receptors, and in some cases

energy-dependent active transport systems. The results of such uptake may

include retention of drugs after inhalation or during the first pass through

the pulmonary circulation after intravenous dosing. In these cases, the lung

becomes a reservoir for slow and persistent release of drugs into the circulation. Pulmonary uptake may also result in high local concentrations of a

drug, which could result in local toxicity, such as is the case with paraquat.

A third pharmacologic function of the lung is the metabolism of some drugs

and endogenous chemicals. The lungs have the potential to metabolize

drugs by the same pathways as the liver. Although the concentrations of

drug-metabolizing enzymes in lung tissues are lower than that in liver, the

blood flow to the lungs (and hence the amount of a drug brought into contact with drug-metabolizing enzymes) is much greater. Therefore, metabo

lism by the lung can contribute substantially to total drug metabolism.

Cytochrome P450 activity is primarily in nonciliated bronchial epithelial

(Clara) and Type 2 alveolar cells. These cells are most vulnerable to injury

as a result of metabolism of certain drugs and chemicals to reactive metabolites. Examples of how these various lung processes contribute to the

pharmacology of inhaled drugs, in particular nicotine, are discussed in subsequent sections.

ABSORPTION AND BIOAVAILABILITY

Nicotine is a tertiary amine composed of a pyridine and a pyrrolidine ring.

Nicotine is a weak base with a pKa of 8.0 soluble both in water and in

lipids. At physiological pH, about 31 percent of nicotine is non-ionized

such that it readily crosses cell membranes. The pH of tobacco smoke is

important in determining the absorption of nicotine from different sites within the body. The pH of smoke from flue-cured tobaccos found in most

cigarettes is acidic (Brunneman and Huffman 1974). At this pH, the nicotine is primarily ionized. In its ionized state, such as in acidic environments, nicotine does not rapidly cross membranes. As a consequence, there

is little buccal absorption of nicotine from cigarette smoke, even when it is

held in the mouth. The pH of smoke from air-cured tobaccos, such as in

pipes, cigars, and a few European cigarettes, is alkaline, and nicotine is

primarily unionized. Smoke from these products is well absorbed through

the mouth (Armitage et al. 1978).

When tobacco smoke reaches the small airways and alveoli of the lung, the

nicotine is rapidly absorbed independent of pH of the smoke. Blood concentrations of nicotine rise quickly during and peak at the completion of

cigarette smoking (figure 1). The rapid absorption of nicotine from cigarette smoke through the lungs, presumably because of the huge surface area

14
Cigarettes

Oral Snuff

Chewing Tobacco

Nicotine Gum (4mg)

N=10

FIGURE 1. Blood concentrations &ring and after cigarette smoking for

9 minutes (one and one-third cigarettes) and use of oral

snuff (2.5 g), chewing tobacco (average 7.9 g), and

nicotine gum (two 2-mg pieces) for 30 minutes

NOTE: Average values for 10 subjects (±SEM).

15
of the alveoli and small airways and dissolution of nicotine into fluid of pH

in the human physiologic range, facilitates transfer across cell membranes.

Blood or plasma concentrations of nicotine sampled in the afternoon in

smokers generally range from 10 to 50 ng/ml (0.6 to 3x10-7 M). The increment in blood nicotine concentration after smoking a single cigarette ranges

from 5 to 30 ng/ml, depending on how the cigarette is smoked. Smoking

is commonly considered to be a process of intermittent dosing of nicotine,

which is in turn rapidly eliminated from the body. There is considerable

peak to trough oscillation from cigarette to cigarette. However, consistent

with a half-life of 2 hours, nicotine accumulates over 6 to 8 hours of regular smoking and nicotine levels persist overnight, even as the smoker sleeps

(Benowitz et al. 1982b). Thus, smoking results not in intermittent exposure

but in exposure to nicotine that lasts 24 hours of each day.

Smoking behavior is complex, and the dose of a drug delivered to the circulation is influenced by how the smoker smokes. Thus, the intensity,

duration and number of puffs, depth of inhalation, degree of mixing of

smoke with air, and other factors influence the dose. Estimation of the

dose of nicotine has been attempted by setting up standardized procedures

of machine testing, where a smoking machine puffs a cigarette in a pattern

similar to that of human smokers. This test is the basis for the cigarette

yield data that were formerly published by the United States Federal Trade

Commission, and that appear in many cigarette advertisements. However,

human smokers smoke differently from machines, and machine deliveries do

not correlate very well with biochemical markers of nicotine absorption in

people (Benowitz et al. 1983).

Bioavailability can be assesed by methods analogous to those used in other

forms of drug testing. For example, we infused nicotine intravenously

either on a separate day or, using stableisotope-labeled nicotine, on the

same day as ad libitum cigarette smoking. Using the clearance estimated

from infusion data and the area under the blood nicotine concentration time

curve while smoking, absolute intake of nicotine from cigarette smoking,

could be estimated (Jacob et al 1988; Benowitz and Jacob 1984). The

intake of nicotine is quite variable, both on the basis of daily intake (range

10 to 80 mg) and on a per cigarette basis (range 0.4 to 1.6 mg per cigarette), among people.

The experimental situation under which smokers are studied may influence

self-determined intake of an inhaled drug. For example, we compared the

intake of nicotine per cigarette in smokers during restriction of cigarette

availability (Benowitz et al. 1986). When smoking was restricted from an

average of 37 to 5 cigarettes per day, intake of nicotine per cigarette

increased 2.7-fold. It is likely for other drugs that the time since the last

use of the drug and other environmental factors will influence the inhaled

dose of the drug and that smokers will vary the dose throughout the day.

16
The bioavailability of a drug from smoking material is in theory influenced

by the amount of drug in the material. For tobacco, we examined this

relationship by measuring the amount of nicotine in unsmoked tobacco and

comparing that to biological markers of absorption of nicotine in smokers

(Benowitz et al 1983). On average, modem American cigarettes contain

9 mg nicotine per cigarette. The average dose inhaled into the systemic

circulation by a smoker is 1 mg. Therefore, the bioavailability averages

11 percent. However, there was very little correlation between the amount

of nicotine in the tobacco or the amount of tobacco in the cigarette burned

with the amount of nicotine absorbed by smokers (Benowitz et al. 1983;

Benowitz and Jacob 1984). The bioavailability of nicotine can be systematically changed by engineering features such as placement of ventilation

holes to allow dilution of tobacco smoke with room air.

In summary, the absorption and bioavailability of inhaled drugs is highly

variable from person to person and potentially from smoke to smoke.

Smoking behavior is complex and permits the smoker considerable latitude

in adjusting the dose to desired levels. Pharmacologic studies of inhaled

drugs of abuse need to consider the extent and sources of individual variability in bioavailability in developing dose-response and dose-toxicity for

such drugs.

DISTRlBUTION KINETICS

Smoking is a unique form of systemic drug administration in that entry into

the circulation is through the pulmonary rather than the portal or systemic

venous circulations. Entry via the lung influences the rate and pattern of

delivery of drugs to body organs. For example, nicotine can be expected

to move quickly from inhaled cigarette smoke to the brain. It is estimated

that it takes 11 seconds or less from the start of a puff to the delivery

of nicotine to the brain and 19 seconds to complete transit through the brain

(table 2). This estimation assumes a 2-second puff, negligible time for

diffusion of nicotine across alveolar membranes, and negligible time for

TABLE 2. Nicotine effects after inhalation: Temporal considerations

Action Seconds

Puff time 2.0

Pulmonary circulation time 7.5

Left ventricle to cerebral circulation time -1.0

Brain transit time 8.5

Total circulation time 19.0

NOTE: Estimates derived from Mapleson (1973).

17
movement from the arterial blood into the brain. With respect to the latter,

nicotine is rapidly and extensively taken up from the blood in its first pass

through the brains of rats (figure 2). Likewise, intravenously injected C14

nicotine is immediately taken up by the brains of mice, reaching a maximum concentration within 1 minute after injection (Stalhandski and Slanina

1970). Similar findings based on positron emission tomography of the brain

were seen after injection of 11C nicotine in monkeys (Maziere et al. 1976).

The time from the start of a puff to delivery of nicotine to the brain is

similar to the time from intravenous injection to arterial circulation,

estimated as from 9 to 16 seconds in healthy people.

FIGURE 2. Concentrations of nicotine in the brain and arterial plasma

of rats after IV injection of 0.1 mg/kg nicotine over

30 seconds

NOTE: Concentration of nicotine peak in the brain at or bcfore the earliest sampling time (30 seconds after the time of injetion). Brain concentrations are much highger than arterial concentrations, particularly in the early minutes after injection.

18
Brain concentrations decline quickly as nicotine is distributed to other body

tissues. Using partition coefficients (the ratio of drug concentration in

tissue compared to blood at steady state) derived from experiments in rabbits, along with organ weights and blood flows taken from people, perfusion

models can be used to stimulate the concentrations of nicotine in various

organs after smoking a cigarette (figure 3). Of note is that the lung has a

steady state partition coefficient of 2.0, suggesting tissue binding. Studies

1.5 mg of nicotine in 10 min given I.Pulm.

_:Art ..:Ven --:Brain -.-:Lu _:Mu ..:Fat

FIGURE 3. Perfusion model simulation of the distribution of nicotine in

various tissues following infusion of 1.5 mg of nicotine into

the lung compartment over 10 minutes

NOTE: The dose, route, and dosing regimen were intended to mimic smoking a cigarette. Typical

human organ weighs and blood flows and partition coefficients from rabbits wcre used for

the simulation.

19
with lung slices demonstrate lung binding, which is somewhat dose dependent (Ludden et al. 1976). There was 50 percent more binding of nicotine

to lung slices at low concentrations than at high concentrations. As a result

of high local concentrations and binding, concentrations of nicotine in the

lung are predicted to be extremely high after intrapulmonary arterial injection and would be expected to be similarly high after tobacco, smoking.

Such high concentrations could conceivably cause local pharmacologic

effects on the lung, such as neutrophil chemcattraction (Totti et al. 1984) at

concentrations that would not be expected to be achieved based on the relatively low concentrations of nicotine in the blood.

Concentrations of nicotine in arterial blood and the brain are seen to increase sharply following exposure, then to decline over 20 to 30 minutes as

nicotine is redistributed to other body tissues, particularly skeletal muscle.

Venous blood concentrations, reflecting outflow of nicotine from body tissues, are predicted to be considerably lower than arterial concentrations for

the duration of the infusion and for several minutes afterward. This discrepancy has been observed in rabbits following rapid intravenous injection

of nicotine (Porchet et al. 1988b) (figure 4). It is seen from figure 3 that

the ratio of nicotine in the brain to that in venous blood is highest during

and at the end of the infusion and gradually decreases as the elimination

phase is entered. The importance of this disequilibrium between venous

and arterial concentrations with respect to pharmacodynamic studies is discussed in a later section.

In summary, inhalation of drugs allows for rapid transfer into the arterial

circulation and into the brain. Rapid passage into the brain provides for the

possibility of rapid behavioral reinforcement from smoking and allows the

smoker to precisely control the concentrations of nicotine in the brain,

hence to modulate pharmacologic effects. To better understand this process,

the relationship between the level of a drug and its effects on the brain

must be examined.

PHARMACODYNAMICS OF NICOTlNE IN TEE BRAIN

Nicotine binds to specific receptors in the brain and affects cerebral metabolism in the areas of specific binding (London et al. 1985). Nicotine is

known to have a complex dose-response relationship. In experimental preparations, nicotine in low doses causes ganglionic stimulation and in high

doses causes ganglionic blockade following brief stimulation. This biphasic

response pattern is observed in the intact organism as well, although the

mechanisms are far more complex. With regard to central nervous system

effects, Ashton has provided evidence that nicotine exerts biphasic actions in

humans (Ashton et al. 1980). When contingent negative variation (CNV, a

component of the evoked EEG potential) was examined, it was seen that

nicotine in low doses increased while high doses decreased the amplitude of

the CNV. It was proposed that low doses produced arousal or increased

20
Femoral Vein

Femoral Artery

Internal Jugular Vein

FIGURE 4. Mean blood concentrations from three vascular sites after an

IV bolus of 25 ug/kg of nicotine given over 1 minute in six

rabbits

KEY: Squares=femoral vein; inverted trianguls=femoral artery; triangles=internal jugular vein.

NOTE: Arterial and internal jugular vcnous concentrations (the latter proportional to brain

concentrations) were much higher than those in femoral venous blood during the first

5 minutes.

SOURCE: Porchet et al. 1988b, copyright 1988. Rockefeller University Press.

attention and vigilance, while higher doses produced relaxation and reduced

stress. Presumably, the smoker, by titrating the level of nicotine in the

brain, can achieve the desired mental state.

The time course of nicotine absorption strongly influences the actions of

nicotine, although it is not clear whether this is a result of a more rapid

21
rate of rise or higher absolute levels of nicotine in the brain. We and

others have found that heart rate acceleration, a reflection of the level of

sympathetic neural discharge and a sensitive physiologic response, tends to

correlate with the magnitude of subjective effect (Rosenberg et al 1980;

West and Russell 1987). Heart rate acceleration appears to be mediated by

the central nervous system either through actions on chemoreceptor afferent

pathways or via direct effects on the brain stem. With repeated intravenous

injections of nicotine, dosed to simulate cigarette smoking, heart rate acceleration and subjective effects were greatest with the first series of injections

and then declined over time with successive injections (Rosenberg et al.

1980).

To examine the importance of rate of dosing of nicotine and resultant

actions and the relationship between effects and brain levels of nicotine, we

compared the consequences of rapid- and slow-loading infusions of nicotine

(Porchet et al. 1988b). The study was performed in seven volunteer smokers who had abstained from smoking overnight. Venous blood concentrations were not markedly different for the two infusion rates except for the

first 5 minutes, when the increase in blood concentration was greater with

rapid infusion (figure 5). Peak concentrations were seen at 30 minutes and

were similar for both infusions. However, the heart rate increased much

more quickly and to a greater level and was associated with transient dizziness and euphoria after rapid infusion, compared to slow infusion (figure 6).

During the slower infusion, heart rate increased slowly, peaking at 30 minutes, and subjective effects were minimal. Using kinetic data derived from

subjects of this study and studies in rabbits and with the use of a perfusion

model, we estimated levels of nicotine in the human brain during the rapid

and slow infusions. Predicted brain concentrations rose much more rapidly

and to a higher level after rapid infusion, with a concentration time course

similar to that seen for heart rate acceleration (figure 7). Brain concentrations versus heart rate acceleration tended to change in parallel These data

suggest that effects of nicotine track brain concentrations over time.

In summary, for drugs that rapidly enter and act upon the brain, drug dosing such as by inhalation results in higher brain concentrations and greater

psychoactive (and possibly cardiovascular) effects than when the same dose

is given more slowly. The possibility of titrating the level of a drug in the

brain to achieve a desired mental state is also facilitated by inhalation as

the route of dosing. These factors may explain why smoking a drug may

be preferred over the oral or nasal routes of administration. In addition, as

mentioned previously in the discussion of drug distribution, venous blood

concentrations substantially underestimate arterial and brain levels after rapid

dosing, and such data cannot (without mathematical transformations) be used

to study concentration-effect relationships.

22
NICOTINE INFUSION

MINUTES

FIGURE 5. Average blood nicotine concentration in seven subjects

NOTE: (On the rapid-loading day, subjects received 17.5 ug/kg/min nicotine for 1.5 minutes, then

1.75 ug/kg/min for 30 minutes; on the slow-loading infusion day, 25 ug/kg/min for 30 minutes. Maintenance infusion on both days was 0.35 ug/kg/min from 30 to 180 minutes.

SOURCE: Porchet et al. 1988b. copyright 1988. Rockefeller University Press.

TOLERANCE

An important issue in drug dependence is the development of tolerance.

Tolerance is defined as the tendency for a given dose of a drug, after

repeated doses, to produce less of an effect. In some cases, increasing the

dose of a drug in the presence of tolerance may achieve the same effect

that was obtained after the first dose. Pharmacodynamic tolerance can be

further defined as the condition that exists when a particular concentration

of a drug at a receptor site (in the intact organism, approximated by the

concentration in the blood) produces less of an effect than it did after a

prior exposure.

23
NICOTINE INFUSION

MINUTES

FIGURE 6. Increase in heart rate in seven subjects

NOTE: On the rapid-loading day, subjects received 17.5 ug/kg/min nicotine for 1.5 minutes, then

1.75 ug/kg/min for 30 minutes; on the slow-loading infusion day, 25 ug/kg/min for 30 minutes. Maintenance infusion on both days was 0.35 ug/kg/min from 30 to 180 minutes.

SOURCE Porchet et al. 1988b, copyright 1988. Rockefeller University Press.

Studies in animals demonstrate rapid development of tolerance to many

effects of nicotine, although tolerance may not be complete (Marks et al.

1985). Smokers know that tolerance develops to some effects of smoking.

Smoking the first cigarette as a teenager is commonly associated with dizziness, nausea, and/or vomiting, effects to which the smoker rapidly becomes

tolerant. Smoking a cigarette after 24 hours of abstinence increases heart

rate, whereas smoking an identical cigarette during the course of the day

fails to increase heart rate (West and Russell 1987). Pharmacodynamic tolerance to heart rate acceleration and subjective effects is seen within the

course of a 30-minute intravenous infusion of nicotine (Benowitz et al.

1982a).

24
NICOTINE INFUSION

MINUTES

FIGURE 7. Predicted mean “brain” concentrations of nicotine over time

(based on a physiological kinetic model, fit to the rabbit

and scaled to humans)

NOTE: lnfusion rates are as described in figure 3. Note that “brain concentrations" peak at the same

time as heart rate (see figure 5).

SOURCE: Porchet et al. 1988b, copyright 1988. Rockefeller University Press.

These and other findings indicate that tolerance is gained rapidly, within

minutes (tachyphylaxis). Tolerance is lost, at least to some degree, after

overnight abstinence. To characterize the time course of tolerance develop

ment and regression, we conducted a study in which subjects received

paired intravenous infusions of nicotine, separated by different time intervals

(Porchet et al. 1988a). Despite higher blood concentrations, heart rate

acceleration and subjective effects were much less when a second infusion

was given at 60 or 120 minutes after the first infusion. At 240 minutes,

the response was fully restored. The pharmacodynamics were modeled

using a pharmacokinetic-pharmacodynamic model. This modeling indicates

a half-life of development and regression of tolerance of 35 minutes. Such

25
modeling suggests that at three half-lives or 1 1/2 hours after a cigarette,

nearly full sensitivity should have been regained.

Because tolerance develops so quickly, the rate of drug dosing influences

the magnitude of effect. That is, the faster the administration, the less time

there is to develop tolerance and the greater the effect for any given dose

or maximal level. The interval at which cigarette smokers smoke cigarettes

may be determined by two factors: the rate of distribution out of the brain

after smoking a cigarette and the kinetics of regression of tolerance to

effects of nicotine.

In summary, tolerance develops rapidly to the effects of nicotine as it does

to effects of other inhaled drugs of abuse. The kinetics of tolerance are

such that tolerance may develop and regress in cycles throughout the day.

The interval at which users smoke their drugs may be influenced by two

factors: the rate of distribution of drug out of the brain after a particular

dose of the drug and the kinetics of regression of tolerance to effects of

that drug. Rapid rate of dosing, as by inhalation, also allows the maximal

drug effect with the least extent of development of tolerance.

PHARMACOKINETICS AND PHARMACODYNAMICS OF

DRUG DEPENDENCE

The reinforcing properties of a drug are expected to be strongest when a

psychoactive effect, usually a pleasant one, follows in close temporal proximity to the self-administration of a drug. In pharmacokinetic or pharmacodynamic terms, after dosing, the drug should enter the bloodstream rapidly

and move rapidly from the bloodstream into the brain. The appearance of

the drug in the brain should be associated with the desired psychoactive

effects. If the effect of a drug is delayed after appearance of the drug in

the brain or if tolerance to the drug effect is already present, the drug is

less likely to be reinforcing.

That the user can easily control the dose of the drug and modulate the resultant psychoactivity would be expected to further strengthen the reinforcing nature of the drug. As discussed previously, nicotine obtained by

cigarette smoking demonstrates these characteristics, as do other drugs of

abuse that are inhaled, such as crack cocaine or marijuana, and drugs that

are intravenously injected, such as heroin and cocaine.

Tolerance is most likely to develop to the effects of a drug when its receptors are continuously exposed to the drug. Frequent and sustained dosing, a

long half-life of a drug, or a combination of these factors, would favor development of tolerance. The half-life of nicotine in the brain following a

single dose exposure is short, probably about 10 minutes in humans, due to

redistribution out of the brain to other body tissues. Some degree of tolerance does develop rapidly (tachyphylaxis), even after brief exposures. Such

26
tolerance also regresses relatively quickly (half-life, 35 minutes) such that a

smoker may learn that smoking a cigarette at particular intervals is more reinforcing than smoking more frequently.

With repetitive dosing, levels of nicotine build up in the body (and in the

brain) in accordance with the terminal half-life of 2 hours. Thus, there is

substantial accumulation of nicotine in the brain and an increasing general

level of tolerance throughout the day. Presumably, however, smoking individual cigarettes still results in peaks of nicotine in the brain, which may

overcome the underlying level of tolerance and produce some of the desired

effects.

Withdrawal symptoms are mast likely to occur when there has been a sustained effect in the brain and the drug is then rapidly removed. Thus, rapid

exit from specific brain regions would favor more severe withdrawal symptoms. Nicotine, as it is dosed by cigarette smokers, exhibits both of these

characteristics. Due to repetitive dosing, the brain is exposed to nicotine

for prolonged periods of time, so that neuroadaptation occurs during the

smoking day. When exposure is terminated, nicotine rapidly exits the brain

and withdrawal symptoms are experienced. It is likely, therefore, that relief

of withdrawal symptoms plays an increasingly important role in determining

smoking behavior as the day progress.

NICOTINE AND THE DAILY SMOKING CYCLE

The pharmacokinetic and pharmacodynamic considerations discussed thus far

help us understand the development of nicotine dependence and human

cigarette-smoking behavior, as well as adverse effects of cigarette smoking.

The daily smoking cycle can be conceived as follows. The first cigarette of

the day produces substantial pharmacologic effects, primarily arousal, but at

the same time tolerance begins to develop. A second cigarette may be

smoked at a later time, at which the smoker has learned there is some regression of tolerance. With subsequent cigarettes, there is accumulation of

nicotine in the body, resulting in a greater level of tolerance, and withdrawal symptoms become more pronounced between cigarettes. Transiently

high brain levels of nicotine following smoking individual cigarettes may

partially overcome tolerance. But the primary (euphoric) effects of individual cigarettes tend to lessen throughout the day. Overnight abstinence

allows considerable resensitization to actions of nicotine. Because of the

dose-response and dose-tolerance characteristics, habitual smokers need to

smoke at least 15 cigarettes and consume 20 to 40 mg nicotine per day to

achieve the desired effects of cigarette smoking and minimize withdrawal

discomfort throughout the day.

27
REFERENCES

Armitage, A.; DoIlery, C.; Houseman, T.; Kohnes, E.; Lewis, P.J.; and

Turner, D.M. Absorption of nicotine from small cigars. Clin Pharmacol

Ther 23:143-150, 1978.

Ashton, H.; Marsh, V.R.; Millman, J.E.; Rawlins, M.D.; Telford, R.; and

Thompson, J.W. Biphasic dose-related responses of the CNV (contingent

negative variation) to i.v. nicotine in man. Br J Clin Pharmacol 10:579-

589,1980.

Bend, J.R.; Serabjit-Singh, C.J.; and Philpot, R.M. The pulmonary uptake,

accumulation, and metabolism of xenobiotics. Annu Rev Pharmacol Tonicol 25:97-125, 1985.

Benowitz, N.L.; Hall, SM.; Herning, R.I.; Jacob, P., III; Jones, R.T.; and

Osman, A.L. Smokers of low yield cigarettes do not consume leas nicotine. N Engl J Med 309:139-142, 1983.

Benowitz, N.L., and Jacob, P., III. Daily intake of nicotine during cigarette

smoking. Clin Pharmacol Ther 35:499-504, 1984.

Benowitz, N.L.; Jacob, P., III; Jones, R.T.; and Rosenberg, J. Interindividual variability in the metabolism and cardiovascuIar effects of nicotine in

man. J Pharmacol Exp Ther 221:368-372, 1982a.

Benowitz, N.L; Jacob, P., III; Kozlowski, L.; and Yu, L. Influence of

smoking fewer cigarettes on exposure to tar, nicotine, and carbon monoxide exposure. N Engl J Med 314:1310-1313, 1986.

Benowitz, N.L.; Kuyt, F.; and Jacob, P., III. Circadian blood nicotine concentrations during cigarette smoking. Chin Phamacol Ther 32:758-764,

1982b.

Brunneman, K.D., and Huffman, D. The pH of tobacco smoke. Food Cosmetics Toxicol 12:115-124, 1974.

Jamb, P., III; Benowitz, N.L.; and Shulgin, A.T. Recent studies of nicotine

metabolism in humans. Pharmacol Biochem Behav 30:249-253, 1988.

London, E.D.; Connolly, R.J.; Szikszay, M.; and Wamsley, J.K. Distribution of cerebral metabolic effects of nicotine in the rat. Eur J Pharmacol

110:391-392, 1985.

Ludden, T.M.; Schanker, L.S.; and Lanman, R.C. Binding of organic compounds to rat liver and lung. Drug Metab Dispos 4:8-16, 1976.

Mapleson, W.W. Circulation-time models of the uptake of inhaled anesthetics and data for quantifying them. Br J Anaesth 45:319-334, 1973.

Marks, M.J.; Stitzel, J.A.; and Collins, A.C. Time course study of the

effects of chronic nicotine infusion on drug response and brain receptors.

J Pharmacol Exp Ther 235:619-628, 1985.

Maziere, M.; Comar, D.; Marazano, C.; and Berger, G. Nicotine-11C: Synthesis and distribution kinetics in animals. Eur J Nucl Med 1:255-258,

1976.

Porchet, H.C.; Benowitz, N.L.; and Sheiner, LB. Pharmacodynamic model

of tolerance: Application to nicotine. J Pharmacol Exp Ther 244:231-

236, 1988a

28
Porchet, H.C.; Benowitz, N.L.; Sheiner, L.B.; and Copeland, J.R. Apparent

tolerance to the acute effect of nicotine results in part from distribution

kinetics. J Clin Invest 80:1466-1471, 1988b.

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.

Stalhandski, T., and Slanina, P. Effect of nicotine treatment on the metabolism of C14-labeled nicotine in mice and rats. Acta Pharmacol Toxicol

(Copenh) 28:75-80, 1970.

Totti, N., III; McCusker, K.T.; Campbell, E.J.; Griffin, G.L.; and Senior,

R.M. Nicotine is chemotactic for neutrophils and enhances neutrophil

responsiveness to chemotactic peptides. Science 223:169-173, 1984.

West, R.J., and Russell, M.A.H. Cardiovascular and subjective effects of

smoking before and after 24 h of abstinence from cigarettes. Psychopharmacology 92:118-121, 1987.

ACKNOWLEDGMENTS

Supported in part by U.S. Public Health Service grants DA 02277,

CA 32389, and DA 016%. These studies were carried out in part in the

General Clinical Research Center (RR-00083) with support of the Division

of Research Resources, National Institutes of Health.

AUTHOR

Neal L. Benowitz, M.D.

Professor of Medicine and Psychiatry

University of California, San Francisco

San Francisco General Hospital Medical Center

Building 30, Fifth Floor

San Francisco, CA 94110

29
The Pharmacology of Cocaine

Smoking in Humans

Reese T. Jones

INTRODUCTION

Smoking crack is smoking cocaine. Why would people go to the trouble of

smoking cocaine when they can so easily snuff it, take it orally, or inject it

parenterally? For many of the same reasons that nicotine is pleasurable to

a tobacco addict and is so much more addicting when smoked in the form

of tobacco cigarettes than when snuffed or chewed, so smoking the alkaloid

cocaine is even more addicting than use by other routes (Benowitz, this volume). For similar reasons, most cannabis-dependent people choose to

smoke marijuana or hashish even though tetrahydrocannabinol (THC) is psychoactive by oral ingestion. Of course, cocaine, nicotine, and THC pharmacology differ, but the aspects of their pharmacology that lead to preference

for the smoked route of ingestion as well as some of the consequences of

that choice are similar. If what is known of other inhaled drugs is kept in

mind (Benowitz, this volume), it may be easier to understand certain conse

quences of cocaine smoking and, perhaps more important, to design proper

experiments to study cocaine smoking.

Smoking cocaine has become popular only in recent years, mostly in North

America and some Latin American countries (Jeri 1984; Siegel 1985). Like

most current patterns of psychoactive drug use, smoking cocaine was not

completely unknown to the ancients. For example, in the United States,

during the early part of this century, the Parke, Davis & Company pharmaceuticals catalog listed, among other forms of coca and cocaine, coca

cheroots and coca cigarettes. The Parke, Davis coca cigars and cigarettes

probably contained between 0.5 and 1.0 percent cocaine and were recommended for therapeutic use. A similar product known as the Coca Leaf

Ball was advertised in England during the same period with the suggestion

that “families, mothers and children” would benefit from the therapeutic

effects of smoked coca. Famous people from England and Europe testified

as to its effectiveness and must have engaged in cocaine smoking, although

there is no indication that addiction to coca smoking was common. In

30
some ways, smoking coca leaf might be analogous to smoking tobacco or

cannabis leaf in that a relatively small concentration (0.5 to 2 percent) of

active substance (cocaine, nicotine, THC) is contained in a relatively large

amount of plant material. Smoking coca leaf never became as popular as

smoking tobacco leaf. Chewing coca leaf remained common only in relatively isolated groups, where it had long been part of their culture (Hanna

and Hornick 1977).

In more recent times, smoking cocaine in the form of a crude plant extract

sometimes called paste and containing about 80 percent cocaine base became popular in Peru and in adjacent countries (Almeida 1978; Jeri 1984;

Flores 1986). Up to then, both in medical applications and illicitly, the

water-soluble salt, usually cocaine hydrochloride, was the form more commonly used. Currently in North America, a readily available and widely

discussed form is cocaine base, usually converted from the cocaine salt by

mixing cocaine hydrochloride and a solution containing an excess of sodium

bicarbonate usually baking soda) and heating and evaporating off the fluid.

This leaves mostly cocaine base in a bicarbonate crystalline mixture. In

most large cities in the United States, the availability of illicit ready-tosmoke base facilitates smoking cocaine. Cocaine base melts at a much lower temperature than the salt (about 80 °C instead of 180 oC) and then boils,

producing an inhalable aerosol (Snyder et al. 1988; Wood, this volume).

Whether forces of the illicit marketplace led to changes in cocaine chemistry and packaging or whether the availability of high concentration, relatively pure cocaine base shaped the market forces to increase consumer demand

is not entirely clear. In any case, a cocaine user who chooses to smoke

can now obtain material containing close to 100 percent cocaine rather than

using coca plant material containing 1 percent of cocaine base. Consider

the likely consequences if THC was readily available to add to smoking

devices or if nicotine addicts began adding additional nicotine to tobacco

cigarettes. Although not a perfect analogy, considering such events may

help to put cocaine smoking in perspective.

PROBLEMS IN STUDYING COCAINE SMOKING

A variety of devices are used for cocaine smoking: pipes of all sorts, or

cigarettes made of admixtures of cocaine base with tobacco, with marijuana,

or with other plant material. The use of nonstandardized smoking devices

contributes to a situation similar to that seen during the early days of

cannabis-smoking research. When programs of controlled laboratory experiments with smoked cocaine are begun, it is not obvious what the typical

laboratory smoking conditions should be, what constraints on smoking behavior are proper, and what physical and chemical considerations will maximize the likelihood that laboratory experiments will be relevant to the real

world of illicit smoked cocaine, but will maintain adequate research subject

safety. In contrast, a researcher of tobacco smoking has the advantages of

great standardization of dose, dosage form, and delivery systems developed

31
by the tobacco industry. Even with that information, smoking behavior procedural issues in tobacco-smoking research, particularly dose regulation, are

complex, and not all are resolved (Biding 1987; Kozlowski et al. 1982).

Relatively little has been published describing the human pharmacology of

cocaine smoking under controlled or semicontrolled laboratory conditions

(Perez-Reyes et al. 1982; Jeffcoat et al. 1989). Experiments in which

experienced volunteers smoked from a glass apparatus heated to 260 oC and

containing 50 mg of cocaine base demonstrated the rapid increase in plasma

cocaine levels during smoking, as compared to intravenous (IV) cocaine

administration, that would be expected from what is known of the smoking

process with nicotine and cannabis (Benowitz, this volume; Perez-Reyes,

this volume). Other than more rapid onset, the resulting cardiovascular and

psychologic changes were not unlike those seen after equivalent IV or nasal

cocaine doses. Other experiments with monkeys (Siegel et al. 1976) and

with smokers of crude extracts of cocaine often called paste noted a generally similar pattern of effect (Paly. et al. 1982).

Laboratory studies with any smoked drug present great methodologic challenges to the investigator. Proper pharmacologic studies must involve control of dose or at least accurate specification of the dose actually absorbed.

The nature of smoking behavior makes dose difficult to regulate and, under

many conditions, difficult even to estimate. Although animals can be made

to smoke under some conditions (Siegel et al. 1976), animals seem reluctant

to smoke the way humans do; thus, human studies are necessary.

Cocaine may present problems that have not been important in tobacco or

cannabis smoking research. The rate of cocaine degradation is very temperature dependent, hence, subject to variability because of smoking-apparatus

design and variation in conditions. Temperature in most cocaine-smoking

devices varies with air flow. Air flow, in the case of humans smoking

under natural conditions, depends on both puff and inhalation volumes and

duration. The importance of puff and inhalation volume in determining

dose is well recognized by tobacco and cannabis researchers (Heming et al.

1983; Benowitz 1986). How to control or regulate dose in psychopharmacologic studies involves compromises, however, and optimal solutions are

not evident yet. When cocaine is smoked, the amount of cocaine pyrolyzed

to other compounds will be related to the temperature in the smoking device. The temperature, in turn, will be related to the smoker’s behavior,

making for a complex equation to determine drug delivery.

When researching nicotine smoking, an investigator can depend on a degree

of industry- and government-derived standardization of the smoking devices

(cigarettes, pipes, etc.). In marijuana smoking research over the years,

some consensus has developed as to smoking material and apparatus, at

least in the laboratory. Cocaine smoking research, however, is nowhere

near that point. The usual and expected “natural” or typical pattern (or

32
more likely, the range of patterns) of cocaine smoking is not at all established; hence, the relevance of laboratory paradigms is still open to question. For example, typical puff and inhalation parameters obtained during

relatively unconstrained cocaine smoking have not been determined as they

have with nicotine and with marijuana.

THE PHARMACOLOGY OF COCAINE SMOKING

In experiments trying to describe the pharmacology of cocaine smoking, we

attempted to control some aspects of smoking behavior but, in these beginning experiments, left options open for the subjects, all of whom claimed to

be experienced cocaine smokers. What is apparent in the laboratory data

collected thus far is great variability; both variability in cocaine smoking

behavior and, under conditions where smoking behavior parameters ate to

some extent constrained, considerable variability in cocaine plasma levels

and effects.

For example, in figure 1 the plasma concentrations are shown from 10 different research subjects smoking cocaine base under similar conditions.

Peak venous cocaine levels ranged from less than 100 ng/ml to approximately 500 ng/ml, with considerable variability in time of peak as well.

These cocaine smokers were allowed to take one or two and occasionally

three inhalations spaced 1 minute apart from an electrically heated l-liter

glass flask, beginning about 45 seconds after 100 mg of finely powdered

cocaine base was dropped into the flask. At that time the cocaine base had

melted, and a fine white aerosol was forming in the flask. The clear puddle of melted cocaine at the bottom of the flask was just beginning to yellow in the process that would turn it into a black tarry mass just a few

minutes later. The temperature of the glass surface at the bottom of the

flask was 260 °C.

The variation in plasma levels is contrasted with those from the same

subjects given cocaine IV at a dose of 0.6 mg/kg of body weight injected

by infusion pump over 1 minute (figure 2). Perhaps noteworthy is the

considerable variability in peak plasma levels of cocaine even with

controlled IV administration. Variability in venous plasma levels is not

unexpected with a rapidly distributed, relatively rapidly metabolized drug,

although it should give pause for thought to those attempting to predict

precise relationships between drug effect and blood levels from a particular

cocaine dose. It should be kept in mind that, particularly by the smoked

route, arterial level of cocaine is more likely to be a much better indicator

of brain level than is venous blood level. As is the case after nicotine

smoking, venous cocaine levels will underestimate brain levels.

When the mean plasma cocaine levels after IV or smoked doses are compared to those of nasal or oral doses of cocaine hydrochloride, reasons for

some of the appeal of the smoked route are evident (figure 3). The time of

33
MINUTES AFTER FIRST INHALATION

FIGURE 1. Plasma levels of cocaine after smoking

MINUTES AFTER DOSE

FIGURE 2. Plasma levels of cocaine: IV administration

34
peak level is significantly earlier after dose in the smoked or IV route.

Similarly, the time of peak subjective effect is earlier by the smoked route

(figure 4). Figure 4 illustrates the peak subjective-high rating in which zero

is defined as a normal, sober state, and 100 is as intoxicated as ever experienced after cocaine administration.

MINUTES AFTER DOSE

FIGURE 3. Plasma levels of cocaine

DEVELOPMENT OF TOLERANCE TO COCAINE

Why should a shorter interval to peak effect be important to a drug user?

probably because neuroadaptation (tolerance) to many psychoactive drug

effects is a rapidly developing process better timed in seconds and minutes

than in hours or days. The slower any drug effect arises, other things

being equal the less intense the effect, because tolerance to the effect is in

a real sense developing concurrently with the appearance of the drug effect.

Rapidly developing tolerance to cocaine is demonstrated in a hysteresis plot

of plasma level vs. subjective intoxication rating in figure 5. If little or no

tolerance (neuroadaptation) developed over the 4 hours summarized in figure

5, the plot following the ascending and descending phases of the time/

plasma level function would be more or less a straight line. One index of

the magnitude of tolerance is the divergence of those ascending and

descending functions (Holford and Sheiner 1981). Note that the subjects

had the option of rating their level of dysphoria with a minus value, and

35
MINUTES AFTER DOSE

FIGURE 4. Subjective high ratings

some did so as the cocaine effects wore off. Figure 6 shows the clockwise

hysteresis plots of subjective intoxication levels vs. cocaine concentrations in

plasma after smoking cocaine and after an IV dose (0.6 mg/kg) in the same

10 research patients whose time vs. concentration data are shown in figures

1 and 2. Quantitative comparisons between the smoking and the IV hysteresis loops involve many assumptions, but a variety of semiparametric methods seem useful for a quantitative pharmacokinetic-pharmacodynamic

descriptive model (Verotta et al. 1989). The similarity to the rapid tolerance that develops to many effects of nicotine is striking (Benowitz, this

volume).

PHYSIOLOGIC AND SUBJECTIVE EFFECTS

Assuming that most of cocaine’s subjective effects are related to its brain

effects, the more rapid transit time from lung to brain (5 to 10 seconds)

after inhalation of cocaine smoke would more likely provide a steeper and

higher gradient of cocaine brain levels than could oral, nasal, or even IV

administration. Table 1 summarizes the magnitude and time of some peak

effects after cocaine was given to the same 10 experienced cocaine users by

various routes, in balanced order, with about 3 days between doses.

36
PLASMA CONCENTRATION (NG/ML)

FIGURE 5. “High” vs. cocaine levels in plasma after nasal cocaine

(20% solution)

PLASMA CONCENTRATION (NG/ML)

FIGURE 6. “High” vs. cocaine levels in plasma after IV and

smoked cocaine

37
That both the peak venous plasma cocaine levels and the peak intoxication

levels are greater when cocaine is taken IV, compared with the smoked

route, might suggest more advantage to the IV route. To make proper

comparisons between smoked dose and effect, the dose actually delivered to

the individual subject must be considered. Therein lies one of the problematic considerations in human research with any smoked material. Traditional procedures to characterize bioavailability do not precisely fit drugsmoking situations. How much of that 100 mg of cocaine base placed in

the pipe actually was absorbed by the subject?

TABLE 1. Mean physiologic and subjective changes after cocaine

Route (Dose)

IV Smoked* Nasal Oral

Changes (0.6 mg/kg) (0.4 mg/kg) (2mg/kg) (2 mg/kg)

Heart rate increase, BPM 46 (10) 32 (2) 26 (40) 20 (55)

(time to max, minutes)

Systolic BP increase 28 (10) 32 (2) 24 (25) 19 (70)

mmHg (time to max)

Diastolic BP increase 16 (10) 22 (1) 11 (25) 14 (75)

mmHg (time to max)

Pupil diameter increase 0.8 (4) 1.1 (5) 0.6 (45) 0.5 (90)

mm (time to max)

Skin temperature decrease -2.8 (30) -1.8 (20) -4.7 (30) -5.1 (75)

°C (time to max)

Subjective high 1-100 48 (4) 35 (1) 18 (20) 18 (70)

scale (time to max)

*Smoked dose is estimate, see text for discussion.

On average, 70 (range 10 to 85) mg of the cocaine base remained in the

flask and was recrystallized on the surface of the tubing after smoking.

Thus, on average, a maximum of about 30 mg of cocaine base was delivered to the mouth of the inhaling research subject. An indeterminant

amount of cocaine was broken down to poorly characterized pyrolytic products in the smoking process. The biological activity of those products is

not well established. Thus, in fact, considerably less than 30 mg of cocaine

base was probably delivered to the mouth of some smokers. Our electrically heated l-liter flask was, of course, a rather special and not a typical freebase pipe, but experience with cocaine pipes brought into the laboratory by

research subject volunteers is similar. Much, perhaps even most, of the

38
cocaine base placed in the pipe does not reach the mouth of the smoker.

How much cocaine aerosol actually reaches lung alveoli depends on heating

conditions, air flow, number of inhalations, and other things determined

largely by the smoker, who only reluctantly yields control to the researcher,

if cocaine smoking is like other smoking behavior.

If all the cocaine not remaining in the pipe was delivered to the lung

alveoli and thus probably completely absorbed, cocaine dose could be better

estimated. However, pyrolysis occurs at variable rates during the smoking

process. Although breakdown is not completely quantified yet, there is

strong evidence that anhydroecgonine methyl ester is one of the major

breakdown products under our smoking conditions. Thus, under the smoking conditions leading to the plasma levels in figures 1 and 2, only a small

fraction of the 100 mg of cocaine base in the smoking apparatus actually

was delivered to the alveolar surface, where rapid absorption is most likely.

The relative percentages of cocaine base delivered, what remains in the

pipe, and what is destroyed vary greatly with the smoking conditions encountered with various smoking apparatus. Although all the 10 subjects in

the experiment discussed claimed to be experienced and reasonably confirmed cocaine base smokers, they exhibited a varied repertoire of smoking

styles ranging from very big puffs with very little and brief inhalation to, at

the other extreme, maximum and rapid forced inhalation and valsalva maneuver with little or no intermediate puff behavior. The variability in smoking behavior we have encountered among cocaine smokers is much greater

than the variability commonly seen in otherwise similar groups of marijuana

or tobacco smokers.

CONCLUSION

Inferring what is likely to be happening in the real world of crack smoking

from laboratory studies is risky until the range of possible cocaine-smoking

behavior in real-life situations is better understood and better described.

Much can be gleaned from what we know of nicotine and THC smoking.

The principles that guide the smoking of those drugs and the unresolved

problems of researching nicotine and THC smoking apply to cocaine as

well.

REFERENCES

Almeida, M. Natural history of dependence of cocaine base paste. Rev

Neuropsiquiatr 41:44-55, 1978.

Benowitz, N.L. Clinical pharmacology of nicotine. Annu Rev Med 37:21-

32, 1986.

Diding, N. Machine smoking results compared to human uptake of cigarette smoke. Int J Clin Pharmacol Ther Toxicol 25:143-147, 1987.

39
Flores, A.R. Drug abuse problems in countries of the Andean subregion.

Bull Narc 38:27-36, 1986.

Hanna, J.M., and Hot-nick, C.A. Use of coca leaf in southern Peru:

Adaptation a addiction. Bull Narc 29:63-74, 1977.

Herning, R.I.; Jones, R.T.; Benowitz, N.L.; and Mines, A.H. How a

cigarette is smoked determines blood nicotine levels. Clin Pharmacol

Ther 33:84-90, 1983.

Holford, N.H.G., and Sheiner, L.B. Understanding the dose-effect

relationship: Clinical application of pharmacokinetic-pharmacodynamic

models. Clin Pharmacokinet 6:429-453, 1981.

Jeffcoat, AR.; 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.

Jeri, F.R. Coca-paste smoking in some Latin American countries: A

severe and unabated form of addiction. Bull Narc 36:15-31, 1984.

Kozlowski, L.T.; Rickert, W.S.; Pope, MA.; Robinson, J.C.; and Frecker,

R.C. Estimating the yield to smokers of tar, nicotine, and carbon

monoxide from the ‘lowest yield’ ventilated filter-cigarettes. Br J Addict

77:159-165, 1982.

Paly, D.; Jatlow, P.; Van, D.C.; Jeri, F.R.; and Byck, R. Plasma cocaine

concentrations during cocaine paste smoking. Life Sci 30:731-738, 1982.

Perez-Reyes, M.; Di, G.S.; Ondrusek, G.; Jeffcoat, A.R.; and Cook, C.E.

Free-base cocaine smoking. Clin Pharmacol Ther 32:459-465, 1982.

Siegel, R.K. New patterns of cocaine use: Changing doses and routes. In:

Kozel, N.J., and Adams, E.H., eds. Cocaine Use in America: Epidemiological and Clinical Perspectives. National Institute on Drug Abuse

Research Monograph 61. DHHS Pub. No. (ADM)85-1414. Washington,

D.C: Supt. of Docs., U.S. Govt. Print. Off., 1985. pp. 204-220.

Siegel, R.K.; Johnson, C.A.; Brewster, J.M.; and Jarvik, M.E. Cocaine selfadministration in monkeys by chewing and smoking. Pharmacol Biochem

Behav 4:461-467, 1976.

Snyder, CA.; 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.

Verotta, D.; Beal, S.L.; and Sheiner, LB. Semiparametric approach to

pharmacokinetic-pharmacodynamic data. Am J Physiol 256(4, Part

2):R1005-1010, 1989.

ACKNOWLEDGMENTS

Supported in part by U.S. Public Health Service grants DA 00053,

DA 02829, DA 04419, DA 01696, and contract no. 271-87-8123.

40
AUTHOR

Reese T. Jones, M.D.

Professor of Psychiatry

Langley Porter Psychiatric Institute

University of California, San Francisco

San Francisco, CA 94143

41
Marijuana Smoking: Factors That

Influence the Bioavailability of

Tetrahydrocannabinol

Mario Perez-Reyes

INTRODUCTION

Marijuana smoking is the most commonly used method for the selfadministration of delta-9-tetrahydrocannabinol (THC) the active principle of

marijuana. The inhalation of marijuana smoke induces subjective effects

(“high”) that are rapidly perceived by the smokers. The perception of these

effects allows smokers the capacity to achieve, within certain limits, their

desired level of high by changing the puff volume, the interval between

puffs, and the number of puffs taken. Therefore, the manner in which

marijuana cigarettes are smoked (smoking dynamics) is probably the mast

important factor in determining the bioavailability of THC. However, the

following factors are also important: (1) the potency of the marijuana

smoked; (2) the amount of unchanged THC present in the smoke inhaled

(i.e., the amount of THC not destroyed by pyrolysis); (3) the amount of

THC lost in side-stream smoke; (4) the method of smoking (cigarette vs.

pipe smoking); and (5) the amount of THC trapped in the mucosa of the

upper respiratory tract. A review of the experiments we have conducted

that permit evaluation of the relative importance of these factors is

presented in this chapter.

METHODS COMMON TO ALL CLINICAL STUDIES

Subjects

Male and female, healthy, paid volunteers who used marijuana no more than

two times per week participated in the studies. They were thoroughly

informed about the experimental procedures, the purpose of investigation,

and the potential risks. All signed an informed consent approved by the

Committee on the Protection of the Rights of Human Subjects of the

University of North Carolina at Chapel Hill.

42
Experimental Variables

Subjective Ratings of Intoxition. The major psychologic effect of marijuana is a temporary euphoric effect with diversely perceived sensory,

somatic, affective, and cognitive changes that are commonly described as

a high. To measure this effect, we asked the subjects to rate their degree

of high at frequent intervals during the experiment on a graph form provided for them. For this rating, the subjects were asked to estimate their

level of high on a scale of 0 to 100; zero represented no effect and 100

represented the highest they had ever been after smoking marijuana. Every

time a rating was to be made, subjects were given their previous rating to

use for comparison. This technique allowed the subjects to rate themselves

as experiencing relatively more, less, or the same effects as those rated in

the previous time interval.

Physiologic Effects. Since the most consistent physiologic effect of marijuana in humans is cardiac acceleration, the ECG was continuously recorded

on a polygraph before the beginning of smoking (baseline) and throughout

the duration of the experiment. The results are reported as. the percentage

of heart rate acceleration over baseline values.

Determination of the THC Plasma Concentration. Blood samples were

drawn through an indwelling needle before the initiation of smoking and at

frequent intervals thereafter. The plasma obtained was analyzed by standard

radioimmunoassay procedures (Owens et al. 1981; Cook et al. 1982).

Statistical Analyses

For comparison, the means of the data plus or minus the standard errors of

the mean (SEM) were calculated and are reported as such throughout the

text. Areas under the curve (AUCs) were calculated by the trapezoidal rule

method. The results were analyzed statistically by means of regression

analysis and t test for either dependent paired data or independent nonpaired

data as appropriate.

MARIJUANA POTENCY

To investigate the influence that marijuana potency per se has on the peak

THC plasma concentrations produced, a regression analysis was done on the

results of experiments in which National Institute on Drug Abuse (NIDA)

marijuana cigarettes of eight different potencies (range 1 percent to

6.24 percent THC) were smoked. The marijuana potencies investigated, the

number of subjects tested, the mean peak THC plasma concentrations, and

the range of individual peak THC plasma concentrations are illustrated in

figure 1. The results indicate the presence of a statistically significant

correlation between the potency of the marijuana smoked and the peak THC

plasma concentrations (r=.269, r=2.27, one-tailed p<.025). However, as

43
shown in this figure, wide individual variations in peak THC plasma

concentrations occurred consistently across all of the marijuana potencies

investigated. This variability in THC plasma concentrations indicates that

factors other than the potency of the marijuana smoked, particularly the

smoking dynamics, influence the magnitude of the resulting THC plasma

concentrations.

MARIJUANA PERCENT THC CONTENT

THC CONTENT SUBJECTS THC (ng/ml) RANGE

1.00%

1.30%

1.32%

1.97%

2.40%

2.40%

2.54%

4.60%

4.84%

6 90.4±20.2

4 71.3±18.4

6 100.0±10.1

6 119.8±10.6

18 63.0±8.6

6 119.0±23.0

6 162.6±18.7

4 146.3±33.1

12 124.2±16.2

45.6-187.8

18.7-99.6

62.8-125.3

44.5-180.9

11.7-137.0

81.0+203.0

107.4-204.7

65.7-227.6

44.8-218.0

FIGURE 1. Relationship between marijuana potency and THC plasma

concentration (top) and marijuana potency and peak THC

plasma concentration (ng/ml) (bottom)*

*r=269; p=.025

NOTE: In the upper part of the figure, the Individual peak THC plasma concentrations of 68 volunteers are plotted against the THC content of the NIDA marijuana cigarettes that they smoked.

In the lower part of the figure, the numerical values measured are summarized.

44
PYROLYTIC DESTRUCTION

When marijuana is smoked, a portion of its THC content is destroyed by

pyrolysis before reaching the mouth of the smoker. Information regarding

the amount of THC destroyed by pyrolysis is derived from both cigarettes

and water-pipe smoking.

Cigarette Smoking Study

Davis and colleagues (1984) conducted a study in which standard NIDA

marijuana cigarettes containing 1.6 percent and 3.1 percent THC were

smoked by a cigarette-smoking machine in a constant draft mode, i.e., the

cigarettes were artificially inhaled in a single puff until nothing was left.

The percent of the original THC not recovered in the smoke condensate

collected and probably lost to pyrolysis was 30.6±1.4 and 30.9±1.2 for the

1.6 percent and the 3.1 percent THC content cigarettes, respectively. Since

no measurement of the THC trapped in the smoking apparatus was done,

the total amount actually lost to pyrolysis, under their experimental conditions, was probably less than 30 percent.

Water-Pipe Smoking Study

Marijuana water-pipe smoking replicates artificial single-draft smoking

because the amount of plant material placed in the bowl of the pipe is

entirely combusted, and the smoke produced is inhaled in a single puff.

One milligram of Vitiated THC was added to 70 mg of placebo marijuana

(0.004 percent THC) and placed in the bowl of a commercially available

water pipe. One bowl was smoked every 2.5 minutes, for a total of 10

bowls. The six volunteers who participated in this study were instructed to

draw air until the total amount of marijuana placed in the pipe’s bowl was

combusted and to inhale the total amount of smoke produced in a single

puff. A Cambridge filter, to trap the THC content of the smoke produced,

was placed between the mouth of the pipe and the mouth of the subjects.

The amount of THC recovered from the pipe, the plant material residues,

and the Cambridge filters was measured by gas liquid chromatography. It

was found, in this unpublished study, that 40.4±1.5 percent of the original

amount of THC was trapped in the Cambridge filters (i.e., approximately

the amount of THC that would have reached the mouth of the subjects if

the marijuana smoke had not been filtered), and that 40.2±4.5 percent of the

original amount of THC remained in the pipe and plant material residues

(figure 2). Therefore, since approximately 80 percent of the original

amount of THC was recovered, it can be concluded that 20 percent was

destroyed by pyrolysis.

The results of these smoking studies, given the large margin of error introduced by differences in methodology, compare favorably and indicate that

between 20 percent and 30 percent of the THC contained in the plant

45
material is destroyed by pyrolysis during single-draft smoking conditions,

irrespective of the marijuana potency smoked.

FIGURE 2. Diagram of the water pipe used in THC studies

NOTE: The amount of THC recovered from the pipe components and the amount of THC that was potentially

inhaled by the subjects (i.e. the THC trapped in the Cambridge filter) are indicated.

SIDESTREAM SMOKE

When marijuana cigarettes are combusted, the smoke produced is either

inhaled when puffs are taken (mainstream smoke) or escapes to the

46
atmosphere during the interval between puffs (sidestream smoke). To have

an approximate measure of the amount of THC present in mainstream

smoke and lost in sidestream smoke when marijuana cigarettes are smoked

by machine, experiments were conducted by Davis and associates (1983).

In these experiments, standard MDA marijuana cigarettes containing

1.6 percent and 3.1 percent THC were smoked by a cigarette-smoking

machine. Two modes of smoking were investigated, continuous inhalation

(i.e., the cigarettes were inhaled in a single puff until nothing was left) and

intermittent inhalation (the cigarettes were inhaled at a rate of two 40-ml

puffs per minute). The results of these experiments indicate that marijuana

potency did not significantly influence the percent of THC present in

mainstream smoke within either mode of artificial smoking, i.e., 69.4±1.4

and 69.1±1.2 percent for continuous smoking and 19.0±1.2 and

16.1±0.6 percent for intermittent smoking, respectively, for the 1.6 percent

and 3.1 percent THC-content cigarettes. Assuming that the amount of pyrolytic destruction of THC is the same between constant and intermittent

smoking, the difference in the amount of THC present in mainstream smoke

between the two modes of smoking indicates that between 40 percent and

50 percent of the original THC content of the cigarettes was lost in side

stream smoke (figure 3).

FIGURE 3. Fate of THC during marijuana cigarette smoking

NOTE: The percent of THC that potentially reach the mouth of the subject (mainstream smoke) and

that lost to pyrolysis and sidestream smokc when marijuana cigarettes are smoked. Percent

values are approximations.

The results of clinical studies of marijuana cigarette smoking (Perez-Reyes

et al. 1982) indicate that, on the average, smokers puff more often (2.6

puffs per minute) than the puff frequency used in the Davis cigarettesmoking study (2 puffs per minute) (Davis et al. 1983). Therefore, it is

reasonable to assume that when subjects smoke marijuana cigarettes, the

amount of THC lost in sidestream smoke is not as large as that found in

the cigarette-smoking-machine experiments. Nevertheless, this loss should

47
still be of considerable magnitude because the cumulative interpuff interval

(the time when the cigarette is idly smoking) accounts for 90 percent of the

total smoking time (table 1).

TABLE 1. Smoking parameters for two modes of marijuana

cigarette smoking

Accelerated Smoking (n=4) Slow Smoking (n=6)

1.51% THC 2.87% THC 1.32% THC 254% THC

Smoking time (min) 5.2±0.9

Number of puffs 18.3±2.3

Puff duration (sec) 2.0 ± 0.2

Interpuff interval (sec) 18.3±2.5

Total puff duration (sec) 31.9±3.8

Total interpuff interval (rain) 4.7±0.9

Plasma THC, time to peak 6.5±l.0

(min)

Plasma THC, peak value 210.8±44.0

(ng/ml)

Mean puff volume (ml) 55.8±10.6

Total puff volume (ml) 889.0±92.0

6.2±l.0

17.0±2.3

1.8±0.l

22.5±4.2

30.4±5.4

5.7±0.9

5.0±1.2

229.6±46.4

49.2±6.8

818.0±106.0

17.9±2.0

30.7±5.1

2.0±0.4

20.7±2.8

56.2±6.3

16.9±2.0

6.7±0.6

100.0±10.1

17.2±2.l

31.5±4.0

2.3±0.3

21.0 ±2.4

74.3±13.8

15.9±2.0

7.7±0.8

162.6±18.7

NOTE: Data obtained in studies in which marijuana cigarettes of comparable potency were smoked by

the volunteers in one instance through a puff-volume measuring instrumcnt (acceleratcd

smoking), and in the other instance without it (slow smoking).

SMOKING DYNAMICS

Marijuana Cigarette Smoking

We have conducted studies in which the number of puffs, the puff duration,

the interval between puffs, and the duration of marijuana cigarette smoking

of similar (Perez-Reyes et al. 1981) or different (Perez-Reyes et al. 1982)

potencies were measured. However, in these studies, the critically important

puff volume parameter was not measured. To fill this gap in the knowledge of the dynamics of marijuana cigarette smoking, we conducted experiments in which the puff volume was measured throughout the period of

smoking.

To measure the puff volume, a cigarette-holder flow-rate transducer based

on the Venturi’s flow meter was used (Hutcheson et al. 1984). The signal

from this pressure transducer was amplified, filtered, and then routed to a

PDP 11/34 Laboratory computer for processing by an online program that

48
computed puff volume, puff duration, inter-puff interval, cumulative puff

volume, and cumulative smoking time.

Using this technology, NIDA marijuana cigarettes containing 1.51 percent

and 2.87 percent THC were smoked at weekly intervals in a double-blind

crossover design by four male volunteers. To imitate natural conditions as

much as possible, the volunteers were instructed to smoke the cigarettes in

their customary fashion and to smoke until they had reached their desired

level of high or for as long as they wished. Blood samples for the determination of the THC plasma concentrations were collected.

Results

Smoking Time. For comparison, the results obtained in this study in which

the cigarettes were smoked through the puff-volume transducer and those

obtained in a study in which cigarettes of comparable potency were smoked

without this device are illustrated in table 1. Examination of the data

shows a profound difference in mean smoking time between the two studies. We hypothesized that the presence of the cigarette-holder puff-volume

transducer in front of the subjects’ faces provoked them into an unintended

accelerated rate of smoking. Accelerated smoking did not affect the mean

puff duration or the mean interval between puffs. The mean number of

puffs was decreased, however, and as a consequence the mean cumulative

interval between puffs was shorter.

Puff Volume. As shown in table 1, the mean puff volume and the mean

cumulative puff volume were not significantly influenced by the potency of

the marijuana smoked. The mean puff volumes observed during the period

of smoking are shown in figure 4. The similarity of the smoking pattern

between the groups is apparent, as is the progressive decrease in puff

volumes over time. To investigate the significance of this pattern, the

cumulative puff volume inhaled by each subject during the first half was

compared to that which occured during the second half of their smoking

period (figure 5). The differences in mean cumulative puff volumes

between cigarette halves are statistically significant: t=3.22, p<.05, and

t=4.53, p<.02 for the 151 percent and the 2.87 percent THC content marijuana, respectively.

THC Plasma Concentration. For each comparable marijuana potency

smoked, the magnitude of the THC plasma concentrations during the time

of observation (figure 6) and the mean peak THC plasma concentrations

measured (table 1) were larger in response to accelerated smoking. This

finding may be due to the large individual variation in THC plasma concentrations (figure l), as different volunteers participated in the studies. This

could also be due to a faster rate of THC delivery during accelerated smoking studies.

49
PUFF ORDER

FIGURE 4. Comparison of mean puff volume

NOTE: Mean smoke volumes inhaled per puff during the period of smoking. It can be seen that the

twofold difference in marijuana potency did not change the smoking profile.

FIGURE 5. Comparison of the cumulative puff volume (total smoking time

divided into halves)

NOTE: A larger mean cumulatlve smoke volume was inhaled during the first half of the cigarettes.

The difference between marijuana potencies is not statistically significant.

50
(SLOW SMOKING)

TIME (Mins)

(ACCELERATED SMOKING)

TIME (Mins)

FIGURE 6. Comparison of the mean THC plasma concentration between

slow and accelerated smoking

The relationship between THC time to peak and the smoking time observed

in response to slow smoking reported in table 1 indicates that THC plasma

concentrations sometimes peaked several minutes before the termination of

smoking. As shown in figure 7, this relationship has been consistently

51
observed in the many marijuana-smoking experiments that we have previously conducted. ThiS observation was always intriguing because it was

contrary to the reasonable expectation that for as long as the smoke produced by the combustion of marijuana cigarettes was inhaled, the resulting

THC plasma concentrations should have continued to increase until smoking

was terminated. Therefore, it was unexpected to find that, during accelerated smoking, the THC plasma concentrations peaked at approximately the

same time that smoking was terminated. We hypothesized that rapid inhalation of marijuana smoke resulted in a rate of delivery of THC to the bloodstream that was faster than the drug’s rate of disappearance into the extravascular compartment. In contrast, when the rate of inhalation of marijuana

smoke was slow, the drug’s rate of disappearance into the extravascular

compartment was faster than the rate of delivery of THC to the

bloodstream.

MEAN THC TIME TO PEAK

MEAN SMOKING TIME

FIGURE 7. Relationship between smoking time and THC time to peak

NOTE: The results of eight separate studies in which marijuana cigarettes were smoked in the

volunteers customary manner are shown. It can be seen that peak THC plasma

concentrations cosistently occurred before the termination of smoking.

52
Marijuana Water-Pipe Smoking

Marijuana is frequently smoked in pipes of different sixes and configurations, particularly in water pipes, because they cool the smoke and facilitate

its inhalation. This method of smoking has become more popular in recent

years because it permits the combustion of smalI portions of high-potency

marijuana (sometimes as high as 13 percent THC in Sinsemilla plant

material).

As discussed, marijuana cigarette smoking of identical THC content results

in large interindividual variations in THC plasma concentrations. This is

because the number of puffs taken, the interval between puffs, and the

volume of smoke inhaled per puff vary greatly from person to person. In

contrast, marijuana pipe smoking permits control of the number of puffs

taken, the interval between puffs, and the volume of smoke inhaled per

puff. Moreover, at least theoretically, the amount of plant material placed

in the bowl of the pipe should be entirely combusted, all of the smoke produced should be inhaled, and practically no THC should be lost in sidestream smoke. We hypothesized that under these controlled smoking circumstances, less interindividual variation in the magnitude of THC plasma

concentrations should occur.

To verify this hypothesis, we conducted three dose-response studies, using a

single-blind crossover design in which marijuana of different potencies was

smoked in a 250-ml capacity water pipe (figure 2). A wire screen was

placed at the bottom of the pipe’s bowl to prevent uncombusted plant

material from dropping into the pipe. A constant amount of water was

used to fill the pipe, which provided a consistent 200-ml volume of air

space between the mouth of the subject and the water level (the puff

volume).

The volunteers were instructed to inhale the total amount of smoke produced by the combustion of the marijuana placed in the pipe’s bowl

(“toke”) and to hold the smoke in their lungs for 10 seconds. Smoke inhalation was facilitated by the drawing of atmospheric air through a small

hole located above the water level. Tokes were taken at 0, 2.5, 5, 7.5, and

10 minutes. This rate of administration was selected because in our previous studies in which radiolabeled ’H-THC was smoked in a water pipe,

the resulting THC plasma concentrations were sustained during the period of

smoking.

Experimental Variables. To determine the peak THC plasma concentrations produced, blood samples were drawn through an indwelling needle at

appropriate intervals. The subjective rating of marijuana high was measured

at frequent intervals throughout the experiments, and the heart rate was

recorded before and after marijuana smoking in only the second and third

studies.

53
Drug Dosage. In our initial study, 0.625 mg/kg of marijuana containing

1.68 percent or 2.51 percent THC was combusted per toke. The amount of

smoke produced by the combustion of this amount of marijuana was large,

and some of the six volunteers who participated in the study had difficulties

combusting the tokes completely or inhaling the total amount of smoke generated. For this reason, a second dose-response study was conducted in

which the amount of plant material combusted per toke was reduced to

0.5 mg/kg for marijuana containing 2.51 percent THC and to 0.4 mg/kg for

marijuana containing 3.12 percent THC. The latter marijuana potency was

obtained by mixing equal parts of 6.24 percent and 0.004 percernt THC.

The amount of smoke produced by the combustion of tokes containing

0.5 mg/kg was still large enough that some of the six volunteers who participated in this study had difficulty inhaling the smoke completely. However, the amount of smoke generated by the combustion of the 0.4 mg/kg

tokes was completely inhaled without difficulty. The pharmacologic effects

obtained in this study were of greater magnitude than those obtained in the

first study, but they were leas pronounced than those produced by the smoking of marijuana cigarettes. In an effort to obtain larger pharmacologic

effects, a third study was conducted in which 0.4 mg/kg of marijuana containing 3.12 percent and 6.24 percent THC was combusted per toke. Originally, as in the previous studies, we planned to teat six volunteers. However, the smoking of the high-potency marijuana induced postural hypotension and bradycardia (vasovagal syndrome) in the third and fourth subjects

tested. Because of this undesirable side effect, the study was terminated

after testing only three subjects. We are perplexed by the occurrence of

this syndrome because we have never observed it in response to marijuana

cigarette smoking. We have, however, observed the occurrence this syndrome in some subjects after the oral administration of high doses of THC

(35 mg) or after the intravenous infusion of THC.

Results

THC Plasma Concentrations. As illustrated in figure 8, the THC plasma

concentrations increased progressively for as long as tokes were taken. As

a result, peak THC concentrations occurred in close proximity to the end of

the smoking period (table 2). This finding suggests that the inhalation of a

large volume of smoke (approximately 200 ml) every 2.5 minutes delivers

enough THC to the blood stream to balance the drug’s rate of disappearance into the extravascular compartment. In this respect, the results of

marijuana water-pipe smoking are comparable to the results of accelerated

marijuana cigarette smoking.

Contrary to our expectations, peak THC plasma concentrations varied widely

among the subjects who participated in the studies (table 2). This result is

disappointing because it demonstrates our failure to reduce the variability in

peak THC plasma concentrations, despite controlling the amount of marijuana combusted per toke, the number of tokes taken, and the interval

54
FIGURE 8. Comparison of the magnitude of THC plasma concentrations

produced by marijuana water-pipe smoking over time

between tokes. It should be noted, however, that smoking factors other

than those controlled in this study may have influenced the magnitude of

the THC plasma concentrations observed. For example, although the subjects were instructed to hold the smoke inhaled for a period of 10 seconds,

this requirement was not enforced. Therefore, according to their custom,

55
many subjects held the smoke for longer intervals of time. Likewise, it was

not possible to maintain a constant puff volume because uncontrolled

amounts of atmospheric air were drawn to facilitate inhalation of the smoke

produced by the combustion of each toke.

TABLE 2. Comparison of the THC plasma concentrations produced by

marijuana water-pipe smoking

Potency

Time to Peak Peak Value Range

(min) (ng/ml) (ng/ml)

1.68% THC

2.51% THC

First Study (n=6)

9.7±0.8 53.7±6.0

8.3±1.0 58.8±7.6

40.9-72.7

34.9-81.8

2.51% THC

3.12% THC

Second Study (n=6)

7.7±0.7 79.7±10.8

9.0±0.7 114.5±10.4

39.0-116.0

80.8-150.0

3.12% THC

6.24% THC

Third Study (n=3)

10.3±0.7 109.6±20.8

8.3±1.8 151.7±37.0

81.0-150.0

90.2-218.0

NOTE: Despite controlled conditions, THC plasma concentrations varied widely among the subjects

tested.

Subjective Effects. The mean subjective rating of high over the time of

observation is illustrated in figure 9. It can be seen that the magnitude of

subjective high increased progressively during smoking and for several minutes after its termination. Peak values occurred approximately 20 minutes

after the beginning of smoking, i.e., approximately 10 minutes after the

occurrence of peak THC plasma concentrations (table 3). Although peak

subjective effects and AUC (0-35’) values increased proportionally to the

magnitude of the THC plasma concentrations obtained, statistical analysis of

this limited amount of data indicates that no significant correlation is present between peak THC plasma concentrations and peak subjective effects

(r=.153, t=0.82, p NS). This lack of correlation is probably due to the

large intersubject variations in both plasma THC concentrations and subjective effects.

Heart Rate Effects. The mean heart rate acceleration over the time of

observation is illustrated in figure 10. The changes in heart rate produced

by marijuana pipe smoking mimic in all respects the subjective rating of

high. Thus, the heart rate accelerated progressively during smoking and for

56
FIGURE 9. Comparison of the magnitude of the subjective rating of high

produced by marijuana water-pipe smoking over time

several minutes after its termination. Peak values occurred approximately

20 minutes after the beginning of smoking i.e., approximately 10 minutes

after the occurrence of peak THC plasma concentrations (table 3).

Likewise, mean peak heart rates and AUC (0-35’) values increased proportionally to the magnitude of the THC plasma concentrations obtained.

57
TABLE 3. Comparison of the pharmacologic effects produced by

marijuana after pipe smoking

Time to Peak Peak Value AUC (0-35’)

18.3±2.5

18.3±1.7

Subjective Rating of High

First Study (n=6)

41.7±10.5

48.3±11.9

20.0±2.2

28.3±4.0

Second Study (n=6)

57.5±8.9

73.0±10.4

Third Study (n=3)

2.0±5.0 50.7±12.9

13.3±3.3 68.3±13.6

980±221

1,071±265

1,457±249

1,754±282

1,270±368

1,595±409

20.8±2.7

21.7±2.1

Percent Heart Rate Acceleration

Second Study (n=6)

34.4±6.2

53.6±5.5

492±151

862±96

14.0±0.0

18.0±2.0

Third Study (n=3)

70.4±3.0

74.9±18.0

1,377±293

1,589322

NOTE: The heart rate was not measured in the first study.

Nonetheless, statistical analysis indicates that no significant correlation is

present between peak THC plasma concentrations and peak heart rate acceleration (r=.385, t=1.67, p NS). This is probably due to the large intersubject variations in plasma THC concentrations and heart rate accelerations.

AMOUNT OF THC TRAPPED IN TEE UPPER RESPIRATORY

TRACT MUCOSA

The results of several independent studies indicate that THC is present in

saliva and that its concentration declines with time after marijuana smoking

(Hawks 1984; Cross et al. 1987; Maseda et al. 1986; Thompson and Cone

1987). The presence of THC in saliva after marijuana smoking is not due

to its secretion by the salivary glands. This is demonstrated by the results

of experiments in which we administered radioactively labeled THC orally

58
FIGURE 10. Comparison of magnitude of mean hem rate acceleration

produced by marijuana water-pipe smoking over time

and intravenously to normal volunteers (Perez-Reyes et al. 1972; PerezReyes et al. 1973). In these experiments we collected timed saliva samples

after drug administration. No radioactivity was detected in any of the saliva

samples assayed. This observation indicates that neither THC nor its me

tabolities are excreted by the salivary glands. Therefore, the THC present in

saliva derives from that sequestered in the buccal mucosa during smoking.

The amount of THC trapped in the buccal mucosa or present in mixed saliva samples is time and dose dependent. This is demonstrated by a study in

which 30 volunteers smoked NIDA marijuana cigarettes containing either

59
2.49 percent (18 subjects) or 4.84 percent (12 subjects) THC blindly. The

mean amount of THC present in swabs of the buccal mucosa and the mean

amount of THC present in 25 microliters of mixed saliva samples are

shown in table 4. Because the THC present in the swabs cannot be accurately related to a volume of saliva, we have reported them as amounts

instead of concentrations. The largest amounts of THC in swab and saliva,

irrespective of the marijuana potency smoked, were observed at the first

time of measurement (i.e., 1 hour after the initiation of smoking). After

this time, mean THC amounts declined progressively to minimal but still

detectable values at the 4-hour end of the study.

TABLE 4. Comparison of the mean amounts of THC (ng) measured in

25 microliters of saliva and swabs of the roof of the

mouth and tongue

2.40% THC 4.84% THC

Swab Saliva Swab Saliva

1 Hour 16.9±7.2 20.9±8.2 59.8±27.5 60.5±24.9

2 Hours 7.8±3.7 5.8±1.8 26.5±12.3 26.7±14.8

3 Hours 7.1±3.8 4.1±1.3 16.6±8.9 6.9±2.7

4 Hours 3.8±1.7 1.9 ±0.8 13.5±7.4 4.2±1.7

Since THC is trapped in the buccal mucosa, it is reasonable to assume that

THC is also trapped in the mucosa of the other parts of the upper respiratory tract, i.e., pharynx, larynx, trachea, and bronchi. Obviously, it is not

possible to know the amount of THC that is trapped in the combined surface. area of these anatomical structures during marijuana smoking. However, judging by the relatively small amounts of THC trapped in the buccal

mucosa (i.e., less than 100 ng in either swabs or 25 microliters of saliva

1 hour after smoking) the total amount of THC trapped in the mucosa of

the upper respiratory tract can be considered to be a factor of minor importance with regard to the bioavailability of THC

CONCLUSION

Although smoking is a practical method for the delivery of marijuana’s

active principle (THC), many factors inherent in the smoking process

decrease the drug’s bioavailability. For example, whether marijuana is

smoked in pipe or cigarette form, 23 to 30 percent of its THC content is

destroyed by pyrolysis; if marijuana is smoked in cigarette form, as much

as 40 to 50 percent of THC can be lost in sidestream smoke.

60
There is a significant correlation between the potency of the marijuana

smoked and the magnitude of the THC plasma concentrations obtained.

However, large interindividual variations in peak THC plasma concentrations

occurred across all of the marijuana potencies investigated. These results

were obtained in experiments in which the volunteers smoked marijuana

cigarettes in their customary fashion (uncontrolled smoking). To reduce this

variability, experiments were conducted in which marijuana was smoked in

a water pipe. This method of smoking permits control of the amount of

marijuana combusted per toke, the number of tokes taken, and the interval

between tokes. Despite controlling these important smoking factors, large

interindividual variations in the magnitude of the THC plasma concentrations obtained continued to be observed.

During marijuana smoke inhalation, THC is trapped in the mucosa of the

upper respiratory tract, thereby reducing the drug’s bioavailability. Judging

by the amounts of THC measured in saliva or swabbings of the buccal

mucosa, however, the total loss of THC by this factor can be considered of

minor importance.

In summary, marijuana smoking is a complex process that does not permit

controlled dosing. Therefore, for research or therapeutic purposes, it is indispensable to measure the THC plasma concentrations to know how efficiently a given individual smoked.

REFERENCES

Cook, C.E.; Seltzman, H.H.; Schindler, V.H.; Tallent, C.R.; Chin, KM.; and

Pitt, C.G. Radioimmunoassays for cannabinoids. In: Hawks, R.L., and

Willette, R.E., eds. The Analysis of Cannabinoids in Biological Fluids.

National Institute on Drug Abuse Research Monograph 42. NTIS Pub.

No. 83-136044. Springfield, VA: U.S. Dept. of Commerce, 1982.

pp. 19-32.

Davis, K.H.; McDaniel, I.A.; Caddell, L.W.; and Moody, P.L. Some smoking characteristics of marijuana cigarettes. In: Agurell, S.; Dewey, W.L.;

and Willette, R.E., eds. The Cannabinoids: Chemical, Pharmacologic,

and Therapeutic Aspects. New York: Academic Press, 1984. pp. 97-109.

Gross, S.J.; Worthy, T.E.; Nerder, L.; Zimmerman, E.G.; Soares, J.R.; and

Lomax, P. Detection of recent cannabis use by saliva 9-THC radio

immunoassay. J Anal Toxicol 9:91-97, 1987.

Hawks, R.L. Developments in cannabinoid analyses of body fluids: Implications for forensic applications. In: Agurell, S.; Dewey, W.L.; and

Willette, R.E., eds. The Cannabinoids: Chemical, Pharmacologic, and

Therapeutic Aspects. New York: Academic Press, 1984. pp. 123-134.

61
Hutcheson, J.S.; Perez-Reyes, M.; and Di Guiseppi, S. An automated

computer-based system for the study of marijuana smoking dynamics.

In: Agurell, S.; Dewey, W.L.; and Willette, R.E., eds. The Cannabinoids: Chemical, Pharmacologic, and Therapeutic Aspects. New York:

Academic Press, 1984. pp. 111-121.

Maseda, C.; Hama, K.; Fukui, Y.; Matsubara, K.; Takahashi, S.; and Akane,

A. Detection of 9-THC in saliva by capillary GC/ECD after marihuana

smoking. Forensic Sci Int 32:259-266, 1986.

Owens, S.M.; McBay, A.J.; Reisner, H.M.; and Perez-Reyes, M. 125I Radioimmunoassay of 9

-tetrahydrocannabino1 in blood and plasma with a solidphase second-antibody separation method. Clin Chem 27:619-624, 1981.

Perez-Reyes, M.; Di Guiseppi, S.; Davis, H.H.; Schindler, V.H.; and Cook,

C.E. Comparison of effects of marihuana cigarettes of three different

potencies. Clin Pharmacol Ther 31:617-624, 1982.

Perez-Reyes, M.; Lipton, M.A.; Timmons, M.C.; Wall, M.E.; Brine, D.R.;

and Davis, K.H. Pharmacology of orally administered 9-tetrahydrocannabinol. Clin Pharmacol Ther 14:48-55, 1973.

Perez-Reyes, M.; Owens, S.M.; and Di Guiseppi, S. The clinical pharmacology and dynamics of marihuana cigarette smoking. J Clin Pharmacol

21:201S-207S, 1981.

Perez-Reyes, M.; Timmons, M.C.; Lipton, M.A.; Davis, K.H.; and Wall,

M.E. Intravenous injection in man of 9-tetrahydrocannabinol and 11-OH9

-tetrahydrocannabinol. Science 177:633-635, 1972.

Thompson, L.K., and Cone, E.J. Determination of 9-tetrahydrocannabinol in

human blood and saliva by high-performance liquid chromatography with

amperometric detection. J Chromatogr 421:91-97, 1987.

ACKNOWLEDGMENTS

Supported by National Institute on Drug Abuse contracts 271-80-3705 and

271-87-8126 and grant DA 04484.

AUTHOR

Mario Perez-Reyes, M.D.

Professor

Department of Psychiatry

The University of North Carolina at Chapel Hill

CB #7175, Medical Research Building A

Chapel Hill, NC 27599-7175

62
Effects of Habitual Use of

Marijuana and/or Cocaine on

the Lung

Donald P. Tashkin, Suzanne Fligiel, Tzu-Chin Wu,

Henry Gong, Jr., Richard G. Barbers, Anne H. Coulson,

Michael S. Simmons, and Theodore F. Beals

INTRODUCTION

Marijuana remains one of the most widely abused substances in our society

(Johnstone et al. 1987). As with tobacco, smoking is the preferred route of

administration of marijuana, so the lungs are the principal organ exposed to

its combustion products. The latter include a number of known respiratory

‘irritants and carcinogens (Hoffman et al. 1975; Novotny et al. 1982), as

well as numerous other compounds the pulmonary effects of which are unknown. Experimental animal (Roy et al. 1976; Fleischman et al. 1975;

Fleischman et al. 1979) and in vitro (Huber et al. 1975; Leuchtenberger and

Leuchtenberger 1976) studies have demonstrated that exposure to marijuana

smoke produces significant toxic, inflammatory, and even carcinogenic

effects on lung tissue and cells. Few studies in man, however, have systematically addressed the long-term effect of real-life habitual marijuana

smoking on the lung. Moreover, the few published clinical studies of the

pulmonary effects of regular marijuana smoking (Rubin and Comitas 1975;

Cruickshank 1976; Coggins et al. 1976; Boulougouris et al. 1976) have

yielded conflicting results, possibly because of small sample sixes, failure to

control adequately for confounding factors, such as concomitant tobacco

smoking, and the cross-sectional nature of these studies, which may not be

sensitive to intraindividual changes in respiratory status. In 1983, a longitudinal study was initiated at the University of California, Los Angeles

(UCLA) in an attempt to determine (1) whether habitual smoking of marijuana alone and with tobacco leads to pulmonary symptoms, lung dysfunction, or respiratory illness and (2) whether such harmful effects persist or

progress with continued (or increased) use and decline with cessation of

use. Another objective of this study was to evaluate the effect of regular

smoking of marijuana with and without tobacco on tracheobronchial

63
histopathology, alveolar cell populations, and the structure and function of

alveolar macrophages, key cells in the lung’s defense against infection and

other noxious insults. Results of this continuing study are presented below.

PULMONARY EFFECTS OF HABITUAL MARIJUANA USE:

RESPIRATORY SYMPTOMS AND LUNG FUNCTION

Cross-Sectional Findings

In 1983 and 1984 a convenience sample of Caucasian men and women

(ages 25 to 49 years) who reported smoking 10 or mote marijuana joints

(or joint-equivalents) per week, on the average, over the previous 5 years or

more was recruited from the greater Los Angeles area by means of newspaper and radio advertisements. Exclusionary criteria included intravenous

drug abuse more than 2 times per lifetime, smoking of illicit substances

other than cannabis more than 12 times per lifetime or within the previous

6 months, significant occupational exposure to dusts or fumes potentially

hazardous to the lungs, a history of chronic respiratory disease, such as

asthma, cystic fibrosis, kyphoscoliosis, or tuberculosis, or previous chest surgery. A comparison sample of 167 nonmarijuana-smoking control subjects

of similar age was recruited from the same general population.

All subjects underwent a detailed respiratory and drug use questionnaire,

modified from the American Thoracic Society/National Heart, Lung, and

Blood Institute questionnaire (Ferris 1978) and the National Institute on

Drug Abuse National Survey on Drug Abuse questionnaire (Fishburne et al.

1980), which included questions concerning respiratory symptoms, general

health, residence, occupational history, socioeconomic status, and current and

past history of use of alcohol, tobacco, marijuana, and other illicit drugs.

In addition, an extensive battery of pulmonary function tests was performed,

including maximal expiratory flow-volume curves breathing air and 80 percent helium:20 percent oxygen, subdivisions of lung volume, single-breath

diffusing capacity for carbon monoxide (DLCO), single-breath nitrogen

washout, plethysmographically determined airway resistance, and specific

airway conductance and methacholine inhalation challenge. Peripheral

venous blood was obtained for measurement of hemoglobin and carboxyhemoglobin (for correction of DLC0 measurements) and for determination of

serum cotinine and 9-tetrahydrocannabinol (THC) and 11-nor-9-carboxyTHC for verification of self-reported daily smoking of tobacco and

marijuana.

Characteristics of the study population are shown in table 1. The smokers

of marijuana (MS) and the control subjects who did not smoke tobacco

(NS) were of similar age, while the tobacco-smoking control subjects (TS)

were 3.4 years older than the smokers of both marijuana and tobacco

(MTS). The MS smoked only slightly more marijuana than the MTS

(p>.6), while the TS smoked significantly more tobacco than MTS (p<.01).

64
TABLE 1. Subject characteristics

Subjects

Mean

Age

N

Marijuana Tobacco

Yrs Joints/Wk Joint-Yrs’ Cigrrettes/D Pk-Yrs'

Marijuana Plus Tobacco

Smokers (MTS)

135 33.6 25.9 54.0 18.8 16.1

Marijuana- Only Smokers 144 31.6 28.0 56.7 0.0 2.7

(MS)

Current Tobacco Smokcrs 70 37.0 0.0 0.1 28.9 22.0

(TS)

Nonsmokers (NS) 97 31.0 0.0

aNumber of joints per day times number of years smoked.

bNumber of cigarcttes per day times number of years smoked.

0.0 0.0 0.0

Respiratory symptom data and results of lung function obtained in this study

population at the time of initial evaluation have been presented previously

(Tashkin et al. 1987a). Prevalence of chronic respiratory symptoms by

smoking category is shown in table 2. Regular smokers of marijuana alone

had significantly more symptoms of chronic bronchitis (cough, sputum,

wheeze) and a significantly greater frequency of episodes of acute bronchitis

than nonsmoking control subjects. No difference in prevalence of chronic

cough, sputum production or wheeze was noted between the marijuana and

tobacco smokers, nor were additive effects of combined smoking of tobacco

and marijuana on the prevalence of acute or chronic respiratory symptoms

apparent.

TABLE 2. Prevalence of chronic respiratory symptoms (percent)

Acute

Bronchitic Shortness

Subjects Cough aSputumaEpisodes b Wheeezec

of Breath

MTS 23.1* 25.6* 14.1* 34.8* 4.4

M S 18.4* 19.7* 13.4* 24.8* 0.7

T S 24.3* 25.7* 10.0* 37.1* 8.6**

N S 1.1 5.6 2.2 7.8 2.2

*p<.01 (comparison with NS; x2).

**p<.05 (comparison with MS; x2).

a

Present on most days for more than 3 months for 2 or more consecutive years.

bMore than 1 episode of increased cough and sputum lasting more than 3 weeks within past 3 years. 

c

Present on more than 21 days during past year.

65
Results of analysis of lung function are presented in tables 3 and 4. Tobacco smokers, irrespective of concomitant marijuana smoking, showed

significantly poorer values and/or a significantly greater frequency of abnormality for several tests of lung function, including DLCO (a measure of

alveolar gas transfer, which is deranged in early emphysema) and Vmax50,

Vmax50, Visoflow, N2/L, CV, and CC (teats reflecting mainly small airways function), in comparison with nonsmokers or with smokers of marijuana alone. On the other hand, marijuana smokers, regardless of

TABLE 3• Significantly different between-group comparisons of lung

function tests from covariance analysis*

Test

Men Women

(n=259) (n=128)

VisoV MTS>NS

MTS>MS

N2L** MTS>NS TS>NS MTS>NS

MTS>MS TS>MS

CV** TS>MS MTS>MS TS>NS

TS>MTS MTS>NS

TS>NS

CC** TS>MS NS>MS TS>NS

TS>MTS TS>MS

TS>MTS

DLCO** MTS<NS TS<NS TS<NS MTS<NS

MTS<MS TS<MS

Ra w

+ MS>NS MTS>NS

MS>TS MTS>TS

SGaw

+ MTS<NS MS<NS

 *p<.05 (least significant difference testing using adjusted group means from ANCOVA).

**Two-way ANCOVA, with age and/or height as covariates: significant tabacco effect (p<.03); no

adverse marijuana effect.

Two-way ANCOVA: significant marijuana effect, males only (p<.03); no tobacco effect.

KEY: VisoV=volume of isoflow; N2/L=change in nitrogen concentration per liter in Phase III of

single-breath nitrogen washout curve; CV=closing volume.; CC=closing capacity; DLCO=singlebreath diffusing capacity of the lung for carbon monoxide; Raw=airway resistance 

SGaw=specific airway conductance.

SOURCE: Tashkin et al. 1987a.

66
concomitant tobacco smoking, demonstrated worse values and a greater frequency of abnormality for airway resistance (Raw) and specific airway conductance (SCaw) (tests of mainly large airways function) in comparison with

nonsmokers or smokers of tobacco alone. No adverse interactive effects of

habitual heavy marijuana use and regular tobacco smoking on lung function

could be demonstrated. These findings indicate that habitual smoking of

marijuana or tobacco causes functional alterations at different sites in the

respiratory tract, with marijuana affecting mainly the large airways and

tobacco predominantly the peripheral airways and alveolated regions of the

lung.

TABLE 4. Significantly different between-group comparisons of frequency

of abnormality on lung function tests

Test Comparison

Vmax50

 Vmax50

VisoV

TS>NS

TS>NS

TS>MS

MTS>NS

CC TS>NS

TS>MS

TM>MTS

DLCO TS>NS MTS>NS

TS>MS MTS>MS

Raw MS>NS

MS>TS MTS>TS

SGaw MTS>NS

p<.05, x2 or Fisher’s exact test (men and women combined).

KEY: Vmax50=flow rate at 50 percent of forced vital capacity; Vmax50=change in maximal expiratory

flow between curves obtained breathing 80 percent helium:20 percent oxygen and breathing air

at 50 percent of the vital capacity.

SOURCE Tashkin et al. 1987a.

Longitudinal Findings

Approximately 50 percent of the cohorts of young habitual smokers of marijuana alone (MS) or with tobacco (MTS) and of control nonsmokers (NS)

67
and smokers of tobacco alone (TS) who were initially tested in 1983 and

1984 (Time 1) returned for retesting in 1985 and 1986 (Time 2) after a 2

to 3 year interval. On repeat testing, the following proportion of retested

subjects remained in the same smoking category at both Time 1 and Time

2: MTS 40 of 54; MS 60 of 71; TS 30 of 32; and NS 56 of 58. Table 5

shows the prevalence, in percent, of abnormal respiratory symptoms at Time

1 and Time 2 in the retested participants, by smoking group, for individuals

who remained in the same smoking category. prevalence of cough, sputum,

and wheeze was significantly higher in all smoking groups (MS, TS, and

MTS) compared to nonsmokers at both Time 1 and Time 2 (p<.05, Chi

square). Among smokers of tobacco or marijuana alone (TS and MS), the

prevalence of all respiratory symptoms tended to decrease in the interval

between the two examinations; in contrast, among the dual smokers (MTS),

frequency of reported cough, sputum, wheeze and shortness of breath tended

to increase. Symptom prevalence was not statistically different, however,

between Time 1 and Time 2 among participants in any smoking category

who remained in the same category (p<.05; McNemar’s test of symmetry).

TABLE 5. Prevalence (percent) of respiratory symptoms at Time 1 (1983

to 1984) and Time 2 (1985 to 1986)

Smoking

Group

Acute

Bronchitic Shortness

Cough Sputum Episodes

T 1 T 2 T 1 T 2 T 1 T 2

Wheeze of Breath

T1 T2 T 1 T 2

MTS 28* 33*+

23* 33* 13* 13* 32* 40*+10 13*

MS 26* 16* 21* 15* 12* 6 28* 19* 4 0

TS 37* 33* 33* 23* 23* 17 37* 20* 17* 10

NS 0 4 4 4 0 0 5 4 2 2

*Significantly greater than values in NS; p<.05; x2 or Fisher’s exact test.

+

Significantly greater than values in MS; p<.05; x2 or Fisher's exact test.

Table 6 shows the prevalence, in percent, of abnormality in several lung

function measures in the retested cohort at both Time 1 and Time 2 for

subjects in each smoking group who did not change smoking categories.

These findings show persistent or increasing prevalence of abnormality in

measures mainly of small airways function (e.g., FEF25-75, CC) and in DLCO

(an indicator of early emphysema) among smokers of tobacco, especially

those who also smoked marijuana, compared to nonsmokers. Over the

same time interval, the higher prevalence of abnormality in measures

reflecting large airways function (e.g., Vmax, Raw among marijuana smokers

compared to tobacco-only smokers and nonsmokers increased even further.

68
TABLE 6. Prevalence (percent) of abnormality in tests of lung function at

Time 1 and Time 2

Group

MTS

MS

TS

NS

a

Significantly greater than values in NS; p<.05, x2 or Fisher’s exact test.

b

Significantly greater than values in TS; p<.05, x2 or Fisher’s exact test.

c

Significantly greater than values in MS; p<.05, x2 a Fisher’s exact test.

KEY: FEF25-75=forced expiratory flow rate between 25 and 75 percent of the forced vital capacity;

Vmax=peak maximal expiratory flow rate; CC=closing capacity; Raw=airway resistance;

DLCO=single-breath diffusing capacity.

The results of this prospective study support previous evidence from crosssectional studies (Tashkin et al. 1980; Tashkin et al. 1987a) of (1) a significant relationship between habitual smoking of marijuana and/or tobacco and

symptoms of chronic bronchitis, (2) a deleterious- effect of tobacco on both

small airways and alveolar function, and (3) an adverse effect of marijuana

primarily upon the large airways. Continuing tobacco and/or marijuana

smoking was associated, in general, with persistent or worse abnormalities

in airway and alveolar function.

One hundred and thirty-eight of the study participants returned for a third

round of testing in 1986 and 1987 after an interval of 3 to 4 years from the

initial examination. Results in this subgroup not only support the above

findings but also suggest an additive effect of habitual smoking of marijuana and tobacco on both respiratory symptoms and lung function, in

agreement with findings from a recent community-based epidemiological

study (Bloom et al. 1987). Thus far, too few subjects discontinued use of

marijuana with or without tobacco to permit meaningful analysis of the possible effect of cessation of marijuana use on respiratory status.

EFFECTS OF HABITUAL SMOKING OF MARIJUANA

ALONE OR WITH TOBACCO ON NONSPECIFIC

AIRWAYS HYPERREACTIVITY

Nonspecific airways hyperreactivity (AHR) has been found to be associated

with regular tobacco cigarette smoking and may be a risk factor for the

69
development of chronic obstructive pulmonary disease in tobacco smokers

(Barter and Campbell 1976; Kanner 1984; Vollmer et al. 1985). Bronchodilator drugs, by inhibiting airway reactivity to nonspecific inhaled

spasmogens, could provide protection against chronic obstructive pulmonary

disease. Marijuana smoke contains many of the Same irritants found in

tobacco smoke (Hoffman et al. 1975; Novotny et al. 1982), in addition to

THC and other cannabinoids. THC causes dose-dependent bronchodilation

(Vachon et al. 1973; Tashkin et al. 1973; Tashkin et al. 1974) and reverses

experimentally induced bronchospasm (Tashkin et al. 1975). THC is also a

bronchial irritant, however, at least when inhaled as an aerosol (Tashkin

et al. 1977). Therefore, because of its bronchodilator or irritant properties,

THC could inhibit or augment the tendency of other noxious components in

marijuana smoke (or in tobacco smoked in addition to marijuana) to cause

AHR. The net influence was therefore examined of heavy, habitual

smoking of marijuana, alone or with tobacco, on nonspecific AHR in

comparison with nonsmoking or smoking of tobacco alone (Tashkin et al.

1988).

Airway reactivity to methacholine was measured in 281 subjects (67 MTS,

116 MS, 34 ‘IS, and 64 NS) using a breath-activated dosimeter and a protocol modified from that of Chai et al. (1975). Nonspecific AHR was considered to be present if the forced expired volume in 1 second (FEV1

)

declined by 20 percent or more from the postdiluent control value after

inhalation of five deep breaths of methacholine in a concentration equal to

or less than 25 mg/ml. The following proportion of subjects in each

smoking category exhibited AHR: MTS 37.3 percent; MS 23.3 percent;

‘IS 29.4 percent; and NS 17.2 percent. Although a greater proportion

of smokers of marijuana or tobacco only exhibited AHR compared to nonsmokers, these differences were not statistically significant. On the other

hand, the proportion of dual smokers of both marijuana and tobacco who

had AHR was more than twice that of nonsmokers, and this difference was

significant (p<.01, x2). While the present small sample of tobacco-only

smokers did not have a significantly higher prevalence of AHR compared to

the nonsmoking control subjects, two-way ANOVA did show a statistically

significant tobacco effect on the degree of airways activity, most pronounced in smokers of both marijuana and tobacco (F=10.14; p=.002).

Therefore synergism between the effects of tobacco and marijuana was suggested, but the interaction did not achieve statistical significance.

It was concluded that the bronchodilator properties of THC do not protect

against the development of AHR in smokers of both tobacco and marijuana.

In contrast, these data suggest that respiratory irritants within marijuana

smoke (including THC and possibly other cannabinoids) augment the effect

of tobacco in increasing airways reactivity. These findings are consistent

with histopathologic evidence of an additive effect of marijuana plus tobacco in causing tracheobronchial epithelial injury.

70
PULMONARY EFFECTS OF HABITUAL MARIJUANA USE:

TRACHEOBRONCHIAL HISTOPATHOLOGY AND ALVEOLAR

MACROPHAGE ULTRASTRUCTURE

Bronchial mucosal biopsies and alveolar macrophages were obtained by

fiberoptic bronchoscopy and bronchoalveolar lavage (BAL) from young,

heavy habitual smokers of marijuana alone (MS, n=30), marijuana plus

tobacco (MTS, n=17), tobacco alone (TS, n=15), and control nonsmokers

(NS, n=11) selected at random from participants in the above study of the

pulmonary effects of habitual marijuana use (Fligiel et al. 1988). For subjects in each smoking category undergoing bronchoscopy, smoking histories

and prevalence of respiratory symptoms and lung function abnormality were

comparable to those for all participants in the main study who belonged to

the same smoking group. During bronchoscopy, mucosa from the primary

carina and randomly selected secondary or tertiary carinae of the right

middle or lower lobe was biopsied (Gong et al. 1987). Biopsy specimens

from each subject were processed for light microscopy and examined by a

single pathologist unaware of the subject’s smoking category. Histopathologic features were assessed according to the criteria suggested by Auerbach

and colleagues (1957; 1961), as indicated in table 7. Results are shown in

table 8. Histopathological alterations in the bronchial mucosa were significantly more common and severe in all groups of smokers than in nonsmokers. Furthermore, most of the mucosal abnormalities (with the exception of

squamous metaplasia and the presence of mitotic figures) were more frequently observed in marijuana-only than in tobacco-only smokers. Most

notable, however, was the finding that virtually all of the epithelial and

basement membrane alterations were most prevalent in subjects who habitually smoked both marijuana and tobacco.

Bronchial biopsies were also examined using scanning electron microscopy

(SEM) and transmission electron microscopy (TEM) for further, indepth

characterization of mucosal morphology. TEM also permitted a thorough

examination of the basement membrane region. The light microscopic findings were confirmed and expanded by both SEM and TEM. TEM revealed

frequent and early squamous metaplastic changes in all types of smokers as

compared to nonsmokers. Moreover, approximately one-third of all the

marijuana smokers (MS and MTS) showed disruption of basement membrane continuity, which most likely occurred as a result of basement

membrane permeation by inflammatory cells from submucosal blood vessels,

a finding not observed in tobacco-only smokers.

The findings are important for several reasons. First, all subjects studied

were young adults, most of whom were asymptomatic with no or minimal

lung function abnormality, indicating that habitual marijuana smoking frequently causes significant, albeit clinically silent, airway injury manifested

by extensive morphologic changes at a relatively early age when symptoms

71
TABLE 7. Criteria for light microscopic evaluation of bronchial biopsies

Epithelial Changes

Basal (reserve) cell hyperplasia: 3 rows of basal cells

Stratification: flattening of surface epithelium and/or absence of cilia

Squamous metaplasia: stratification involving any portion or all of

the mucosa as well as the total loss of cilia

Goblet cell hyperplasia: goblet cells constituting >25% of all

epithelial cells

Variation of nuclear size, shape, and chromatin content

Presence of mitotic figures

Increased nuclear-to-cytoplasmic ratio (>1:4)

Intraepithelial inflammation: 4 or more inflammatory cells per high

power field

Basement Membrane Thickening >7 μm

Submucosal Inflammation 8 inflammatory cells per high power field

and pulmonary dysfunction are absent or uncommon. Furthermore, the

marijuana smokers we studied (MS and MTS) were several years younger

than the TS, and the MTS smoked nearly half as much tobacco as did the

TS subjects, suggesting that habitual marijuana smoking may be at least as

damaging, if not more damaging, to the epithelium of the central airways

than the smoking of tobacco. The latter possibility is further suggested by

the similar, if not greater, frequency of histopathologic abnormalities among

MS compared to TS despite a far smaller daily number of marijuana joints

smoked by the MS (3 to 4 per day) than the daily number of tobacco cigarettes smoked by the TS (more than 20 per day). Finally, the observation

that bronchial mucosal changes were most prevalent in MTS suggests an

additive effect of marijuana and tobacco smoking on airway injury, consistent with findings from the only other study of histopathologic abnormalities

in the airways of chronic cannabis smokers (Tennant 1980). It is of interest, however, that while Tennant (1980) found extensive histopathologic

changes in airways of heavy hashish smokers, all of his subjects had

respiratory symptoms and many had abnormal lung function, in contrast to

our subjects, most of whom were asymptomatic with normal lung function.

72
TABLE 8. Histopathologic changes in bronchial biopsies examined by

light microscopy

Epithelial Changes

Basal Cell

Hyperplasia

Stratification

Squamous

Metaplasia

Goblet Cell

Hyperplasia

Cellular

Disorganization

Nuclear Variation

Mitotic Figures

Increased Nuclearto-Cytoplasmic

Ratio

Inflammation

Basement

Membrane

Thickening

Submucosal

Inflammation

15 88

14 82

11 65

12 71

13 76

14 82

4 24

8 47

8 47

14 82

3 18

22 73

16 53

8 27

25 83

14 47

12 40

4 13

13 43

10 33

22 73

7 23

6 40

6 40

6 40

8 53

4 27

7 47

4 27

6 40

6 40

5 33

4 27

0 0

0 0

0 0

3 2 7

1 9

0 0

0 0

0 0

0 0

0 0

4 36

MS>NS .00002

MTS>NS .000003

MTS>TS .005

MS>NS .0014

MTS>TS .00002

MTS>NS .0006

MTS>MS .010

MS>NS .001

MTS>NS .0007

MTS>TS .006

TS>NS .010

MTS>NS .00002

MTS>MS .005

MS>NS .007

MTS>NS .008

MTS>NS .008

MS>NS .00002

MTS>NS .00002

MS>TS .010

MTS>TS .006

*By Fisher's exact test (one-tailed).

**Only values within the 99% confidence levels are reported.

SOURCE :FIigiel et al. 1988, copyright 1988, Australian Government Press.

The true potential for the type and extent of the bronchial epithelial histopathology noted in the marijuana smokers examined to produce clinically

significant chronic airway disease or to progress to malignant change is

unknown, but certainly possible (Auerbach 1957; Knudtson 1960). Prolonged followup of these subjects may help answer this question.

73
Alveolar macrophages obtained by BAL from the same three groups of

smokers (MS, n=15; MTS, n=8; TS, n=7) and nonsmokers (NS, n=7) were

compared with respect to morphologic and morphometric ultrastructural cellular features (including an evaluation of cell membrane, nucleus, and cell

plasma contents, especially cytoplasmic inclusions). Compared to NS, both

TS and MS had frequently larger cytoplasmic inclusions, most of which

contained needlelike structures. While TS showed an increased nuclear

contour index and decreased cell membrane ruffling, MS demonstrated

increased blebbing and an increased ruffling index. Macrophages of NS

showed the greatest ruffling index and frequently contained surfactant material. These findings indicate definite ultrastructural differences between

alveolar macrophages of smokers in general and nonsmokers, as well as be

tween the macrophages of MS and TS. These morphological differences

may indicate differences in functional activity of these important cells.

The numbers of alveolar macrophages and other inflammatory cells (lymphocytes, neutrophils, and eosinophils) recovered in BAL fluid from

16 MTS, 14 MS, 13 TS, and 19 NS were determined from Giemsa-stained

cytopreps (Barbers et al. 1987). Results are indicated in table 9. These

findings indicate that smoking of marijuana or tobacco alone induces an

inflammatory cellular response in the alveoli consisting mainly of increased

numbers of alveolar macrophages and that habitual smoking of both marijuana and tobacco causes a much greater inflammatory response, implying

an adverse effect of marijuana smoking on the lung that is independent of

and additive to that of tobacco. ‘The mechanism of the inflammatory

response in the lungs of marijuana and/or tobacco smokers is not known but

may include cellular stimulation by physicochemical irritants or antigenic

substances within the smoke leading to release of mediators with chemoattractant properties, release of enzymes, or generation of toxic oxygen

radicals.

TABLE 9. Mean number ± SEM of each type of white blood cell

(WBC/mlx104) recovered in bronchoalveolar lavage from

smokers of marijuana and/or tobacco and from nonsmokers

Subject Alveolar

Group Macrophages Lymphocytes Neutrophils Eosinophils

MTS 57.9±6.4abc 3.0±0.7a

1.16±0.42a0.11±0.11

MS 27.1±5.1a

1.9±0.7 0.50±0.18 0.04±0.03

TS 33.3±4.2a

2.0±0.5 0.28±0.05 0.02±0.02

NS 10.6±1.5 0.9±0.1 0.07±0.03 0.04±0.02

a

Significantly different from NS; p<.01, unpaired Student’s t test.

b

Significantly different from MS; p<.01, unpaired Student’s t test

c

Significantly different from TS; p<.01, unpaired Student’s t teat.

74
RELATIONSHIP BETWEEN SMOKING DYNAMICS AND

RESPIRATORY DELIVERY OF INSOLUBLE PARTICULATES

(TAR) DURING THE SMOKING OF MARIJUANA VS. TOBACCO

As already noted, the frequency of chronic respiratory symptoms and tracheobronchial epithelial histopathology observed in young smokers of only 3 to

4 marijuana joints a day was similar to that of smokers of more than 20

tobacco cigarettes a day (Tashkin et al. 1987a). One possible explanation 

for these findings is that more particulates and gaseous irritants may be

delivered to the respiratory tract in the smoke from marijuana than in the

smoke from a comparable amount of tobacco, perhaps due, in part, to differences in the dynamics of smoking these two substances. To evaluate this

possibility, the dynamics of smoking a single tobacco cigarette were compared in 11 habitual smokers of both marijuana and tobacco, 9 habitual

smokers of marijuana only, and 10 regular smokers of tobacco only. At the

same time, the amount of particulates delivered to and retained by the

smoker following the smoking of each type of cigarette was quantified. Wu

and colleagues previously reported results obtained in dual smokers of both

marijuana and tobacco in relation to smoking marijuana of low potency

(0.004 and 1.24 percent THC) (Wu et al. 1988). Since “street” marijuana

is currently estimated to be of higher potency, further experiments were

performed involving the smoking of marijuana containing 2.74 percent THC.

In addition, these studies were extended to habitual smokers of marijuana

only or tobacco only to ascertain whether or not concomitant tobacco or

marijuana smoking affects the smoking dynamics and delivery of smoke

particulates for each of the other types of smoked substance.

Subjects were studied on one or two separate days at the same time of day.

They were asked to refrain from smoking tobacco for at least 2 hours and

cannabis for at least 6 hours before visiting the laboratory. During the first

visit, the dual smokers of marijuana and tobacco initially smoked their own

brand of tobacco, followed, in a single-blind fashion, by a placebo marijuana joint (0.004 percent THC) and then by a marijuana cigarette containing 1.24 percent THC. On the second visit., subjects smoked placebo

marijuana followed by a joint containing 2.74 percent THC. An interval of

about 30 minutes separated the smoking of each cigarette. The habitual

smokers of marijuana only followed the same protocol except that they did

not smoke tobacco. The regular smokers of tobacco only were studied in a

single session during which they smoked a tobacco cigarette of their own

brand. All subjects were asked to smoke each cigarette in a manner as

similar as possible to their customary method of smoking each type of

substance.

Characteristics of each type of cigarette smoked during these sessions are

shown in table 10. Tobacco cigarettes were invariably filter tipped and

consisted of different brands with varying FTC-estimated yields ranging

from low to high nicotine and tar contents. The tobacco cigarettes on the

75
average were slightly longer and weighed slightly more than the marijuana

joints. The tar yields, measured in mainstream smoke generated by a

syringe with a 50-ml puff volume, 2-second puff duration and 30-second

inter-puff interval to butt lengths of 35 mm for tobacco and 25 mm for

marijuana, indicated an average 2.2-fold greater tar delivery from marijuana

than from tobacco. The draw resistance for marijuana was about one-third

that for tobacco because of a much looser packing of the marijuana

cigarette.

TABLE 10. Cigarette characteristics

FTC Yield Drawb

Length Weight Nicotine Tar Tara Resistance

(mm) (43) (mg) (mg) (mg) (cm H2O/ml/sec)

Tobacco

(various brands,

filter tipped)

80-120 878-1,126 0.4-1.4 4.6-23 14.1-20.9 0.51

(89.6±10.0) (986±81) (0.84±0.32) (12±5.7) (17.8±2.9)

Marijuana

0% THC 85 741-940 - - 38.5±0.8

1.24% THC 85 849-985 - - 46.3±2.2 0.17

2.74% THC 85 710-795 - - 33.6±3.4

Measured in mainstream smoke generated by syringe with a 50-ml puff-volume, 2-second puff duration,

and 30-second inter-puff interval to butt lengths of 33 mm for tobacco and 25 mm for marijuana.

The pressure difference between two ends of the cigarette (between mouth and the distal end).

Puff volume, duration, number, and interval were measured using a sensitive

Fleisch pneumotachograph (linear between 5 and 100 ml per second) connected through Tygon tubing to the distal end of a glass cylinder that contained two ventilation ports and was sealed at its proximal end by a rubber

stopper (figure 1). A tobacco or marijuana cigarette was held in a small

plastic holder inserted through the rubber stopper. Between puffs, the ventilation ports prevented excessive accumulation of carbon monoxide and extinction of the lighted cigarette. During a puff, the ventilation holes were

occluded to allow the air drawn through the cigarette to be measured by the

pneumotachograph. Inhaled volume and smoke retention time were measured using inductive plethysmography. Volume changes were calculated

from the weighted sums of the chest wall and abdominal signals by the

“least-squares” calibration method and showed agreement with spirometric

measurements to within plus or minus 10 percent.

A proportional smoke-trapping device interfaced to the puff-volume measuring apparatus measured the amount of smoke particulates delivered to the

smoker’s mouth (figure 1). In this device, the mainstream smoke split into

two parallel pathways, a low resistance pathway containing several parallel

capillary tubes (pathway B) and a high resistance pathway with only one

76
FIGURE 1. Puff volume measuring apparatus interfaced to proportional

smoke trapping device for measuring delivery of

smoke particulates

capillary tube (pathway A). A Cambridge filter pad trapped the smoke

passing through pathway A. The amount of tar trapped by the filter was

extracted with methanol and analyzed spectrophotometrically at a wave

length of 400 nm. Gravimetric and spectrophotometric studies have shown

that a fairly constant fraction of the tar (approximately 12.5 percent or oneeighth) is retained in the Cambridge filter over a range of puff volumes,

durations, and flow rates (Rose et al. 1987). Therefore, the actual quantity

of smoke particulates delivered to the mouth can be calculated simply by

multiplying the amount of particulates trapped in the filter pad in pathway

A by 7. Smoke exhaled after each puff was collected using another filter

attached to the distal end of a megaphone device connected to a vacuum

system, as described by Hinds et al. (1983). The amount of smoke deposited in the respiratory tract can then be calculated by simple subtraction and

expressed as a percentage of the amount inhaled.

77
Mean data for smoking dynamics are shown in table 11 for MIS. Compared to tobacco, marijuana smoking was associated with a 30 to 40 percent

smaller number of puffs, a 30 to 78 percent greater puff volume, a 33 to

50 percent longer puff duration, a 40 to 54 percent greater inhaled volume

and a 3.9- to 5.2-fold longer retention time. Similar differences were noted

comparing MS with TS, suggesting that among habitual MTS, the dynamics

of smoking one type of substance does not influence smoking topography

for the other substance. It is also noteworthy that, despite marked differences in the degree of intoxication, the different strengths of marijuana were

associated with a similar smoking profile, except for puff volume, which

was smaller during the smoking of 2.74 percent marijuana compared to the

lower strengths of marijuana, but still larger than the puff volume associated

with tobacco smoking.

TABLE 11. Mean smoking topographic data for smokers of marijuana

plus tobacco (n=11)

Marijuana

Parameter Tobacco 0%THC 1.24% THC 274% THC

Puff No. 12.6±4.7 7.4±2.2b8.3±3.1b8.4±3.2b

Puff Volume, ml 47.8±14.4 84.9±34.0b,c 82.6±20.4b,c 62.4±23.1

Puff Duration, sec 2.4±l.l 3.6±2.1b

4.4±2.2b,c 3.1±2.1

Puff Interval, sec 32.1±16.9 38.8±14.2 43.1±18.4 36.6±12.6

Inhaled Volume, 1 1.24±0.31 1.91±0.74b

1.74±0.60b 1.73±0.72b

Retention lime, sec 3.0±0.8 14.0±11.1b

15.7±11.6b11.6±8.0b

No Smoking Interval, sec 28.9±17.4 22.2±9.5 24512.3 22.7±5.7

Butt Length, mm 38±14 28±17 29±17 27±10

“High,” % (maximum 100%) - 17±14 69±17d

74±18d

a

Significant differences across tobacco and marijuana cigarettes (0-2.74 percent THC); p<.006,

two-way ANOVA.

b

Significantly different from tobacco; p<.03.

c

Significantly different from 2.74 percent THC; p<.02.

d

Significantly different from 0 percent THC; p<.001.

For the dual smokers, the mean quantity of inhaled particulates measured in

optical density (OD) units and the percentage of inhaled particulates deposited in the respiratory tract for each type of cigarette are shown in table 12.

Compared to tobacco, marijuana was associated with the delivery of an

average of 2.9 times more particulates and a 40 percent greater retention of

the inhaled particulates in the respiratory tract. Similar differences were

noted comparing MS with TS.

In summary, in smokers of both marijuana and tobacco, a nearly threefold

greater delivery of particulate matter to the smoker’s mouth was found

78
during the smoking of a single marijuana joint of varying potency up to

27.74 percent THC compared to a single tobacco cigarette of the smoker’s

own brand. We also found the respiratory retention of particulates inhaled

from marijuana smoke to be 40 percent greater than that of particulates

inhaled from the smoke of tobacco. Therefore, the net respiratory burden

of particulates was approximately fourfold greater during marijuana than

tobacco smoking. Similar differences were noted comparing smokers of

marijuana alone with smokers of tobacco alone.

TABLE 12. Mean quantity of inhaled particulates and percent of inhaled

particulates retained in the respiratory tract &ring the

smoking of marijuana or tobacco by habitual smokers of

both substances (n=11)

Marijuana

Parameter Tobacco O%THC 1.24% THC 274% THC

Inhaled Particulates, 4.8±2.l 12.9±5.8* 14.9±5.8* 13.7±4.6*

Optical Density

Particulate Deposition 59.3±19.6 84.3±15.6+84.4±17.4+

86.7±13.2+

* Significantly different from tobacco, p<.001, ANOVA multiple comparison test.

Factors that might account for the greater particulate burden to the lungs

from smoked marijuana than from a comparable quantity of smoked tobacco

include differences in the combustion characteristics of the two plant

substances and a greater filtration of the tobacco cigarettes, which were

filter tipped and more densely packed than the marijuana joints. These differences could largely account for the more than twofold greater tar yield

from marijuana than tobacco that was measured using syringe-simulated

puffs of similar volume and duration (table 10). Another possible factor

involves consumption of a larger proportion of the marijuana than tobacco

cigarettes, leaving a smaller butt length. Nonetheless, although marijuana

was smoked to a smaller average butt length (28 mm) than tobacco

(38 mm), the tobacco cigarettes were longer and heavier to begin with, so

that a similar quantity of the two types of cigarette was consumed. A

fourth factor, differences in smoking dynamics, probably contributed significantly to the observed differences in tar delivery to and retention by the

smoker. Differences in smoking dynamics included a 30 to 78 percent

larger puff volume, a 40 to 54 percent greater inhaled volume, and a 3.9 to

5.2 times longer retention time for marijuana than for tobacco, which

appeared to more than offset any effect of the 30 to 40 percent smaller

number of puffs of marijuana than of tobacco. The deeper inhaled volumes

and, in particular, the much longer breathholding times during marijuana vs.

tobacco smoking could have accounted for the greater fractional deposition

79
of inhaled marijuana smoke particulates in the respiratory tract due to gravitational sedimentation and interception of surfaces in the lower airways and

air spaces during molecular diffusion.

In conclusion, the fourfold greater respiratory burden of particulates

observed during the smoking of a single marijuana cigarette compared to a

single tobacco cigarette may partly explain earlier findings that daily smoking of only a few joints of marijuana without tobacco was associated with

chronic respiratory symptoms and tracheobronchial epithelial histopathology

of frequency and extent similar to that noted in daily smokers of more than

20 cigarettes of tobacco without marijuana. These observations provide further support for the concept that the habitual smoking of only a few marijuana cigarettes a day can have adverse long-term effects on the lung.

On the other hand, the mechanism of the relatively greater impact of marijuana than tobacco smoking on central airway function and the apparently

greater frequency of some histopathologic changes in the epithelium of the

central airways among marijuana-only than tobacco-only smokers, in contrast to the predominant effect of tobacco smoking on the peripheral airways, remains unclear. Possible explanations for these differences could be

a relatively greater fractional deposition of marijuana smoke particulates in

the central airways due to inertial impaction, greater absorption of irritating

gases within marijuana smoke by the mucosa of the central airways leading

to proportionally less particulate deposition or gaseous absorption more

peripherally in the respiratory tract, or a combination of these factors.

Since particle size is an important determinant of the mechanism and rate of

deposition of particulates in the respiratory tract (Brain and Valberg 1974),

the aerodynamic size of particulates was measured in the smoke of filtered

and unfiltered tobacco cigarettes (Marlboro) and marijuana joints containing

0.004, 1.24, and 2.75 percent A9-THC using a multistage cascade impactor

(Andersen, Series 290). Puff volumes and durations of air similar to those

observed in smokers of marijuana and tobacco (100 ml, 4 sec and 50 ml,

2 sec, respectively, at a flow rate of 25 ml/sec) were delivered through the

marijuana or tobacco cigarette in a combustion chamber. The cigarette

smoke was immediately diluted 10 to 30 times by a high volume of

ambient air at 21 °C and 15 percent relative humidity in a mixing chamber

to prevent agglomeration of smoke particulates. The smoke was drawn into

the cascade impactor at a flow rate of 4.0 liters per minute. The excess

smoke mixed with air was exhausted to the atmosphere through a vent.

Methanol extracts of the impaction plates were assayed spectrophotometrically at 400 nm. Measurements were performed in quintuplicate for each

type of cigarette. Mass mean aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated based on the assumption of

a log normal distribution of particle sixes by the least squares method.

Results are shown in table 13.

80
#TABLE 13. Mean mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of smoke particulates from

tobacco and marijuana cigarettes

Type of Number of MMAD

Cigarettes Measurements Mean GSD

Standard

Deviation

Tobacco, Filtered 5 0.490 1.71 0.039

Tobacco, Nonfiltered 5 0.539 1.79 0.061

Marijuana, 0% THC* 5 0.579 1.85 0.032

Marijuana, 1.24% THC** 5 0.927 1.71 0.039

Marijuana, 2.74% THC** 5 0.957 1.66 0.072

* Significantly larger than filtered tobacco (p<.05).

**Significantly larger than filtered or unfiltered tobacco and 0 percent ‘MC marijuana (p<.001).

The findings indicated that marijuana (1.24 and 2.74 percent THC) smoke

particulates, although approximately twice as large as particles in tobacco

smoke, are still in the submicronic range that favors peripheral deposition in

the respiratory tract by gravitational sedimentation and molecular diffusion

(Brain and Valberg 1974). Another factor that could account for excessive

central deposition of smoke particulates due to inertial impaction is a rapid

rate of inhalation of the smoke mixed with air (Valberg et al. 1978). However, although the smoking topography studies did not permit precise measurements of inhaled flow rates, estimates of the latter from inductive

plethysmographic tracings did not suggest a substantially greater inhaled

flow rate during marijuana compared to tobacco smoking. Thus it appears

unlikely that excessive central airway deposition of marijuana smoke particulates occurs during marijuana smoking (compared to tobacco smoking) as a

result of either large particulate sixes or unusually rapid rates of inhalation

of the smoke mixed with air. Further studies involving lung imaging of the

respiratory distribution of deposited smoke particulates labeled with a suitable gamma-emitting radionuclide are required to further evaluate this

question.

PULMONARY EFFECTS OF FREEBASE COCAINE

Because of the increasing prevalence of cocaine smoking (freebasing) and

reports in small numbers of cocaine freebase users of respiratory symptoms

and abnormalities in diffusing capacity of the lung (Weiss et al. 1981;

Itkonen et al. 1984), the relationship between self-reported cocaine use by

freebasing and respiratory symptoms and lung dysfunction was analyzed.

Subjects included habitual smokers of marijuana with and without tobacco

who denied intravenous drug abuse and were participants in a study of the

chronic pulmonary effects of habitual marijuana use (Tashkin et al. 1987a).

81
Of 356 marijuana smokers in the study sample, 84 (67 males, 17 females,

mean age 30 years) reported freebasing cocaine from 1 to 992 times in

their lifetimes. Comparisons were made between tobacco-smoking cocaine

freebase users (CTS, n=36) and tobacco-smoking, cocaine nonusers (TS,

n=136) and between tobacco-nonsmoking, cocaine-freebase users (CS, n=48)

and total nonusers (NS, n=36). “Moderate” (more than 10 times, mean 131

times) and “mild” (fewer than 10 times, mean approximately 3 times)

cocaine users were also analyzed separately. Marijuana use was similar (52

to 53 joint-years) but tobacco use less (12 vs. 17 pack-years) in CTS than

T’S, marijuana use was less in CS than NS (43 vs. 62 joint-years), and

cocaine smoking was comparable in CTS and CS (lifetime use frequency 56

vs. 57 times). No significant differences in frequency of chronic cough,

sputum, wheeze, or shortness of breath were noted across any of the above

groups. However, moderate freebase cocaine users reported hemoptysis

(14 percent), black sputum (43 percent) and chest pain (78 percent) after

smoking cocaine. Also, moderate freebase cocaine use had an adverse

effect on several tests of airway function (FEV1, FEV1/FVC, Vmax, Visoflow, N2/1, closing volume, and RV), which was significantly greater than

that which could be attributed to tobacco (p<.05). On the other hand,

moderate freebase cocaine use, unlike tobacco, had no demonstrable effect

on DLCO. These findings suggest that, among habitual marijuana smokers,

moderate cocaine smoking (1) does not appear to have an effect on chronic

respiratory symptoms that is additive to that of marijuana alone; (2) not

uncommonly causes acute respiratory symptoms shortly after smoking

cocaine; (3) irritates large and small airways, as reflected by functional

changes that are not obscured by concomitant marijuana use and appear to

he at least additive to those produced by tobacco; and (4) has no demonstrable effect on the pulmonary microcirculation.

Preliminary data in 39 additional cocaine smokers who reported heavy use

of cocaine (mean of 11.7 grams per week) with, a history of regular freebase smoking for an average of 34 months reveal a high proportion of subjects with acute cardiopulmonary symptoms temporally related to cocaine

smoking. Of this group of heavy cocaine smokers, 44 percent reported

. coughing up black sputum from their chests and 48 percent noted hemoptysis within 12 hours after smoking cocaine, while 60 percent reported chest

pain (mostly pleuritic in quality) and 40 percent experienced palpitations

within 1 hour after freebasing.

Of the 272 non-freebase users in the sample of heavy, habitual marijuana

smokers, 242 admitted to “snorting” cocaine. However, analysis of respiratory symptoms and lung function in nonsmokers of cocaine classified into

groups of “snorters” and “nonsnorters” of cocaine who did and did not

smoke tobacco failed to show any significant relationship between snorting

cocaine and either respiratory symptoms or lung dysfunction among either

smokers or nonsmokers of tobacco. More detailed studies are needed to

examine the impact of frequent cocaine smoking on lung structure and

82
function and the relationship between frequency, intensity, recency, and

method of cocaine freebase use on respiratory alterations, adjusting for

effects of concomitant use of other drugs that can have a damaging effect

on the lungs.

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ACKNOWLEDGMENTS

This work was supported by United States Public Health Service grant

RO1 DA 03018 from the National Institute on Drug Abuse.

AUTHORS

Donald P. Tashkin, M.D.

Professor of Medicine

UCLA School of Medicine

University of California, Los Angeles

10833 Le Conte Avenue

Los Angeles, CA 90024-1736

Suzanne Fligiel, M.D.

Head, Electron Microscopy

Department of Laboratory Medicine

Veterans Administration Medical Center

Allen Park, MI 48101

and

Associate Professor of Pathology

Department of Pathology

Wayne State University Medical Center

Detroit, Ml 48202

Tzu-Chin Wu, M.D.

UCLA School of Medicine

Henry Gong, Jr., M.D.

UCLA School of Medicine

Richard G. Barbers, M.D.

Assistant Professor of Medicine

University of Massachusetts School of Medicine

Worcester, MA 01605

Anne H. Coulson

UCLA School of Public Health

Michael S. Simmons

UCLA School of Medicine

86
Theodore F. Beals, M.D.

Director, Clinical Electron Microscopy

Veterans Administration Medical Center

Ann Arbor, MI 48105

and

Assistant Professor of Pathology

University of Michigan Medical School

Ann Arbor, Ml 48109

87
Marijuana Effects and Urinalysis

After Passive Inhalation and Oral

Ingestion

Edward J. Cone

INTRODUCTION

The psychoactive properties of marijuana in humans have been the basis of

considerable scientific scrutiny. Over the last two decades, numerous

studies have been performed on the pharmacokinetic and pharmacodynamic

properties of marijuana and tetrahydrocannabinol (THC), the major active

constituent of marijuana. The majority of these studies involve drug administration by subjects smoking marijuana plant material or orally ingesting

pure THC. Recently, the effects of passive exposure to marijuana smoke

and the oral ingestion of marijuana plant material have been the subject of

considerable speculation as a result of extensive urine testing of individuals

who claim exposure by these alternate routes of administration.

Absorption of sufficient THC by oral ingestion of marijuana plant material

to produce behavioral effects and detectable concentrations of urine metabolites was considered likely if free THC was present or conversion of THC

precursor acids in the plant material to THC occurred. Wall and colleagues

(1983) found that the bioavailability of orally administered THC was 10 to

20 percent for male and female college students. Also, Law and associates

(1984) reported detection of cannabinoid metabolites in blood and urine

after administration of cannabis resin to human subjects in a meat sandwich.

Studies of subjects who were passively exposed to marijuana smoke also

indicated the possibility for absorption of sufficient THC to test positive by

urinalysis for cannabinoid metabolites (Perez-Reyes et al. 1983; Mason et al.

1983; Morland et al. 1935; Law et al. 1984; Ferslew et at 1983).

However, two basic pharmacologic questions remained essentially unanswered regarding the effects of marijuana administered by these two routes

of administration. These questions were:

88
1. Ate behavioral effects produced and are they dose related?

2. What is the urine excretion profile of cannabinoid metabolites?

Clinical studies at the Addiction Research Center, in collaboration with the

U.S. Navy, were designed to answer these and related pharmacologic and

toxicologic questions. In separate studies, male subjects either were passively exposed to marijuana smoke under highly controlled conditions or

ingested marijuana-laced brownies. Behavioral measures and all urines were

collected and analyzed. This chapter summarizes the results from these

studies (Cone and Johnson 1986; Cone et al. 1987; Cone et al. 1988).

METHODS

Subjects

Subjects were healthy, drug-free males with a history of marijuana use.

Criteria to enter the study included a documented period of cannabinoidnegative urines (EMIT dau, 20 ng/ml cutoff). Four negative days were

required for the brownie study and 14 days for the passive-inhalation study.

During the study, subjects were housed on a closed research ward under

close surveillance. Two additional male subjects, from the staff, with no

history of marijuana use participated in one phase of the passive-exposure

study. The study was conducted under the guidelines for the protection of

human subjects (45CRF46), and each subject gave informed consent.

Passive Marijuana-Exposure Conditions

Five subjects were passively exposed under double-blind conditions to side

stream smoke from 16 marijuana cigarettes (2.8 percent THC) for 1 hour

each day (0830 to 0930) for 6 consecutive days. Preceding and following

the days of marijuana smoke exposure, the subjects were exposed for

2 days to the smoke of 16 placebo cigarettes (0 percent THC). During

exposure, the subjects wore goggles and sat quietly in a small, unventilated

room (2.1 m x 2.5 m x 2.4 m). The approximate room volume after

adjustment for contents was 12,225 L. Right of the cigarettes were burned

during the first 12 minutes, and the remaining eight were burned 30 to

42 minutes into the exposure session. The cigarettes were burned in a

constant-draft smoking apparatus designed to release only sidestream smoke.

Flow rates for the smoking apparatus were adjusted for an average cigarette

bum time of 12 minutes. Room air samples were withdrawn at timed intervals and analyzed for THC content. A second exposure study was performed with the subjects for 6 consecutive days under similar conditions but

with exposure to the smoke of four marijuana cigarettes (2.8 percent THC).

This was preceded and followed by only 1 day of placebo smoke exposure.

A third study was performed with two marijuana-naive subjects who were

exposed for 6 consecutive days to the smoke of 16 marijuana cigarettes

89
(2.8 percent THC) without blind conditions to placebo smoke. Individual

urine specimens were collected from all subjects throughout the entire study

and were frozen until analyzed.

Marijuana-Laced Brownie Study

Each of the five subjects who participated in the study received single treatments of the following three doses of marijuana brownies, assigned in random order under double-blind conditions: two brownies containing an

amount of cannabis plant material equivalent to two marijuana cigarettes

(2.8 percent THC); two brownies containing the equivalent of one marijuana

cigarette (2.8 percent THC) and one placebo marijuana cigarette (0 percent

THC); and two brownies containing the equivalent of two placebo marijuana

cigarettes (0 percent THC). The brownies were consumed within 5 minutes

starting at 0830. All individual urine specimens were collected and frozen

until analyzed.

Behavioral, Physiological, and Chemical Measures

Subjects completed subscales of the Addiction Research Center Inventory

(Haertzen 1974) as follows: marijuana scale (Mar 15); morphine-benzedrine

group (MBG) scale, which reflects feelings of euphoria and well-being;

pentobarbital-chlorpromazine-alcohol group (PCAG) scale, which reflects

sedation and intoxication; and the lysergic acid diethylamide (LSD)-specific

scale. Additional measures collected included scales from the Single-Dose

Questionnaire (Feel Drug and Liking) and a visual analog scale (VAS) for

measurement of good feelings and physiological effects (pulse, blood

pressure). All measures were taken at 0730 and 0800 before each test

session (0830 to 0930) and thereafter at 0930, 1030, 1130, and 1230. For

the marijuana brownie study, additional measures also were collected at

1430,1630, and 2030.

Urinalysis

All urines were analyzed for cannabinoids by EMIT dau with a 20 ng/mL

cutoff and by Abuscreen radioimmunoassay (RIA) with a 10 ng/mL level of

quantitation. Urines also were analyzed quantitatively for ll-nor- 9

-THC-9-

carboxylic acid (9-carboxy-THC) by gas chromatography/mass spectrometry

(GC/MS) with a 5 ng/mL level of quantitation.

RESULTS

Behavioral Effects and Urinalysis After Passive Inhalation of

Marijuana Smoke

Mean responses after passive exposure to the smoke of 16 marijuana cigarettes were significantly (p<.05) elevated when compared to placebo smoke

exposure (day 2) on the MAR 15, LSD, VAS, Feel Drug and Liking scales

90
(figure 1). Responses appeared to be elevated on the PCAG scale, but were

not significantly different from placebo and were unchanged on the MBG

scale (not shown). Modest elevations also were obtained on these scales

with the same subjects after exposure to the smoke of four marijuana cigarettes. The mean response on these scales by the same subjects after

actively smoking one marijuana cigarette (2.8 percent THC) is included in

figure 1 for comparison.

FIGURE 1. Subjective effects induced by passive inhalation of

marijuana smoke

*Significant difference (p<.05) compared with day 2 response.

**Significant difference (p<.05) compared with day 3 response.

NOTE: Mean AUC data (n=5) for the response (minus control) over time are presented after exposure

to the smoke of marijuana (MJ) or placebo (P). Mean (±SE) drug (D)

AUC data in the same subjects also are shown after active smoking of one marijuana and one

placebo cigarette and after active smoking of two placebo cigarettes (A).

Source: Cone and Johnson 1986, copyright 1986, C.V. Mosby Company.

91
The behavioral effects after passive exposure to marijuana smoke were time

related, with peak behavioral responses being obtained immediately at the

end of the smoke-exposure session. Responses were diminished thereafter

and generally were completely dissipated within 3 hours. Generally, neutral

or negative responses were obtained to the placebo-smoke-exposure sessions.

Assay of urine specimens by immunoassay for cannabinoids and by GC/MS

for 9-carboxy-THC provided information on the amount of THC absorbed

during the passiveexposure sessions with marijuana smoke and the time

course of excretion of marijuana metabolites. Following exposure to the

smoke of 16 marijuana cigarettes, 6 of the 7 subjects in the 2 separate

experiments produced positive EMIT urine specimens after the first exposure session. The remaining subject produced his first positive after the

second exposure session. A high percentage (50 to 100 percent) of all

specimens produced thereafter during the 6 consecutive passive-exposure

days were positive for cannabinoids. Following exposure to the smoke of

four marijuana cigarettes, the results were more variable, with only two subjects producing positive EMIT specimens after the first exposure. Several

more positive specimens were produced during the remaining sessions by all

subjects with the exception of one subject who produced no positive specimens throughout exposure at the lower passive-smoke-exposure dose. The

ranges in concentration of 9-carboxy-THC in urines collected during the

consecutive smoke-exposure days, determined by GC/MS assay, are shown

in table 1 in comparison to those determined for the same subjects after

actively smoking one or two marijuana cigarettes. Following the 6 consecutive days of passive marijuana-smoke exposure, subjects continued to

excrete cannabinoids in urine for varying periods, as shown in table 2.

Behavioral Effects and Urinalysis After Oral Ingestion of MarijuanaLaced Brownies

After the ingestion of brownies containing cannabis plant material equivalent

to one or two marijuana cigarettes (2.8 percent THC content), subject

responses on behavioral measures for Feel Drug, Liking, and the VAS

scales were elevated over that of placebo (figure 2). Heart rate was elevated after administration of the two marijuana-cigarette-equivalent brownies.

The AUC measures for Feel Drug and Liking scales were significantly

(p<.05) different from placebo drug administration. The onset of behavioral

responses was somewhat slow and variable, with some subjects reporting

elevated responses after 30 minutes, but peak responses generally occurred

2.5 to 3.5 hours after dosing. The responses were prolonged and continued

to be elevated above placebo through 11.5 hours.

Cannabinoid metabolites were detected in the first urine voids after oral

ingestion of marijuana plant material and continued to be detectable for 3 to

14 days. Peak concentrations of 9-carboxy-THC were equivalent to or

92
higher than those measured after smoking (table 1). Mean detection times

to the last positive specimen after dosing are shown in table 2.

TABLE 1. Individual ranges in peak urinary concentration of 9-carboxyTHC by GC/MS assay after marijuana exposure

Marijuana Number of 9-carboxy-THC

Exposure Dose1

Subjects (ng/ml)

Marijuana brownie2

1 5 108-325

(Oral) 2 5 177-436

Marijuana smoke34 5 0-12

(Passive inhalation) 16 5 15-35

16 1;1 10;87

Marijuana cigarettes31 5 9-140

(Active smoking) 2 5 19-152

1Dose is expressed in marijuana cigarette equivalents; each cigarette contained 2.8 percent THC. The

average cigarette weight was 875 mg.

2Data are from Cone et al. 1988.

3Data are from Cone et al. 1987.

TABLE 2. Hours to detection of last positive urine specimen for

cannabinoid metabolites after marijuana exposure

Marijuana Number of Mean Hours + SEW

Exposure Dose’ Subjects EMIT (20) RIA (10) GUMS (5)

Marijuana brownie’ 1 5 121.6±27.9 140.3±31.9 149.0±36.2

(Oral) 2 5 142.6±34.3 190.3±42.7 156.3±49.0

Marijuana smoke’ 4 5 1.7±1.3 30.9±10.4 9.4±5.8

(Passive inhalation) 16 5 44.2±6.0 129.4±27.5 65.2±15.4

16 1;1 25.4;141.0 69.2;144.6 25.4;141.0

Marijuana cigarettes’ 1 10

(Active smoking) 2 10

53.0±12.2

69.1±13.5

1Dose is expressed in marijuana cigarette equivalents; each cigarette contained 2.8 percent THC. The

average cigarette weight was 875 mg.

2

The cutoffs (ng/ml) for each assay are shown in parentheses

3Data are from Cone et al. 1988.

4Data are from Cone et al. 1987.

Unpublished data.

93
FIGURE 2. Mean behavioral and physiological effects produced in jive

subjects after consumption of marijuana-laced brownies

NOTE: Marijuana brownie content is shown in inset. 1MJ CIG=brownie dose that contained plant

material equivalent to one marijuana cigarette (2.8 percent THC) and one placebo cigarette

(0 percent THC); 2MJ CIG=brownie dose that contained plant material equivalent to two

marijuana cigarettes (2.8 percent THC).

SOURCE Cone et al. 1988 copyright 1988, Preston Publications.

CONCLUSION

Dose-Related Behavioral Effects Produced After Passive MarijuanaSmoke Exposure and After Ingestion of Marijuana Plant Material

Behavioral responses on the Feel Drug and Liking scales were significantly

elevated over placebo responses after passive exposure to the smoke of 16

marijuana cigarettes and after the oral ingestion of the equivalent marijuana

plant material of 1 or 2 cigarettes. Responses on these measures after

lower doses were elevated, but did not differ significantly from placebo. It

is clear from these studies that as the level of exposure to marijuana increased, so did the behavioral response. In addition, the behavioral

response observed after passive exposure to the smoke of 16 marijuana

cigarettes in a small, unventilated room was both qualitatively and quantitatively equivalent to that observed after active smoking of 1 marijuana

cigarette. Behavioral response after oral ingestion was both qualitatively

and quantitatively similar to that observed after active smoking, but with a

delay of 1 to 3 hours.

94
Urine Excretion Profile of Cannabinoid Metabolites After Passive

Marijuana-Smoke Exposure and After Ingestion of Marijuana Plant

Material

Passive exposure to the smoke of 16 marijuana cigarettes led to excretion of

substantial amounts of cannabinoid metabolites in urine after each exposure

with some evidence of accumulation after multiple exposures. Occasionally,

levels in urine were sufficiently concentrated to exceed the 100 ng/mL cutoff calibrator commonly used in drug testing. After passive exposure to the

smoke of 4 marijuana cigarettes, an exposure level that seems more likely

to be encountered in social environments than is the extreme exposure to

the smoke of 16 marijuana cigarettes for 1 hour, urines tested positive for

cannabinoid metabolites at levels only slightly above the 20 ng/mL cutoff.

None of the urines collected after exposure to the smoke of four marijuana

cigarettes tested positive by the 75 ng/mL EMIT calibrator.

Oral ingestion of marijuana plant material in cooked brownies led to excretion of highly concentrated amounts of cannabinoid in urine, as seen in

table 1. Although marijuana smokers potentially can titrate their drug effect

and thus their amount of marijuana exposure, the effects of oral ingestion of

marijuana are slow in onset and do not allow the subject sufficiently rapid

feedback for dosage adjustment. Hence, the amounts of 9-carboxy-THC in

urine after oral ingestion were, at times, substantially higher than those

observed after smoking equivalent doses.

REFERENCES

Cone, E.J., and Johnson, R.E. Contact highs and urinary cannabinoid excretion after passive exposure to marijuana smoke. Clin Pharmacol Exp

Ther 40(3):247-256, 1986.

Cone, E.J.; Johnson, R.E.; Darwin, W.D.; Yousefnejad, D.; Mell, L.D.;

Paul, B.D.; and Mitchell, J. Passive inhalation of marijuana smoke: Urinalysis and room air levels of delta-9tetrahydrocannabinol. J Anal Toxicol 11:89-96, 1987.

Cone, E.J.; Johnson, R.E.; Paul, B.D.; Mell, L.D.; and Mitchell, J. Marijuana-laced brownies: Behavioral effects, physiologic effects, and urinalysis in humans following ingestion. J Anal Toxicol 12:169-175, 1988.

Ferslew, K.E.; Manno, J.E.; and Manno, B.R. Determination of urinary

cannabinoid metabolites following incidental exposure to marijuana smoke.

Res Commun Subst Abuse 4:289-300, 1983.

Haertzen, C.A. An overview of Addiction Research Center Inventory

Scales (ARCI): An appendix and manual of scales. DHEW Pub. No.

(ADM)74-92. Washington, DC: Supt. of Docs., U.S. Govt. Print. Off.,

1974.

Law, B.; Mason, P.A.; Moffat, AC.; Gleadle, R.I.; and Ring, L.J. Forensic

aspects of the metabolism and excretion of cannabinoids following oral

ingestion of cannabis resin. J Pharm Pharmacol 36:289-294, 1984.

95
Mason, A.P.; Perez-Reyes, M.; McBay, A.J.; and Foltz, R.L. Cannabinoid

concentrations in plasma after passive inhalation of marijuana smoke.

J Anal Toxicol 7:172-174, 1983.

Morland, J.; Bugge, A.; Skuterud, B.; Steen, A.; Wethe, G.H.; and Kjedlsen,

T. Cannabinoids in blood and urine after passive inhalation of cannabis

smoke. J Forensic Sci 30:997-1002, 1985.

Perez-Reyes, M.; Di Guiseppi, S.; Mason, A.P.; and Davis, K.H. Passive

inhalation of marijuana smoke and urinary excretion of cannabinoids.

Clin Pharmacol Ther 34:36-41, 1983.

Wall, M.E.; Sadler, B.M.; Brine, D.; Taylor, H.; and Perez-Reyes, M.

Metabolism, disposition, and kinetics of delta-9-tetrahydrocannabinol in

men and women. Clin Pharmacol Ther 34:352-363, 1983.

ACKNOWLEDGMENTS

Rolley E. Johnson, William D. Darwin, and David Yousefnejad of the

Addiction Research Center assisted in the design and execution of the study,

and Leroy D. Mell, Buddha D. Paul, and John Mitchell of the Navy Drug

Screening Laboratory assisted with GC/MS analysis. Partial financial support was provided by the U.S. Navy.

AUTHOR

Edward J. Gone, Ph.D.

Laboratory of Chemistry and Drug Metabolism

Addiction Research Center

National Institute on Drug Abuse

Baltimore, MD 21224

96
Pyrolytic Degradation of Heroin,

Phencyclidine, and Cocaine:

Identification of Products and Some

Observations on Their Metabolism

C. Edgar Cook and A. Robert Jeffcoat

INTRODUCTION

Smoking of drugs offers certain advantages to the user, Purity of the drug

is less important for smoking than it is for intravenous use, the extensive

capiIlary network in the lungs facilitates rapid absorption, and the blood circulation pathway ensures that absorbed substances will be rapidly transported to the brain, where they can exert their effects. Thus, rapid onset of

action (immediate, positive feedback) combined with some ability to exert

control over the dosage and rate of onset of activity combine to produce

powerful incentives for smoking of abused drugs. These incentives have

long been obvious to smokes of marijuana, opium, and tobacco, but have

in recent years also led to smoking of other drugs, such as phencyclidine

(PCP) and cocaine. As drug users become more aware of the dangers of

acquired immunodeficiency syndrome (AIDS) associated with intravenous

drug use, it is possible that smoking will increase in popularity as a means

of drug taking.

The advantages enumerated above are also accompanied by disadvantages

that may not be so readily apparent to the smoker. Many organic compounds undergo pyrolytic or oxidative reactions when exposed to high temperatures in the presence of air. The resulting compounds may have acute

toxicities differing from those of the parent drug, as well as having more

long-term effects, such as the induction of lung cancer associated with

tobacco smoking. It thus becomes necessary to learn as much as possible

about’ the nature of these pyrolytic products, their structures, their absorption

into the body, and their routes of metabolism as well as toxicity associated

with them. Over the past few years, our laboratory has been involved in

studies of this type. In this chapter we summarize our work on the

97
pyrolysis of three drugs of abuse-heroin, cocaine, and PCP. This work

demonstrates the diversity of structures that can be obtained and illustrates

that smoking of drugs can lead to demonstrable absorption of pyrolysis

products.

PYROLYSIS PRODUCTS OF HEROIN

Opium has been smoked for centuries in the Middle and Far East. Therefore, when in the early 1900s heroin was introduced into China and started

replacing opium as a narcotic, it also was usually smoked (Huizer 1987).

Although in western countries intravenous injection is a frequent mode of

heroin abuse, there are still many reports of its continued use by smoke

inhalation. These have been reviewed by Cook and Brine (1985) and by

Huizer (1987). Smoking of heroin-suffused marijuana or tobacco cigarettes

has been reported, but apparently the inhalation of smoke from heroin

heated on aluminum foil (chasing the dragon) is currently the most popular

method of administration (Huizer 1987). A number of cases of leukoencephalopathy have been attributed to a toxic compound or compounds

obtained from heroin smoking (Walters et al. 1981; Walters et al. 1982),

but no specific compound has yet been implicated. The problem could

have arisen from either pyrolysis products of heroin or of diluents used in

the particular heroin smoked. Since relatively little was known about the

pyrolysis of heroin or its salts, we investigated the structure of some of the

major products of this process (Cook and Brine 1985).

Materials and Methods

Chemicals. Heroin hydrochloride and not-morphine hydrochloride were

obtained from the National Institute on Drug Abuse. Free heroin was

obtained by base conversion of the hydrochloride and extraction into butyl

chloride solution. The resulting product was shown to be pure by high performance liquid chromatography (HPLC). Normorphine was similarly

obtained. It was acetylated with acetic anhydride in pyridine to N-acetylnor heroin. Codeine, pseudocodeine, and isocodeine were acetylated in a

similar manner. Methylation was carried out by use of diazomethane in

ether containing 10 percent methanol. Allopseudocodeine acetate was prepared from ß-bromocodide by refluxing in acetic acid (Cook and Brine

1985).

Pyrolysis. Pyrolysis was carried out in a quartz boat placed in a quartz

furnace tube and connected to a dry trap and two bubbler traps containing

100 mL of absolute ethanol, each at -70 “C. Air was passed through the

system at 30 mL per minute. A tube furnace was heated to the desired

temperature and placed quickly around the quartz tube. Pyrolysis products

were isolated from the various parts of the apparatus by dissolving in

solvent and were chromatographed on HPLC. Products were isolated on a

Partisil-10/PAC column, eluted with a linear gradient of hexane/ethanol.

Like fractions were combined and analyzed by HPLC on Partisil-10/ODS-2

98
and, if necessary, purified further in this system. The solvent was a mixture of ammonium acetate buffer and acetonitrile. For final purification,

ammonium acetate and acetic acid were removed by absorbing material on a

C-18 SepPak cartridge (Cook and Brine 1985).

Results and Discussion

Four major ultraviolet (UV)-absorbing peaks and a large number of minor

UV-absorbing peaks were observed when heroin hydrochloride pyrolysate

was subjected to HPLC (figure 1). One peak (F of figure 1) was attributable to heroin itself. Six other components were isolated in purities

exceeding 90 percent. The three major pyrolysis products (peaks C, D, and

G of figure 1) were then identified conclusively by means of physicochemical properties and comparison with standard compounds.

Solvent: Linear gradient of 10-70% EtOH in hexane

Column:’ Partisil 10 / PAC

FIGURE 1. HPLC of a pyrolysate of heroin hydrochloride

NOTE: Partisil-10 PAC column, eluted with a linear gradient of hexane/ethanol.

SOURCE: Cook and Brine 1985, copyright 1985. American Society for Testing and Materials.

99
As figure 2 shows, product G was 6-0-acetylmorphine. The UV spectrum

showed a slight bathochromic shift from heroin consistent with conversion

of the 3-acetate to a phenol. Proton and 13C nuclear magnetic resonance

(NMR) revealed that the N-methyl of heroin was still present and the 6aacetoxy group was unchanged. There were, however, changes in the NMR

spectrum of the C-2 to C-4 positions, and only one acetyl group was present. (The 1H NMR spectrum of 6-0-acetylmorphine is quite sensitive to the

presence of impurities such as ammonium acetate or acetic acid obtained

from buffer salts used in the purification process. Only when these were

removed by adsorbing the compound on a C-18 SepPak column, washing

with water, and eluting with organic solvent could we obtain consistent

proton NMR spectra identical with those of genuine 6-0-acetylmorphine.)

FIGURE 2. Identification of heroin pyrolysis product G as

6-0-acetylmorphine

High-resolution mass spectrometry showed a molecular formula identical to

that of 6-0-acetylmorphine and the loss of a fragment corresponding to acetate (or loss of acetic acid from a protonated molecular ion). Treatment of

the compound with diazomethane yielded a product identical with that obtained by acetylation of codeine but differing in properties from iso-,

pseudo-, or allopseudocodeine acetate.

100
Pyrolysis product C (figure 3) was identified as N-acetylnorheroin resulting

from formal elimination of the N-methyl group and acetylation of the resulting secondary amine. Its UV spectrum closely resembled that of heroin.

Proton and 13C NMR spectra showed the absence of the N-methyl group of

heroin and the presence of three acetyl groups. Both spectra showed little

change from that of heroin for carbons 1 to 8, thus indicating that this portion of the molecule was essentially unchanged. The mass spectrum

showed the addition of one carbon and one oxygen atom to heroin and the

loss of various combinations of ketene and acetic acid on fragmentation.

All of these data pointed to N-acetylnorheroin, and, when not-morphine was

treated with acetic anhydride, product C was obtained.

FIGURE 3. Identification of heroin pyrolysis product C as

N-acetylnorheroin

As figure 4 shows, pyrolysis product D was identified as N, 6-0-diacetylnormorphine, the 3-desacetyl analog of product C. As was the case with 6-0-

acetylmorphine, there was a small bathochromic UV shift from that of

heroin. NMR spectra of product D, however, showed no N-methyl group.

The presence of two acetyl groups was observed from the 13C and proton

NMR, and the region from C-l to C-8 was very similar in its NMR properties to that of 6-O-acetylmorphine. Mass spectrometry indicated the formal loss of CH2

 from heroin, and the fragmentation pattern showed loss of

ketene from the molecular ion. These properties again are consistent with

N, 6-0-diacetylnormorphine, and acetylation of D gave the same product as

that obtained when normorphine was acetylated.

Thus the three major products were conclusively identified. Product A

(figure 5) was obtained in much smaller quantities. It exhibited a strong

UV chromophore. Its high-resolution mass spectrum showed there was no

101
Small Bathochromic UV shift from heroin

FIGURE 4. Identification of heroin pyrolysis product D as

N, 6-0-diacetylnormorphine

nitrogen in the molecular ion and indicated a formula of C18H14O4. The

mass spectrum also indicated fragmentation involving the loss of two

CH2CO residues, consistent with the presence of two acetate functions. The

degree of unsaturation revealed by mass spectrometry could be accommodated by three aromatic rings together with two acetoxy groups, leading us to

tentatively identify this product as 3, 4-diacetoxyphenanthrene. A search of

the literature showed that this compound had been reported in 1886 by

Fischer and Gerichten from heating of heroin methiodide with silver acetate

in acetic acid; and it appears that this indeed is product A, although an

authentic sample was not available for comparison. (Dr. Fischer, unfortunately, was not in a position to provide us with such a sample.)

Strong UV chromophore

FIGURE 5. Tentative identification of heroin pyrolysis product A as

3,4-diacetoxphenanthrene

102
The pryrolysis products identified are consistent with anticipated pyrolytic

mechanisms shown in figure 6. In this figure, we see the initial formation

of a quaternary N-acetyl intermediate by interaction of two molecules of

heroin. Attack of a nucleophile is most likely to occur on the N-methyl

group leading to its loss and the generation of the N-acetyl compound.

Although nucleophilic attack could also take place on the other carbon substituents of the nitrogen, it is less likely to occur there for steric reasons.

Alternatively, removal of a proton in the position ß to the nitrogen would

lead to formation of a double bond and opening of the piperidine ring.

Alternatively, in the case of heroin hydrochloride, nucleophilic attack on the

methyl group of the protonated amine would lead to the formation of norheroin, which could undergo acetylation by reaction with other molecules of

heroin.

FIGURE 6. Possible pyrolytic mechanisms for heroin

103
That some of the reactions involving ß elimination are also occurring is

indicated by the data for pyrolysis products B and E. Product E had a formula indicating formal addition of CH2

, to heroin. The structures of figure

7, where R=CH3, would fit this molecular formula. Such structures could

derive from N-methylation followed by olefin formation. Since product E

did not have a styrene nor a conjugated diene chromophore, opening of the

piperidine ring to give the nonconjugated diene appears the most likely explanation at this time. Pyrolysis product B has a molecular ion at m/z 411,

which is consistent with the N-acetyl N-methyl formulas shown in figure 7.

FIGURE 7. Possible structures for heroin pyrolysis products B and E

Products involving rearrangements or inversions of the allylic acetate system

did not appear to play a major role in the pyrolysis, although such compounds could certainly exist as some of the minor products shown in the

HPLC chromatogram. Finally, a large amount of unidentified tarry material

was produced, which could have resulted from further reaction of some of

the initially formed pyrolysis products.

The pyrolysis of heroin hydrochloride and heroin free base was studied at

several temperatures ranging from 200 to 800 °C (Jones and Davis, personal

communication). As figure 8 shows, decomposition was much more extensive with the hydrochloride salt. Less than 10 percent of the heroin

remained as the parent compound after 10 minutes at 300 “C. At this time

and temperature, almost two-thirds of the heroin free base was recovered as

the parent compound. 6-0-Acetylmorphine was the major product at

250 °C in the pyrolysis of heroin hydrochloride. About 20 percent of the

original heroin was converted to this product, together with 14 percent of

N-acetylnorheroin and about 6 percent of N,6-0-diacetylnormorphine. Pyrolysis of heroin free base at somewhat higher temperatures yielded similar

patterns and amounts of these products.

104
Temperature (C)

FIGURE 8. Products from pyrolysis of heroin and heroin hydrochloride at

various temperatures

NOTE Data on breakdown products arc from pyrolysis of the hydrochloride. (Unpublished data

obtained from K. Davis and L. Jones; mean of two experiments.)

Huizer (1987) has reported on the decomposition of heroin at varying temperatures and in the presence of a number of possible diluents. Some of

these, such as caffeine and barbital, enhance the recovery of heroin from

the hydrochloride under the conditions of “chasing the dragon,” whereas

others, such as procaine hydrochloride, decreased the yield of heroin in the

pyrolysis condensate. Most of the diluents decreased the recovery of heroin

base, and none actually enhanced its recovery. Thus Huizer concluded that

the available yield of heroin is strongly dependent upon the presence of

contaminants or diluents and the nature of these substances.

In summary, extensive degradation occurs at temperatures that might be

involved in inhalation of heroin by smoking. Even heroin free base undergoes significant decomposition under these conditions. 6-0-Acetylmorphine,

which is formed on smoking and is also a metabolite of heroin, has narcotic

activity and would be expected to contribute to the pharmacological effect.

N-Acetylnormorphine derivatives, also formed in significant amounts, are

probably unlikely to have intrinsic opiatelike activity but could have other

pharmacological effects or perhaps be metabolically converted to normorphine. Since a large number of other compounds were apparently produced

105
in minor amounts by heating of heroin, and since diluents used in street

samples may also undergo pyrolysis to potentially toxic products, it appears

that the smoking of heroin could have significant toxicological consequences. Further information on the pharmacological and toxicological

implications of this type of heroin administration is needed.

PYROLYSIS STUDIES OF COCAINE

The smoking of cocaine free base has become an increasingly popular

method of self-administration. The ready availability of crack (a form of

cocaine free base) in particular has raised major concerns about this route

of administration. Since esters undergo pyrolytic elimination reactions, it

seemed plausible to expect cocaine to be degraded by heat. We, therefore,

undertook a study to identify the pyrolysis products of this compound (Cook

et al. 1985; Jeffcoat et al. 1985).

Materials and Methods

Unlabeled cocaine and cocaine containing tritium in the N-methyl position

or on the C-4 methylene group were obtained from the National Institute

on Drug Abuse and diluted with unlabeled cocaine. The radiochemical

purity was greater than 92 percent by HPLC analysis.

Pyrolysis. Tritiated cocaine in quantities of 25 to 200 mg was pyrolyzed

by an apparatus similar to that used for the pyrolysis of morphine. Pyrolysis products in the quartz tube and those collected in the U-tube were

recovered by dissolving them in acetonitrile.

Human Studies. Cocaine was administered to human subjects by the intravenous, smoking, and nasal insufflation routes. The clinical and analytical

procedures have been described (Jeffcoat et al. 1989; Perez-Reyes et al.

1982).

Results and Discussion

Cocaine hydrochloride is almost completely destroyed at 800 °C (the temperature of the glowing end of a tobacco cigarette) and cocaine itself is

extensively degraded with only 16 percent of the pyrolysis product being intact cocaine. At lower temperatures, however, cocaine is much more stable,

although the percentage of intact cocaine recovered appears to be strongly

dependent on exact experimental conditions. Separation of pyrolysis product

mixtures by HPLC showed four major peaks of radiolabeled material. The

third of these peaks had the same retention time as cocaine, and its mass

spectrum was identical with authentic cocaine.

Since the materials in the first two peaks were too volatile to permit their

isolation from the HPLC solvents, the crude pyrolysis product mixture was

106
examined by combined capillary gas chromatography/mass spectrometry

(GC/MS). Three major types of pyrolysis products in addition to cocaine

were observed. One set of three peaks with similar retention times relative

to cocaine had almost identical mass spectra, which were also identical to

the published spectra of methyl ecgonidine (Lowry et al. 1979; Lukaszewski

and Jeffery 1980). Although this compound is reported to be formed when

cocaine hydrochloride is introduced into a heated injection port of a gas

chromatograph, we found no such occurrence when cocaine free base was

injected at the temperature used in this work The presence of three gas

chromatography peaks indicates that other isomeric products are formed in

addition to methyl ecgonidine. One of these could be the ß, y isomer.

Internal ring opening could also lead to compounds isomeric with methyl

ecgonidine (Cook et al. 1985).

A second set of four peaks with similar but not equivalent mass spectra was

also observed. The presence of molecular ions at m/z 150 was consistent

with identification of these as the doublebond isomers of methylcycloheptatrienecarboxylate. This assignment was further supported by a base peak at

m/z 91 corresponding to benzyl or cycloheptatrienyl ion with loss of carboxymethyl residue and transfer of a hydrogen. The compounds also underwent loss of methyl and methoxy residues (Cook et al. 1985).

In addition to these major peaks, significant amounts of methyl benzoate,

benzoic acid, and N-methylbenzamide were also observed (Cook et al.

1985). These products are explained by elimination of benzoic acid and by

mechanisms involving acylation of the tertiary nitrogen of cocaine to form

intermediates, which undergo nucleophilic attack much as was described in

the case of morphine.

The effectiveness of smoking as a route of administration is readily seen by

comparison of plasma concentrations of cocaine in human subjects who

either smoked the drug or were given an intravenous injection of cocaine

hydrochloride (Jeffcoat et al. 1989). The who curves are essentially comparable, and it was observed that the plasma levels of cocaine had reached

a maximum by approximately 6 minutes after the beginning of smoking

(figure 9). Table 1 shows calculations of pharmacokinetic parameters and

bioavailability calculated after different routes of administration in studies

from our laboratory (Jeffcoat et al. 1989). Average elimination half-lives

ranged from 56 minutes for the subjects who smoked cocaine to 80 minutes

for those who snorted the drug. Bioavailability based on the amount of

radiolabeled material actually inhaled showed a range of 32 to 77 percent.

Since simulated smoking studies indicated that only about 44 percent of the

material inhaled was cocaine, this suggests that most of the cocaine actually

inhaled reaches the circulation unchanged. However, marked interindividual

variation in the amount of cocaine obtained by smoking is likely.

107
FIGURE 9. Cocaine and benzoylecgonine concentrations in the plasma of

subjects who received cocaine by smoking or intravenous

injection

SOURCE Cook et al. 1985, copyright 1985, Biomedical publications.

A somewhat smaller proportion of the inhaled cocaine was excreted in the

urine than was the case for intravenous cocaine. This suggests that some of

the pyrolysis products are eliminated in the bile and feces. Examination of

the urinary excretion of cocaine metabolites (figure 10) showed little difference in the average amount of ecgonine methyl ester excreted whether

the drug was given intravenously, by smoke inhalation, or by snorting; but

the amount of benzoyl ecgonine appeared significantly less in the case of

the smoking route. Figure 11 shows the ratio of benxoyl ecgonine to ecgonine methyl ester in the urine. During the period 0 to 24 hours, there is a

significantly (p<.05) lower average ratio for the smoking route than for the

other two routes of administration. This difference is even more pronounced in the period from 24 to 72 hours after administration. However,

little (about 14 percent) of the total urinary excretion occurred during this

latter period, with 86 percent of the excretion occurring in the 0- to 24-hour

period. Furthermore, there was considerable overlap in the ranges in the 0-

to 24-hour period. Data in a recent paper by Ambre et al. (1988) show a

range of ratios of benzoylecgonine to ecgonine methyl ester excretion of 0.8

to 8.7 after intravenous administration. Thus, although an average difference in metabolism based on route appeared to be present in our population

of cocaine users, this ratio could not be used as a means of distinguishing

whether a given individual had smoked or snorted cocaine.

108
TABLE 1. Pharmacokinetic analysis of cocaine in plasma

a a

Route Number (hr-1) (hr-1)

B

(hr-1)

Bioavailability

Percent

IV 4 — 3.71a

±1.73 0.532±0.127 100

Snorting 6 3.54±2.12 — 0.523±0.113 80±13

68-l03b

Snorting c 6 2.28±0.39 d

 — 0.535±0.04d

Snortingc 6 3.92±0.38 d

 — 0.465±0.017d

Smoking 6 38.O±16.0 3.14f

0.740±0.274 47±198

(32-77)b,g

Smokingc 6 31.0±8.3 d

 — 0.718±0.083d

Overall 16 — — 0.607±0.210

KEY: aThree subjects; brange; 

c

simultaneous fit of all data; dSE; efit of mean plasma data; fone subject

only; gbased on inhaled radiolabeled material.

NOTE Unless otherwise noted, date are unweighted means ± SD of Individual subjects.

SOURCE: Based on data in Jeffcoat et al, 1989.

METABOLISM OF 1-PHENYLCYCLOHEXENE—A PYROLYSIS

PRODUCT OF PCP

The increase in popularity of PCP as a drug of abuse has been credited to a

shift in its mode of use from oral ingestion to smoking (Stillman et at

1980). As a tertiary benzylamine, PCP is a likely candidate for elimination

reactions under pyrolytic conditions, and in 1980 Martin and colleagues

reported that in a burning cigarette 1-phenylcyclohexene (PC) is a major

product from PCP (Martin et al. 1980; Freeman and Martin 1981). The

production, absorption, pharmacokinetics, and metabolic fate of this compound are thus of interest.

Materials and Methods

Pyrolysis apparatus and conditions have been described (Cook et al. 1981),

as have the in vitro metabolism of 1-phenylcyclohexene and synthesis of

metabolites (Cook and Brine 1984). Details of the clinical and analytical

portion of studies involving smoking of PCP have also been published

(Cook et al. 1982). Pharmacokinetic parameters were fit to the mean PC

concentrations in plasma by the nonlinear least squares regression procedure

109
FIGURE 10. Percent of dose of cocaine excreted as parent drug or

metabolites in urine as a function of route of

administration (average 3-day excretion)

FIGURE 11. Ratio of benzoylecgonine to ecgonine methyl ester excreted

in urine as a function of route of administration and time

from drug administration

NOTE: Error bars show ranges. Number of subjects four for intravenous injection (IV), six each for

nasal insufflation (NI) and smoking (SI).

110
in the program RS/l (Bolt, Beranek, and Newman, Cambridge, MA).

Points were weighted as the reciprocal of variance.

Results and Discussion

When heated either as the free base or its hydrochloride salt, PCP is converted to 1-phenylcyclohexene (Martin et al. 1980; Freeman and Martin

1981; Cook et at 1981). Even at the relatively low temperature of 300 °C,

the hydrochloride salt is converted in 93 percent yield to l-phenylcyclohexene and piperidine hydrochloride. These products were identified

by 1H and 13C NMR analysis, and the structures were confirmed by GC/MS

analysis of 1-phenylcyclohexene and of piperidine as its benzoyl derivative

(Cook et al. 1981). The free base of PCP undergoes considerably less de

composition at 300 “C, and more than half of the material is recovered as

PCP. In contrast to simple pyrolytic conditions, both the hydrochloride salt

and free base give similar yields of PCP and PC when smoked in a parsley

cigarette, with almost 1.5 times as much PCP as PC being found in the

smoke under simulated smoking conditions (Cook et al. 1981).

When human subjects, using an apparatus that permitted the collection of

sidestream smoke and exhalant, smoked parsley cigarettes containing

100 μg of tritium-labeled PCP hydrochloride, we could estimate the dose of

PCP and PC actually inhaled by the subjects by measuring the radioactivity

not inhaled and using the results of the simulated smoking studies (figure

12) (Cook et al. 1981; Cook et al. 1982; Cook et al. 1983).

Sidestream 16 (±4)%

Inhaled 69%

(39% PCP, 30% PC)

FIGURE 12. Fate of PCP hydrochloride on parsley cigarettes smoked by

human volunteers

NOTE: Values in mode % of PCP on cigarette. Inhaled percentage based on simulated smoking

studies.

SOURCE Cook et al. 1985, copyright 1985. Biomedical publications.

111
High-performance liquid radiochromatography demonstrated that both PCP

and PC were present in plasma. Our calculations showed that PCP teaching

the lungs was well absorbed with excellent bioavailability and that, in general, the pharmacokinetics and metabolism of PCP were similar to those

observed after intravenous administration (Cook et al. 1982). The terminal

half-life was 24±7 (SE) hours in five individuals who smoked PCP and

16±2 (SE) hours in six who were given the drug intravenously. If we estimated the dose of PCP actually inhaled as described above, then the calculated bioavailability averaged 110±47 percent. This suggests both good

absorption of unchanged PCP and considerable variability in individual

smoking (table 2).

TABLE 2. Pharmacokinetics and bioavailability of phencyclidine by

intravenous, oral, and smoking routes

Volume of

Terminal Total Renal Distribution

Route Dose (V4 8 )

Compound (Number) (μg/kg) (hr)

Clearance Clearance Bioavailability

(liter/min) (liter/min) (liter/kg) (Percent)

*Apparent value.

NOTE: Data are mean (SE). Renal clearance is pH-dependent; average urine pH was -65.

SOURCE: Bused on data In Jeffcoat et al. 1989.

As figure 13 indicates, PC was also rapidly absorbed. The average time to

peak was 8 minutes. The subjects actually showed the highest concentration of PC at 5 minutes after beginning of smoking, the earliest time point

for which plasma samples were available. Thus, it was not possible to

determine absorption half-life, but it is very rapid. A standard multiexponential pharmacokinetic equation was fit to the data by means of nonlinear

regression analysis. Where necessary, points before the peak plasma concentration were ignored in this analysis. In three subjects, a biexponential

equation best fit the data, whereas a triexponential equation gave a better fit

for two of the subjects (Cook et al. 1982). When the mean plasma data

112 
FIGURE 13. Mean plasma concentrations (±SEM) of PC in five subjects

who smoked parsley cigarettes containing 100 μg of PCP

hydrochloride

NOTE: Line is beat fit of a triexponential equation to data points from 10 minutes to 72 hours. Top

panal shows from 0 to 2 hours, bottom panel from 2 to 48 hours.

were used (figure 14), the data points from 10 minutes to 72 hours after

smoking began were best fit by a triexponential equation (F test, p<.01)

113
with half-lives of 0.131, 1.44, and 17.0 hours for the three phases,

respectively.

FIGURE 14. Pharmacokinetic parameters calculated from mean of plasma

concentrations of PC

*Apparent value.

NOTE: Dose was calculated from percent of dose inhaled and fraction of PC from simulated smoking

studies. Data represent mean ± SE (n=5). a ß, and Y derived from nonlinear regression

analysis of mean data.

A word of caution is in order with regard to the pharmacokinetic calculations for PC. Because of the very low concentrations, long counting times

had to be used, and, even with analysis of 3 ml of plasma, the mean value

at 72 hours represents only about 3 decompositions per minute above background. The standard errors of the parameters were quite reasonable, however, and the calculated values of the rate constants were changed less than

10 percent if the data points only to 30 hours were used in the calculation.

We also converted the picograms per milliliter values to percent of dose per

liter of plasma (based on the individual estimates of dose inhaled), normalized to an 80-kg person. This had almost no effect on the variance of the

values or the fitted rate constants.

PC had a very large apparent volume of distribution (approximately

35 L/kg) if it is assumed that all of the estimated dose of PC reached the

circulation. Renal clearance of PC was extremely low in comparison with

total clearance, indicating that extensive metabolism occurred before excre

tion. Fecal excretion may also play a major role in the elimination of PC

metabolites, since the amount of PC metabolites identifiable in urine constituted less than 10 percent of the estimated dose of PC (Cook et al. 1982).

In many respects, the pharmacokinetics of PC thus resembles that of the

very lipophilic compound 9-tetrahydrocannabinol (Agurell et al. 1986;

114
Chiang and Barnett 1984; Wall et al. 1983; Hunt and Jones 1980; Barnett

et al. 1982)—perhaps not a surprising conclusion.

The small fraction of PC metabolites excreted in urine and the low doses

used made identification of metabolites in humans very difficult. We therefore made an initial study of the in vitro metabolism of this compound by

rat liver. Our results (Cook et al. 1984) confirmed and extended the results

of Martin et al. (1982) on metabolism by mouse liver. A large number of

metabolites were identified by comparison of physical data with those of

standards whose structures were established by synthesis and rigorous physical methods (Cook et al. 1984). These studies showed that PCP undergoes

oxidative metabolism at multiple sites on the molecule and that multiple

oxidations also occur (figure 15). The double bond may be epoxidized and

hydrolyzed to a diol. Oxidation may occur at the 3 position to yield an

alcohol or ketone. Further hydroxylation may occur at the 4 position, and

oxidation may also occur at the 6 position. In several of the metabolites,

an asymmetric center is present, thus raising the possibility of stereoselective metabolism. Further research is needed on this feature of PC

metabolism.

Good separation of many of these metabolites may be achieved by HPLC

on a Pattisil-PAC column, and reasonable separations are also achieved

on a reverse phase chromatography system.

Extraction experiments showed that PC metabolites in urine were highly

polar and, therefore, likely consisted of conjugated substances. Thus, for

identification of metabolites, urine samples were incubated with a mixture of

B-glucuronidase and sulfatase. Extraction with butyl chloride then gave an

organic-soluble phase from which acid-soluble PCP metabolites could be extracted with aqueous HCl. A subsequent extraction with aqueous sodium

hydroxide removed basesoluble PC metabolites, accompanied by some of

the more water-soluble neutral metabolites. We found little evidence for the

presence of phenolic metabolites of PC. The neutral metabolites were purified by small-scale preparative HPLC, generally on a reverse-phase column.

Plasma extracts were also examined by HPLC in an attempt to identify PC

metabolites present in plasma. Metabolites were designated a, ß,

in order of their polarity. Little, if any, of metabolite a was present in

urine.

Only small amounts (generally less than a microgram) of these metabolites

could be isolated from urine of a single subject. Therefore, we relied on

techniques such as derivatization and isotope dilution experiments to identify

the metabolites. Thus, metabolite ß was suspected to be l-phenylcyclohexen-3-o1, based on its retention time on HPLC. To confirm this, the

isolated metabolite was mixed with a known amount of standard compound,

and the specific activity was determined by HPLC analysis. The weight of

the material in the HPLC peak was determined by comparison of UV

115
FIGURE 15. In vitro metobolites of phenylcyclohexene

NOTE: Arrows represent chemically reasonable, but speculative, metabolic pathways. Broken and

solid lines show relative stereoechemical relationships, not absolute configuration. The position

of the hydroxyl at C-3 In PC-1,2,3-triol and BP-3-ol was not conclusively established. M

designates metabolites Identified from mouse (Martin et al. 1982); R designates rat

metabolites.

SOURCE Cook et al. 1984, copyright 1984, American Society for Pharmacology and Experimental

Therapeutics.

absorption with a standard curve, and the radioactivity was measured by

liquid scintillation spectrometry. Other aliquots of the mixture were derivatized and subjected to this same procedure. As derivatives, we chose the

acetyl and p-nitrobenzoyl esters, to achieve a range of retention times on

HPLC, thus maximizing the probability of separating closely related substances. Since the specific activity of the two derivatives coincided very

closely with that of the parent metabolite, we concluded that metabolite ß is

indeed 1-phenylcyclohexen-3-o1.

Metabolite y on treatment with methoxylamine underwent a shift in retention time on HPLC, thus indicating the presence of a keto group. Benzoylation also resulted in a change in retention and suggested the presence of a

116
hydroxyl group. Since the metabolite had the same retention time as 4-

hydroxy-1-phenylcyclohexen-3-one, we suspected that it was this compound.

To confirm this, a mixture of isolated metabolite and standard was reduced

with sodium borohydride to give a mixture of diols separable by chromatography. The major product, the trans-3,4-diol, was then recystallized to

constant specific activity. Very little of the radioactivity associated with

metabolite was lost during recystallization, thus confirming that this metabolite was 4-hydroxy-1-phenylcyclohexen-3-one.

Figure 16 summarizes the basis for the firm identification of two metabolites of PC (ß and y) in human urine. Evidence for the identity of four

other metabolites was also obtained (figure 17). l-Phenylclohexen-3-one

cochromatographed with a major metabolite (a) in plasma and is a reasonable candidate for a. Metabolite o from urine was separated into two

components with the retention times of cis- and trans-1-phenylcyclohexene3,4-diol. Only one third to one-half of the radioactivity associated with

these metabolites was found when they were converted to ester derivatives;

we regard this identification as less conclusive than the ones above. lPhenylcyclohexene-3,6-diol cochromatographed with radioactivity from the

metabolite mixture, and this compound may also be present in hydrolyzed

urine.

Isotope dilution / HPLC of parent + 2 esters

Reacts with methoxyamine / HPLC

Reacts with benzoyl chloride / HPLC

Isotope dilution / recrystallization of

NaBH4 product

FIGURE 16. Summary of evidence for identification of PC metabolites ß

and from human urine

CONCLUSION

It has been shown that the drugs PCP, heroin, and cocaine undergo fairly

extensive decomposition when heated. A variety of pyrolytic products have

been isolated and identified. These compounds may exert their own

117
HPLC (plasma)

(c) HPLC

HPLC of acetate (35% pure)

(t) HPLC

HPLC of acetate and benzoate (47-50% pure)

HPLC

FIGURE 17. Summary of evidence for tentative identification of PC

metabolites a, o, and E in human subjects

pharmacological activity or may prove to have toxic effects. Much yet

remains to be learned about these potential hazards of smoking of abused

drugs.

REFERENCES

Agurell, S.; Halldin, M.; Lindgren, J.E.; Ohlsson, A.; Widman, M.;

Gillespie, H.; and Hollister, L Pharmacokinetics and metabolism of

-tetrahydrocannabinol and other cannabinoids with emphasis on man.

Pharmacol Rev 38:21-44,1986.

Ambre, J.; Ruo, T.I.; Nelson, J.; and Be&nap, S. Urinary excretion of

cocaine, benzoylecgonine, and ecgonine methyl ester in humans. J Anal

Toxicol 12:301-306, 1988.

Barnett, G.; Chiang, C.N.; Perez-Reyes, M.; and Owens, S.M. Kinetic

study of smoking marijuana. J Pharmacokinet Biopham 10:495-506,

1982.

118
Chiang, C.N., and Barnett, G. Marijuana effect and delta-9-tetrahydrocannabinol plasma level. Clin Pharmacol Ther 36:234-238, 1984.

Cook, C.E., and Brine, D.R. Pyrolysis products of heroin. J Forensic Sci

30(1):251-261, 1985.

Cook, C.E.; Brine, DR.; Quin, G.D.; Perez-Reyes, M.; and Di Guiseppi,

S.R. Phencyclidine and phenylcyclohexene disposition after smoking

phencyclidine. Clin Pharmacol Ther 31:635-641, 1982.

Cook, C.E.; Brine, D.R.; Quin, G.D.; Wall, M.E.; Perez-Reyes, M.; and

Di Guiseppi, S.R. Smoking of phencyclidine: Disposition in man and

stability to pyrolytic conditions. Life Sci 29:17-1972, 1981.

Cook, C.E.; Brine, D.R.; and Tallent, C.R. Identification of in vitro rat

metabolites of l-phenylcyclohexene. Drug Metab Dispos 12(2):186-192,

1984.

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.; Perez-Reyes, M.; Jeffcoat, AR.; and Brine, D.R. Phencyclidine

disposition in humans after small doses of radiolabeled drug. Fed Proc

42(9):2566-2569, 1983.

Fischer, O., and Gerichten, E.V. Zur Kenntniss des morphins. Chemische

Berichten 19:792-794, 1886.

Freeman, A.S., and Martin, B.R. Quantification of phencyclidine in mainstream smoke and identification of phenylcyclohex-l-ene as pyrolysis

product. J Pharm Sci 70:1002-1004, 1981.

Huizer, Henk. Analytical studies on illicit heroin. V. Efficacy of volatilization during heroin smoking. Pharmaceutisch Weekblad Scientific Edition 9:203-211, 1987.

Hunt, C.A., and Jones, R.T. Tolerance and disposition of tetrahydrocannabinol in man. J Pharmacol Exp Ther 215:35-44, 1980.

Jeffcoat, A.R.; Cook, C.E.; and Perez-Reyes, M. Psychoactive drugs. In:

Barnett, G., and Chiang, C.N., eds. Pharmacokinetics and Pharmacodynamics of Psychoactive Drugs. Foster City, CA: Biomedical Publications, 1985. pp. 441-455.

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 Disp 17:153-159, 1989.

Lowry, W.T.; Lomonte, J.N.; Hatchett, D.; and Garriott, J.C. Identification

of two novel cocaine metabolites in bile by gas chromatography/mass

spectrometry in a case of acute intravenous cocaine overdose. J Anal Tox

3:91, 1979.

Lukaszewski, T., and Jeffery, W.K. Impurities and artifacts of illicit

cocaine. J Forensic Sci 25:499-507, 1980.

Martin, B.R.; Bailey, B.B.; Awaya, H.; May, E.L.; and Narasimhachari, N.

In vitro metabolism of I-phenyl-l-cyclohexene, a pyrolysis product of

phencyclidine. Drug Metab Dispos 10:585-689, 1982.

119
Martin, B.R.; Freeman, A.S.; Kelly, K.L.; Clayton, V.M.; Vincek, W.C.; and

Harris, L.S. Fate of phencyclidine in a burning cigarette. Fed Proc

39:302 (A189), 1980.

Perez-Reyes, M.; DiGuiseppi, S.; Ondrusek, G.; Jeffcoat, AR.; and

Cook, C.E. Free-base cocaine smoking. Pharmacol Ther 32(4):459,

1982.

Stillman, R.; Barnett, G.; and Petersen, R. Phencyclidine (PCP): Epidemiology, pharmacology, and pharmacokinetics. In: Mello, N.K., ed. Advances in Substance Abuse: Behavioral and Biological Research. Vol. 1.

Greenwich, CT: JAI Press, 1980. pp. 289-303.

Wall, M.E.; Sadler, B.M.; Brine, D.; Taylor, H.M.; and Perez-Reyes, M.

Metabolism, disposition and kinetics of 9-tetrahydmcannabinol in men

and women. Clin Pharmacol Ther 34:352-363, 1983.

Wolters, E.Ch.; Van Wyngaarden, G.K.; Stam, EC.; Rengelink, H.;

Lousberg, R.J.; Schipper, M.E.; and Verbeeten, B. Leucoencephalopathy

after inhaling ‘heroin’ pyrolysate. Lancet 2:1233-1237, 1982.

Wolters, E.Ch.; Van Wyngaarden, G.K.; Stam, EC.; Rengelink, H.;

Lousberg, R.J.; Schipper, ME.; and Verbeeten, B. ‘Heroin’ leucoencephalopathy; spongiform leuco-myelo-encephalopathy after inhalation of

the pyrolysate of contaminated heroin. Ned Tijdschr Geneeskd 126:508-

514, 1981.

ACKNOWLEDGMENTS

Work was carried out under National Institute on Drug Abuse contract

271-80-3705. Dr. Mario Perez-Reyes carried out the clinical portion of the

described studies. Dolores Brine and Judith Hill provided technical assistance with the pyrolysis and analytical studies. Ken Davis and Larry Jones

provided the quantitative data on pyrolysis of heroin.

AUTHORS

C. Edgar Cook, Ph.D.

A. Robert Jeffcoat, Ph.D.

chemistry and Life Sciences

Research Triangle Institute

P.O. Box 12194

Research Triangle Park, NC 277092194
Chemical and Biological Analysis

of Marijuana Smoke Condensate

Charles M. Sparacino, Patricia A. Hyldburg, and

Thomas J. Hughes

INTRODUCTION

The various products derived from Cannabis sativa L., generally lumped

together under the descriptive name “marijuana,” are ingested for recreational purposes by a sizeable portion of the population. Most popularly, the

leaves and flowering parts of the plant are smoked, producing a variety of

physical and psychomimetic effects commonly referred to as being “high.”

Owing to the large number of people using marijuana, an intensive effort

has been under way since about 1968 to define its chemistry, the metabolism of its major active ingredients, and the possible health effects of its

use. Although much knowledge has been gained about the chemistry,

pharmacology, metabolism, and pharmacokinetics of pure compounds from

Cannabis, an insufficient amount is known about these subjects as they

relate to marijuana smoke because of its complex chemistry.

The general goal of our work was the development of methodology for the

comprehensive analysis of the biologically active fractions of marijuana

smoke condensate (MSC). Biological activity was assessed through the use

of the Ames Salmonella mutagenesis assay (Maron and Ames 1983). This

“mutagenesis directed” research was designed to focus the analytical effort

on the most biologically active fraction(s) to increase the possibility of identifying potential mutagens or carcinogens in marijuana smoke condensate.

Chemical and biological screening of smoke condensate fractions was conducted for both marijuana and tobacco to allow for comparisons.

The complex chemistry of marijuana smoke involves not only the major

cannabinoids, but also a large number of combustion and pyrolytic products.

Many compounds found in marijuana smoke are known to have adverse

biological properties. For example, one study (Leuchtenbeyer et al. 1973)

showed that exposure of human lung explants to fresh marijuana smoke

resulted in alterations in DNA and chromosome complement. But-stein

121
et al. (1973; Burstein et a1. 1975) have presented evidence that certain

phenolic components present in the extracts of Cannabis sativa L. are strong

inhibitors of prostaglandin biosynthesis. Polycyclic aromatic hydrocarbons

(PAH) are formed during any organic combustion process and have been

isolated from MSC and tobacco smoke condensate (TSC) (Lee et al. 1976).

These compounds are among the most studied of any class with regard to

their mutagenic and carcinogenic properties. Novotny et al. (1980),

Maskarinec et al. (1976), and Merli et al. (1981) have identified many toxicologically important species in various MSC fractions and have, in some

cases, compared these data with similar information from TSC factors.

Some studies on the mutagenicity of MSC have been reported (Busch et al.

1979; Wehner et al. 1980), but no research has yet combined mutagenic

studies with a concerted, comprehensive effort to identify specific compounds responsible for the biological activity.

Historically, a tremendous amount of work has been done to determine the

composition and pharmacological activity of tobacco smoke. Parameters

(puff frequency, volume, duration, and holding time) governing the generation of TSC are well documented in the literature (Ogg 1964). Although it

is easy to assume that such methodology is directly applicable to the marijuana situation, this assumption in fact is not correct (Peterson 1979).

Information found in the literature partially describing the physicodynamics

of marijuana smoking and the observation of research subjects smoking

marijuana cigarettes indicate that people smoking marijuana tend to do so in

a much more vigorous and aggressive manner than is pursued with tobacco.

While this information illustrates that a single set of parameters describing

the physicodynamics of marijuana smoking cannot be defined easily, it

clearly shows that marijuana smoking parameters are more rigorous than

those describing the tobacco smoking process.

GENERATION OF SMOKE CONDENSATES

The particular smoking parameters used in governing the production of

marijuana smoke are important since the composition of the resulting MSC

will vary, depending on the conditions under which it is produced. In a

previous study performed at Research Triangle Institute (Davis et al. 1984),

the burning temperature and other values associated with the collection of

MSC in a constant draft apparatus operating at varying flow rates were

determined. These data demonstrated that the total mass of smoke condensate produced, as well as the recovery of 9-THC, decreases with increasing flow rate. The cannabinoid profile, however, remained relatively

constant, and the burning temperature of the marijuana did not appear to be

greatly dependent on flow rate. Also, the burning temperature of standard

National Institute on Drug Abuse cigarettes being smoked by human subjects was measured. A range of burning temperatures of 730 to 800 °C

was determined in these experiments.

122
Methods and Results

Selection of Marijuana Samples for Study. Several distinctly different

types of marijuana are available on the illicit market and thus were potential

subjects for study under this program. With respect to 9-THC potency,

these range from essentially nonpotent hemp-type marijuana, which is found

on rare occasions, to the common Colombian or Mexican marijuana of 3 to

4 percent 9-THC content, to extremely potent sinsemilla of Colombian,

Hawaiian, or Californian origin containing 6 to 12 percent 9-THC. Governed by the practical limitations of this program, we chose to study two of

these types, producing MSC from them and carrying out biological testing

and analysis as described later. Although a number of pairs could be

chosen from among the types listed above, we felt that samples of Mexican

marijuana containing, respectively, 1.3 percent 9-THC (low dose) and

4.4 percent 9-THC (high dose) represented the most practical choices,

bracketing most marijuana available on the street in this country. For

comparison, a typical cigarette tobacco was studied along with the two

marijuana samples. This tobacco was obtained as reference cigarettes from

the Tobacco and Health Research Institute (University of Kentucky,

Lexington, KY).

Constant Draft and Intermittent Puff Smoking Systems. Realizing that

there is no smoking system available that exactly duplicates the way

humans smoke marijuana, we felt that the best approach to the production

of MSC was to use two types of smoking systems: first, the constant draft

apparatus to produce smoke condensate representative of the total smoke

produced at the burning temperature of marijuana cigarettes being smoked

by human subjects; and, second, intermittent puff smoking systems operating

under the less rigorous tobacco smoking parameters, equivalent to the least

stringent conditions under which marijuana might be smoked by humans.

By using each extreme of actual smoke conditions, the evaluation of smoke

condensates from these two systems could provide a qualitative and quantitative range within which the various components of the marijuana smoke

actually experienced by human smokers might be found.

Cigarettes of 70 mm length were prepared from high-potency marijuana and

low-porosity “street” cigarette papers with a manual cigarette-rolling

machine. Low-potency marijuana and research tobacco cigarettes were machine rolled, cut to 70 mm length, and rerolled in low-porosity papers.

Each of the three cigarette types was cornbusted under puff and constant

draft modes on a 30-port cigarette smoking machine (figure 1) to produce

six different smoke condensate types. The cigarette smoking was conducted

at flow rates of 1,200 mL per minute for all constant draft combustion runs,

40 mL per 2-second puff (1 puff per minute) for puff mode combustion

runs with marijuana, and 35 mL per 2-second puff (1 puff per minute) for

puff mode combustion runs with tobacco cigarettes. Mainstream smoke was

collected in a three-element trapping system consisting of an ethanol

123
bubbler, a packed sand trap, and an impinger trap arranged in series. The

volume of each smoke condensate was reduced in vacuo at room temperature to less than 200 mL. At least 10 grams dry weight of each type of

smoke condensate were produced. Six smoke condensates were thus produced: MSC-low potency by puff and by constant draft mode, MSC-high

potency by puff and by constant draft, and TSC by puff and by constant

draft.

FIGURE 1. Cigarette smoking machine for generation of

smoke condensates

FRACTIONATION OF SMOKE CONDENSATES

MSC and TSC are known to be highly complex matrices containing up to

several thousand compounds at levels that may vary over several orders of

magnitude (Gudzinowicz and Gudzinowicz 1980). Adequate characterization

(chemical and biological) of these components requires prefractionation to

(1) increase the relative mass of particular components and thereby the sensitivity of biological and analytical techniques for characterization, and

(2) allow accurate assessment of biological and chemical characterization for

significant constituents with minimum interference from other components in

the matrix. The latter becomes a particularly important consideration with

respect to the testing of mutagenic activity, since toxicity resulting from a

few specific components in complex sample mixtures often becomes the

limiting factor.

Methods and Results

Fractionation of smoke condensates (MSC and TSC) was accomplished

using an acid-base liquid to liquid partition sequence, Similar sequences

have been used in many studies of smoke condensates (Merli et al. 1980;

124
Merli et al. 1981) as part of the fractionation methodology due to the facile

separation of major classes. Liquid-liquid partition provides a good means

of dealing with the relatively large mass of crude smoke condensate that

must he used to obtain adequate levels of individual constituents for later

characterization.

The acid-base. partition scheme used in the fractionation of smoke condensates is shown schematically in figure 2. The procedure is based on that of

Petersen et al. (1982) as applied to diesel soot extract.

FIGURE 2. Acid-base partition scheme

In summary, the smoke condensates were taken to dryness and redissolved

in methylene chloride (100 mL). Aliquots were removed for total mass

determination. Portions of the condensate solution were then extracted in a

125
separatory funnel three times with 5 percent aqueous sulfuric acid. The

three combined aqueous fractions were back-extracted once with 20 mL of

methylene chloride. This methylene chloride was returned to the original

smoke condensate solution.

To recover bases from the aqueous phases, the pH of the aqueous phase

was adjusted to 13 with 40 percent aqueous potassium hydroxide. This

aqueous phase was then extracted in a separatory funnel three times with

methylene chloride. The base fraction (methylene chloride) was dried with

sodium sulfate.

Next, acids were recovered from the original condensate solution (now in

methylene chloride) by extraction with 5 percent aqueous sodium hydroxide.

The combined aqueous base solutions were back-extracted with one aliquot

of methylene chloride and the aliquot returned to the original methylene

chloride smoke condensate. The acids were recovered by adjusting the pH

of the sodium hydroxide solution to acidic values and extracting the solution

with methylene chloride. Thus, methylene chloride solutions were obtained

for the crudes (C), neutrals (N), acids (A), and bases (B). Aliquots were

removed from all phases for total mass determination.

The neutral fraction (N) was further subfractionated by silica gel column

chromatography. Prior to subfractionation, a portion of the neutral fraction

was solventexchanged into hexane. A 20 g silica gel column was used.

The neutral fraction was introduced at the top of the column and then

eluted. Four eluting solvents were used to collect four fractions. Fraction

1 was eluted with 60 mL of hexane, fraction 2 with 100 mL of 1:l toluene

and hexane, fraction 3 with 100 mL of methylene chloride, and fraction 4

with 200 mL of methanol. Each fraction was collected separately; further

fractionation has not been conducted.

The base fractions (B) of the smoke condensates were further fractionated

by normal phase, gradient HPLC. A cyanopropyl column with 5-micron

spherical packing was used. Three solvent mixes were used:

Solvent A: 0.05 percent methanol/hexane

Solvent B: 0.05 percent methanol/methylene chloride

Solvent C: 20 percent methanol/methylene chloride

The gradient was as follows:

100 percent solvent A for 5 minutes

100 percent solvent A to 100 percent solvent B over 20 minutes

100 percent solvent B for 8.5 minutes

100 percent solvent C for 6 minutes

126
The flow rate was maintained at 4.0 mL per minute. Seven subfractions

were collected based on the time elapsed from the start of the run. The cut

points were selected arbitrarily, based on the appearance of the

chromatogram. Fractions were collected as follows:

Fraction 1: 0 to 5 minutes

Fraction 2: 5 to 9 minutes

Fraction 3: 9 to 12 minutes

Fraction 4: 12 to 15.5 minutes

Fraction 5: 15.5 to 23.5 minutes

Fraction 6: 23.5 to 33 minutes

Fraction 7: 33 to 39 minutes

Multiple runs were performed and like fractions from each run were

combined.

CHARACTERIZATION OF SMOKE CONDENSATES

Biological Characterization

The objectives of the bioassay testing were (1) to determine and compare

the mutagenic activity of the chemical class fractions from TSC and MSC

in the Ames Salmonella mutagenicity assay (Maron and Ames 1983), (2) to

determine the effect of smoking mode (puff vs. constant draft) and marijuana batch on mutagenicity, and (3) to identify the most mutagenic fractions so that effective analytical chemical characterization techniques could

be developed for identification of mutagenic and carcinogenic compounds.

The mutagenic potential of the various fractions was evaluated using the

preincubation modification of the standard plate incorporation technique of

the Ames Salmonella assay. Fractions were tested for mutagenicity under

the following conditions: Salmonella typhimurium strains TA98 and TAlOO

at five dose levels (10 to 2.50 μg per plate), triplicate plates per dose, with

and without Aroclor 1254-induced hamster liver S9. Preincubation was performed for 20 minutes at 37 °C with shaking.

The following fractions were tested: crude (C), neutral (N) acid (A), and

base (B) fractions of the six smoke condensates, and the seven subfractions

(base fractions 1 to 7) of the base fraction of the high-dose MSC generated

by constant draft (see above). Appropriate solvent and negative controls

were conducted concurrently with each ‘assay. Bioassay results were analyzed for mutagenic activity by slope analysis modeling. Slopes are reported

as revertants per μg of sample. Slope values were given a ranking for mutagenic activity based on slope analysis of an historical data base of mutagens and nonmutagens (table 1). The higher the slope value, the more

mutagenic the fraction was. Slope analysis for crude and chemical class

127
TABLE 1. Ranking of slope values from mutagenic fractions

Slope* Mutagenic Ranking

(Revertants/μg) in Ames System

0.01 to 0.1 negative/equivocal

0.1 to 1.0 weak

1.0 to 5.0 moderate

above 5.0 strong

* Nonlinear regression analysis of Myers et al. (1981).

fractions (A, B, N) of marijuana and tobacco smoke condensates is provided

in table 2. Slope analysis of HPLC subfractions of the basic fraction is

shown in table 3.

Chemical Characterization

Mass Determinations. Dry weight determinations were made on aliquots

of the various chemical class fractions and HPLC fractions of the crude

condensates. All weight determinations were made in triplicate by withdrawing 100 μL aliquots from the previously measured total volume of the

fraction in solvent. These aliquots were placed in a tared container and

taken to dryness in a drying oven (50 “C). Each container was alternately

weighed and dried until a constant weight was obtained.

Table 4 shows the mass distribution obtained from the chemical class fractions (A, B, N) of the crude condensates. Most of the material was found

in the neutral fraction.

Table 5 shows the mass distribution of the HPLC fractions of the base fraction of three of the crude condensates. As can be seen from the table, the

bulk of the material was found in the last three fractions.

Thermogravimetric Analysis. The base fractions of the crude smoke condensates were subjected to thermogravimetric analysis. In this technique,

the weight of a sample is continuously recorded while the sample is heated

using a specific temperature program. The rate and extent of physical and

chemical changes that are accompanied by an increase or decrease in mass

can be determined in this manner. Weight losses can be attributed to drying, volatilization, or combustion. Weight losses occurring below 100 °C

are generally due to drying.

Samples of 20 to 50 mg were heated, from ambient temperature, at

2 °C per minute to approximately 600 °C at atmospheric pressure. A

128
TABLE 2. Slope analysis of chemical class fractions from tobacco and

marijuana smoke condensate in the Ames Assay

Fraction

Slope

(Revertants/μg)

TAlOO TA98 Mutagenic Ranking*

TSC-Puff-Crude 1.39

TSC-Puff-Neutrals 0.03

TSC-Puff-Acids 0.00

TSC-Puff-Bases 0.00

weak

TSC-Constant-Crude 0.14 0.53 weak

TSC-Constant-Neutrals 0.68 0.11 weak

TSC-Ccnstant-Acids 0.04 0.08 equivocal

TSC-Ccnstant-Bases 1.16 2.08 moderate

MSC-Low-Puff-Crude 0.16

MSC-Low-Puff-Neutrals 0.21

MSG-Low-Puff-Acids 0.12

MSG-Low-Puff-Bases 0.19

MSG-Low-Constant-Crude 0.17 0.25

MSG-Low-Constant-Neutrals 0.52 0.09

MSG-Low-Constant-Acids 0.10 0.09

MSG-Low-Constant-Bases 1.54 3.79

MSG-High-Puff-Crude

MSG-High-Puff-Neutrals

MSG-High-Puff-Acids

MSG-High-Puff-Bases

MSG-High-Constant-Crude

MSC-High-Constant-Neutrals

MSG-High-Constant-Acids

0.11

0.14

0.05

MSG-High-Constant-Bases 1.54

0.60 weak

0.61 weak

0.18 weak

7.16 strong

*see table 1.

KEY: TSC=tobacco smoke condensate.

MSC=marijuana smoke condensate.

Puff/Constant smoking mode used in condensate generation.

NOTE: Salmonella typhimurium TA98 and TAl00 with Aroclor-induced hamster liver S9, five dose

levels, triplicate plates per dose. preincubation with shaking for 20 minutes at 37 °C

0.28

0.15

0.00

1.92

0.66

0.17

0.31

0.92

A E

0.23

0.89,

138 moderate

0.36 weak

0.46 weak

7.04 strong

129

weak

weak

weak

weak

weak

weak

weak

weak

equivocal

equivocal

moderate
thermogravimetric analyzer coupled to a data analyzer was used. A flow of

50 mL per minute of inert gas was maintained through the sample chamber

throughout the test.

TABLE 3. Slope analysis of HPLC subfractions of base fractions from

tobacco and marijuana smoke condensate in the Ames assay

Fraction

Slope

(Revertants/μg)

TSC-Constant-Base Fraction 1 0.75

TSC-Constant-Base Fraction 2 0.84

TSC-Constant-Base Fraction 3 0.37

TSC-Constant-Base Fraction 4 1.28

TSC-Constant-Base Fraction 5 4.81

TSC-Constant-Base Fraction 6 1.95

TSC-Constant-Base Fraction 7 4.00

MSC-Puff-Base Fraction 1 1.89

MSC-Puff-Base Fraction 2 3.36

MSC-Puff-Base Fraction 3

MSC-Puff-Base Fraction 4 12.89

MSC-Puff-Base Fraction 5 14.98

MSC-Puff-Base Fraction 6 16.48

MSC-Puff-Base Fraction 7 18.68

MSC-Constant-Base Fraction 1 1.01

MSC-Constant-Base Fraction 2 3.52

MSC-Constant-Base Fraction 3 4.04

MSC-Constant-Base Fraction 4 9.68

MSC-Constant-Base Fraction 5 13.38

MSC-Constant-Base Fraction 6 14.23

MSC-Constant-Base Fraction 7 11.43

KEY: TSC=tobacco, smoke, and condensate.

MSC=marijuana, smoke, condensate, high dose.

Puff vs. Constant=smoking mode used in condensate generation.

NOTE: Salmonella typhimurium TA98 with Aroclor-induced hams&r lives S9, five dose levels,

triplicate plates per dose, preincubation with shaking for 20 minutes at 37 °C.

Table 6 shows the results from this experiment. Minor weight losses due

to drying were observed in all samples. Most of the sample material was

volatilized at temperatures below 300 °C. This indicates that the bulk of

the material is amenable to gas chromatography (GC) or gas chromatography/mass spectrometry (GC/MS) analysis.

130
TABLE 4. Weight percents of chemical class fractions

Condensate

Fraction (Percent by Weight) Total

Acid Base Neutral Insoluble Recovery

MSC-Low-Puff 5.5 7.2 38.0 2.9 54

MSC-Low-Constant 9.5 4.0 22.5 9.5 47

MSC-High-Puff 5.8 5.7 48.8 2.3 63

MSC-High-Constant 6 3 6.9 51.7 2.5 67

TSC-Puff 7.4 11.9 54.7 5.7 80

TSC-Constant Draft 8.2 6.6 34.9 7.3 51

KEY: MSC=marijuana smoke condensate.

TSC=tobacco smoke condensate.

Insoluble=emulsion formed during solvent partition.

Puff vs. Constant=smoking mode used in condensate generation.

TABLE 5. Weight percents of HPLC fractions of bases

Condensate HPLC Fraction Percent by Weight

MSC-Low-Puff 1

2

3

4

5

6

7

MSC-Low-Constant 1

2

3

4

5

6

7

TSC-Constant 1 30.0

2 3.5

3 12.2

4

5

9.9

16.9

6 8.4

5.2

2.6

5.6

8.4

25.4

13.1

39.6

9.7

2.3

5.0

9.9

31.1

10.7

313

7 19.1

KEY: MSC=marijuana smoke condensate.

TSC=tobacco smoke condensate.

Puff vs. Constant=smoking mode used in condensate generation.

131
TABLE 6. Thermogravimetric analysis of marijuana and tobacco smoke

condensate bases

Condensate 70 110 150

Percent Volatile (°C)

190 230 270 310 350 600

MSC-Low-Puff a 53 66 71 75 78 79 80 82

MSC-Low-Constant 3 13 22 37 51 63 71 75 83

MSG-High-Puff 2 10 25 44 59 68 74 18 82

MSC-High-Constant 5 14 26 44 62 76 135 89 94

TSC-PUff 3 7 30 68 79 86 91 93 97

TSC-Constant 11 37 66 76 84 87 90 93

KEY: MSC=Marijuana smoke condensate.

TSC=Tobacco smoke condensate.

Puff vs. Constant=smoking mode used in condensate generation.

Elemental Analysis. All crude smoke condensates were submitted to Galbraith Laboratories, Inc., Knoxville, TN, for elemental analysis. Elements

determined were carbon, hydrogen, nitrogen, sulfur, and oxygen. Results

are presented in table 7 as percent of total. No significant differences were

found between any of the condensates analyzed. The atomic carbon/hydrogen ratios for all samples were low (~0.6), indicating a “paraffinic” sample,

rather than an “aromatic” one (C/H ratios for aromatic systems are typically

greater than 1.0).

TABLE 7. Elemental analysis results

Condensate %C %H %N %S %O

MSC-High-Puff 6653 8.43 6.48 033 17.84

MSC-High-Constant 64.92 8.71 6.82 0.40 18.42

MSC-Low-Puff 63.18 9.14 4.17 0.26 20.65

MSC-Low-Constant 61.78 8.37 4.29 031 23.77

Tobacco-Puff 63.11 8.33 4.70 0.25 19.48

Tobacco-Constant 63.70 854 4.46 0.18 18.74

KEY: MSC=Marijuana smoke condensate.

TSC=Tobacco smoke condensate.

Puff vs. Constant=smoking mode used in condensate generation.

Gas Chromatography/Mass Spectrometry of Base HPLC Fractions. The

base fraction of high-dose marijuana smoke condensate (constant draft) was

analyzed by GC/MS using electron impact ionization. A 60 m DB-1701

132
capillary column was used with the following program: 40 °C for 5 minutes, then 2 °C per minute to 265 °C, then 265 °C for 5 minutes. The

source temperature was held at 180 °C. A skilled MS interpreter analyzed

the resulting spectra and identified the compounds listed in table 8. The

table also shows the HPLC fraction in which those compounds appeared.

TABLE 8. GC/MS compound identification for HPLC fractions 2 to 7 for

the base fraction of MSC batch B (constant draft)

Compound Identification Fraction

propionamide

butyroamide

cyclopentadiene

phenol

methylpyrazine

1-methyl imidazole

2-vinyl pyridine

dimethyltrisulfide

3,3dimethyloxetase

3,3-dimethylcyclobutanecarbonitrile

methyl ethyl pyrrole

dimethylpiperazine

1,2-dimethylimidazole

alkyl amide

N-methyl-2-pyridinamine

1,3,5-trimethylpytazole

dimethylethylpyrrole

valeramide

methyl pyrimidine

3-acetylpyridine

2-methoxy-3-methylpyrazine or C6H8N2O

dimethylethanamine imidazole

tropolone

nitropicoline

C7 alkyl amine

dimethylphenol

G3-alkyl pyrazole

dimethylpyrimidone

methyl acetyl pyrrole

1,4-benzoquinone

C9H14O isomer

alkyl amide

m-aminophenol

6

5,7

3

3,4

3,4,5

5,6

7

7

3

2,3

6,7

3,4,5

2,3,7

5,7

6

3

6

6,697

4

3,4

3

4

6

4,6,7

2,3

4,5,7

5

5

NOTE: Relatively high levels of compounds are indicated by repeated fraction numbers.

133
TABLE 8. (continued)

Compound Identification Fraction

C8H12O isomer

C3-alkyl pyrazole

1H-indazole, 4,5,6,7-tetrahydro or (C7H10N2) isomer

1-butoxy-2-propanol

methylpropionyl furan

benzimidazole

C7H6N2 isomer

3-methyl-5-triazolo (4,3-a)pyrazine

N-(a-picolidene)-n-propylamine

5-hydroxyindole

C8-alkyl amine

quinoline

dimethyl tetrazine

C4-alkyl pyrazole isomer

C9-alkyl amine

C5-alkyl pyrazole isomer

3-methyl-4-ethylpyrrole

C9H12O

C9H14O

C10H14O

C8H12O

C8H10O

methyl ethyl pyrazine

phenoxy ethanol

nicotine

aminobenzamide

phenyl urea

methyl benzimidazole

methylthiopyridine

methylquinoline or C10HgN isomer

C6,-alkyl pyrazole

dimethylbenzimidazole or

ethylbenzimidazole isomer

methoxybenzaldehyde

4-methyl carbostyril

C4,-alkyl pyrazine or C8H12N2

(C10H14O2) propyl methoxy phenol isomer

CnH2n+1OH (alcohol)

3-methyl-l,8-naphthyridine or isomer

pyridine carboxylic acid, methyl

benzoic acid, 3-methyl

5

6

3,5,6

3

4,5

3,4

4,4

2

3

2

3

2

7

4,6,6,6,7,7,7

5,7

5,6,6,6,6,6,7,7,7

4

4

5,7

4

7

4

5,7

3,4,5

4,5

4

4

2,2,3,3,4,4,6

7

2,2,2,2,6

6

2,2,2,2,3,3,3,4,4,4,6,6

3,3,6

2

5,6,6,6,6,7,7,7,7

5,5

5

2

3,4

3

NOTE: Relatively high levels of compounds are indicated by repeated fraction numbers.

134
TABLE 8. (continued)

Compound Identification Fraction

phenyl pyrazoline

3,4-dimethylbenzoic acid

benzyl acetate

C1 0H1 6O

1,2-dihydro-3-isobutyl-l-methylpyrazin-2-one

ethyl hydroxy acetophenone

2,4-dimethylquinazoline

phenyl methyl urea

phenyl pyridine

propylbenzimidazole

aminoquinoline or C9H8N2,

dimethyl naphthyridine

N-phenyl acrylamide

methoxy propyl pyrazine

phenyl alcohol

ethoxy benzaldehyde

tolyl azide

phenyl methyl quanidine

C6,-alkyl phenol

C3,-alkyl benzimidazole

1-decanol

C5,-alkyl pyrazine

alkyl amide

dimethylbenzimidazone isomer

trimethyl-2-oxo-1,2,3,4-tetrahydropyrimidine

dimethoxybenzene isomer

aminodimethylpyrimidine

hydroxymethylquinoline

methylbenzoxazole

tert-butyl-parahydroxybenzoate

C10H1202 (ester)

methyl-n(pyrid-2-yl)dihydropyrrole

C12H18O

methylaminonaphthyridine

diphenylamine

C9H10O3

tert-butylparahydroxybenzoate

ethoxyquinazoline or isomer

diethylphenylene diamine

C5H5N5 isomer

N,N-dimethyl-N-(p-methoxyphenyl) formamide

4

3

4

7

3,5

4

2,3

3

553

4

3

4

3

4,5

3

4,5

3

5

3,4

4

4,5,6,7

7

5,6,6

5

7

5

7

7

7,7

4

7

3

3,4

3

2,2,2,3

4,5

NOTE: Relatively high levels of compounds are indicated by repeated fraction numbers.

135
TABLE 8. (continued)

Compound Identification Fraction

nitroacetanilide 4

2,2,4-trimethylpenta-1,3-diol di-isobutyrate 2,3,4,5,6,7

C11H16O (alcohol) 3,5,7

(C12H20N2) N,N’-dimethyl-N,N’-diethyl- 3,4

p-phenylene diamine

dimethylbenzimidazole 6

diethyl biphenyl 2

N-benzy 1-4-aminobutyronitrile (C11H14N2) 2

N-methyl diphenylamine 2

1-undecanol 2,3,4,5,6,7

dimethylnaphthyridine or C10H10N2, isomer 6,6

trimethylnaphthyridine or C11H12N2 isomer 4,5,5,6,6

alkyl amide 4

hexanenitrile 3(pyrrolidinylmethylene) or 3,494

(C11H18N2) isomers

aminodiphenylene oxide 4

methyl-pteridinone isomer 5

alkyl nitrile 4

alcohol 5,6

2-(propylamino)benzothiazole 4

C13H22N2 isomer 3,3

phenyl benzothiazole 4

aminomethylquinoline 4

tetramethylcyclopentane dione 5

1-methyl-dihydro-ß-carboline 2

alkyl amine 4,6,6,6

alkylthiopyridine 6

lystrin 3

N,N-dicyano-4-methylphenylene diamine 4

alkyl thiopyridine 7

7,8-benzoquinoline 2

tetramethylcyclopentanedione 4

5,5-diphenylimidazolid-4-one or C15H14ON2 2

1-methylphenazine 2

aliphatic alcohol (n-dodecanol?) 5,6,7

alkyl amide 5,5,7

alkyl amine 7

methyl palmitate 7

dimethylnaphtho (2,3,6-) thiophene 4

homologous aliphatic alcohol (n-tridecanol?) 7

1-methyl-ß-carboline 3,4,5,6

NOTE: Relatively high levels of compounds are indicated by repeated fraction number.

136
TABLE 8. (continued)

Compound Identification Fraction

ß-carboline

p-cumyl phenol

dibutyl phthalate

benzyl acetophenone

homologous aliphatic alcohol (n-tetradecanol?)

diphenylpyridine isomer

alkyl ester

dihydroxymethyl phenyl quinazoline

ditolyl ethane

1-azido naphthalene

1-phenyl decane

dimethyl-ß-carboline isomer

alkyl amide

phenylbenzimidazole

2,6-diterbutylnaphthalene or isomer

C14H10O3 isomer

homologous aliphatic alcohol (n-tetradecanol?)

methylthiazolopyrimidine

8-acetoxy-pyrazolobenzo-as-triazine or C11H8N2O4

methyl stearate

methyl phenyl cinnoline or C15H12N2, isomer

2-thiocyanatodiphenylamine

methylpyriloindole

alcohol (n-pentadecanol?)

naptho-sydinone

n -hexadecanol

n-C22H46 (docosane)

alkyl amine

C12H10N2O4 isomer

methylphenyl quinoxaline or C15H12N2

n-C23H48, tricosane

homologous aliphatic alcohol (n-heptadecanol?)

n-C24H50 (tetracosane)

DL-cannabichromene

n-C25H52 (pentacosane)

3-n-pentyl-delta-9-tetrahydrocannabinol

dioctyl phthalate

n-C26H54 (hexacosane)

3-n-pentyl cannabinol

n-C27H56 (heptacosane)

alkyl amide

5,6,7

3

2,3,7

3

6,7

2

2

4

3

6

6

5,5,6

6

3

3

3,4

3,5,6,7

2,3

2,3,6

3,7

3

4

4

5,6

6

6

3,5,7

5,7

3,3,4

3

3,4,5,6,7

5

3,4,5,6,7

2,3

3,4,5,6,7

2,3,7

3,4,5,6,7

4,5,7

2,3,4,6,7

4,5,6,7

3,4,5,6,7

NOTE: Relatively high levels of compounds are indicated by repeated fraction numbers.

137
TABLE 8. (continued)

Compound Identification Fraction

n-C28H58 (octacosane)

saturated hydrocarbon

n-C29H60 (nonacosane)

alkyl phthalate

saturated hydrocarbons

n-C3 0H6 2

3,4,5,7

5,5

2,5,7

6

5,5,6

4,5,6

NOTE: Relatively high levels of compounds are indicated by repeated fraction numbers.

CONCLUSION

Bioassay results showed that (1) the crude fractions were weakly mutagenic,

(2) the acidic fractions were not significantly mutagenic, (3) the neutral

fractions were weakly mutagenic, (4) the base fractions were significantly

mutagenic, (5) the marijuana high-dose base fraction was sevenfold more

mutagenic than either the tobacco or the low-dose marijuana base fraction,

(6) constant draft base fractions were more mutagenic than puff mode basic

fractions for both marijuana and tobacco, and (7) the more polar subfractions (numbers 4 to 7) of the base fraction were more mutagenic than the

less polar subfractions.

Thermogravimetric analysis was performed on the base fractions of the

crude smoke condensates to provide an indication of the applicability of GC

or GC/MS analysis to these fractions. Most of the material was found to

volatilize at temperatures below 300 “C, indicating GC as an appropriate

technique for analysis of these fractions.

Elemental analysis of the crude smoke condensates for carbon, hydrogen,

nitrogen, sulfur, and oxygen revealed no significant differences between the

various condensates. The carbon/hydrogen ratios indicated that the crude

samples were “nonaromatic.”

Approximately 200 compounds were tentatively identified by mass spectrometry. Of this total, about half were amines, with about half of these

being aromatic amines. Pyrazines, pyrimidines, pyrroles, pyridines, and

isoxazoles were the predominant compound classes. Some alkylated pyrazoles and pyrazines were detected in very large amounts, as was an alkylated benzimidazole. Some THC derivatives were also detected. It should

be noted that the mutagenicity of the vast majority of these compounds has

not been determined.

138
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Inhibition of biosynthesis by the naturally occurring cannabinoids. Biochem Pharmacol 22:2905-2910, 1973.

Burstein, S.; Varanelli, C.; and Slade, L Prostaglandins and cannabis—III.

Inhibition of biosynthesis by essential oil components of marijuana. Biochem Pharmacol 24:1053-1054, 3975.

Busch, F.; Said, D.; and Wi, E. Mutagenic activity of marijuana smoke

condensate. Cancer Lett 6:319-324, 1979.

Davis, K.H.; McDaniel, I.A.; Caddell, L.W.; and Moody, P.L. Some smoking characteristics of marijuana cigarettes. In: Willett, R.; Agurell, S.;

and Dewey, W., eds. The Cannabinoids: Chemical, Pharmacologic and

Therapeutic Aspects. Orlando, FL: Academic Press, 1984. pp. 97-109.

Gudzinowicz, B., and Gudzinowicz, M. Analysis of Drugs and Metabolites

by Gas Chromatography/Mass Spectrometry. Vol. 7. Natural, Pyrolytic

and Metabolic Products of Tobacco and Marijuana. New York: Marcel

Dekker, 1980.

Lee, M.; Novotny, M.; and Battle, K. Gas chromatography/mass spectrometry and nuclear magnetic resonance spectrometric studies of carcinogenic

polycyclic aromatic hydrocarbons in tobacco and marijuana smoke condensate. Anal Chem 48:405-416, 1976.

Leuchtenbeyer, C.; Leuchtenbeyer, R.; Ritter, V.; and Inui, N. Effects of

marijuana and tobacco smoke on DNA and chromosomal complement in

human lung explants. Nature (London) 242:403-404, 1973.

Maron, D., and Ames, B. Revised methods for the Salmonella mutagenicity

test. Mutat Res 113:173-215, 1983.

Maskarinec, M.; Alexander, G.; and Novotny, M. Analysis of the acidic

fraction of marijuana smoke condensate by capillary gas chromatography/mass spectrometry. J Chromatogr 126:559-568, 1976.

Merli, F.; Novotny, M.; and Lee, M. Fractionation and gas chromatographic analysis of aza-arenas in complex mixtures, J Chromatogr 199:371-

378, 1980.

Merli, F.; Wiester, D.; Maskarinec, M.; Novotny, M.; Vassilanos, D.; and

Lee, M. Characterization of the basic fraction of marijuana smoke condensate by capillary gas chromatography/mass spectrometry. Anal Chem

53:1929-1935, 1981.

Myers, L.; Sexton, N.; Southerland, L.; and Wolff, T. Regression analysis

on Ames test data. Environ Mutagen 3:575-586, 1981.

Novotny, M.; Kump, R.; Merli, F.; and Todd, L Capillary gas chromatography/mass spectrometric determination of nitrogen aromatic compounds

in complex mixtures. Chem 52:401-406, 1980.

Ogg, C. Determination of particulate matter and alkaloids (as nicotine) in

cigarette smoke. JAOAC 47:357-362, 1964.

139
Petersen, B.; Nishioka, M.; and Chung, C. Nitroaromatics in air particulate

extract. Presented at the 3rd Biannual Symposium on Application of

Short Term Bioassays to Complex Environmental Mixtures, Chapel Hill,

NC, January 1982.

Peterson, R. Importance of inhalation patterns in determining effects of

marihuana use. Lancet March:727-728, 1979.

Wehner, F.; VanRensburg, S.; and Thiel, P. Mutagenicity of marijuana and

Transkei tobacco smoke condensate in the Salmonella/microsome assay.

Mutat Res 77:135-142, 1980.

ACKNOWLEDGMENT

This work was supported by grant R01 DA 03417 from the National

Institute on Drug Abuse.

AUTHORS

Charles M. Sparacino, Ph.D.

Assistant Director

Analytical and Chemical Sciences

Patricia A. Hyldburg, Ph.D.

Research Analytical Chemist

Analytical and Chemical Sciences

Thomas J. Hughes, MS.

Senior Research Genetic Toxicologist

Chemistry and Life Sciences

Research Triangle Institute

3040 Comwallis Road

Research Triangle Park, NC 27789

140
Pyrolysis and Inhalation Studies

With Phencyclidine and Cocaine

Billy R. Martin and Joseph Boni

INTRODUCTION

Smoking has been the major mode of use for many substances (including

tobacco and marijuana) for centuries. In the past decade, the list of substances has been enlarged to include phencyclidine (PCP) and cocaine free

base. A particularly important reason for the surge in smoking of these

substances is the rapid absorption that occurs with inhalation, which results

in a rapid onset of behavioral effects that are deemed pleasurable. In the

case of PCP, it has been suggested that this rapid onset of effects allows

the user to titrate his drug and therefore minimize serious side effects.

However, smoking or inhalation of abused substances creates a spectrum of

problems from minor behavioral problems to death. Despite the fact that

many substances are abused either by smoking or inhalation, they are frequently administered by other routes of administration when they are characterized pharmacologically. Obviously, smoking or inhalation studies are

fraught with difficulties that can be avoided by simply injecting the drug.

The question then arises as to what factors must be considered when extrapolating from the drug injection studies to the human abuse situation.

It is essential to understand the fate of abused substances during the smoking process as well as the absorption, biodisposition, and metabolism of

these substances by the inhalation route. Moreover, we must be able to

determine whether the pharmacological effects of the drugs are quantitatively and qualitatively similar after different routes of administration. Our

strategy has been to develop an apparatus and procedures to (1) produce

smoke or drug vapor in a way that mimics the drug abuser’s behavior, and

(2) expose animals to this smoke or vapor in sufficient quantities to produce

a pharmacological effect. Initially, we determine the extent of pyrolysis that

occurs and identify the pyrolysis products. Then, conditions are established

for exposing animals to the smoke or vapor, followed by biodisposition

studies of the parent compound and pyrolysis products. Finally, behavioral

studies are conducted to assess the correlation with the biodisposition

141
studies. Difficulties that are unique for each drug arise when conducting

inhalation and smoking studies, although some of the problems are common

to all drug studies. One common difficulty is establishing dosimetry in

inhalation and smoking experiments. This difficulty can be overcome by

employing radiolabeled drug and subsequent quantitation of total body radioactivity in the animal after drug exposure.

PYROLYSIS OF PHENCYCLIDINE

PCP has been abused by several routes of administration, which include

smoking, snorting, oral ingestion, and intravenous (IV) injection. However,

it is thought that smoking became very popular because this route permitted

a precise control of dosage and thus reduced the probability of overdose.

PCP is frequently applied to parsley or marijuana dry leaf material to be

smoked, possibly the most common method of abuse. The quantity of drug

present in mainstream smoke and pyrolysis products is an important factor

in determining pharmacological effects. That PCP was heat labile was

shown by the earlier work of Lin and colleagues (1975), who reported the

formation of 1-phenyl-1-cyclohexene when PCP was analyzed by gas-liquid

chromatography. Given the fact that the tip of a burning cigarette can

reach temperatures of 600 it °C was predicted that PCP would be extensively pyrolyzed when smoked.

To investigate the pyrolysis of PCP, an alcoholic solution of 3H-PCP-HCI

(radiolabeled in the phenyl ring) was injected into cigarettes made of commercially processed parsley leaves, along the longitudinal axis (Freeman and

Martin 1981). The ethanol was allowed to evaporate by air drying at room

temperature. The cigarettes, which contained 50 mg of 3H-PCP-HCl in

most experiments, were smoked in an apparatus designed to mimic human

smoking conditions. The apparatus, depicted in figure 1, consisted of a

cigarette holder connected to a series of traps to collect the constituents of

the mainstream smoke. Air was drawn through the entire apparatus by a

water aspirator or vacuum pump. In addition, puffing was simulated by a

valve that opened and closed the system at designated intervals. Air flowed

only when the valve was closed. An air flow regulator, located between

the last trap and the switching valve, was set to deliver 45 ml of smoke

during a 7-second puff duration. At the end of the pyrolysis experiment,

the entire apparatus was disassembled and washed with methanol. Radio

activity was quantitated in these washes of the apparatus and glass wool

trap, as well as the liquid traps. A cigarette requires approximately 6 to

8 minutes to bum under these conditions. The major portion of radioactivity was collected on the glass wool trap, and only trace quantities of

radioactivity were found in the cigarette butt and the remainder of the

apparatus. The glass wool trap has been found to be the most effective

means of collection of PCP and other drug vapors, including the organophosphates (Scimeca et al. 1985) and cocaine. Using liquid nitrogen traps

and bubbling the vapor or smoke through methanol traps proved to be less

142
effective. Analysis of the glass wool traps by thin-layer chromatography

and gas chromatography/mass spectrometry (GC/MS) revealed that the PCP

was readily converted to 1-phenyl-1-cyclohexene (PC). Actually, comparable quantities of PCP and PC were delivered in mainstream smoke, and

little material was lost in the side-stream smoke (Freeman and Martin

1981). It was assumed that each mole of PCP that was cleaved to form PC

resulted in the formation of an equal quantity of piperidine. However, the

volatile nature of piperidine and the fact that this portion of the PCP

molecule was not radiolabeled made quantitation difficult. Therefore,

studies were conducted with 3H-PCP-HCl that was radiolabeled in the

piperidine ring. Analysis by both thin-layer chromatography and GC/MS of

the glass wool extracts revealed that only 6 percent of the starting material

was recovered as 3H-pipetidine. As in the previous experiments, 3H-PCP

and PC accounted for the major portion of the material, while almost

30 percent of the starting material could not be identified (Martin and

Freeman 1983).

FIGURE 1. First generation PCP pyrolysis apparatus

One of the important questions was whether there was a constant delivery

of PCP during the smoking of a cigarette through which PCP was evenly

distributed. To assess the rate of delivery, the glass wool traps were

changed as each third of the cigarette was smoked. It was clear from these

studies that the major portion of the PCP was delivered during the smoking

of the last third of the cigarette, which suggested that the PCP was

condensing in the cigarette (Freeman and Martin 1981). Although we

verified that the burning tip of the cigarettes reached temperatures in excess

of 600 °C (Lue et al. 1986), it was thought that PCP was most likely being

vaporized before the burning tip of the cigarette reached it (figure 2). Cook

et al. (1985) had demonstrated that PCP was completely destroyed when

143
heated to 600 °C in a quartz oven. Therefore, it appeared that PCP was

being volatilized at a temperature lower than that of the burning tip, since

PCP is effectively delivered in mainstream smoke.

FIGURE 2. Recovery of PCP from a burning cigarette

In the above studies, the emphasis was on the delivery of PCP during the

smoking process and the formation of PC Questions remained regarding

the fate of the piperidine moiety. Beaver and Jones (1984) later reported

that the pyrolysis of PCP in a quartz oven at high temperatures resulted in

the formation of significant quantities of polynuclear aromatics, which

included styrene, a-methyl styrene, naphthalene, 2-methyl naphthalene,

l-methyl naphthalene, biphenyl, cyclohexylbenzene, acenaphthene, phenanthrene, and anthracene. The pyrolysis of PCP was evaluated further to

determine whether these polynuclear aromatics, in addition to other pyrolysis

products, were formed during smoking (Lue et al. 1936). For these studies,

marijuana placebo cigarettes (easier to use than parsley cigarettes) were

impregnated with 100 mg of PCP-HCI along with 0.5 to 5.0 μCi of phenyl-

[

3H]PCP and piperidino-[3H]PCP and burned under the conditions described

144
above. Mainstream smoke was passed through glass wool filters as well as

through acidic and basic traps. Greater than 90 percent of the starting

material could be accounted for, with the major portion (77 percent) in the

first glass wool trap.

Methanolic extracts of the glass wool traps were analyzed by both high

pressure liquid chromatography (HPLC) and GC/MS. For quantitative

GC/MS analysis, concentration curves were obtained for 1, 5, 7.5, and

10 ng of each polynuclear aromatic standard using single-ion monitoring

analysis. The limit of detectability for each of the standards was 0.5 ng per

injection or 1 ug/10 ml of methanol extract. Because of the possibility that

the polynuclear aromatics could be formed during smoking but might not be

collected on the glass wool filters, control experiments were conducted in

which the marijuana placebo cigarettes were treated with 4.8 mg of

3H-PCP-HCl with and without 0.1 mg of each of the polynuclear aromatic

standards. The methanol extracts of the glass wool filter condensates from

these cigarettes were analyzed by GC/MS using single-ion monitoring. The

recoveries of the polynuclear aromatic standards ranged from 33 to 100 percent. None of the polynuclear aromatics was detected in the condensates

from cigarettes impregnated with 3H-PCP-HCl alone. It appears that these

compounds are formed from PCP only under conditions that do not mimic

smoking.

In addition to single-ion monitoring analysis, total ion chromatograms were

also obtained for all of the methanol extracts. Four peaks were observed in

the condensates from the 3H-PCP-HCl-spiked cigarettes that were not evident in those from the placebo cigarettes. The spectra and retention times

of these compounds were coincident with those of PCP, l-phenyl-l-cyclohexene, piperidine, and N-acetylpiperidine, which represented 39, 46, 7, and

9 percent, respectively, of the starting material (figure 3). These results

suggest that PCP is not exposed to temperatures during smoking that are

high enough to produce polynuclear aromatics.

PYROLYSIS OF 1-(1-PIPERIDINO) CYCLOHEXANECARBONITRILE

(PCC)

PCC is a major contaminant in illicit PCP because the most common clandestine synthesis of PCP employs PCC as the intermediate; the PCC is not

always removed from the final product (Helisten and Shulgin 19776; Giles

et al. 1977). The presence of PCC in PCP represents a potential health risk

given the fact that it is considerably more toxic than PCP (Baker 1982).

PCC is readily degraded to 1-(1-piperidino)-l-cyclohexene, piperidine, and

cyclohexanone during either GC or thin-layer chromatogmphic (TLC) analysis or on standing in solution (Helisten and Shulgin 1976; Giles et al.

1977). As the stability of PCC during smoking had not been investigated,

despite the potential contribution its pyrolysis products may make to the

pharmacology and toxicology of illicit PCP, a study was conducted to

145
FIGURE 3. Pyrolytic fate of PCP during smoking

determine the quantity of PCC that is delivered in mainstream smoke and to

identify and quantify pyrolysis products (Lue et al. 1988).

[

3H]Piperidino-[14C]cyano-PCC was synthesized to facilitate the quantitation

of PCC in mainstream smoke and the identification of pyrolysis products.

Marijuana placebo cigarettes were impregnated with this double-labeled PCC

and smoked with the apparatus depicted in figure 1. The mainstream

smoke was passed through the glass wool, acidic, and basic traps. Approximately 75 percent of the tritium was collected in these traps, while 46, 11,

and 5 percent of the starting 14C was found in the glass wool, H2SO4 and

NaOH traps, respectively. The constituents of the glass wool trap were

analyzed by GC/MS, which revealed the presence of l-(l-piperidino)-

1-cyclohexene, PCC, piperidine, and N-acetylpiperidine. Cyanide ion was

detected in the glass wool, acidic, and basic traps by a titration procedure.

It appeared that approximately 47 percent of the PCC was delivered intact

in mainstream smoke. Approximately 58 percent was cleaved to form cyanide and 1-(1-piperidino)-1-cyclohexene. The latter was further broken

down to cyclohexanone (representing 21 percent of the starting material),

piperidine (29 percent), and N-acetylpiperidine (7 percent); about 2 percent

remained intact. The entire pyrolytic profile is shown in figure 4.

146
Cyclohexanone Piperidine N-Acetylpiperidine

FIGURE 4. Pyrolytic fate of PCC during smoking

PYROLYSIS OF COCAINE

Although the abuse of cocaine has generated considerable attention in the

past few years, cocaine has been used for religious, medicinal, and recreational purposes for thousands of years. Inhalation of burning coca leaves or

smoking coca evolved gradually and remained major modes of medicinal

and recreational use until enactment of the Pure Food and Drug Ad of

1906 and the Harrison Act of 1914, which curtailed the distribution and use

of coca leaves as well as coca products. It is ironic that these laws probably contributed to the diversion of cocaine use from coca smoking to intranasal and IV administration of cocaine-HC1, because the latter product

became more readily available. Cocaine-HCl has been smoked with marijuana, tobacco, or herbs either as a cigarette or in a pipe. The relatively

modest effects that were derived from smoking the salt soon led to smoking

coca paste (crude extract containing free alkaloids) and finally to “free

base,” which is cocaine-HCl that has been converted to the free base (Jeri

et al. 1978; Siegel 1985). The physical properties of the different preparations have dictated their mode of use. Cocaine-HCl has a melting point of

195 °C, is not very volatile, and decomposes with heat, but it is highly

water soluble, therefore readily absorbed via the mucous membranes. Conversely, cocaine free base has a melting point of 95 °C making it readily

volatile. As a result of these properties, cocaine-HCl is usually administered IV or intranasally, whereas cocaine free base is volatilized and

147
absorbed through the large surface area of the lungs after inhalation. One

of the greatest problems facing us, as summarized by Siegel (1985), is the

emergence of new patterns of use, such as the inhalation or smoking of free

base, which has increased the risks of dependency and toxicity.

The toxicity of cocaine depends upon several factors: the route of administration, dose, drug combinations, etc. (Cohen 1985). The general features of

acute cocaine intoxication include extensive central nervous system (CNS)

stimulation progressing to convulsions followed by cardiovascular and respiratory failure (Jones 1984). Although cocaine has long been considered a

potentially dangerous drug of abuse, the popularity of free basing has led to

the realization of these dangers. The recent upsurge of “crack” has increased the availability and decreased the cost of cocaine. Additionally

catastrophic is the fact that “crack” is a rapidly addicting form of cocaine

that easily results in lethal overdose. At present, the underlying factors for

this increased toxicity resulting from free basing are unclear. The influence

of route of administration on addictive potential and lethality of cocaine

undoubtedly involves pharmacokinetic differences. However, there may be

other contributory factors. For example, Pearman (1979) suggested that factors that increase the absorption of cocaine may be more important considerations for toxicity than simply overdose. Several investigators have found

that the bioavailability and intensity of many cocaine effects are similar

after oral and nasal cocaine (Wilkinson et al. 1980; Van Dyke et al. 1978;

Jones 1984), while the profiles of cocaine effects are similar after IV and

smoked routes. Bioavailability by the nasal route was found to be 30 to

40 percent (Jones 1984) and 80 percent (Cook et al. 1985) of that after IV.

Biodegradation of cocaine (through oral (PO), IV, and intranasal routes) has

been reported to form benzoylecgonine and ecgonine methyl ester (methylecgonine) (Lukaszewski and Jeffery 1980; Chinn et al. 1980; Wallace et al.

19776; Wallace et al. 1977; Jindal and Vestergaard 1978).

Although previously published studies have dealt with the pharmacological

and pharmacodynamic properties of cocaine abuse, little attention has been

paid to the volatilized products of free basing cocaine or crack (Gawin

1980; Isner et al. 1986; Novak and Salemink 1984). Some of the important

aspects of cocaine inhalation include the purity of street cocaine, imprecision of dosing by variability in volatilization and pyrolysis of cocaine,

smoking technique, rate of inhalation, naive vs. chronic use, target tissue,

and underlying pathology. Previous studies have shown that cocaine can be

thermally degraded to benzoic acid and methylecgonidine (Cook and Jeffcoat 1986; Cook et al. 1985) as well as methyl-4-(3-pytidinyl)butyrate

(Novak and Salemink 1984). The species and quantities of volatilized products of cocaine formed under conditions simulating free basing have not

been thoroughly investigated, however, despite their pharmacological and

toxicological importance. Therefore, the present study was conducted to

identity and quantify the delivery of cocaine and to investigate the formation of pyrolysis products during volatilization (Martin et al. 1989).

148
Protocol

The method of volatilizing 3Hcocaine (levo-(-)-[benzoyl-3,4-3H(N)]-cocaine)

represented a modification of the procedure described above for PCP. A

U-shaped pipe was constructed from glass tubing (6 mm inside diameter

and 24 cm in length) and placed in a Lindberg Crucible Furnace filled with

glass beads, as depicted in the bottom of figure 5. After the pipe had

reached the desired temperature, 50 mg of cocaine base containing 1 uCi of

[

3H-benzoyl]-cocaine was inserted into the pipe with the cocaine delivery

system. Cocaine was placed between two glass wool plugs in a glass tube,

which was inserted into the glass pipe at the start of the experiment. The

cocaine and glass wool plugs were pushed out of the tubing and into the

pipe. Vapor was drawn through the volatilization apparatus at 8-second

puff intervals with intermittent 4-second rest periods (no flow). Cocaine

and its pyrolysis products were recovered from the pipe with ethanol

(10 ml), and they were extracted from the glass wool with three l0-ml portions of dichloromethane. The pH of the acid trap was adjusted to approximately 8.5 using sodium bicarbonate prior to extraction with three l0-ml

portions of dichloromethane. The pH of the alkaline trap was adjusted to

4.5 to 5.0 prior to extraction with three l0-ml portions of dichloromethane.

The multiple extractions were combined and concentrated to 10 ml under a

gentle stream of nitrogen at room temperature. In some experiments, the

vapor was sampled repetitively using l-ml air-tight syringes. The vapor

was added to ethanol, and the ethanol was analyzed by liquid scintillation

spectrometry and by GC/MS.

Air Flow Rate

The rate of air flow through the system had a dramatic effect on volatilization of the cocaine. At a flow rate of 100 ml per minute, only 7 percent

of the starting material was recovered in the glass wool trap, and only trace

quantities were found in the other traps after a 5-minute volatilization

at 260 °C temperature. When the flow rate was increased to 400 ml per

minute, approximately 40 percent of the radioactivity remained in the pipe

and approximately 40 percent was recovered in the glass wool trap. As

with the lower flow rate, only trace quantities of radioactivity were

observed in the remaining traps. The contents in the pipe, glass wool, and

traps were analyzed by mass spectrometry either by acquiring the total ion

chromatogram or by selected-ion monitoring. Both procedures revealed the

presence of only cocaine, methylecgonidine, and benzoic acid (figure 6).

There was considerable pyrolysis of cocaine at the low flow rate, so only

small quantities of cocaine remained in the pipe and in the glass wool trap.

The major component was methylecgonidine in both the pipe and the glass

wool. At the higher flow rate of 400 ml per minute, there was little pyrolysis, as evidenced by the fact that high concentrations of cocaine were

found in the pipe as well as in the glass wool trap.

149
FIGURE 5. Smoking-exposure apparatus for PCP and cocaine

Temperature

The effect of temperature on pyrolysis was examined by maintaining the

flow rate of 400 ml per minute during a IO-minute volatilization. Increasing the temperature from 260 °C to 400 °C produced a linear increase in

the formation of methylecgonidine and benzoic acid. In the temperature

range of 350 to 400 °C, approximately 50 percent of the cocaine was de

graded, whereas only trace quantities of cocaine remained intact at 650 °C

150
Benzoic acid Methylecgonidine

FIGURE 6. Pyrolytic profile of cocaine

Because pyrolysis and volatilization of cocaine are highly dependent upon

the experimental condition, studies were conducted to determine optimal

conditions for delivery of a constant concentration of cocaine for subsequent

animal studies. The results, shown in figure 7, reveal the time course

of radioactivity in the air stream as the air flow rate is altered. Most of the

radioactivity is delivered in the first minute at a flow rate of 400 ml per

minute, whereas the delivery period is extended to approximately 3 minutes

when the flow rate is reduced to 100 ml per minute. In a separate series

of experiments, the flow rate was held constant at 400 ml per minute, and

the temperatures were varied between 200 and 260 °C. A relatively constant cocaine delivery of 5 minutes duration was obtained at 200 °C. At

higher temperatures, the duration of a plateau phase was shortened to the

point where maximal cocaine delivery was observed within a l-minute time

frame (figure 8).

151
FIGURE 7. The effect of flow rate on the delivery of W-cocaine

NOTE The results are presented as means from three experiments in which the flow rates were

100 200 300 or 400 ml per minute.

It is clear that the experimental conditions are an important determinant in

the volatilization of cocaine as well as in its degradation. The toxicity

associated with inhalation of cocaine remains to be determined, but it may

well depend upon the conditions under which cocaine is vaporized. It is

important that future inhalation studies with either humans or animals be

conducted under carefully controlled conditions, so that the delivery of

cocaine, as well as the extent of degradation, can be determined (Snyder

et al. 1988).

PHARMACOLOGICAL CONSEQUENCES OF INHALATION OF

DRUGS OF ABUSE

One of the major concerns regarding the smoking of drugs of abuse is

whether the pharmacological and toxicological properties of the drug are

similar to those found after other routes of administration. One important

aspect of smoking drugs is that the smoker is exposed to the parent compound as well as to pyrolysis products. In addition, the metabolism and

biodisposition of the drugs may well be different following inhalation.

152
120

100

80

6 0

4 0

2 0

0

FIGURE 8. The effect of temperature on the delivery of 3H-cocaine

NOTE: The results are presented as means from three experiments in which the themperatures were

200 220 240 or 260 °C.

Studies conducted in our laboratories have shown that 1-3H-pheny1-1-

cyclohexene (3H-PC), the major pyrolysis product of PCP, is metabolized

extensively when incubated in vitro with crude microsomal membranes from

mouse livers (Martin et al. 1982). The major routes of metabolism were

allylic hydroxylation, oxidation of the allytic alcohol, and epoxidationhydrolysis. The following metabolites were identified: l-phenyl1-cyclohexen-3-o1 (major metabolite), 1-phenyl-1-cyclohexen-3-one (PC-3-

one) (major), 1-phenyl-1-cyclohexen-6-o1 (minor), l-phenyl-l-cyclohexen6-one (minor), and l-phenylcyclohexane-1,2-diol (PC-1,2-diol) (minor).

Pharmacological evaluation of PC clearly showed that it produced less behavioral effect and was less toxic than PCP (Holsapple et al. 1982). The

ED, and confidence limits for PCP in the inverted-screen test were 2.5 (1.6

to 2.9) umo1/kg, with a duration of action of approximately 30 minutes after

IV administration. PC’s effects lasted less than 5 minutes after IV administration, and the ED, (confidence limits) was 325 (294 to 360) μmo1/kg.

The behavioral activity of PC-3-ol, PC-3-one, and PC-1,2-diol (PC-6-ol and

PC-6-one were not synthesized in quantities sufficient for testing) also lasted

less than 5 minutes (Martin et al. 1982). The diol was considerably less

active than PC, and PC-3-o1 and PC-3-one were somewhat mote active than

153
PC. Nonetheless, all the metabolites were considerably less active than PCP

and undoubtedly contributed little to the latter’s behavioral activity.

To determine whether the behavioral properties of the drug are the same

after smoking and other routes of administration, studies were carried out in

our laboratories. Male Sprague-Dawley rats were trained to discriminate

intraperitoneal (IP) injections of 3.0 mg/kg PCP from saline under a 2-lever

fixed-ratio 32 schedule of food presentation (Wessinger et al. 1985). After

reliable discriminative control of lever choice was established, a dosedependent increase in PCP-lever selection and dose-dependent decreases in

rates of responding were determined after IP injections. When doses of

PCP were administered by exposure to smoke from cigarettes containing

PCP, a dose-dependent increase in PCP-lever responding was also observed.

Tetrahydrocannabinol (THC) administered via smoke exposure, up to

doses that markedly suppressed response rates, did not result in PCPappropriate responding, demonstrating the specificity of the PCP stimulus by

the inhalation route. Brain levels and distribution of 3H-PCP were determined in rats administered doses calculated to result in 50 percent generalization by the IP injection or smoke inhalation route. By both routes of

administration, roughly equivalent brain levels were attained, and the distribution was relatively even across the seven brain areas analyzed.

CO-ABUSE OF MARIJUANA AND PCP

There is also the possibility of drug interactions by the smoking route. One

obvious interaction is PCP and -THC, since PCP is sometimes smoked in

marijuana cigarettes. The behavioral and pharmacological interactions be

tween THC and PCP were studied following coadministration of the

drugs in smoke to mice (Freeman and Martin 1983). Although THC

(25, 50, or 100 mg per cigarette) had little effect on spontaneous motor

activity, all doses attenuated the hyperactivity elicited by PCP•HCL (25 and

50 mg per cigarette). THC produced a dose-related hypothermia, whereas PCP•HCL (50 mg per cigarette) had no effect on body temperature.

However, PCP enhanced the hypothermia produced by smoke from cigarettes containing 25 mg of THC. THC (100 mg per cigarette) had no

effect on the biodisposition of 3H-PCP and its pyrolytic product, 3H-phenylcyclohexene (3H-PC), when examined immediately after 3H-PCP•HCl (50 mg

per cigarette) exposure. At 30 minutes, however, brain, liver, lung, and

plasma contained higher concentrations of ‘H-PC, and fat and plasma contained lower concentrations of 3H-PCP in the mice exposed to both drugs,

compared to those that were exposed to 3H-PCPmHCl alone. It appears,

therefore, that THC has the potential for altering the behavioral, pharmacological, and pharmacokinetic sequelae of PCP abuse.

CO-ABUSE OF COCAINE AND PCP

Chitwood (1985) reported that most cocaine users are polydrug users who

frequently use cocaine in conjunction with CNS depressants. There is also

154
co-abuse with heroin, commonly known as “speedball,” which represents a

highly toxic combination (Finkle and McCloskey 1978). There appears to

be less co-abuse with stimulants. In one report, cocaine users reported

simultaneous use of PCP and cocaine, albeit infrequently (Chitwood 1985).

There have been recent reports in the media of intoxication with “space

base,” which is a combination of PCP and cocaine. The behavioral and

pharmacological profiles of cocaine and PCP are similar in many respects,

as discussed by Cunningham and Appel (1982) and Bagachi (1985). They

also exert similar effects on the sympathomimetic and serotonergic systems,

which may or may not suggest a common mechanism of action (Cunningham and Appel 1982). It is noteworthy that Colpaert et al. (1979) found

cocaine and PCP to have similar discriminative properties. Cunningham and

Appel (1982) found PCP to partially substitute for cocaine in cocaine-treated

animals. More important, Thompson and Winsauer (1985) found that cocaine potentiates the disruptive effects of PCP on complex operant behavior

in monkeys. Although the similarities and dissimilarities of cocaine and

PCP remain to be established unequivocally, it is becoming evident that coabuse of cocaine and PCP could have dire consequences. An understanding

of the pharmacokinetics and pharmacological effects of combinations of

these agents following inhalation would provide not only insight into the

potential dangers of such co-abuse but a basis for devising treatment

strategies.

CONCLUSION

Inhalation represents one of the most effective modes of self-administration

of drugs that are relatively easily volatilized. In some cases, inhalation

represents a relatively safe way to use some drugs, such as PCP, in that the

user is better able to titrate the dosage to achieve a desired behavioral

effect. Certainly fewer deaths are reported for smoking than for oral administration of PCP. Also, the user does not have to resort to IV injection

to achieve a rapid onset. Nonetheless, inhalation presents the user with

dangers that are not associated with other modes of administration. Usually

the drug must be volatilized by heating, which can result in degradation of

the drug to products that may be dangerous. Inhalation that is achieved by

smoking leafy material results in the introduction of foreign material into

the lungs, the health consequences of which are well documented. A major

concern is that the pharmacological profile of the drug obtained after smoking or inhalation may be different from that produced after other routes of

administration. There is no doubt that far greater addiction is produced

when cocaine is smoked than when coca leaves are chewed. The greater

pharmacological effects that are associated with inhalation of cocaine are

accompanied by greater toxicity. It has been well established that smoking

and inhalation represent effective means of self-administering PCP and

cocaine. It remains to be determined whether drug users will attempt to

use other drugs by this mode of administration.

155
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administration by injection and smoke inhalation. Pharmacol Biochem

Behav 23:607-612, 1985.

Wilkinson, P.; Van Dyke, C.; Jatlow, P.; Barash, P.; and Byck, R. Intranasal and oral cocaine kinetics. Clin Pharmacol Ther 27:386-394. 1980.

ACKNOWLEDGMENTS

Mr. Edward Dimen designed and constructed the inhalation apparatus. This

research was supported by the U.S. Public Health Service grant DA 023%

from the National Institute on Drug Abuse and the Virginia Commonwealth

Center on Drug Abuse Research.

AUTHORS

Billy R. Martin, Ph.D.

Joseph Boni, Ph.D.

Department of Pharmacology and Toxicology

Medical College of Virginia

Virginia Commonwealth University

Richmond, VA 23298-0613

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Animal Models of Drug SelfAdministration by Smoking

Ronald W. Wood

INTRODUCTION

Animal models of drug self-administration have made major contributions to

understanding the behavioral and pharmacologic determinants of substance

abuse practices. Intravenous (IV) administration has been the predominant

route employed in such studies; the limited self-administration literature on

organic solvents and volatile anesthetics demonstrates that the inhalation

route is useful and practical. The literature on animal models of smoking is

less persuasive because the experimental designs have been limited and

because the technical challenges offered by these experiments can be substantial. Description of the dose delivered by smoking may be complex

because of the number of compounds present and the differences in deposition and absorption associated with gases and particulates. The pattern of

particulate deposition is species and particle-size dependent. Combustion

products may be aversive because of eye or airway stimulation; behavior of

the user may minimize aversive stimulation. In some species, the trigeminal reflex limits exposure to acutely aversive atmospheres. Combustion or

pyrolysis need not occur if cocaine base is heated gently; the smoke

observed under these conditions is a condensation aerosol that may not be

aversive. Special generation techniques will be required to generate:

(1) reliable exposure concentrations with stable particle size distributions;

(2) test atmospheres of low irritancy that are appropriate for the species

under study; and (3) exposures tailored specifically for studies of selfadministration, behavioral and physiological effects, pharmacokinetics, or

acute or chronic toxicity.

The chemicals subject to abuse by inhalation may be divided into two

classes, those that are volatile at room temperature (inhalants) and those that

require heat for self-administration, i.e., smoking. This chapter will

examine the commonalities and differences between these substance abuse

practices in a search for the major determinants of the process and for the

implications for developing useful animal models of the process.

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The inhalation route offers a number of advantages; only the simplest equipment is needed, no needles are required, and the rapid onset of effects is

unmatched. A wide variety of chemicals are self-administered by inhalation. Inhalant abuse continues as an unrelenting substance abuse problem

that is global in scope; organic solvents, fuels, gases, and volatile anesthetics continue to be abused to such an extent that the lifetime incidence of

inhalant abuse among high school seniors ranks fifth behind alcohol, tobacco, marijuana, and stimulants. Smoked compounds include tobacco products

(cigars, cigarettes, pipes), narcotics (opium, heroin), cannabis products (marijuana leaf, hashish, extracts), hallucinogens (dimethyltryptamine, the arylcyclohexylamines), and cocaine base (crack, freebase). A drug or chemical

can be “smoked” if it displays substantial vapor pressure below temperatures

at which pyrolysis occurs.

Forcible exposure to combustion products by chamber or tracheostomy are

useful for some toxicologic purposes, but they yield no information on selfadministration, and they may vary in their ability to provide adequate identification or characterization of the health consequences of smoking. The test

atmospheres generated in such studies do not necessarily resemble the products generated by human smoking topographies, because the products of a

burning cigarette are complexly determined, as are the properties of the

aerosol (Davies 1988). In such studies, animals are exposed either continuously or during a fixed proportion of breaths, and the spectrum of toxic

effects has been observed to differ from that in humans (Larson and Silvette

1971; Auerbach 1967; Hammond et al. 1970).

There are many anecdotal reports of animals that smoke. Darwin (1892)

reports

Many kinds of monkeys will, as I have myself seen,

smoke tobacco with pleasure . . . The same tastes are

common to some animals lower in the scale. Mr. A

Nichols informs that he kept in Queensland in Australia

three individuals of the Phaseolarstus cinereus [koala]

that . . . acquired a strong taste for rum and smoking

tobacco. (Darwin 1892, p. 7)

Tracheostomized dogs have been used extensively in studies of lung

diseases induced by cigarette smoke, and the animals seem to adapt to these

procedures quite well:

After a few weeks, the [tracheostomized] dogs became

habituated to cigarette smoking and seemed to enjoy it as

indicated by tail wagging and jumping into the ‘smoking

box’ voluntarily. Thereafter, the use of the pump was discontinued and the dogs voluntarily inhaled smoke by drawing on the cigarettes . . . (Hammond et al. 1970, p. 742)

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By and large as he becomes more used to the smoking, in

humanoid fashion, he seems to relish the habit. (Cahan and

Kirman 1968, p. 573)

The scientific literature on drug self-administration by smoking, however, is

restricted and of limited value. There is no doubt that performances can be

engendered that resemble human smoking topographies; monkeys can be

trained to puff on cigarettes to obtain a liquid reinforcer, and this has been

used as an exposure technique to study the various consequences of exposure to the test smoke atmosphere. Although these puffing performances

can be characterized as responseproduced exposures, they are not necessarily demonstrations of self-administration, i.e., behavior maintained by the

drug as a reinforcer. Few studies have conducted any of the control procedures necessary to document self-administration (Pickens and Thompson

1968; Wood 1979). These control procedures include

acquisition of an arbitrary response with access to the agent as the

reinforcer,

decease in the frequency of the response when access to the purported

reinforcer is discontinued;

generation, by schedules of reinforcement, of typical patterns of

behavior;

maintenance by the agent of frequency of responding that is an

inverted-U-shaped function of concentration; and

discrimination by the subject between those responses that produce the

agent and those that do not, that is, the behavior must not result from a

general rate-increasing effect.

The last control procedure is a critical requirement, since a responseproduced exposure to a psychoactive substance may increase its own frequency of occurrence. For example, an animal may develop a stereotypy

on or have a seizure near the response sensor; in the case of puffing, the

drug exposure may alter the frequency of oral activities.

Further information supports inhalation, but not self-administration. Patent

compounds or metabolites in body fluids, such as, in cigarette smoking, the

presence of large amounts of cotinine or nicotine in the urine, indicates

inhalation, because the acidic smoke is not absorbed through the oral

mucosa (Armitage 1970). For puffing response topographies, the volume of

smoke drawn past the subjects lips should be greater than that of the buccal

cavity and/or typical puffing responses. Finally, exposure-related physiological changes support inhalation, but provide no evidence of selfadiministration.

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TOBACCO SMOKING MODELS

Several investigators have produced smoke puffing by encouraging animals

to drink through a straw, i.e., to pull a column of water up from a reservoir, intermittently switching from water to smoke and subsequently requiring puffing for liquid reinforcers. Ratner et al. (1974) generated puffing in

a cebus monkey in this manner. Jarvik (1967) was able to train fewer than

half of his monkeys to suck water through a tube. To improve this percentage, he obtained a group of maternally deprived and semi-isolated monkeys

with a high rate of sucking their digits. They puffed cigarettes freely without any other reinforcing consequences; they also puffed on formaldehyde,

glacial acetic acid, and isobutyric acid. These early studies relied on observation, lip contact, or water bubbling for response definition; the investigators occasionally observed nasal expulsion of smoke. The monkeys

preferred cigarette smoke over hot air, tobacco smoke over tobacco vapor,

and very fragrant pipe tobacco over cigarette smoke. No preference was

demonstrated between high- and low-nicotine cigarettes, or between cigar

and cigarette smoke; one would expect differential control by nicotine if it

is the reinforcer maintaining the puffing behavior. In the case of cigar

smoking, Armitage et al. (1970) demonstrated that nicotine in cigar smoke

is readily absorbed through the oral mucosa, whereas it is not from cigarette

smoke, and that this absorption was associated with the high pH of cigar

smoke. Jarvik attributed the lack of preference to the duration of the drug

effect, which may have made it difficult to discriminate between the consequences of the two smoke sources, assuming that nicotine was maintaining

the puffing. Jarvik also demonstrated that water reinforcement could produce tenfold to twentyfold increases in the rate of puffing.

Glick et al. (1970) used lip contact on either of two mouthpieces as a measure of puffing and maintained this performance on a fixed ratio (up to 30

lip contacts per water reward); this baseline was used to evaluate the effects

of drug treatment on puffing rates. Mecamylamine, a ganglionic blocking

agent, irreversibly eliminated smoke puffing in two animals, had no effect

on a third, and produced reduction followed by recovery of the smoke puffing preference in a fourth. The third and fourth animals were then administered varying doses of pentobarbital, hexamethonium, scopolamine, and

amphetamine, in that order. Pentobarbital decreased the rate of puffing but

did not alter the preference for smoke over air. Hexamethonium, another

ganglionic blocking agent, reversed the preference while decreasing rate at

large doses. Scopolamine and d-amphetamine reversed the preferences and

greatly decreased the fixed ratio puffing rates. The pentobarbital effects

indicated that preference reversal was not simply an artifact of rate reduction. A further experiment suggested that the scopolamine and amphetamine

effects might be attributable to increased thirst, since water deprivation may

increase the aversiveness of tobacco smoke. The hexamethonium and mecamylamine reversals of puffing preferences suggest that the reinforcing

properties of cigarette smoke might represent pharmacologic effects other

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than olfactory and gustatory effects. A subsequent study (Robinson et al.

1974) utilizing radioactive smoke indicated that some smoke was inhaled

into the lungs with this training procedure.

Rucker (1970) maintained cigarette puffing in monkeys with a water reinforcer and examined the effects of fixed ratio size on puff volume, peak

flow rate, and puff duration. An appendix to this thesis documents numerous attempts and strategies to produce inhalation. He instituted a progressive puff volume requirement that increased by 10 percent with each puff

and decreased by 10 percent after four failures. He then changed the

requirement to an increase equal to 10 percent of the amount by which the

monkey exceeded the preceding requirement, with no decrement in the

requirement. These procedures produced puffs that would not exceed 100

milliseconds in duration; the animals were sucking vigorously. Water deprivation was increased; attempts were made to reinforce blowing and, by

manipulating the characteristics of the spout, to make puffing impossible.

He differentially reinforced longer puff durations, which rarely exceeded 300

milliseconds and added resistance to the air flow, again without successful

production of inhalation, as judged by the volume of the response. The

monkeys failed to continue puffing in the absence of liquid reinforcement.

A baboon model of cigarette smoking also has been developed, again by

using water as a reinforcer for maintaining a minimum negative pressure

and duration on a mouthpiece (McGill et al. 1978). The criterion for

smoke inhalation was elevated blood carbon monoxide levels in comparison

to a presmoking baseline; CO levels in nonsmoking animals in the smoking

room were not reported. Animals obtained their entire water ration in this

manner, puffing on up to 48 cigarettes per day at spaced intervals throughout a 12-hour day. One attendant could maintain 20 animals on this

regime, making Large chronic studies feasible.

This preparation has been used to examine the effects of nicotine content on

puffing performance (Rogers et al. 1985), and to study bronchial reactivity

following cigarette (Roehrs et al. 1981) or nicotine aerosol puffing (Wallis

et al. 1982). The preparation has also been used for an extensive series of

studies on thyroid hormone levels (Sepkovic et al. 1988), on atherosclerosis

induction (Rogers et al. 1980, Rogers et al. 1988), on urinary mutagen formation (Marshall et al. 1983), on alveolar macrophage migration (Fine et al.

1981), and on bronchoalveolar lavage fluids (Rogers et al. 1981; Kolb et al.

1981; Radhakrishnamurthy et al. 1983).

Ando and Yanagita (1981) trained 14 rhesus monkeys to suck liquids

through a tube; they then maintained air sucking at a tube using sweet

fluids as a reinforcer at an adjacent spout. Animals were given prolonged

periods of schedule-controlled puffing, where either a progressively increasing puff duration was required to produce liquid, or a tandem fixed-interval

fixed-ratio schedule of puffing was in effect; in either circumstance, the

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animals were given access to cigarettes in the afternoon without any other

consequence for periods that were gradually lengthened to 20 hours. At the

time of their report, only 2 of the 14 animals had developed sustained “voluntary smoking” that lasted for 2 or more years; in the face of great variability, the authors reported that low-nicotine cigarettes seemed to lead to

decreases in smoking. In two animals that did not sustain voluntary smoking, schedule-controlled smoking was established using a sweet reinforcer,

and control of the performance by the schedule of reinforcement was

demonstrated. Schedule-controlled smoking was associated with elevated

blood levels of nicotine. Yanagita et al. (1983) reported that 10 of the 14

monkeys continued to puff for a month or so after termination of the

reinforcement contingency, and that the two chronic voluntary smokers continued to smoke from 1976 through the time of the later report.

Because the success rate was low with these procedures, Ando et al. (1986)

tried two other techniques. In the first experiment, with two monkeys,

smoking was established with a progressive puff duration requirement; the

animals were then shifted to see if smoking could be maintained adjunctively by reinforcing nozzle licking at a separate tube with different values of a

random interval schedule. One animal’s puffing waned and did not recover;

in the other animal, performance was maintained fairly well at short

random-interval values. In the second experiment, four other monkeys were

shifted from a random-interval smoking contingency with a 0.2 second minimum response duration to a modified Sidman avoidance schedule. When

this performance stabilized at a low shock-delivery rate, the avoidance contingency was terminated; under these extinction conditions, two quit within

15 sessions, but the remaining two persisted for 50 and 80 sessions.

CANNABIS (MARIJUANA, DELTA-9-THC, AND HASHISH)

Cole et al. (1971) attempted to study the effects of delta-9-tetrahydrocannabinol (THC) on spaced responding performance by engendering smoking be

havior in two chimpanzees and an orangutan. These great apes were reinforced for successively longer puff durations on unlighted cigarettes, the

duration ranging from 0.2 to 5.4 seconds. Lights served as feedback for

puffing, and M&M’s were used as reinforcers. The reinforcer was presented at the time the criterion was exceeded (Pieper and Cole 1973). Criteria

were increased between sessions in approximately 250 millisecond increments. When stable, long-duration puffing was established, smoke was

gradually introduced into the air stream by reducing the number of holes

punched at the base of the lit cigarette. The consistent increases in the rate

of the spaced responding performance that occurred immediately following

the smoking sessions with THC in the cigarette were consistent with drug

intake, but the dose effect functions were equivocal. IV self-administration

of delta-9-THC is difficult to engender as evidenced by the heroic but unsuccessful effort of Harris et al. (1974). Pickens et al. (1973) induced IV

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delta-9-THC self-administration in animals with a recent history of phencyclidine self-administration.

Pickens and Thompson (1972) reported being able to obtain fixed-ratio

schedule control of puffing, maintained by presentation of hashish smoke.

Performance was not maintained by heated air. In disagreement with this

early report, Pickens et al. (1973) subsequently reported being unable to

maintain hashish smoking in the absence of food delivery. Hashish was

removed from the burning chamber and the animal was placed on a concurrent (FR3 sucking) (FR5 lever pulling) schedule of food presentation.

These values were selected because they produced approximately equal response frequencies in the absence of hashish. When the animal was given

hashish for sucking, the frequency of sucking increased (resembling Jarvik’s

earlier finding with tobacco). An ADA reversal design was executed for

two animals. In this design, hashish was available, removed, and made

available again, demonstrating a reversal in preference; that is, in the

absence of smoke, the animals preferred lever pressing maintained by food.

The authors interpreted these finding cautiously, indicating that a decrease

in the probability of lever pressing during hashish availability may have

been an effect of hashish smoke on lever pressing, rather than a true preference; taste was also a complicating factor.

DlMETHYLTRYPTAMINE

Siegel and Jarvik (1980), using animals with tobacco- and cocaine-puffing

histories, established puffing on lettuce cigarettes by requiring a l-second

puff duration for access to 1.5 ml water, the monkeys earned all of their

daily water in this manner in a 1-hour session. Termination of the water

deprivation eliminated puffing. The subject was then. placed in a dark

soundproof chamber with free access to food and water; this did not

increase lettuce puffing. Animals were then removed from the chamber

and puffed on dimethyltryptamine (DMT) cigarettes for access to water;

“ . . . after a few puffs on DMT cigarettes, monkeys frequently exhibited

aggressive displays, threats, and barks directed at the smoking tube . . .”

(Siegel and Jarvik 1980, p. 120). Termination of water deprivation eliminated DMT puffing. Animals were then placed on free access to food and

water in the dark chamber, and DMT puffing increased “dramatically”; one

animal puffed greatly during the first session, displayed convulsions and

spasms, and did not approach the spout again. The other two animals gradually commenced puffing across several days, and clusters of puffs occurred

at intervals of about 1 half-hour, approximating DMT’s duration of action.

Observers recorded increases in behaviors similar to those observed following injection of DMT to animals in darkened environments. Puffing

produced some illumination of the chamber under both lettuce and DMT

conditions, but was only sustained in the presence of DMT. The authors

suggested that “. . . more complete tests of DMT’s reinforcing properties in

isolation sessions would require either challenges with forced injections of

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DMT or choice trials with cigarettes containing short-acting nonhallucinogenic stimulants . . .” (Siegel and Jarvik 1980, p. 120). Since some puffing

occurred spontaneously in the absence of drug or the water reinforcement

contingency, DMT puffing may have been due to an agonistic property of

the drug on puffing, rather than to a reinforcing property of the drug. If an

arbitrary response had been required to produce the opportunity to puff on a

lighted cigarette, then a comparison of the frequency of this response with a

response with no drug consequence would argue against an agonistic effect

of the drug. A subsequent reversal of which arbitrary operant was effective

in producing drug access would demonstrate discriminative control of this

behavior. A demonstration of control of the frequency of this responding

by some parameter of delivered dose would then conclusively demonstrate

reinforcing properties of DMT by inhalation. The truism that hallucinogens

do not engender self-administration by laboratory animals apparently has

arisen because nonarylcyclohexylamine hallucinogens have received limited.

attention, and those that have been studied tend to have delayed onset or

prolonged duration of action. Thus, studies of DMT self-administration by

either the intravenous or inhalation route would be of interest.

COCAINE

Siegel at al. (1976) prepared lettuce cigarettes with cocaine base. Three

monkeys were trained to puff as in the Jarvik (1967) study, and then were

permitted for an hour to smoke lettuce or lettuce-cocaine base cigarettes

while under water deprivation or not, and were finally given a choice

between a lettuce or a lettuce-cocaine base cigarette under conditions of

water deprivation. Although the authors did not perform the t-tests of most

interest, under nonwater-deprived conditions the monkeys, by any measure,

smoked more cocaine than lettuce cigarettes. Whether this performance was

induced by a rate-increasing effect of cocaine or by a reinforcing effect of

the drug is not known.

Siegel and Jarvik (1980) repeated these observations in a different order,

finishing the experiment with nonwater-deprived cocaine smoking; this final

condition provided 23-hour access to cocaine cigarettes rather than just

1 hour. Two monkeys tended to distribute puffing fairly evenly across the

first few hours and then quit, only to resume after the daily l-hour break

for service. The third animal displayed high rates of puffing that was not

associated with increased urinary benzoylecgonine. The authors reported

that the rate of puffing did not attenuate across this block of 20 sessions.

THE INFLUENCE OF AEROSOL PHENOMENA ON THE DESIGN

OF SUITABLE ANIMAL MODELS

The pattern and efficiency of airway aerosol deposition are dependent on

the size of the aerosol and the species exposed (Schlesinger 1985;

Schlesinger 1988). We recently characterized the size of crack smoke

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(cocaine base aerosol) to be about 2.3 microns with a geometric standard

deviation ranging from 1.68 to 2.22 (Snyder et al. 1988). As one would

expect from human experience, these particles are small enough to achieve

alveolar deposition following oral inhalation. These observations on cocaine

particle size have important implications for the development of animal

models if comparable patterns of absorption are to be achieved in other

species. Large particles impact on surfaces in the upper respiratory tract;

because of their high velocity through and the tortuosity of the upper airways of the rodent, even relatively small particles impact in the noses of

these species. The rat, for example, is an obligate nose breather, and to

achieve penetration to the lung with this species will require the generation

of cocaine aerosols with a much smaller particle size.

Smoke, as we think of it in this context, is a condensation aerosol of the

drug of interest. As the drug is heated, it vaporizes, only to cool relatively

rapidly; as it cools, the vapor condenses spontaneously or upon particles

(condensation nuclei) too small to be seen with optical techniques. In the

case of drug smoking using a torch and pipe, the torch provides very large

numbers of these nuclei. Given a constant airborne drug concentration, the

number of nuclei are the predominant determinant of the size of the resultant particles. At the butt of a tobacco cigarette, one might expect 3x109

particles per cubic centimeter (Davies 1988). At such high particle numbers, the likelihood of particle collision and subsequent coagulation is great

and proceeds very rapidly; Smoluchowski (1917) was the first to assert that

the rate of particle coagulation is proportional to the square of the particle

count. Thus, particles grow as the smoke ages, and the number of smaller

particles decreases by as much as 40 percent in 1 second (Davies 1988).

It should be apparent that substantial technical problems are associated with

the rapid generation and delivery of aerosols that are small enough to reach

the lungs of small animals at the high air concentrations associated with

drug smoking. The higher the desired concentration, the larger the particle

tends to be. To decrease the size of the particle, the number of condensation nuclei can be increased; as the number of either condensation nuclei or

condensed particles increases, the duration that the particle exists becomes

dramatically shorter. Thus, there are practical and engineering limits to

what can be achieved. Fortunately, a number of condensation aerosol generators have been designed since the initial LaMer-Sinclair generators of

World War II (Muir 1965; Swift 1967). All consist of the same basic

design: condensation nuclei flowing through a heated vapor generator into

a reheating zone, which assures complete volatilization and subsequent uniform aerosol condensation in a downward flow chimney. Prodi (1972)

demonstrated the feasibility of generating solid particles ranging in size

from 0.22 to 2.8 microns from materials with melting points in the same

range as cocaine base; his innovation provided independent control of vapor

concentration and nucleus count. His condensation nucleus generator, however, is not capable of generating high enough counts to maintain these

167
small particle sixes at high airborne concentrations. Other condensation

nucleus generation techniques may be suitable (Swift 1967).

Once such an atmosphere is made., characterizing the material can commence. Most continuous-particle-sizing instruments are designed for monitoring particles in the “clean” rooms of the semiconductor industry, and

cannot accommodate the concentrations found at the end of a cigarette or in

a burning building. Most such instruments rely on light scattering from

single particles and require extraordinarily small measuring volumes and

precise optics. Many have small physical paths that cannot accommodate

high particle concentrations, bias particle measurement because of sampling

artifacts (anisokinetic sampling), or may run at sufficiently high temperatures to alter the particle under measurement. Noncontinuous devices rely

on particle impaction and subsequent recovery and quantitation of the

impacted material. This process can be complicated by the presence of

pyrolysis products; their presence may be difficult to accommodate without

the use of sophisticated analytical devices, extensive control calibrations

with the pyrolysate, or radioactive materials. The measurement of the air

concentration of the chemical of interest poses the same challenge in addition to the special problems associated with trapping particles in contrast to

vapors, e.g. cold fingers do not trap particulate materials. Thus, the initial

generation and characterization of aerosol test atmospheres requires the

scientific ingenuity of a multidisciplinary team; once this effort is under

control, the pharmacology and toxicology can commence.

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ACKNOWLEDGMENTS

This work was supported by grants DA 00623, DA 04438, and DA 05080

and Research Scientist Development Award DA 00117.

AUTHOR

Ronald W. Wood, Ph.D.

Department of Environmental Medicine

New York University Medical Center

550 First Avenue

New York, NY 10016

171
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96 DRUGS OF ABUSE: CHEMISTRY, PHARMACOLOGY, IMMUNOLOGY, AND AIDS. Phuong Thi Kim Pham, Ph.D., and Kenner Rice, Ph.D.,

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97 NEUROBIOLOGY OF DRUG ABUSE: LEARNING AND MEMORY.

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98 THE COLLECTION AND INTERPRETATION OF DATA FROM

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178

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