Drugs of Abuse:

Chemistry,

Pharmacology,

lmmunology,

and AIDS

U. S. DEPARTMENT OF HEALTH AND HUMAN SERVICES • Public Health Service • Alcohol, Drup Abuse, and Mental Health Administration
Drugs of Abuse: Chemistry,

Pharmacology, Immunology,

and AIDS

Editors:

Phuong Thi Kim Pham, Ph.D.

Research Technology Branch

Division of Preclinical Research

National Institute on Drug Abuse

Kenner Rice, Ph.D.

Laboratory of Medicinal Chemistry

National Institute of Diabetes and Digestive and Kidney Diseases

NIDA Research Monograph 96

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, MD 20857
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, DC 20402
Drugs of Abuse: Chemistry,

Pharmacology, Immunology,

and AIDS
ACKNOWLEDGMENT

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

review on “Current Chemical and Pharmacological Advances on Drugs of

Abuse Which Alter Immune Function and Their Impact Upon HIV Infection” held on October 31 to November 2, 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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in the text. Further reproduction of this copyrighted material is permitted

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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)90-1676

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.

iv
Contents

Page

Introduction 1

Phuong Thi Kim Pham

The Bivalent Ligand Approach in the Design of

Highly Selective Opioid Receptor Antagonists 3

Philip S. Portoghese

Opium Alkaloids and Affinity Labels 21

Sydney Archer, Jean Bidlack, and

G. Keith Mulholland

Novel Thebainelike Morphinandienes and Their

Diels-Alder Adducts 35

Leendert Maat

Opioid Antagonists: Structure Activity Relationships 50

Dennis M. Zimmerman and J. David Leander

Effect of Fluorine Substitution on the Anti-HIV

Activity of Dideoxynucleosides 61

Victor E. Marquez

Opiate Antagonists and Anti-HIV Agents 80

Elliot F. Hahn

v
Page

Classical and Nonclassical Cannabinoids:

Mechanism of Action—Brain Binding 100

Allyn C. Hewlett, M. Ross Johnson, and

Lawrence S. Melvin

Cocaine Receptor-Design of Ligands 112

F.I. Carroll, Abraham Philip, and

Anita H. Lewin

Structural and Functional Similarities of

Opiate Receptors of Neural and Immune Origin 122

Daniel J.J. Carr and J. Edwin Blalock

Immunomodulatory Effects of Chronic Morphine

Treatment: Pharmacologic and Mechanistic Studies 131

Henry U. Bryant, Edward W. Bernton, and

John W. Holaday

Morphine-Induced Immune Modulation: Does It

Predispose to HIV Infection? 150

Prince Kumar Arora

A Murine Retrovitus Model for Studies of the Role

of Cofactors and Ethanol in AIDS Development 166

Ronald R. Watson

Drug Abuse and AIDS: Causes for the Connection 181

Robert M. Donahoe

Immune Function in Heroin Addicts and Former Heroin

Addicts in Treatment: Pre- and Post-AIDS Epidemic 192

Mary Jeanne Kreek

Opioid Peptides, Receptors, and Immune Function 220

Hemendra N. Bhargava

Summary of Recommendations for Future Research 234

Louis Harris

List of NIDA Research Monographs 237

vi
Introduction

Phuong Thi Kim Pham

Thousands of pharmaceutical drugs line the shelves of apothecaries across

the Nation. They are designed to treat a host of diseases and ailments

ranging from heart disease and diabetes to allergies and strep throat. But,

of all these drugs, only two (methadone and naltrexone) are available to

treat narcotic addiction. The cocaine epidemic of the last few years,

coupled with the spread of acquired immunodeficiency syndrome (AIDS) in

the drug-abusing population, make the development of effective pharmacological treatments for drug addiction more critical than ever before.

While the prevalence of human immunodeficiency virus (HIV) seropositivity

in the United States is highest for homosexual males (ranging from 20 percent to 70 percent), the incidence of seropositivity among intravenous (IV)

drug abusers may be even greater, with prevalence in certain areas reaching

75 percent. Of all seropositives, IV drug abusers are generally regarded as

posing the most substantial risk of spreading the disease to heterosexuals,

with the risk being greater in urban areas. Also at risk are infants born to

parents who are IV drug users. Current statistics suggest that one out of

two children born to seropositive women develop and almost certainly die

of AIDS before the age of 5. Therefore, does the high rate of seroprevalence among drug abusers reflect some factor predisposing them to AIDS?

Both clinical and preclinical studies suggest that chronic morphine treatment

depresses the immune function, predisposing an individual to higher incidence of infections. By altering immunocompetence, abused drugs, such as

morphine, heroin, and possibly cocaine, represent a potential source of

immunodeficiency independent of HIV infection, thus making the addicts

susceptible to HIV and the development of AIDS.

The first direct evidence that heroin addiction may influence the immune

system came from the observation that lymphocyte mitogenesis was depressed in heroin addicts. Opiate addiction produced a significant depression in the absolute number of T lymphocytes in peripheral blood. The

immune modulatory capacity of the opioid receptor agonist morphine led investigators to hypothesize the existence of opioid receptors on cells of the

1
immune system. Recent studies have now identified such receptors. Binding sites for both classical opioids, i.e., naloxone-reversible agonists, and

nonclassical opioids, insensitive to naloxone reversibility, have been detected

on lymphocytes. In addition, recent observations describing the production

of opioid peptides by cells of the immune system have led to the hypothesis

of bidirectional pathways shared between the immune and neurcendocrine

systems based on the commonality of ligands and receptors.

The intention of this technical review was to bring together a group of

internationally recognized medicinal chemists, pharmacologists, and immunologists working on both drug addiction and AIDS to discuss recent

advances. Researchers from areas of pharmacology, medicinal chemistry,

and immunology presented their work, interacted, and discussed several new

ideas as well as areas of emphasis for future research.

It is hoped that investigations of the effects of drug abuse on the immune

system may facilitate our understanding of the basic mechanisms underlying

the AIDS syndrome. This in turn will help us to determine the feasibility

of treatment modalities for AIDS and AIDS-related syndromes.

ACKNOWLEDGMENT

The author wishes to thank Richard Weber, Ph.D., of the National Institute

of Diabetes and Digestive and Kidney Diseases for providing advice and

guidance in the preparation of the technical review meeting and the

monograph.

AUTHOR

Phuong Thi Kim Pham, Ph.D.

Program Manager and Organic Chemist

Research Technology Branch

Division of Preclinical Research

National Institute on Drug Abuse

Parklawn Building, Room 10A-13

5600 Fishers Lane

Rockville, MD 20857

2
The Bivalent Ligand Approach in

the Design of Highly Selective

Opioid Receptor Antagonists

Philip S. Portoghese

INTRODUCTION

Significant advances in opioid research rely heavily on the use of selective

ligands as tools. Although the opioid antagonists naloxone 1 and naltrexone

2 have been employed extensively for this purpose (Sawynok et al. 1979;

Gold et al. 1982) their usefulness in sorting out the effects mediated by

different opioid receptor types is limited because they are not highly selective. The multiplicity of opioid receptors, their varied distribution in neural

tissue, and the array of physiologic and pharmacologic effects produced by

endogenous and exogenous opioid ligands have created a need for highly

selective antagonists for use as research tools (Herz 1987). In this regard,

the opioid antagonist should be potent, highly selective, and be useful both

in vitro and in vivo for optimal utility as receptor probes. Also, such antagonists should be relatively stable to enzymatic degradation and be capable

of penetrating the central nervous system (CNS). All of these properties

are desirable in the same molecule so that the in vivo pharmacology can be

more reliably correlated with in vitro studies. This chapter reviews two

convergent variations of the bivalent ligand approach (Portoghese 1987) that

have been employed in the design of highly selective opioid antagonists.

DESIGN CONSIDERATIONS AND STRUCTURE-ACTIVITY

RELATIONSHIP (SAR) STUDIES

The rationale employed to design selective opioid antagonists emanated from

two different concepts that bear certain elements of similarity. In the first

approach, there was a possibility of bridging two vicinal opioid receptors by

means of ligands that contain two opioid antagonist pharmacophores linked

through a spacer (Portoghese 1987). The second approach (Portoghese

et al. 1988a) utilized the “message-address” concept (Schwyzer 1977) that

involved the linkage of an opioid antagonist pharmacophore (the “message”)

3
1, R=CH2CH=CH2

; and 2, R=CH2CH(CH2)2

through a spacer to a nonpharmacophore recognition unit (the “address”).

These two different approaches are covered separately, and their convergence under certain conditions is discussed in this chapter. The two

approaches share the feature of simultaneous occupation of two vicinal

recognition sites by the ligand. In this regard, the general term “bivalent

ligand” has been given to ligands that are involved in such interactions.

The opioid antagonist pharmacophore component of bivalent ligands based

on the double pharmacophore approach and the message-address concept

was derived from the potent opioid antagonist naltrexone 2. This antagonist

is selective for the μ opioid receptor type, but it also blocks and receptors (Takemori and Portoghese 1984). It was chosen as the pharmacophore because it (a) is compact, (b) is conformationally defined, (c) is relatively stable to enzymatic degradation, (d) has a ketone group that can be

modified for attachment of a spacer, and (e) can penetrate the blood-brain

barrier.

Double Pharmacophore Approach

The basic premise of this approach was that enhanced potency or selectivity

may be conferred by simultaneous occupation of proximal recognition sites

(bridging) by both recognition units in a single bivalent ligand (Portoghese

1987). These sites may be (a) two neighboring opioid receptors or (b) two

different sites on a single opioid receptor. The author’s initial studies were

of a range-finding nature in order to evaluate the feasibility of changing

selectivity and enhancing affinity through this approach. Subsequent studies

were designed in an effort to distinguish between cases a and b.

The ligands 3 and 4 were synthesized in initial studies contained glycyl

units (Portoghese et al. 1986a; Portoghese et al. 1986b). This permitted

varying spacer length by changing the number of glycyl units, and it

provided easy access to different-length spaces using standard peptide

4
chemistry. Another reason for selecting glycyl units rather than a hydrocarbon chain was the desire to avoid incremental increases in hydrophobic

properties upon lengthening the spacer. Symmetry was introduced into the

spacer by a succinyl or fumaryl group. Both groups were employed in

order to study the relationship between spacer flexibility and antagonist

activity.

Series 3

Series 4

In order to factor out the effect of the spacer on potency, ligands 5 and 6

containing a single pharmacophore and an appropriate spacer were synthesized as reference compounds.

The structure-activity profiles of series 3 and 4 are presented in figure 1.

These studies were carried out on the guinea pig ileum (GPI) preparation

that contains μ and receptors. The graphs illustrate the relative effectiveness of members of the series to antagonize either morphine (μ-selective

agonist) or ethylketazocine ( -selective agonist) as a function of the number

of glycyl units (n) in the spacer.

5
5

6

Significantly, the SAR profile of the succinyl series 3 for antagonism of

morphine was substantially different form that of ethylketazocine (figure 1,

panel A); peak antagonism of morphine was observed at n=2, whereas

maximum antagonism of ethylketazocine was seen with the bivalent ligand

having the shortest spacer length (n=0). The fact that antagonism of the

agonist effect of morphine and ethylketazocine was greatly enhanced at

specific spacer lengths is consistent with bridging of either receptor subsites

or vicinal opioid receptors. Moreover, the fact that the spacer lengths differed for maximum antagonism at μ and receptors suggested that these

receptor types may have different recognition characteristics or may be

organized differently from one another.

While such potency enhancements are in harmony with the bridging of vicinal opioid receptors (case a), it also is consistent with bridging between an

opioid recognition site and a neighboring subsite that is part of the same

receptor (case b). In an effort to distinguish between these two possibilities, a bivalent ligand containing a combination of (-) and (+)-enantiomeric

pharmacophores has been synthesized (figure 2, meso isomer) (Portoghese

et al. 1985). This ligand possesses the same spacer (n=2) that afforded

peak antagonism at μ receptors [threo isomer, series 3 (n=2)]. If the

6
(Gly) n

FIGURE 1. Relationship between relative opioid antagonist potency

and the number of glycol units, (Gly)n, in each half of

the spacer

NOTE: Panel A illustrates this for the succinyl series 3; panel B is for the fumaryl series 4. Note

the different profiles for antagonism at kappa receptors in series 3 and 4.

bivalent ligand containing two identical (-)-pharmacophores (threo isomer)

bridges vicinal opioid receptors, then replacement of one of the pharmacophores with its inactive (+)-enantiomer (meso isomer) should afford a potency decrease. It was found that the meso isomer and the monovalent ligand

5 possessed nearly equal antagonist potencies, but had approximately onethirtieth the threo isomer, thereby confirming that the neighboring site has

an enantio-preference characteristic of an opioid receptor site.

The closely related series 4 in which the spacer contains a fumaryl moiety

was synthesized and the μ and antagonist potencies were evaluated on the

GPI preparation (figure 1, panel B). The fumaryl moiety was employed in

order to determine the effect of conformational restriction of the spacer on

opioid receptor selectivity. It can be seen that the fumaryl series 4 possesses an SAR profile at μ receptors that is very similar to that of the succinyl series 3. However, the interaction of each of these two series at

receptors differed substantially from the other. This difference was characterized by the significantly longer spacer requirement for peak -antagonist potency in the fumaryl series relative to the succinyl series. We have

postulated (Portoghese et al. 1986b) that the conformational restriction

imposed by the fumaryl group in a short spacer (n=0) prevents effective

interaction of both pharmacophores with vicinal recognition sites of the

7
FIGURE 2. Meso and threo [3, n=2, R=CH2CH(CH2

)2] bivalent ligands

with identical spacers

-receptor system. As the spacer is lengthened (n=2) and becomes more

flexible, the simultaneous occupation of proximal sites can then occur with

greater facility.

These results led to investigation of bivalent ligands whose pharmacophores

are immobilized by a rigid spacer. This approach was based on the idea

that immobilization of the antagonist pharmacophores in the proper orientation might facilitate simultaneous occupation of proximal recognition sites.

Since their prior studies suggested that short spacers favored opioid receptor antagonist activity, we chose pyrrole as a spacer based upon its synthetic

accessibility via the Piloty synthesis (Lipkowski et al. 1986a). This heterocyclic ring should restrict conformational mobility of both pharmacophores

by virtue of fusion at positions C-6 and C-7 of the morphinan structure.

Some of the members of this series 7 arc listed in table 1 (Portoghese

et al. 1987; Portoghese et al. 1988b). The parent member of the series,

8
nor-binaltorphimine 7a (nor-BNI), possessed exceptionally high -opioid receptor antagonist potency and unprecedented K-antagonist selectivity. This

high in vitro antagonist selectivity of nor-BNI is paralleled by its high binding selectivity for opioid receptors (Ki

 ratios: μ/ =169, =153). It is

noteworthy that this selectivity arises from an increase in affinity at receptors and an affinity decrease at μ and receptors.

TABLE 1. Opioid antagonist activities of series 7 in the GPI and MVD

aKe=(antagonist]/(IC50 ratio-1).

bAntagonism of ethylketazocine in the GPI.

cAntagonism of morphine in the GPI.

dAntagonism of [D-Ala2

,D-Leu5]enkephalin in the MVD.

eKe not calculated because IC50 ratio is not significantly greater than 1.

The minimum requirements for selectivity are (1) at least one free

phenolic OH group and (2) at least one N-cyclopropylmethyl or N-allyl

substituent. Several compounds (7c, 7d) possessed selectivity that was as

good or better than nor-BNI 7a. The fact that the ligand 8, which contains

only one morphinan with a fused pyrrole, is not selective, suggests that

the pyrrole moiety indeed functions as a spacer. Moreover, the data

(table 1) suggested that only one antagonist pharmacophore may be required

for selectivity and that the other morphinan portion of the molecule

confers selectivity by interacting with a unique proximal subsite on the

same receptor.

9
This question was addressed further by the synthesis of the meso isomer 9

of nor-BNI (Portoghese et al. 1988c). The logic for the synthesis of 9 was

based on the fact that it contains a combination of the antagonist pharmacophore derived from (-)-naltrexone and its inactive (+)-enantiomer. The

perspective formulas (figure 2) corresponding to 7a and 9 illustrate the different geometry of these molecules. Thus, if opioid antagonist selectivity

is retained, it could not be ascribed to bridging two vicinal opioid receptors.

In smooth muscle preparations, 9 was approximately five times more potent

than nor-BNI (7a) but possessed reduced selectivity, presumably as a consequence of more effective interaction with μ and opioid receptors. This

confirmed the idea that only one of the two antagonist pharmacophores of

nor-BNI is required for opioid antagonist selectivity. Accordingly, one of

the antagonist pharmacophores of nor-BNI was postulated to serve as the

“message” and the second pharmacophore to function as the “address” at

the same opioid receptor. It can be noted that although the portions of the

molecules 7a and 9 that contain the postulated address component are enantiomeric, the basic nitrogen function is in approximately the same location

(figure 3). Is it possible that this nitrogen mimics the Arg6 or Arg7 residues

in the address sequence of dynorphin?

8 and 9

Nor-BNI has become widely used in opioid research. In addition to its use

in vitro, it also has been effective in vivo (Takemori et al. 1988). The fact

that the same ligand can be employed both in vivo and in vitro is a distinct

advantage in its use as a research tool.

Message-Address Approach

The message-address concept was developed by Schwyzer (1977). In

essence, this concept proposes that the message comprises those amino acid

residues of a peptide hormone that are responsible for triggering a specific

1 0
FIGURE 3. Perspective formulas of nor-BNI (left) and its meso

isomer (right)

biological effect. The address component is composed of amino acid residues that contribute to the specific binding force between the ligand and

receptor without being able to trigger the biological effect. In some cases,

the message and address components appear to be confluent (e.g., insulin),

while, in other cases, the message and address peptide sequences are clearly

separated (e.g., ACTH). The latter case is schematically illustrated in

figure 4.

The endogenous opioid peptides appear to conform to the latter case (figure 4) in that they contain an N-terminal tetrapeptide sequence Tyr-GlyGly-Phe that is an important requirement for opioid activity (Morley 1980).

It has been proposed that this N-terminal tetrapcptide sequence carries the

“message” responsible for mediating the opioid effect and that segments of

these peptides that differ in amino acid sequence play an “address” role in

conferring selectivity (Chavkin and Goldstein 1981). That is, its function is

to bind to a unique recognition locus that is complementary to a specific

receptor type.

Lipkowski and colleagues have reported that attachment of a peptide

“address” segment to an opiate alkaloid pharmacophore (figure 5) changes

selectivity by increasing the affinity for the target receptor type (Lipkowski

et al. 1986b; Lipkowski et al. 1988). Thus, a C-terminal dynorphin-related

peptide fragment attached to an opiate increased selectivity, while a

C-terminal enkephalin dipeptide enhanced selectivity. The Phe4 residue

was considered to be part of the address sequence in that study because in

opiate alkaloids, such as morphine. clearly only one aromatic ring is part of

the message. The results of these studies suggested that the address subsite

is proximal to the message-recognition locus and that it might be possible

to modulate selectivity by attachment of a suitable molecular “appendage”

other than a peptide to the opiate pharmacophorc.

11
LIGAND

MESSAGE : ADDRESS

LIGAND - RECEPTOR COMPLEX

RECEPTOR TYPE A

RECEPTOR TYPE B

RECEPTOR TYPE C

FIGURE 4. A schematic illustration of the message-address concept

NOTE: Note that the message is identical. while the address sequence differs. Selectivity at different

receptor types is conferred by the address sequence.

Subsequent investigation of the nor-BNI series (table 1 and figure 2) discussed earlier in this chapter indicated that the selectivity of these ligands

may involve a message-address mechanism. The possibility of designing

nonpeptide opioid receptor antagonists was investigated using a

naltrexone-derived pharmacophore joined to a compact “address” moiety.

An important consideration in this design was the conformational restriction

of this moiety. The desire to build conformationally restricted ligands, particularly with respect to the address component, was based upon the idea

that a rigid address moiety might confer greater selectivity because this

would preclude possible conformational adaptation in the binding to other

opioid receptor types.

A logical choice of the address component in the target compounds was a

flat aromatic system whose conformation is restricted by virtue of fusion at

positions 6 and 7 of the morphinan structure. This aromatic ring was

12
FIGURE 5. Structural comparison of dynorphin(1-8)OMe (top) and

alkaloid-dynorphin address hybrid (bottom)

envisaged to mimic the phenyl group of the Phe4 residue of enkephalin (figure 6). Several aromatic heterocyclic systems were employed for this purpose and were synthesized from naltrexone 2. These compounds include

the indole, benzofuran, quinoline, and quinoxaline rings (Portoghese et al.

1988a). In this series, the pyrrole or other hetero-substituted monocyclic

components in these bicyclic aromatic systems represents the rigid spacer

that connects the benzene ring (the address) to the morphinan nucleus (the

message).

Early on, the indoles 10 (table 2) were considered reasonable candidates

because they fulfilled the criteria discussed above and were readily accessible from naltrexone 2 via the Fischer indole synthesis (Portoghese et al.

1 3
FIGURE 6. Naltrexone-derived pharmacophore fused through a spacer to

a benzene ring -address component

1988d; Portoghese et al. 1988e). Testing of the indoles on the electrically

stimulated GPI longitudinal muscle and mouse vas deferens (MVD) preparations revealed that they were devoid of opioid agonist activity and possessed

a unique opioid antagonist profile. All of the compounds were very poor

antagonists of morphine and ethylketazocine (EK), which act selectively in

the GPI at μ and opioid receptors, respectively. Two members in the

series potently antagonized the effect of the agonist [D-Ala2,DLeu5

]enkephalin (DADLE) in the MVD. These were naltrindole 10a (NTI)

and the 7’-fluoro derivative 10d, with the values in the 0.2 to 0.4 nM

range. The receptor binding of NTI was consistent with these results. NTI

10a was greater than 100 times more effective in reversing the agonist

effect of DADLE relative to morphine or EK. Substitution of the indole

nucleus in the 5’-position with electron-donated or electron-withdrawing substituents (10c, e, g, h) reduced potency and selectivity at receptors. In

this regard, the data suggest that size is the most important factor that contributes to this reduction of antagonist potency. Substitution at the

4’-position (10b) similarly reduced antagonist activity at receptors. The

7’-position appears to be the least affected by substitution in view of the

higher potency of the fluoro and methyl congeners (10d and 10f) relative to

the other positional isomers. Methylation of the indole nitrogen (10i)

afforded a reduction of -antagonist potency (Ke=1 nM) and reduced selectivity ratios ( ~20, ~35) relative to NTI 10a. Thus it appears that

aromatic substitution may sterically interfere with the interaction of the

address component with a unique subsite that is proximal to the opiate

recognition locus at the opioid receptor.

The question of the necessity of the indole nucleus for antagonist selectivity was addressed through the preparation of 11, since the benzofuran

ring system is isostcric with that of indole. The benzofuran 11 is a

1 4
TABLE 2. Opioid antagonist activities of naltrindole 10a and its analogs

10

Compound R

1 R

2 DADLE

Ke(nMa)

Morphine EK

10a (NTI)

10b

10c

10d

10e

10f

10g

10h

10i

H H

H 4’-F

H 5’-F

H 7’-F

H 5’-Me

H 7’-Me

H 5’-OMe

H 5’-NO2

Me H

0.21 32 58

4.2 125 160

2.0 61 46

0.35 23 >300

4.2 160 250

1.5 19 >300

5.7 63 1 3

168 >300 >300

1.5 35 22

NOTE: =[antagonist]/(DR-1) where DR=the agonist IC50 in presence of antagonist divided by

control agonist IC50.

-selective antagonist but with reduced potency (Ke=1.5 nM) and selectivity

( =21, =35) relative to NTI. It is noteworthy that this decreased antagonist selectivity is a consequence of the decreased efficiency with which

11 blocks the effect of DADLE. These results suggested that the isosteric

nature of the indole and benzofuran ring systems confers selectivity.

The data support the possibility that pyrrole or furan ring in 10 or 11

functions simply as a spacer in holding the benzene moiety in a specific

conformation relative to the naltrexone-derived pharmacophore. This is

consistent with the observation that the naltrexone-pyrrole derivative 8 is not

selective. Presumably, this rigid spacer permits the aromatic ring to

associate with a subsite on the “address” recognition locus of the receptor

while reducing the affinity for μ and receptors. Further evidence for this

was obtained with the quinoline 12 and quinoxaline 13 analogs, as these

1 5
11; 12, X=CH; 13, X=N

ligands are not isosteric with 10 and 11 but yet have the benzene moiety

fixed in a similar coplanar orientation with respect to the C-ring. Both 12

and 13 are antagonists but with potencies and selectivities lower than that

of benzofuran 11, a finding that is in harmony with the aforementioned

model. Also, the fact that 12 and 13 were not as potent as NTI or the

benzofuran 11 is consistent with the fact that the benzene ring is oriented

somewhat differently because the spacer is a six-membered ring.

Interestingly, the NTI analog 14, derived from the μ agonist oxymorphone,

possessed no opioid agonist or antagonist activity in the GPI and was a

partial agonist (70 percent maximum) in the MVD preparation. This suggested that its agonist activity may be mediated primarily through receptors. The apparent qualitatively similar recognition characteristics for NTI

and 14 may mean tha opioid receptor antagonism and agonism are mediated through identical or similar recognition sites. Thus, it is conceivable

the Try’ and Phe4 aromatic rings of enkephalin bound to the receptor may

approximate the orientation of those in NTI or 14 (figure 7).

16
The most potent antagonist (NTI) in the series was evaluated in mice

(Portoghese et al. 1988e) for its effectiveness in antagonizing the antinociceptive effect of -, μ-, and -selective agonists using the writhing assay.

When administered SC, it effectively blocked the -selective agonist Tyr-DSer-Gly-Phe-Leu-Thr (Gacel et al. 1981) without blocking the effects of μor -selective agonists. Thus, NTI exhibited an in vivo pharmacologic

profile that was qualitatively similar to that found in smooth muscle and

receptor-binding studies.

NTI is the first example of a highly selective and potent opioid antagonist that is not a peptide. Pentapeptides related to the enkephalins (e.g.,

ICI 174864) have been reported (Cotton et al. 1984). However, the affinity

and potency of ICI 174864 are approximately two orders of magnitude

lower than NTI, and it has the disadvantage of low in vivo potency when

administered peripherally due to poor penetration into the CNS.

FIGURE 7. Possible similarity between NTI (A) and the opioid

receptor-bound conformation of enkephalin (B)

CONCLUSION

The bivalent ligand approach has led to the design and synthesis of highly

selective opioid receptor antagonists. One version of this approach, which

involved the synthesis of ligands that contained two pharmacophores connected by a rigid spacer, resulted in the -selective antagonist nor-BNI.

Further studies revealed that only one pharmacophore is required for

selectivity. This suggested that part of the second pharmacophore confers

selectivity by simulating a portion of the “address” sequence in dynorphin, a

-selective opioid peptide. In this context, peptides that conform to the

message-address concept can be viewed as bivalent ligands because they

contain two recognition units, only one of which functions as a pharmacophore; the second recognition unit, the “address,” provides selectivity

through interaction with an address subsite on the receptor. This principle

1 7
was employed to design the -selective antagonist NTI and related ligands.

In this case, the address is a benzene ring attached to the pharmacophore

(the message) through a rigid spacer. As a consequence of its conformational rigidity, this address component has been postulated to bind to the

-address subsite and is incapable of conformationally adapting to address

subsites associated with other opioid receptor types. The success of these

studies suggests that it may be possible to alter antagonist selectivity in a

predictable way by simulating a portion of the address peptide component

with a rigid nonpeptide moiety.

REFERENCES

Chavkin, C., and Goldstein, A. Specific receptor for the opioid peptide

dynorphin: Structure-activity relationships. Proc Natl Acad Sci USA

78:6543-6.547, 1981.

Cotton, R.; Giles, M.G.; Miller, L.; Shaw, J.S.; and Timms, D. A highly

selective antagonist for the opioid delta receptor. Eur J Pharmacol

97:331-332. 1984.

Gacel, G.; Fournie-Zaluski, M.C.; and Roques, B.P. FEBS Lett 118:245-

246, 1981.

Gold, M.S.; Dackis, C.A.; Pottash. A.L.C.; Sternbach, H.H.; Annitto, W.J.;

Martin, D.; and Dakis, M.P. Naltrexone, opiate addiction, and endorphins. Med Res Rev 2:211-246, 1982.

Herz, A. The multiplicity of opioid receptors and their functional significance. In: Mutschler. E., and Winterfeldt, E., eds. Trends in Medicinal

Chemistry. Weinheim: VCH Verlagsgesellschaft, 1987. pp. 337-350.

Lipkowski, A.W.; Misicka, A.; Portoghese, P.S.; and Tam, S.W. Peptides

as receptor selectivity modulators of opiate alkaloids. In: Shiba, T., and

Sakakibara, S., eds. Peptide Chemistry 1987. Osaka: Protein Research

Foundation, 1988. pp. 709-712.

Lipkowski, A.W.; Nagase, H.; and Portoghese, P.S. A novel pyrrole synthesis via the reaction of ketones with N-aminoamides. Tetrahedron Lett

27:4257-4260, 1986a.

Lipkowski, A.W.; Tam, S.W.; and Portoghese. P.S. Peptides as receptor

selectivity modulators of opiate pharmacophores. J Med Chem 29:1222-

1225, 1986b.

Morley, J.S. Structure-activity relationship of enkephalin-like peptides.

Annu Rev Pharmacol Toxicol 20:81-110, 1980.

Portoghese, P.S. Bivalent ligands in the development of selective opioid

antagonists. In: Mutschler, E., and Winterfeldt, E., eds. Trends in

Medicinal Chemistry. Weinheim: VCH Verlagsgcsellschaft, 1987.

pp. 327-336.

Portoghese, P.S.; Larson, D.L.; Yim, C.B.; Sayre, L.M.; Ronsisvalle, G.;

Lipkowski, A.W.; Takemori, A.E.; Rice, K.C.; and Tam, S.W.

Stereostructure-activity relationship of opioid agonist and antagonist

bivalent ligands. Evidence for bridging between vicinal opioid receptors.

J Med Chem 28:1140-1141, 1985.

18
Portoghese, P.S.; Larson, D.L.; Yim, C.B.; Sayre, L.M.; Ronsisvalle, G.;

Tam, S.W.; and Takemori, A.E. Opioid agonist and antagonism bivalcnt

ligands. The relationship of spacer length and selectivity at multiple

opioid receptors. J Med Chem 29:1855-1861, 1986a.

Portoghese, P.S.; Lipkowski, A.W.; and Takemori, A.E. Benaltorphimine

and nor-binaltorphimine, potent and selective kappa opioid receptor

antagonists. Life Sci 40:1287-1292, 1987.

Portoghese, P.S.; Nagase, H.; Lipkowski, A.W.; Larson, D.L.; and

Takemori, A.E. Binaltorphimine-related bivalent ligands and their

opioid receptor antagonist selectivity. J Med Chem 31:836-841, 1988b.

Portoghese, P.S.; Nagase, H.; and Takemori, A.E. Only one pharmacophore

is required for the opioid antagonist selectivity of norbinaltorphimine. J

Med Chem 31:1344-1347, 1988c.

Portoghese, P.S.; Ronsisvalle, G.; Larson, D.L.; and Takemori, A.E.

Synthesis and opioid antagonist potencies of naltrexamine bivalent ligands

with conformationally restricted spacers. J Med Chem 29:1650-1653,

1986b.

Portoghese, P.S.; Sultana, M.; Nagase, II.; and Takemori, A.E. The

message-address concept in the design of highly selective and potent nonpeptide delta opioid receptor antagonists. In: Melchiorre, C., and

Giannella, M., eds. Recent Advances in Receptor Chemistry.

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of the message-address concept in the design of highly potent and

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ACKNOWLEDGMENT

This research was supported by the National Institute on Drug Abuse.

19
AUTHOR

Philip S. Portoghese, Ph.D.

Department of Medicinal Chemistry

College of Pharmacy

University of Minnesota

Minneapolis, MN 55455

20
Opium Alkaloids and Affinity Labels

Sydney Archer, Jean Bidlack, and G. Keith Mulhollund

INTRODUCTION

The first direct evidence that heroin addiction may influence the immune

system came from the observation that lymphocyte mitogenesis was

depressed in heroin addicts (Brown et al. 1974). Street opiate addiction

produced a significant depression in the absolute number of total T lymphocytes in peripheral blood (McDonough et al. 1980). Binding sites for both

classical opioids, i.e., naloxone-reversible agonist binding agents, and nonclassical opioids insensitive to naloxone reversibility have been detected on

lymphocytes (Lopker et al. 1980; Hazum et al. 1979; Schweigerer et al.

1985; Mehrishi and Mills 1983; Dave et al. 1985). However, the functions

of these multiple receptors are unknown. To the best of the authors’

knowledge, affinity labels derived from the opium alkaloids have not been

applied to such studies.

METHODS

Affinity labels are generally derived from reversibly binding ligands that are

so modified that they can be made to bind covalently to the receptor itself

or to an insoluble matrix such as omega-aminohexyl Sepharose to form a

material that can be used to purify a receptor by means of affinity chromatography. There are affinity ligands that can perform one of these functions

well but can be unsuitable for the other. For example, the affinity ligand

FIT was prepared from a μ ligand, yet it selectively acylated receptors

prepared from NG 108-15 membranes (Klee et al. 1982). Yet, under reversible conditions FIT was a nonselective μ and agonist and for this reason was not suitable for use in affinity chromatography. On the other hand,

14 -bromoacetamidomorphine (BAM I), served well as a ligand for affinity

chromatography but did not bind irreversibly to the μ receptor at 25 °C unless dithiothreotol (DTT) was present.

BAM 1 and its companion 14 -bromoacetamidomorphine (BAMO) 2 were

prepared as shown in figure 1 (Archer et al. 1983). Thebaine 3 was treated

with chloronitrosocyclohexane to give an adduct that, after reduction with

21
zinc dust in the presence of methanol, gave a mixture of the 14 -aminocodeinone 4 and the corresponding ketal 5. The latter was treated with

bromoacetyl bromide to furnish the ketal 6, which upon treatment with

boron bromide followed by mild acid hydrolysis gave BAMO 2. The codeinone 4 gave dihydrocodeinone 9 by catalytic hydrogenation. In order to

reduce the ketone to 14 -aminocodeine 7, sodium borohydride was used as

the reducing agent. Treatment with boron bromide gave 14 -aminomorphine 8, which, when allowed to react with bromoacetyl bromide, gave a

mixture of amide esters, which on mild acid hydrolysis gave the desired

BAM 1 (figure 1).

FIGURE 1. Preparation of BAM and BAMO

The ability of BAM and BAMO to inhibit the binding of [

3H]-dihydromorphine in rat neural membranes was determined using seven different concentrations of the ligands in three different experiments. The rat neural

22
membranes were prepared as described by Bidlack and Abood (1980), and

the receptor binding assays were carried out according to the procedure described by Pert and Snyder (1973). Under these conditions, the IC50 value

of morphine was 4 nM, whereas that for BAM and BAMO were 15 nM

and 10 nM, respectively.

The ability of the two ligands to bind covalently to the opioid receptor was

carried out by first incubating rat neural membranes with 20 nM concentrations of 1 and 2 at 37 °C for 30 minutes. An equal concentration of morphine served as a control. The membranes were centrifuged at 105Xg for

15 minutes. The pellet was resuspended in 50-mM Tris buffer, pH 7.5, and

the washing procedure was repeated four times to remove any unbound

ligands. [

3H]-Dihydromorphine and [3H]-naloxone were used at a final concentration of 4 nM to determine the inhibition of binding. The results are

summarized in table 1.

TABLE 1. Inhibition of binding by BAM and BAMO

Compound

Percent Inhibitiona

[

3H] Dihydromorphine [

3H] Naloxone

BAM 19±4 37±3

BAMO 35±3 36±2

Morphine 1±3 2±2

aThe ability of the neural membranes to bind 4 nM [3H]-dihydromorphine and [3H]-naloxone was

measured by the glass-fiber filter technique in the presence of either 10-6 M dextrophan or 10-6 M

levorphanol. The data are reported as the mean percent inhibition plus or minus standard error for five

experiments.

Both BAM and BAMO partially inhibited the binding of the two radiolabeled ligands, with BAMO being slightly more effective against the

agonist than BAM. Morphine had no inhibitory effect. It is noteworthy

that even to achieve this relatively modest inhibition of ligand binding, the

neural membranes had to be incubated with BAM and BAMO at 37 °C for

30 minutes.

To demonstrate that BAM was a selective reversible blocker of the μ receptor, a series of experiments on the inhibition of opioid binding were performed. DAGO was used as the highly selective μ agonist; DDDPE was

used as the specific agonist; and bremazocine, in the presence of DAGO

and DADLE, μ and blockers, respectively, was used as the agonist.

The procedures used were those reported earlier (Bidlack and Abood 1980;

Pert and Snyder 1973). The results are summarized in table 2. The selectivity of BAM for the μ site was at least seventyfold greater than for the

site and almost two hundredfold greater than for the site. The

23
insertion of a bromoacetamido group at C-14 of morphine did not have an

effect on receptor selectivity and only decreased slightly the affinity of this

ligand for the opioid receptors.

TABLE 2. Comparison of the inhibition of opioid binding to rat brain

membranes by morphine and BAMa

Radiolabeled Ligand

IC50 (nM)

Morphine BAM

0.25 nM [

3H]-DAGO

3.0 nM [

3H]-DDDPE

0.2 nM [

3H-Bremazocine]

plus μ and blockers

2 nM [

3H]-naloxone

1 nM [125I]- -endorphin

1.1±0.2 5.0±0.7

137±7 345±33

324±33 950±116

11±2 37±10

6±4 46±5

aRat membranes were incubated with 12 different concentrations of morphine or BAM in the presence

of the radiolabeled opioid in 50 mM Tris HCI. pH 7.5 at 2.5 °C. IC50 values were calculated by least

squares fit to a log-probit analysis. The results are expressed as the mean IC50 value ± SEM from

three experiments performed in triplicate.

Under the above conditions, BAM behaved as a selective reversible μ ligand. However, when similar experiments were carried out in the presence

of dithiothreitol (DTT), a different picture emerged. Rat membranes were

incubated with either 20 nM of DTT or Tris HCI buffer (pH 7.5) for

20 minutes at 25 °C. After this preincubation period, 10 μM of BAM or a

buffer (to act as a control) was added, and incubation at 25 °C was continued for an additional 30 minutes. The membranes were diluted to 40 ml

with Tris buffer and washed six times by centrifugation at 48,000Xg for

15 minutes to remove all noncovalently bound BAM. The specific binding

of 0.25 nM of [

3H] DAGO was measured. The results are summarized in

table 3.

In contrast to the results reported in table 1, in which only modest irreversible inhibition of binding by BAM was achieved by incubation at 37 °C in

the absence of DTT, in the presence of the latter almost complete irrevetsible binding was observed at 25 °C. Apparently there is a disulfide bond

present near the active site of the μ receptor that must be reduced to sulfhydryl groups, which are then alkylated easily by BAM. Even the presence

of a large excess of DTT, which itself may be susceptible to alkylation,

does not interfere with the alkylation of the receptor by RAM. Apparently

the ligand binds too rapidly to the active site, so that alkylation of DTT

does not occur.

24
TABLE 3. The effect of BAM and DTT on [

3H] DAGO binding to rat

neural membranes

Conditions Percent Control Binding

10 μM BAM 93±6

20 mm DTT 106±4

20 mm DTT + 10 μM BAM 10±3

Very Little alkylation of and receptors occurs under conditions wherein

the binding of [

3H] DAGO is prevented by BAM as shown in figure 2.

N-Ethyl maleimide (NEM), a reagent that binds avidly to sulfhydryl groups,

has been shown to decrease [

3H]-dihydromorphine binding to rat neural

membranes and to suppress the effect of GTP on opioid binding (Childers

1984). Studies such as these suggest that there are free sulfhydryl groups

in the vicinity of opioid binding sites in neural membranes of the rat brain.

However, at 25 °C, BAM does not appear to alkylate these group, since in

the absence of DTT no irreversible inhibition of opioid binding occurs.

DTT (mM)

FIGURE 2. Comparison of the inhibition of μ, , and binding to rat

brain membranes as a result of incubating membranes with

DTT and BAM

Source: Ridlack et at. 1989. coyright 1989, American Chemical Society.

25
BAM was also used as a ligand for affinity chromatography, and the affinity column prepared by coupling BAM with omega aminohexyl Sepharose

4B was used to partially purify the μ receptor (Bidlack et al. 1981). Rat

brains minus the cerebellum were homogenized, and the opioid receptors

present were solubilized with the aid of the detergent Triton X-100. After

centrifugation the supematant was treated with Biobeads SM-2. The supernatant was removed, concentrated until the protein concentration was 30 to

50 mg/ml, and then was applied to the affinity column. The opiate receptor

was present in the fractions eluted with either 1 μM of levorphanol or

1 μM of etorphine. These fractions were combined and dialyzed against

Tris-HC1 buffer to remove the opiate ligands (i.e., levorphanol or etorphine),

and the viability of the receptor fractions was tested by equilibrium dialysis

using [

3H]-dihydromorphine as the opiate ligand. NaDodSO4

/polyacrylamide

gels prepared from the active fractions showed the presence of three bands

of apparent molecular weights 43,000, 35,000, and 23,000 daltons.

This procedure was repeated in the presence of protease inhibitors, because

it was suspected that material of higher molecular weight may have escaped

detection owing to degradation by proteases in the initial crude isolates.

Again, fractions were obtained that bound [

3H]-dihydromorphine, but

NaDodSO4/polyacrylamide gels revealed the presence of a new band with an

apparent molecular weight of 58,000 daltons.

Another thebaine-derived opiate ligand Hybromet, 13, was used by

Maneckjee et al. (1985) to isolate and purify an opiate receptor. The

synthesis of this ligand is shown in figure 3.

FIGURE 3. Synthesis of hybromet

26
The ketone 10, a Diels-Alder condensation product of the reaction of thebaine and methyl vinyl ketone, was allowed to react with the lithium

reagent 11 to give the tertiary carbinol 12, which on methoxymercuration

gave Hybromet 13. An affinity column was prepared from 13 and Affigel

401, a sulfhydryl-containing matrix.

The homogenate derived from Sprague-Dawley rat brains minus the cerebellum was solubilized with the detergent CHAPS. This suspension was centrifuged at 105,000Xg for 60 minutes at 4 °C, and the supematant was

applied to the affinity column. After elution first with Tris-HCI buffer

containing CHAPS, the opiate receptor was eluted with 1 μM normorphine

in 10-mM Tris-HCl buffer. Fractions exhibiting [

3H]-labeled opiate-binding

activity were pooled and dialyzed against 10-mM Tris-HCI buffer.

[

3H]-etorphine binding to the partially purified receptor was studied as a

function of radioligand concentration. Half-maximal binding was observed

at about 1 nM radioligand concentration. Scatchard analysis revealed a

class of high-affinity sites with an apparent Kd=1.3 nM and =2800

fmol/mg protein. Studies using [

3H]-ethylketocyclazocine as a selective

agonist and DADLE, a agonist, showed that the partially purified receptor

was primarily of the μ type. Electrophoresis under denaturation conditions

revealed the presence of three bands of apparent molecular weights 94,000,

49,000, and 35,000 daltons. Two of these bands corresponded in approximate molecular weight to those reported by Bidlack et al. (1981).

While Hybromet appears to be a useful ligand for affinity chromatography,

care must be exercised when attempting to use the ligand to bind to crude

preparations of the opiate receptor because of the avidity of this ligand for

sulfhydryl groups. For example, the presence of DTT or other exogenous

sulfhydryl-containing compounds must be avoided because Hybromet, in

contrast to BAM, will react with them instantly before binding to the

receptor.

Rapoport and Sheldrick (1963) reported that when thebaine and dimethylacetylene dicarboxylate (DMAD) were heated in benzene for 1 hour at 50

°C, the normal Diels-Alder adduct 14 was obtained in 90 percent yield, but

under comparable conditions, ethyl propiolate (EP) furnished the adduct 15

in only 6 percent yield. Despite the low yield, 15 appeared to be an attractive candidate for conversion to an affinity ligand because (1) the carboethoxy group could easily be transformed to a functionality suitable for use in

irreversible binding and (2) the newly generated double bond could be reduced with tritium to yield a radioligand with high specific activity. With

these considerations in mind, the authors decided to reinvestigate this reaction. Surprisingly, the interaction of thebaine with acetylenic dienophiles

turned out to be far more complex than originally reported (Singh et al.

1986).

27
When thebaine was allowed to react with methyl propiolate (MP) in tetrahydrofuran for 24 hours at 35 °C, none of the expected Diels-Alder product

16 was obtained. Instead the enol ether 17 was obtained in 30 percent

yield accompanied by some unreacted thebaine. The infrared (IR) and nuclear magnetic resonance (NMR) spectra are in agreement with the assigned

structure. The outstanding features of the NMR spectrum of 17 were the

signals for the H-9 proton, which appeared as a doublet of doublets at

5.92, and for the H-18 proton, which was a singlet at 7.35, which is

characteristic of the proton in -aminoacrylic esters. When the acetylenic

dienophile 3-butyne-2-one was used instead of MP, the analogous compound

18 was obtained. Hydrolysis of 17 using 6N HCI resulted in the formation

of the ketone 19. In the NMR spectrum of 19 the vinyl signal for the H-7

proton disappeared, but the 7.35 signal was still present. The structure of

19 was secured by means of a single crystal x-ray determination.

When the same reaction was carried out in methanol at room temperature,

the adduct 17 was obtained in 32 percent yield accompanied by an etherinsoluble ketal 20 isolated in 62 percent yield. The NMR spectrum showed

signals for four methoxy groups: the coupled H-7 and H-8 vinyl protons

(J=10Hz), the H-9 proton at 8 5.94, and the H-18 proton at 7.35 coupled

to the H-19 proton at 4.40 (J=13 Hz). This coupling constant suggested

that the H-18 and H-19 protons were trans to each other. When DMAD

and thebaine were allowed to react in tetrahydrofuran, the normal DielsAlder adduct 14 was obtained as previously reported (Rapoport and

Sheldrick 1963). When the reaction was carried out in methanol, the ketal

21 was obtained in 35 percent yield. Whether the two carbomethoxy

groups are trans to each other as shown is not certain. When 3-butyne2-one reacted with thebaine in methanol, the open-chain ketone 22 was

obtained along with the enol ether 18, which was isolated as an oil

(figure 4).

14, R

1

 = R

l

= COOCH3 17, R

l

 = OCH3 19

15, R

1

 = COOC2H5, R2 = H 18, R

1

 = CH3

16, R1

= COOCH3, R

2

 = H

FIGURE 4. Reaction of thebaine with acetylenic dienophiles

28
When the ketal 20 was treated briefly with a slight excess of a strong acid,

such as hydrochloric or p-toluenesulfonic, it cyclized almost immediately to

give the enol ether 17. Longer exposure to strong acid furnished the ketone

19. Heating the ketal under reflux with a dilute solution of aqueous methanolic sodium hydroxide resulted in the formation of a yellow isomeric ketal

whose ultraviolet (UV) and NMR spectra were in agreement with the

assigned structure 23. The UV spectrum of 17 showed one maximum at

280 nm and a shoulder at 225 nm, whereas that of 23 showed two maxima

at 282 nm and 326 nm. The H-10 proton in the spectrum of 23 appeared

as a doublet at 6.30 coupled to the H-9 proton.

Prolonged basic hydrolysis of 23 gave the amine 24 as an oil that furnished

a crystalline fumarate. Treatment of 24 with MP converted it back to 23

(figure 5).

20, R1

 = H, R

2

 = OCH3

21, R1 = COOCH3, R

2

 = OCH3

22, R1

 = H, R

2

 = CH3

FIGURE 5. Ketals derived from thebaine and acetylenic dienophiles

Sodium borohydride reduction of the ketone 19 gave the alcohol 25 characterized as the acetate 26. Catalytic reduction of 26 resulted in the

reduction of the double bond to give the dihydro ester 27.

If the catalytic reduction of 19 is carried out in acetic acid over a period of

25 hours, followed by treatment with acetic anhydride in pyridine, the ester

27 is obtained in modest yield. However, if the reduction is carried out for

only 10 minutes and the base isolated, then dissolved in methanol, and the

solution allowed to stand exposed to air for a period of about 2 days, a

new substance separates from solution in 40 percent yield. The NMR spectrum showed a signal at 6.06, which appeared as a doublet and was

assigned to H-9 as well as two other singlets at 5.82 and 6.38, which

were assigned to the C-18 protons. The UV spectrum showed a maximum

29
at 284 nm, and the mass spectrum indicated that the molecular weight was

381. Structure 31 was assigned to this unusual product by means of singlecrystal x-ray analysis (Singh et al. 1986). A possible mechanism to account

for the formation of 31 is shown in figure 6.

FIGURE 6. A possible mechanism for the conversion of 19 to 31

Brief hydrogenation of 19 gives the dihydroketo ester 28. When this ester

is allowed to stand in air for 2 days, the N-oxide 29 is formed, which then

undergoes a ring opening, as shown in 29, to give the hydroxylamine 30,

which undergoes a dehydrative cyclization to afford the observed product

31. Theuns et al. (1984) isolated the base 32 from Papaver bracteatum and

commented that it may be an artifact. They postulate that it resulted from

rearrangement of one of the thebaine N-oxides present in the plant extracts

by a process analogous to that shown in figure 6.

Over the past several years, the authors’ group has shown how thebaine, a

companion of morphine and codeine in Papaver somniferum, can be converted to affinity chromatography and also as irreversibly binding ligands

for the μ receptor. An unusual feature of BAM is that it binds irreversibly

to the μ receptor under mild conditions only under reducing conditions (i.e.,

in the presence of dithiothreitol). We have succeeded in preparing a radiolabeled analog of BAM with high specific activity, in order to couple such

30
a ligand with the undenatured, partially purified μ receptor, to facilitate

isolation of the pure receptor.

Lopker et al. (1980) have shown that human granulocytes and monocytes

show stereospecific high-affinity binding for dihydromorphine, a highly selective μ agonist, and also for 3-quinuclidinyl benzilate (QNB) (figure 7) a

specific ligand for muscarinic cholinergic receptors. Muscarinic binding

occurred in viable cells as welt as in cell fragments.

FIGURE 7. Muscarinic agents

Several years ago, the authors prepared 2-tropanyl benzilate (TRB)

(Atkinson et al. 1977) and compared its central and peripheral antimuscarinic activity with QNB. These ligands were equipotent on peripheral muscarinic receptors, but TRB was about five times more potent than QNB on

central muscarinic receptors (antitremorine assay).

31
More recently they have had occasion to compare TRB with QNB and a

variety of other muscarinic acetylcholine receptor ligands (m AChR), using

[

3H]-QNB in competitive binding assays in mouse-brain homogenates

(table 4).

TABLE 4. IC50 values for various m-AChR ligands obtained from

competitive binding assays using [

3H] QNB in mouse-brain

homogenates

Drug IC50 (nM)

QNB

Scopolamine

nor-Scopolamine

PB

NMPB

FEPB

TRB

nor-TRB

TRFDA

FENTRB

0.8±0.2

1.3±0.3

6.9±0.9

17.3±2.7

1.8±2.7

6.4±0.4

0.7±0.3

0.7±0.3

8.4±0.1

2.5±0.1

It is interesting to note that nor-scopolamine and PB are significantly less

potent than their N-methylated counterparts, whereas TRB and nor-TRB are

equipotent, and both have the same potency as QNB in this assay. The

major advantage of TRB over QNB is that the former can easily be labeled

with a positron-emitting isotope. [

11C]-TRB was easily prepared from norTRB. Excellent visualization of muscarinic receptors in the cerebellum and

striatum of monkeys was achieved. [

11C]-TRB is an excellent candidate for

studying muscarinic receptors in man, including heroin addicts using noninvasive techniques. The short half-life of the 11C isotope makes the chemical

synthetic procedure more difficult, but the advantage of 11C- labeled drugs is

that the very short half-life of this carbon isotope makes repeated administrations possible.

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Mehrishi, J.N., and Mills, I.H. Opiate receptors on lymphocytes and platelets in man. Clin Immunol Immunopathol 27:240-249, 1983.

Pert, C.B., and Snyder, S.H. Properties of opiate-receptor binding in rat

brain. Proc Natl Acad Sci USA 70:2243-2247, 1973.

Rapoport, H., and Sheldrick, P. The Diels-Alder reaction with thebaine.

Thermal rearrangement of some adducts from acetylenic dienophiles.

J Am Chem Soc 85:1636-1642. 1963.

33
Schweigerer, L.; Schmidt, W.; Teschemacher, H.; and Gramsch.

-Endorphin: Surface binding and internalization in thymoma cells.

Proc Natl Acad Sci USA 82:5751-5758, 1985.

Singh, A.; Archer, S.; Hoogsteen, K.; and Hirschfield, J. Thebaine and

acetylenic dienophiles. J Org Chem 48:173-177, 1986.

Singh, A.; Kullnig, R.K.; Seyed-Mozaffari, A.; and Archer, S. Methyl 9,14-

didehydro-4,5-epoxy-17-methyl-2-methylene-6-oxothebinan -acetate.

J Org Chem 51:3378-3380, 1986.

Theuns, H.G.; Janssen, R.H.A.M.; Biessels, H.W.A.; Menichini, F.; and

Salemink, C.A. A new rearrangement product of thebaine from Papaver

bracteatum Lindl. Structural assignment of thebaine N-oxides. J Chem

Soc Perkin Trans 1:1701-1706. 1984.

ACKNOWLEDGMENTS

This work was supported by grants DA 01674 and DA 03742 from the

National Institute on Drug Abuse; by 2-DO1-NS-5655 from the National

Institute of Neurological and Communicative Disorders and Stroke; and DE

FG02-S7ER 60561 and DE FG02 87ER 60528 from the Department of

Energy.

AUTHORS

Sydney Archer, Ph.D.

Cogswell Laboratory

Department of Chemistry

Rensselaer Polytechnic Institute

Troy, NY 12180-3590

Jean M. Bidlack, Ph.D.

Department of Pharmacology

School of Medicine and Dentistry

University of Rochester Medical Center

601 Elmwood Avenue

Rochester. NY 14162

G. Keith Mulholland, Ph.D.

Division of Nuclear Medicine

University of Michigan Medical Center

Ann Arbor, MI 48109

34
Novel Thebainelike Morphinandienes and Their Diels-Alder

Adducts

Leendert Maat

INTRODUCTION

Since the first isolation of morphine from opium, much chemistry has been

done, and numerous derivatives and analogs have been prepared, primarily

to obtain more specifically active central nervous system (CNS) analgesics.

Nevertheless, new compounds with the morphinan skeleton can still be discovered. Their chemistry is interesting, and they may contribute to the

knowledge of structure-activity relationships. Several of the new compounds have been developed on the basis of a Diels-Alder reaction with the

opium alkaloid thebaine. Fifty years ago the first publications on this subject appeared (Schöpf et al. 1938), predicting the pharmacological importance of these cycloaddition compounds (Sandermann 1938). Some classes

of new Diels-Alder adducts have been studied, and developments regarding

them are reviewed in this chapter.

PEPTIDE-MORPHINANS BASED ON DIELS-ALDER ADDUCTS

WITH ENKEPHALIN MOIETIES

Since the identification of the enkephalins, a new lead for molecules with

opiate activity has been explored. The enkephalins should be able to

assume conformations in aqueous solution that, at least partly, resemble

morphine, the “classical” CNS analgesic (Aubry et al. 1988). Furthermore,

there is some similarity between the C-terminal tail of the enkephalins and

the lipophilic part of the highly potent Diels-Alder modified morphinans,

such as etorphine, studied by Bentley (1971). Compounds have been synthesized that combine such a morphinan structure with a C-terminal residue

of leucine-enkephalin (figure 1) (Beyennan et al. 1982). Work began with

the cycloadduct of thebaine with ethyl actylate. Coupling of the acid group,

obtained by hydrolysis of the ester substituent at position 7 with a residue

of leucine-enkephalin, resulted in a molecule with the peptide part attached

35
to the rigid C-ring system. The couplings were carried out by reaction of

the freshly prepared acid chloride with, respectively, the ethyl ester of Lleucine, L-phenylalanyl-L-leucine, and glycyl-L-phenylalanyl-Lleucine. ODemethylation at position 3 yielded the compounds that resemble the

tyrosine moiety of both enkephalin and the A-ring of morphine.

FIGURE 1. Thebaine adducts coupled with enkephalin moieties

Efforts to demethylate the 3-methoxy group selectively while maintaining

the other one in position 6 were unsuccessful. Therefore, only the 3,6-

dihydroxy compounds were prepared by a procedure using hydrogen bromide in glacial acid. In order to obtain the final products, the C-terminal

carboxylic acid, formed by partial hydrolysis of the ester, was reesterified.

In two cases, the leucinol derivatives were also prepared (figure 1), because

the corresponding enkephalin reduction products showed a considerable

increase in potency. The synthesis of more peptide-morphinans based on

novel Diels-Alder adducts is in progress.

Some of the compounds were potent antinticeptives in the mouse hotplate

assay (subcutaneous (SC) injection). The ethenoisomorphinan-3,6-diol with

R=Leu-OC2H5 proved to be quite potent in the opiate receptor assay in rat

brain membranes, in electrically stimulated guinea pig ileum, and in mouse

vas deferens preparations. Recent investigations showed that the diols with

R=Phe-Leu-OEt and R=Phe-leucinol are delta receptor agonists and that the

diol with R=Gly-Phe-Leu-OEt is a potent antagonist of mu agonists. All

three peptide morphinans were potent displacers of 3H-etorphine in rat

36
cerebral membranes. The latter compound was less potent than naltrexone

as a displacer. The first two diols were extremely potent displacers of

3H-etorphine, with EC 50’s in the range of etorphine (Smith et al. 1986).

DIELS-ALDER ADDUCTS WITH A NOVEL PATTERN OF

OXYGEN-CONTAINING SUBSTITUENTS

Bentley (1971) studied the cycloaddition products starting from thebaine,

which contains three oxygen functionalities on the morphinan skeleton.

Extensive chemical and biological studies in the aromatic oxygenated

morphinan-6-one series by Schmidhammer et al. (1983) revealed that the

pharmacological activity of morphinans is strongly dependent on the number

and position of oxygen-containing substituents in the aromatic nucleus as

well as at C-6. The author has started to combine these two modifications

of the morphine molecule. There are in principle two routes starting from

thebaine: the synthesis of the cycloadduct followed by deoxygenation

reactions or conversely beginning with a deoxygenated thebaine derivative

followed by a Diels-Alder reaction.

For the latter route, the author developed two convenient syntheses of

6-demethoxythcbaine (Beyerman et al. 1984), one starting from the minor

opium alkaloid neopine and one from codeine (figure 2). All simple dehydration reactions of neopine failed to give 6-demethoxythebaine. Methanesulfonyl (mesyl) chloride in pyridine gave the 6-mesyl derivative of neopine,

which could be converted into 6-demethoxythebaine by treatment with potassium tert-butoxide. Starting with the easily accessible codeine, the

6-mesyl derivative was obtained quantitatively in the presence of 0.75

equivalent of tricthylamine instead of pyridine. Treatment with lithium bromide in boiling toluenc gave the 8 -bromo morphinan that was converted

into 6-demethoxythebaine with the aid of potassium tert-butoxide. Treatment of 6-O-mesylcodeine with sodium hydride gave directly 6-demethoxythebaine, but the overall yield was lower and the workup procedure was

more laborious than the synthesis via the 8 -bromo morphinan.

6-Demethoxythebaine was the first morphinan-6,8-diene, which has been

converted into Diels-Alder adducts with fewer oxygen-containing substituents

(Hutchins et al. 1981; Crabbendam et al. 1981). With ethyl acrylate or

methyl vinyl ketone, it gave predominantly the 7 -substituted 6,14-ethenoisomorphinan in analogy to the reaction with thebaine. Conversion of the

7 -acetyl group with propyl magnesium bromide into a tertiary alcohol substituent yielded compounds closely related to etorphine (figure 3).

Selective conversion of the 7 -ethoxycarbonyl substituent into a methyl

propyl carbinol substituent, which was feasible with the thebaine derivative,

failed with this 6-demethoxythebaine compound. Treatment with methyl

magnesium bromide gave the dimethyl carbinol (figure 3, R

1

=R2=Me), a

diprenorphine analog, which possesses the advantage of not having the extra

chiral center of the carbinol group.

37
FIGURE 2. Syntheses of 6-demethoxythebaine

6-Demethoxythebaine

R

1

 =Me, R2=Pr

R

1

 =Pr , R2=Me

FIGURE 3. Diels-Alder adducts of 6-demethoxyrhebaine

The pharmacological activity of 6-demethoxyetorphine-3-O -methyl ether

(R1=Me, R

2

=Pr) was shown to be approximately 40 times that of morphine

38
in the mouse hotplate assay (SC injection). The 6-demethoxyetorphine

epimer (R1=Pr, R

2

=Me) with the (S)-configuration of the tertiary alcohol

function at C-7 was only 2 times more potent (Crabbendam et al. 1984).

Further deoxygenation at position 3 of the 7 -dimethylcarbinol (R1

=R2=Me)

derivative was accomplished by catalytic hydrogenation (Pd-C) of the

3-(1-phenyltetrazol-5-yl) ether. It yielded a compound that proved to be a

strong agonist (approximately 200 times that of morphine) in the mouse tailflick assay (Linders et al. 1988b).

SCISSION OF THE EPOXY RING IN 4,5 -EPOXYMORPIIINANS

In order to obtain the series of 4-hydroxyethenomorphinans, several epoxy

ring opening procedures were tried on 4,5 -epoxyethenomorphinans. A

selective ring opening was difficult. Therefore, it was necessary to start

from a morphinandiene. The reductive opening of the epoxy ring in

thebaine with retention of the conjugated 6,8-diene function had been

studied extensively (Razdan et al. 1978). The results were poor, mainly

because the ring opening is accompanied by double-bond isomerization,

giving the isomeric 5,8-diene (table 1).

TABLE 1. Reductive scission of the 4,5 -epoxy bridge of thebaine

Thebaine -Dihydrothebaine Dihydrothebsine-ø

(Percent) (Percent)

1899 Freunda Na/EtOH 50

1939 Smalla

 Na/EtOH 50

1951 Karrera LiA1H4/benzene 42

1952 Bentleya Na/NH3 95

1978 Portlocka K/NH3 34

1984 Kanematsub

 Fe(CO)5 / Zn/HOAc 47 (4-MeO-)

1984 DelftCZn/NH4C1/EtOH,aq 70 (6-H-) 30

1986 Delftd Zn/2% KOH 98 1

aRazdan et al. 1978; b

Fujii et al. 1984; CCrabbendam et al. 1984; dLinders et al. 1986

39
When 6-demethoxythebaine was treated with lithium aluminum hydride,

according to one of the methods described for the epoxy ring opening of

thebaine, a mixture of three major compounds was obtained, the third compound being the 5,7-diene, desoxycodeine-A. To prove the structure of the

latter compound, it was prepared via bromocodide, starting from codeine in

a way somewhat more convenient than the older procedure via chlorocodide.

It is remarkable to note that the 5,7-diene system in desoxycodeine-A failed

to give Diels-Alder adducts. The reaction was studied with desoxycodeineA itself and some 4-phenolic ethers and esters under both atmospheric and

elevated pressures. Although the torsion angle around the C6-C7 bond,

according to both x-ray analysis and molecular modeling calculations, does

not differ much from that around the C7-C8 bond in morphinan-6,8-dienes,

no cycloaddition product could be detected.

Finally, it was found that a simple treatment of thebaine in boiling

2-percent aqueous potassium hydroxide with zinc powder afforded almost

exclusively and quantitatively -dihydrothebaine (Linders et al. 1986). This

reaction was then applied successfully to 6-demethoxythebaine, yielding

6-demethoxy- -dihydrothebaine. However, the scission of the epoxy ring in

6-demethoxynorthebaine according to this procedure is still accompanied by

approximately 15 percent of 5,8-diene formation.

DIELS-ALDER ADDUCTS OF 6-DEMETHOXYDIHIYDROTHEBAINE

Diels-Alder reaction of thebaine with monosubstituted ethenes may afford,

in principle, eight isomeric adducts. This was first recognized by Schöpf

et al. (1938), who proposed on the basis of methylation reactions that the

cycloadditions he studied took place from the P-side and that the oxygencontaining substituents were directed to the newly formed (C7-C8) double

bond, in agreement with the so-called endo-rule according to Diels. Indeed,

all further cycloaddition reactions described until recently yield predominantly one isomer with the dienophile approaching from the p-face of thebaine and with the substituent at position 7 (figures 1 and 3). These

compounds were initially named 7 -substituted 6,14-endo-ethenomorphinans.

Heating 6-demethoxy- -dihydrothebaine in an excess of methyl vinyl ketone

for 60 hours yielded two new adducts in a ratio of 3:2, according to high

performance liquid chromatograph (HPLC). The reaction when performed

under conventional conditions caused extensive polymerization, which made

the workup and the isolation of the adducts cumbersome. A dramatic improvement was achieved when the reaction was carried out using a modified

microwave oven (Lindens et al. 1988a). Recently, the use of microwave

heating in organic synthesis has been reported (Emsley 1988). The reactions described were carried out in closed vessels, which resulted in high

reaction pressure and temperature. In the author’s setup, under atmospheric

conditions, the reaction, surprisingly, was complete within 24 hours with

40
substantially less formation of polymeric material, although the reaction temperature must have been similar to that in his earlier experiments. The

usual workup procedure involving acid-base extraction gave the pure adducts

after fractional crystallization. In contrast to all previous Diets-Alder reactions, the cycloaddition to 6-demethoxy- -dihydrothebaine, lacking the

epoxy bridge, occurred exclusively from the a-face of the diene function

(figure 4).

65% 35%

FIGURE 4. Diels-Aider reaction of Memethoxy- -dihydrothebaine with

methyl vinyl ketone

At first, structure elucidation of the new addition products met with difficulties. From the shifts of the vinylic protons H-18 and H-19, it was

concluded that the etheno bridge is at the p-face of the molecule. The

vinylic protons of the -face etheno-bridged compounds show a downfield

shift that can be attributed to the deshielding effect of the aromatic nucleus.

The signals of the protons at C-7 and C-8 coincide with those of the acetyl

and N-methyl groups. Mass spectrometry showed the expected molecular

ions at m/z 353. The presence of a fragment m/z 57 (C3H5O) in the second

compound can be explained by a McLaffetty rearrangement of the acetyl

group. Molecular models show that this rearrangement can only occur in

two 6 ,14 -ethenomorphinans, viz, 7 - and 8 -acetyl, and in two

6 ,14 -ethenomorphinans. However, the latter two can be ruled out on the

basis of the nuclear magnetic resonance (NMR) data. From these spectral

data, again with the application of the Diets endo-rule, the author tentatively

assigned the 8 -acetyl-6 ,14 -ethenomorphinan structure to the amorphous

second compound. For x-ray analysis, it was converted into the crystalline

4-O-phenyl ether (R=C6H5). Detailed NMR as well as mass spectrometty

did not result in an unequivocal proof of the two structures. Therefore,

single-crystal x-ray analyses were used for conclusive evidence. Simultaneously with the author’s findings, Ghosh et al. (1983) reported on the DielsAlder reaction of the O-phenyl ether of -dihydrothebaine with methyl vinyl

ketone also giving rise to -face cycloaddition.

41
It is obvious that a new class of etheno-bridged morphinans is now accessible starting from the 4-hydroxymorphinandienes. As target molecule,

reminiscent both of diprenorphine and of compounds previously prepared by

the author, 3-hydroxy- 17-trimethyl-6 ,14 -ethenomorphinan-7 -methanol was chosen for pharmacological testing, starting from the first cycloadduct (figure 4). Reaction of the 4-hydroxyl group with bromobenzene in

boiling pyridine in the presence of potassium carbonate and copper powder

gave the 4-O-phenyl ether. The 7 -acetyl group was converted into the dimethyl carbinol substituent with methyl magnesium bromide. The phenoxyl

group was then easily removed by means of reduction with sodium in liquid

ammonia. The compound appears to show activity in only one of the biological assays (SC injection in mice). In the paraphcnylquinone (PPQ)

stretching assay, it is morphinelike. In the tail-flick test at 1.0, 10.0, and

30.0 mg/kg, the compound is inactive as it is in the mouse hotplate assay.

Since it has been previously noted that the PPQ assay is less discriminating

with respect to the opioidlike activity, the compound will be examined

further at the receptor level (Linders et al. 1988a).

The naming and numbering of the novel morphinans is confusing because

different conventions are in use. The author suggests naming the “Bentleytype of so-called endo-etheno adducts” 6,14-ethenoisomorphinans and the

new class of compounds 6,14-ethenomorphinans. In both cases, an etheno

bridge is added to a distinguishable molecule, namely, isomorphinan and

morphinan respectively. This leaves the usual numbering of the ring system

unaltered at the positions 7 and/or 8, together with the statement of a and

with respect to the “phenanthrene projection” (figure 5).

N-FORMYLMORPHINANDIENES IN REACTlON WITH

NITROETHIENE

The Diels-Alder reaction of thebaine has been studied with a great variety

of dienophiles. However, nitroethene has not received any attention. Although nitro compounds are not biologically important, the nitro group can

be converted into the versatile amino substituent, interesting, for example,

for opioid receptor studies (Lessor et al. 1984).

Direct reaction between thebaine and nitroethene did not lead to cycloaddition. Under alkaline conditions nitroethene undergoes polymerization.

Therefore, an indirect route was chosen via the neutral N-formylnorthebaine

(Maat et al. 1985). Now the addition proceeded quantitatively, and the

product was, as expected, almost exclusively the 7 -nitro-6,14-ethenoisomorphinan (figure 6). The N-formyl substituent can be easily replaced by

the N-methyl substituent via the northebaine derivative, which has the advantage that here any other useful alkyl group can also be introduced.

Reduction of the nitro group with reagents such as lithium aluminum hydride and formamidinesulfinic acid stopped at the oxime stage. However,

42
MORPHINAN ISOMORPHINAN

CA: MORPHINAN (14 )-MORPHINAN

7 X-ETHENOMORPHINAN 7 X-ETHENOISOMORPHINAN

CA: 7 X-6 14 -ETHENOMORPHINAN 7 X-6.14-ETHENOMORPHINAN

"-exo-" "-endo-"

FIGURE 5. Naming the different etheno-bridged morphinans

treatment with aluminum amalgam in an aqueous mixture of ether and

methanol gave the desired 7 -amine-6,14-ethenoisomorphinan (figure 6).

The cycloaddition of nitrcethene to 6-demethoxy-N-formylnorthebaine seems

to be more peculiar. Preliminary experiments show that at least three

cycloaddition products can be isolated (figure 7). Separation of the isomers

was achieved by column chromatography. The major product (~ 50 percent) is the 8 -nitro-6,14-ethenoisomorphinan. Also, the usual 7 -nitro6,14-ethenoisomorphinan is present (~ 10 percent). However, for the first

time, an -face addition to a 4,5 -epoxymorphinan-6,8-diene occurs in a

fairly high yield (Prazeres et al. 1986). The structure was based on the 1H

NMR data of the N-H compound, obtained after acid hydrolysis. The signals in the 200-MHz 1H NMR spectrum were assigned with the use of a

homonuclear 2D correlation (COSY). From the magnitude of the vicinal

couplings between H-7 and H-7 and H-8 (J 8.5 Hz and J 4.0 Hz),

it can be concluded that the nitro group is at the 8 -position. The 6 ,14

orientation of the etheno bridge with respect to the isomorphinan skeleton

was established by the long-range coupling between H-5 and H-7

(1.3 Hz), which is in agreement with the W-arrangement of the respective

protons in that structure. Moreover, long-range couplings between H-5 and

H-18 and between H-7 and H-18, as present in the case of 6 ,14

etheno-bridged adducts, were not observed in this compound. In addition, it

43
FIGURE 6. Synthesis of 7 -nitro-6,14-ethenoisomorphinans and their

reduction products

is to be noted that proton H-8 is shielded (1.2 ppm) with respect to the

corresponding H-7 in the 7 -nitro-6 ,l4 -ethenoisomorphinan, which

might be explained by the shielding effect as a result of the proximity of

the benzene ring in the 6 ,14 -etheno adduct. A downfield shift in the

resonance of the vinyl protons H-18 and H-19 of 0.4 and 0.8 ppm respectively was also observed. In conclusion, the structure of this compound is

4,5 -epoxy-3-methoxy-8 -nitro-6 ,14 -ethenomophinan (~ 30 percent).

In order to see if this phenomenon also occurs with the usual dienophiles,

the Diels-Alder reaction of 6-demethoxy-N-formylnorthebaine was carried

out with methyl vinyl ketone. Chromatographic analysis showed only one

major product, and the expected 7 -acetyl-6,14-ethenoisomorphinan could be

isolated in 80-percent yield (figure 8).

The regio- and site-selectivities of the Diels-Alder reaction of morphinan

6,8-dienes have been explained as a combination of steric and electronic

effect. Apparently, the strongly regio-directing properties of the small and

44
FIGURE 7. Diels-Alder reaction of 6-demethoxy-N-formylnorthebaine

with nitroethene

FIGURE 8. Diels-Alder reaction of 6-demethoxy-N-formylnorthebaine with

methyl vinyl ketone

planar nitroethene govern the site selectivity of the Diels-Alder reaction

when the 6-methoxy substituent is absent, giving rise to 8-substituted cycloadducts. Flattening at the nitrogen by replacing the N-methyl group by an

N-formyl group, together with the removal of the 6-methoxyl group, also

makes a-face approaching of nitroethene possible.

Until now the author synthesized the N-formylmorphinan-6,8-dienes by demethylation of the corresponding N-methyl derivatives followed by formylation. However, the demethylation with diethyl azodicarboxylate proceeds

sometimes in low to moderate yields. Therefore, an alternative route was

studied. The author started from N-formylnorcodeine, which was prepared

by treatment of codeine with 2,2,2-trichloroethyl chloroformate in the

presence of potassium hydrogen carbonate, a modification of a known procedure, which effected complete conversion into the carbamate. The latter

45
compound was reduced using zinc dust and, subsequently, formylated with

ethyl formate. Along the lines of the pathway via the 6-O-mesylcodeine

analog, depicted in figure 2, it was found that treatment of the 8 -bromo

intermediate with an excess of potassium tert-butoxide gave at 0 °C, after

1 minute, the desired N-formylmorphinan-6,8-diene. Due to the instability

in strong alkaline medium, careful control of the reaction conditions is necessary. When 6-O-mesyl-N-formylnorcodeine was treated with lithium bromide for a prolonged period, the initially formed 8 -bromo intermediate

rearranged to (+)-(Z)-7-formyl-8,9-dihydro-l-hydroxy-2-methoxy-7H-dibenz[ d,

f]azonine (figure 9), the structure of which was proven by means of a

single-crystal x-ray analysis.

FIGURE 9. (R)(Z)-7-Formyl-8,9-dihydro-l-hydroxy-2-methoxy-7Hdibenz[d,f]azonine

CONCLUSION

Although many Diels-Alder reactions of thebaine and thebainelike morphinandienes have been studied, it is expected that still more reactions can

be discovered, all affording new compounds that may add new data on

structure-activity relationships. The N-formylmorphinans, especially, form

an interesting group of compounds. They are not only useful in synthetic

pathways, but they also can be easily converted into N-substituted

morphinans.

From the data available it is clear that the cycloaddition to 4,5 -epoxymorphinan-6,8-dienes takes place from the -face. Only in the case of the

reaction with nitroethene does a substantial -face addition of approximately

46
30 percent co-occur. Morphinan-6,8-dienes that lack the 4,5 -epoxy ring

give rise to -face addition, as the few examples demonstrate (figure 10).

The latter reaction needs further investigations and the reaction products,

eventually converted into further modified compounds, will constitute new

classes of morphinans.

FIGURE 10. Cycloadditions to morphinan-6,8-dienes

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Prazeres, M.A.; Peters, J.A.; Linders, J.T.M.; and Maat. L. 4,5 -Epoxy-3-

methoxy-8 -nitro-6 ,14 -ethenomorphinan. A novel type Diels-Alder

adduct from 6-demethoxy-17-formylnorthebaine and nitroethene. Recl

Trav Chim Pays-Bas 105:554-555, 1986.

Razdan, R.K.; Portlock, D.E.; Dalzell, H.C.; and Malmberg, C. Synthesis

of -dihydrothebaine. J Org Chem 43:3604-3606, 1978.

Sandermann, W. Diën-Anlagerungsverbindungen des Thebains. Ber Dtsch

Chem Ges 71:648-650. 1938.

48
Schmidhammer, H.; Jacobson, A.E.; and Brossi, A. Chemical and biological study of aromatic oxygenated 6-ketomorphinans. Med Res Rev

3:1-19, 1983.

Schöpf, C.; Von Gottberg, K.; and Petri, W. Uber Thebainmaleinsäureanhydrid, Thebainchinon, Thebainhydrochinon und dessen

Säureumlagerungsprodukt, das Flavothbaon. Liebigs Ann Chem

536:216-257, 1938.

Smith, C.B.; Medzihradsky, F.; and Woods, J.H. Dihydromorphine-peptide

hybrids have mu receptor antagonistic and delta receptor agonistic activity

on the mouse vas deferens and bind with high affinity to opioid receptors

in rat brain. In: Holaday, J.W.; Law, P.-Y.; and Herz, A., eds.

Progress in Opioid Research. Proceedings of the 1986 International

Narcotics Research Conference. National Institute on Drug Abuse

Research Monograph 75. DHHS Pub. No. (ADM)87-1507. Washington,

DC: Supt. of Docs., U.S. Govt. Print. Off., 1986. pp. 189-192.

ACKNOWLEDGMENTS

The management of Diosynth, B.V., Apeldoom, The Netherlands, is

acknowledged for the gift of chemicals. The U.S.A. Committee on

Problems of Drug Dependence, Inc., and Dr. A.E. Jacobson, Chairman,

Drug Testing Program, are acknowledged for the results of the

pharmacological studies.

AUTHOR

Leendert Maat, Ph.D.

Department of Organic Chemistry

Delft University of Technology

Julianalaan 136

2628 BL Delft

THE NETHERLANDS

49
Opioid Antagonists: Structure

Activity Relationships

Dennis M. Zimmerman and J. David Leander

INTRODUCTION

Considerable progress has been made toward understanding the functions of

the various opioid receptors. Three opioid receptors have been well characterized (the mu, kappa, and delta), and substantial evidence has indicated

the existence of other opioid receptor subtypes. Endogenous ligands for

these receptors have been identified, and this has resulted in the realization

that the endogenous opioids and their receptors have important physiological

functions beyond the modulation of pain. One such function may be

immune system modulation.

Much of what is now known concerning the opioid receptor system can be

attributed to extensive structure activity relationship (SAR) studies where

rank correlations between pharmacological potencies and binding affinities

have been carefully compared. In such studies, opioid antagonists have

played important roles. and it is likely that similar pharmacological comparisons will be equally important for the delineation of any endogenous opioid

regulation of the immune system. Therefore, an understanding of the SARs

of the opioid antagonists may be useful in the characterization of any opioid

involvement in immune system regulation.

BACKGROUND

Opioid antagonist activity was first discovered with N-allyl derivatives of

rigid opiate agonists. Nalorphine, the N-allyl derivative of morphine, figure 1, was the first antagonist approved to treat narcotic overdose (Robinson

1974). It was soon discovered that nalorphine was not a “pure” antagonist

but had partial agonist activities, which caused nalorphine to be referred to

as having mixed agonist-antagonist properties (Martin et al. 1976). In addition, following the characterizations of the different opioid receptor subtypes, it was discovered that nalorphine had relatively high affinities for all

three classes of opioid receptors (Magnan et al. 1982).

50
FIGURE 1. Comparison of the molecular structures of morphine,

nalorphine, and naloxone

The first opioid antagonist discovered to have no opioid agonist effects

(zero intrinsic activity) was naloxone (Blumberg and Dayton 1974). Like

nalorphine, naloxone has significant affinity for the mu, kappa, and delta

receptors; however, it has highest affinity for the mu receptor (Magnan

et al. 1982). Compared to other neurotransmitter receptors, it has been difficult to discover opioid antagonists devoid of agonist properties. In opioid

pharmacology, the ability to measure agonist activity of a partial agonist is

test dependent. The most sensitive means of detecting opioid agonist activity is to determine a compound’s ability to block the electronically stimulated contractions of smooth muscles, particularly the isolated guinea pig

ileum and mouse vas deferens tissues (Kosterlitz et al. 1974; Leslie 1987).

Compared to the number of opioid agonists and partial agonists that have

been identified, relatively few opioid antagonists with zero intrinsic activity

are known. It is the SARs of these pure antagonists that are the focus of

this review.

IMPACT OF STRUCTURE ON ACTIVITY

Naloxone is the N-allyl derivative of oxymorphone (figure 2). The

N-cyclopropyImethyl derivative naltrexone is also an opioid antagonist but is

more potent and longer acting than naloxone (Blumberg and Dayton 1974).

The SAR shown in figure 2 for oxymorphone and derivatives is typical of

those observed with other morphine-based structures. There is generally a

loss of intrinsic activity, in an approximate stepwise manner, through replacement of the N-methyl with dimethylallyl and the other groups as

shown. The allyl and cyclopropylmethyl derivatives generally have the lowest relative intrinsic activities, and this relationship is usually found for all

three opioid receptors. With the oxymorphone molecule, these derivatives

have no opioid agonist properties (Blumberg et al. 1967). However, with

other rigid opioid molecules, the N-allyl and cyclopropylmethyl derivatives

often have partial agonist activities. For example, cyclazocine (a benzomorphan derivative) (Eddy and May 1966) cyclorphan (a morphinan

51
R

C H 3

Oxymorphone Agonist

CH2 CH = C

C H 3

C H 3

Partial agonist

C H 2 C=C—H Partial agonist

CH2

Partial agonist

CH2 CH = CH2 Naloxone Antagonist

C H 2

Naltrexone Antagonist

FIGURE 2. Antagonist effects of N-substituted oxymorphone analogs

derivative) (Hellerbach et al. 1966), and LY 113878 (a phenylisoquinoline derivative) (Zimmerman et al. 1988a) (figure 3) are potent, highaffinity, partial agonists. Much is now known concerning the impact of the

various structural features of naloxone or naltrexone on intrinsic activity.

The C-14 hydroxyl substituent of the oxymorphone molecule is essential for

pure antagonist activity. For example, Compound 1, which is the naltrexone analog without a C-14 hydroxyl (figure 3) is a potent opioid partial

agonist (Kosterlitz et al. 1973). Similarly, addition of a hydroxyl

substituent to the same relative position of cyclorphan (figure 3) giving

oxilorphan (originally referred to as BC2605), results in a dramatic loss of

opioid intrinsic activities without having a significant effect on opioid

receptor affinites. Oxilorphan, however, still has significant partial agonist

activities (Kosterlitz et al. 1973; Pachter 1974).

Other structural modifications of rigid opiate structures have led to the

discovery of new opioid antagonists (figure 3). MR2266 (figure 2) a

N-furylmethylbenzomorphan derivative, is a well-characterized opioid antagonist. It has, at times, mistakenly been referred to as a selective kappa

antagonist. However, MR2266 has approximately equal affinity for mu and

52
FIGURE 3. Structural comparisons of various opioid antagonists

kappa receptors. It also has weak opioid partial agonist properties in

smooth muscle bioassays (Magnan 1982). Diprenorphine, an oripavine

derivative, has highly potent antagonist activities. It has at times been referred to as a universal antagonist, with the implication that it is a pharmacologically pure antagonist; however, it is known to have significant kappa

agonist activity (Traynor et al. 1987). WIN 44,441-3 is a potent, nonselective, opioid antagonist devoid of agonist activities (Michne et al. 1978;

Wood 1984). Remarkably, it has a methyl-substituted nitrogen, and replacement of the cyclopentane group at the terminus of the 3-alkanone substituent with other phenyl or other alkyl groups imparts partial agonist activities

to the molecule.

Naltrexone and naloxone have served as precursors for the synthesis of

several novel pure opioid antagonists through structural modification of the

53
C-6 keto group (figure 4). These would include the opioid antagonists

nalmefene (Hahn et al. 1975), naloxonazine (Pastemak and Wood 1986),

naltrindole (NTI) (Portoghese et al. 1988), and norbinaltorphimine (nor-BNI)

(Portoghese et al. 1987). NTI and nor-BNI are recently discovered selective

antagonists of delta and kappa receptors, respectively. Their discovery

represents a significant advance in SARs of the opioid antagonists.

FIGURE 4. C-6 antagonist analogs of naltrexone

Structural alteration of the enkephalins has led to the discovery of the peptide antagonist ICI174864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH; Aib=

aminoisobutytic acid), which has high selectivity for the delta receptor

54
(Cotton et al. 1984). Although it does have weak agonist activity (Cohen

et al. 1986), the selective antagonist properties of ICI174864 have proven

highly useful for investigating delta receptor activities. Its utility in vivo is

limited, however, because it crosses the blood-brain barrier poorly. Recently, the discovery of a somostation analog CTP (D-Phe-Cys-Tyr-D-Trp-LysThr-Pen-Thr-NH2

), a selective mu receptor antagonist, was reported (Shook

et al. 1987). Its selective reversible antagonist properties likely will make it

a useful pharmacological tool.

The authors have reported the discovery of potent opioid antagonist activity

in a series of trans-3,4-dimethyl-4-phenylpiperidines (figure 5) (Zimmerman

et al. 1978). Quite surprisingly, in this series, the antagonist activity was

shown to be a consequence of methyl substitution at the three position of

the piperidine ring. LY99335 was a pure opioid antagonist, while its des-3-

methyl analog, LY25506, was an opioid agonist. With the trans-3,4-

dimethyl-4-phenylpiperidines, antagonist potency is not significantly affected

by replacement of the N-methyl with an N-allyl or a cyclopropylmethyl substituent; however, antagonist activities were significantly increased with

other substitutions at nitrogen. LY117413 is a pure opioid antagonist with

affinities for the opioid receptors comparable to naloxone.

FIGURE 5. Structures of the trans-3,4-dimethyl-4-phenylpiperidine

antagonists

55
Recently, at the Lilly Research Laboratories, the SAR of this 4-phenylpiperidine antagonist series has been further explored with the focus of this

effort being directed to the possible use of an opioid antagonist as an appetite suppressant in the treatment of obesity. An extensive study was undertaken in an attempt to maximize the appetite-suppressant effect for possible

clinical investigation and also to further characterize the opioid receptors

involved in this effect (Zimmerman et al. 1988b). Opioid receptor affinities

were compared to opioid antagonist potencies against mu (morphine) and

kappa (U50,448) analgesia using the mouse writhing test and against

bremazocine-induced diuresis (kappa effect) in rats. Selected compounds

were evaluated for their effects on food and water consumption in meal-fed

obese Zucker rats.

Some 75 different N-substituted analogs were synthesized, and many highly

potent pure antagonists were discovered. All were devoid of opioid agonist

effects, which further confirmed the antagonist pharmacophore nature of the

trans-3,4-dimethyl-4-phenylpiperidine molecule. A few exhibited exceptional potency as appetite suppressants, but, surprisingly, the effects on food

consumption were not well correlated with antagonist activities at either the

mu, kappa, or delta receptors. Compound LY255582 (figure 5) emerged as

having the best overall activity profile (Leander et al. 1988). As an appetite suppressant, it was at least 40 times more potent than naltrexone on an

ED, basis (table 1). Furthermore, the maximum reduction achieved with

LY255582 was significantly greater than that achieved with naltrexone (data

not shown). Neither of these properties could be attributed to an increase

in a particular opioid receptor antagonist activity.

CONCLUSION

In summary, until recently, only a small number of opioid pure antagonists

were available for pharmacological studies. Most of these are nonselective

for the three opioid receptors; however, they still serve as very useful

probes to study possible opioid receptor-mediated effects. Recently, selective antagonists with affinities for the mu, kappa, and delta receptors have

been discovered, and their use offers considerable promise for the further

characterization of the functions of the endogenous opioids and their receptors. Pharmacological comparisons of the 4-phenylpiperidine antagonists as

appetite suppressants indicate that other ways for the characterization of

selective opioid receptor effects may lead to unexpected findings. It may

be reasonable to expect other inconsistent findings in future pharmacological

comparisons, and it is important to realize that we still have only a limited

understanding of the opioid receptor system.

56
TABLE 1. Opioid receptor affinities and antagonist effects of LY255582 compared to naloxone and naltrexone

Opioid Receptor Affinities

Ki-Values (nM)

Antagonism of

Opioid Analgesia

Mouse Writhing Test

AD50

d

; (mg/kg, SC)

Antagonism of Effects on

Kappa Diuresis Food Consumption

in Rats Obese Zucker Rats

3H-NALa 3H-EKCb 3H-DADLc mu kappa AD50

e ED20

f

(mu) (kappa) (delta) (morphine) (U50,488) (mg/kg, SC) (mg/kg, SC)

LY255582 0.41 2.0 5.2 0.015 0.05 0.38 0.05

Naloxone 3.7 66 32 0.08 1.1 3.5 1.4

Naltrexone 0.56 6.0

aNaloxone, using crude membranes from rat brain.

3.9 0.05 0.06 2.5 2.1

bEthylketocycluocine, using crude membranes from guinea pig cortical tissue with fentanyl and DADL added to inhibit binding to mu and delta receptors

cD-Ala*-D-LeuJ-enkephalin, using crude membranes from rat brain.

dDose required for SO-percent reduction in the analgesic response to either morphine (1.25 mg/kg, SC) or U50,488 (2.5 mg/kg, SC).

eDose required to decrease the 5-hour bremazocine-induced (0.08 mg/kg, SC) urination by 50 percent.

fDose required to reduce 4-hour food intake by 20 percent in rats trained to eat daily for 8 hours.
REFERENCES

Blumberg, H., and Dayton, H.B. Naloxone, naltrexone and related noroxymorphones. In: Braude, M.C.; Han-is, L.S.; May, E.L.; Smith, J.P.; and

Villarreal, J.E., eds. Advances in Biochemical Psychopharmacology.

Vol. 8. New York: Raven Press, 1974. pp. 33-43.

Blumberg, H.; Dayton, H.B.; and Wolf, P.S. Analgesic and narcotic

antagonist properties of noroxymorphone derivatives. Toxicol Appl

Pharmacol 10:406, 1967.

Cohen, M.L.; Shuman, R.T.; Osborne, J.J.; and Gesellchen, P.D. Opioid

agonist activity of ICI174864 and its carboxypeptidase degradation

product, LY281217. J Pharmacol Exp Ther 238:769-772, 1986.

Cotton, R.; Giles, M.G.; Miller, L.; Shaw, J.S.; and Timms, D. ICI174864:

A highly selective antagonist for the opioid delta-receptor. Eur J

Pharmacol 97:331-332, 1984.

Eddy, N.B., and May, E.L. Synthetic Analgesics, Part IIb, 6,7-Benzomorphans. Barton, D.H.R., and von Doering, W., eds. New York:

Pergamon, 1966.

Hahn, E.F.; Fishman, J.; and Heilman, R.D. Narcotic antagonists. Carbon6 derivatives of N-substituted noroxymorphones as narcotic antagonists.

J Med Chem 18:259-262, 1975.

Hellerbach, J.; Schnider, O.; Besendorf, H.; and Pellmont, B. In: Barton,

D.H.R., and von Doering. W., eds. Synthetic Analgesics, Part IIa,

Morphinans. New York: Pergamon, 1966.

Kosterlitz, H.W.; Waterfield, A.A.; and Berthoud, V. Assessment of the

agonist and antagonist properties of narcotic analgesic drugs by their

actions on the morphine receptor in the guinea-pig ileum. In:

Braude, M.C.; Harris, L.S.; May, E.L.; Smith, J.P.; and Villarreal, J.E.,

eds. Advances in Biochemical Psychopharmacology. Vol. 8. New York:

Raven Press, 1974. pp. 319-334.

Kosterlitz, H.W.; Waterfield, A.A.; and Berthoud, V. Assessment of the

agonist and antagonist properties of narcotic analgesic drugs by their

actions on the morphine receptor in the guinea-pig ileum. In: Proceedings of the 35th Annual Scientific Meeting of the Committee on Problems

of Drug Dependence, Chapel Hill, NC, 1973. pp. 131-151.

Leander, J.D.; Shaw, W.N.; Mitch, C.H.; and Zimmerman, D.M. Opioid

antagonist and appetite suppresant pharmacology of LY255582.

Abstracts of the International Narcotic Research Conference, ALBI, 1988.

p. 45.

Leslie, F.M. Methods used for the study of opioid receptors. Pharmacol

Rev 39:197-249, 1987.

Magnan, J.; Paterson, S.J.; Tauani, A.; and Kosterlitz, H.W. The binding

spectrum of narcotic analgesic drugs with different agonist and antagonist

properties. Naunyn Schmiedebergs Arch Pharmacol 319:197-205, 1982.

58
Martin, W.R.; Eades, C.G.; Thompon, J.A.; Huppler, R. E.; and

Gilbert, P.E. The effects of morphine- and nalorphine-like drugs in the

nondependent and morphine-dependent chronic spinal dog. J Pharmacol

Exp Ther 197:517-532, 1976.

Michne, W.F.; Lewis, T.R.; and Michalec, S.J. Novel developments of

N-methylbenzomorphan narcotic antagonists. In: Van Ree, J., and

Terenius, L., eds. Characteristics and Functions of Opioids. New York:

Elsevier/North-Holland Biomedical Press, 1978. pp. 197-206.

Pachter, I.J. Synthetic 14-hydroxymorphinan narcotic antagonists. In:

Braude, M.C.; Harris, L.S.; May, E.L.; Smith, J.P.; and Villarreal, J.E.,

eds. Advances in Biochemical Psychopharmacology. Vol. 8. New York:

Raven Press, 1974. pp. 57-62.

Pastemak, G.W., and Wood, P.W. Minireview: Multiple mu opiate

receptors. Life Sci 38:1889-1898, 1986.

Portoghese, P.S.; Lipkowski, A.S.; and Takimori, A.E. Binaltorphimine and

nor-binaltorphimine, potent and selective -Opioid receptor antagonists.

Life Sci 40:1287-1292, 1987.

Portoghese, P.S.; Sultana, M.; Nagase, II.; and Takemori, A.E. Application

of the message-address concept in the design of highly potent and selective non-peptide delta opioid receptor antagonists. J Med Chem 31:281-

282, 1988.

Robinson, F.M. Chemistry of narcotic antagonists of the nalorphine type.

In: Braude, M.C.; Harris, L.S.; May, E.L.; Smith, J.P.; and

Villarreal, J.E., eds. Advances in Biochemical Psychopharmacology.

Vol. 8. New York: Raven Press, 1974. pp. 21-31.

Shook, J.E.; Pelton, J.T.; Lemcke, P.K.; Porreca, F.; Hruby, V.J.; and

Burks, T.F. Mu opioid antagonist properties of a cyclic somatostatin

octapeptide in vivo: Identification of mu receptor-related functions.

J Pharmacol Exp Ther 24:1-7, 1987.

Traynor, J.R.; Corbett, A.D.; and Kosterlitz, H.W. Diprenorphine has

agonist activity at opioid K-receptors in the myenteric plexus of the

guinea-pig ileum. Eur J Pharmacol 137:85-89, 1987.

Wood, P.L.; Pilapil, C.; Thakur, M.; and Richard, J.W. WIN44,441: A

stereospecific and long acting narcotic antagonist. Pharm Res 1:46-48,

1984.

Zimmerman, D.M.; Cantrell, B.E.; Swartzendruber, J.K.; Jones, N.D.;

Mendelsohn, L.G.; Leander, J.D.; and Nickander, R.C. Synthesis and

analgesic properties of N-substituted trans-4a-atyldeathydroisoquinolines.

J Med Chem 31:555-560. 1988a.

Zimmerman, D.M.; Mitch, C.M.; Mendelsohn, L.G.; Shaw, W.;

Cantrell, B.E.; Reel, J.; Snoddy, J.; and Leander, J.D. Structure activity

relationships of the trans-3,4-dimethyl-4-pheuylpiperidine antagonists: The

discovery of the potent appetite suppressant LY255582. Abstracts of the

International Narcotic Research Conference, ALBI, 1988b. p. 154.

Zimmerman, D.M.; Nickander, R.; Horng, J.S.; and Wong, D.T. New

structural concepts for narcotic antagonists defined in a 4-phenylpiperidine

series. Nature 275:332-334, 1978.

59
AUTHORS

Dennis M. Zimmerman, Ph.D.

J. David Leander, Ph.D.

Lilly Research Laboratories

Eli Lilly and Company

Lilly Corporate Center

Indianapolis, IN 46285

60
Effect of Fluorine Substitution on

the Anti-HIV Activity of

Dideoxynucleosides

Victor E. Marquez

INTRODUCTION

Fluorine substitution has been extensively investigated in drug research and

biochemistry as a means of enhancing biological activity and increasing

chemical or metabolic stability (Goldman 1969; Ciba Foundation Symposium 1972; Filler 1979; Filler and Naqvi 1982; Bamette 1984). The most

important factors considered for the synthesis of fluorine-containing compounds are: (1) the relative small size of fluorine, which is comparable to

that of hydrogen; (2) the powerful electron-withdrawing properties of fluorine; (3) the increased stability of the carbon-fluorine bond relative to the

carbon-hydrogen bond; and (4) the increased lipophilic character of fluorine

relative to hydrogen.

In terms of size, fluorine has a small van der Waals radius (1.35 Å) that

closely resembles that of hydrogen (1.20 Å) (Pauling 1960). Therefore,

substitution of a hydrogen by fluorine in a molecule is expected to cause

minimal steric perturbations with respect to the molecule’s mode of binding

to a receptor or an enzyme. In contrast, since fluorine is the most electronegative of the elements (Pauling 1960), its powerful electron-withdrawing

properties can profoundly affect chemical reactivity. When attached to a

reaction center, fluorine is a moderately good leaving group, and when

placed near a reaction center, it can dramatically change chemical reactivity

at that center via its strong inductive effect (Chambers 1973).

The combined effect of small size and powerful electron-withdrawing properties, for example, has been successfully exploited in the steroid field in

optimizing the corticoid activity of 11-ß-hydroxy steroids by increasing the

acidity of the 11-ß-hydroxyl group as in 9- -fluoro-ll-ß-hydroxyprogesterone (1, figure 1) (Wettstein 1972).

61
FIGURE 1. Structures of some fluorinated bioactive molecules

The higher energy of the C-F bond (108 kcal/mol) relative to that of C-H

(104 kcal/mol) is another important property that is responsible for the

increased metabolic inertness of some fluorine-containing compounds. A

classical example in this category is the antitumor drug 5-fluorouracil (2,

figure 1). The chemotherapeutic utility of 2 is due to its conversion to the

active metabolite 5-fluoro-2’deoxyuridylate (3, figure 1), which behaves as

a powerful inhibitor of thymidylate (dTMP) synthetase. This inhibition is

caused by the unreactive fluorine at C-5, which blocks the ensuing addition

of formate after a reversible complex between 3 and the enzyme is formed

(Pogolotti and Santi 1977).

As mentioned previously, when the fluorine atom is attached to a reactive

center, it undergoes facile elimination of fluoride, and the resulting electrophilic species can irreversibly inhibit enzymatic activity through the formation of a covalent bond to the enzyme (Johnston et al. 1979; Muehlbacher

and Poulter 1985). For example, the glycosylase inhibitor 2-deoxy-2-fluoroß-D-glucosyl fluoride (4, figure 1) undergoes rapid elimination of the glycosylic fluorine to give an oxocarbonium ion capable of reacting very rapidly

with the enzyme (Withers et al. 1988). The resulting 2-deoxy-2-fluoroglycosyl-enzyme complex is then stabilized by the fluorine substituent at

C-2. The combination of these two effects leads to enzyme inactivation

through the generation of a more stable intermediate that traps the enzyme

and prevents it from completing the final transformation (Withers et al.

1988). In other cases, the reactive center beating the fluorine atom can be

unmasked at the active site by the normal enzymatic process, and the resulting inactivation of the enzyme is referred to as “suicide inhibition.” The

62
well-known antimetabolite ß-fluoro-D-alanine (5, figure 1) is a good example of a “suicide inhibitor” that contains fluorine (Kollonitsch 1982).

After the initial “normal,” enzymatic-catalyzed reaction between ß-fluoro-Dalanine and pytidoxal phosphate, the resulting intermediate aldimine loses

fluoride ion and generates an electrophilic species capable of alkylating the

bacterial enzyme (alanine racemase) irreversibly (Kollonitsch 1982).

Another important property of fluorine, which is directly related to its high

electronegativity, is that it can function effectively as a hydrogen bond

acceptor (figure 2). This property of fluorine, allowing it to operate as a

substitute for oxygen in a hydroxyl function, has not been extensively

studied in drug design. In the area of carbohydrate chemistry, however, the

systematic replacement of hydroxyl functions by fluorine has permitted the

accurate mapping of important hydrogen-bonding interactions with the various receptors involved in the transport of sugars (Bamctt 1972). As seen in

table 1 (Barnett 1972), the similar size and electronegativity of fluorine and

oxygen suggest that fluorine may indeed be better compared as an isostere

of oxygen rather than of hydrogen because of the similarity of the C-F and

C-OH bonds in polarity and bond length. It is possible, therefore, that

fluorinated analogs of hydroxyl-containing compounds will retain biological

activity or behave as powerful inhibitors of the enzymes that process the

nonfluorinated substrates. All of these properties are potentially exploitable

in drug design.

FIGURE 2. Possible hydrogen bonds between protein and drugs

The various properties of the C-F bond discussed above have provided the

necessary incentive to investigate the effects of fluorine substitution on the

antiretroviral activity of dideoxynucleosides.

63
TABLE 1. Comparison of the size and electronegativity of some elements

Element

Bond Length Van der Waals

(CH3-X) (Å) radius (Å)

Total

(Å) Electronegativity

H 1.09 1.20 2.29 2.1

F 1.39 1.35 2.74 4.0

0 (in OH) 1.43 1.40 2.83 3.5

CL 1.77 1.80 3.57 3.0

S (in SH) 1.82 1.85 3.67 2.5

Source: Table based on data in Barnett 1972.

DIDEOXYNUCLEOSIDES AS ANTIRETROVIRAL AGENTS

The unique mode of replication of retroviruses, requiring virion-encoded

reverse transcriptase (RT), provides a virus-specific target for effective

chemotherapy. Dideoxynucleosides have been shown to inhibit the replication of human immunodeficiency virus (HIV) and other retroviruses by

inhibiting RT (Mitsuya and Broder 1986; Dahlberg et al. 1987). The inhibition is believed to result from the incorporation and consequent chain

termination effect of the dideoxynucleosides, which must be metabolically

activated from the monophosphate to the triphosphate level by cellular

rather than viral enzymes (Johnson et al. 1987; Johnson et al. 1988).

The anti-HIV activity of these compounds is normally determined in vitro

by measuring the increase in cell viability after cells infected with HIV

have been treated with various concentrations of the test drugs. In one

experimental approach, the anti-HIV activity is measured in MT-4 cells, and

the ED50, defined as the concentration of the test agent capable of conferring 50-percent protection to HIV-infected cells, is reported along with the

equivalent parameter for cytotoxicity (CD50) (Pauwels et al. 1987; Baba et

al. 1987). Another protocol uses an immortalized helper-inducer T-cell

clone (ATH8). and the results are normally expressed in percent protection

vs. percent cytotoxicity for a given dose (Mitsuya and Broder 1986; Kim et

al. 1987). Aside from the inherent difficulties in comparing data from different laboratories that use different assay systems, the in vitro tests give an

end-point result that encompasses a complex sequence that spans biochemical events from activation (three cellular enzymes) to interaction with viral

RT. Consequently, drawing clear-cut structure-activity conclusions from

these studies is not possible unless one looks at each step individually by

preparing and testing each metabolite. Despite these shortcomings, the in

vitro assay is the more practical and economical method for rapid screening.

64
EFFECT OF FLUORINE SUBSTITUTION IN

DIDEOXYNUCLEOSIDES

This problem can be divided into two general areas dealing with the effects

of fluorine substitution on either the sugar or the aglycon moieties of the

dideoxynucleosides. Each of these areas, in turn, could be further divided

into pyrimidines and purines.

Sugar-Modified Dideoxynucleosides

In looking at the activation process from the nucleoside level to the triphosphate form (figure 3), it is possible to envision that a fluorine substituent at C-3' on the sugar might be a hydrogen-bonding equivalent of a

hydroxyl group (figure 2). In addition, one could anticipate that for this

3'-fluoro substituent to be an effective isostere of oxygen, it should have

a-stereochemistry. In the case of a 2'-fluoro substitution, however, in

which fluorine functions as a steric isostere of hydrogen, it is more difficult

to predict which stereochemical orientation will preserve substrate recognition by the different enzymes involved in the activation process.

FIGURE 3. Nucleoside activation steps

Pyrimidines. The biological evaluation of some of the first fluorinesubstituted pyrimidine dideoxynucleosides (Herdewijn et al. 1987; Balzarini

et al. 1988) confirmed the anticipated results mentioned above. When the

anti-HIV activities of 3'- -fluoro-2',3'-dideoxythymidine (7, figure 4) and

2'-ß-fluoro-2',3'-dideoxythymidine (8, figure 4) were compared to those of

2',3'-dideoxythymidine (ddTh, 9, figure 4) and the prototype drug used clinically against AIDS, AZT (3'-azido-2',3'-dideythymidine, 6, figure 4), the

results confirmed the importance of the 3'- -hydroxyl group at some stage

of the activation process or at the level of interaction with RT. In MT-4

cells, isosteric fluoro and azido groups both appeared to be equipotent,

albeit the fluoro analog was more toxic. In the ATH8 system, however, the

two compounds were nearly equipotent and equitoxic (Herdewijn et al.

65
FIGURE 4. Anti-HIV activity (MT-4 ceils) of AZT vs. fluorinated and

nonfluorinated thymine nucleosides

1987). This near equivalence between fluoro and azido groups is not too

surprising since the azido group is often considered to be a pseudohalogen

(Treinin 1971). At the 2'-position, however, fluorine substitution above the

plane of the sugar ring destroyed activity (figure 4). A more striking effect

at 3' was observed for the dideoxyuridine case (Van Aerschot et al. 1988).

As seen in figure 5, the compound with the 3'-fluoro substituent in the

-configuration (11, figure 5) displayed a 5,000-fold increase in anti-HIV

potency over the parent unsubstituted compound (10, figure 5) (Van

Aerschot et al. 1988).

When the pyrimidine base was cytosine, both test systems (ATH8 and

MT-4) revealed that the same pattern of fluorine substitution on dideoxycylidine (ddC, 12, figure 6) was in general detrimental and with opposite

effects to those observed in the dideoxyuridine and dideoxythymidine cases

(Herdewijn 1987; Van Aerschot et al. 1988). This is probably a reflection

of the fact that the two drugs are activated by different cellular kinases,

deoxycytidine kinase and thymidine kinase, respectively (Baizarini et al.

1988). In this instance (figure 6), the 2'-ß-fluoro substituted analog (14,

figure 6) was slightly superior to the 3'- -fluoro compound (13, figure 6)

(Van Aerschot et al. 1988). When two fluorine atoms in the -configuration were simultaneously introduced at the 2' and 3' positions (compounds

15 and 16, figure 7). there was no difference between the dideoxyuridine

and dideoxycytidine series, and both classes of compounds were completely

devoid of anti-HIV activity (Van Aerschot et al. 1988). Compounds with

the substitution pattern of 17 and 18 (figure 7) have not yet been reported.

66
10 11

ED50 (μM) CD50 (μM) SI

10 210 >625 > 3

11 0.04 16 400

FIGURE 5. Anti-HIV activity (MT-4 cells) of fluorinated vs.

nonfluorinated uracil nucleosides

12 13 14

ED50 (μM) CD50 (μM) SI

12 0.3 40 120

13 16 26 1.6

14 9.8 117 12

FIGURE 6. Anti-HIV activity (MT-4 cells) of fluorinated vs.

nonfluorinated cytosine nucleosides

67
inactive

not reported

FIGURE 7. Structures of polyfluorinated nucleoside analogs

It is worth noting that, contrary to the pronounced increase in potency

brought about by fluorine substitution on an sp3-hybridized carbon, as observed with compounds 7 and II, substitution at the same relative position

on an sp

2

-hybridized carbon was detrimental to the anti-HIV activity.

Fluorine substitution on the very potent 3'-deoxy-2',3'-didehydrothymidine

(19, D4T) caused a 1,000-fold reduction in potency (compound 20, figure 8)

(Van Aerschot et al. 1988).

68
19 2 0

ED50 (μM) CD5 0 (μM) SI

19 0.01 1.2 120

2 0 10-50 232 4.5-2.3

FIGURE 8. Anti-HIV activity (MT-4 cells) of fluorinated vs.

nonfluorinated D4T

Purines. Among the dideoxypurine nucleosides, 2',3'-dideoxyadenosine (21,

figure 9) is a compound in clinical trial that shows potent activity and

better selectivity than AZT in the lymphocyte ATH8 in vitro test system

(Johnson et al. 1988). In addition to the changes that fluorine substitution

would be expected to have on the activation of this substance by its two

operational kinases (adenosine kinase and deoxycytidine kinase), the possibility of altering the chemical properties of this drug through fluorine

substitution became very attractive. Although all dideoxynucleosides are

hydrolytically less stable than their patent nucleosides, this difference

reaches staggering proportions in the case of dideoxyadenosine, which

undergoes acid-catalyzed cleavage of the glycosylic linkage at a rate 40,000

times faster than adenosine (York 1981). The half-life of this drug at pH 1

and 37 °C was determined to be 35 seconds (Marquez et al. 1987). Clearly, this precludes any intended use of this drug by the oral route.

69
21, W=X=Y=Z=H, R=NH2

22, W=X=Y=Z=H, R=OH

23, W=X=Z=H, Y=F, R=NH2

24, W=Y=Z=H, X=F, R=NH2

25, W=Y=Z=H, X=F, R=OH

26, W=X=Y=H, Z=F, R=NH2

27, X=Y=Z=H, W=F, R=NH2

FIGURE 9. Structures of fluorinated and nonfluorinated dideoxy purine

nucleosides

Since the acid-catalyzed hydrolysis of purine nucleosides is thought to proceed by an Al mechanism in which the protonated nucleoside dissociates in

the rate-controlling step to a glycosyl carbonium ion and free purine, it was

felt that introduction of an electronegative fluorine atom adjacent (C-2') to

the reaction center should destabilize the resulting oxocarbonium ion and

decrease the rate of hydrolysis (figure 10).

The syntheses of both - and ß-fluoro dideoxyadenosine analogs (23 and

24, figure 9) permitted confirmation of this hypothesis by showing that both

compounds were completely inert to acid hydrolysis under the same conditions that rapidly hydrolyzed dideoxyadenosine (Marquez et al. 1987).

Although minimal steric changes were expected to occur after fluorine substitution in both isomers, it was impossible to predict beforehand the effect

that the stereochemical orientation of the fluorine would have on biological

activity. Testing of both drugs in the ATH8 system indicated that only the

ß-fluoro analog 24 was an effective anti-HIV agent, virtually indistinguishable from its parent compound, dideoxyadenosine (Marquez et al. 1987).

Another important consequence of fluorine substitution and the increased

chemical stability of the glycosylic bond was manifested in the enzymatic

70
FIGURE 10. Mechanism of acid hydrolysis of dideoxynucleosides and acid

stabilization rationale

inertness of the dideoxyinosine metabolite 25 towards cleavage by purine

nucleoside phosphorylase (Marquez et al., unpublished) (figure 11). Dideoxyinosine (22), as well as its fluorinated analog 25, is formed from the

parent adenosine nucleosides via adenosine deaminase. However, such

metabolic transformation has little or no bearing on the resulting anti-HIV

activity, since both dideoxyadenosine and dideoxyinosine, as well as the

corresponding 2'-ß-fluoro analogs, are equally effective against HIV. On the

other hand, resistance to purine nucleoside phosphorylase is a desirable

FIGURE 11. Metabolism of 2’- -fluoro-dideoxypurines

feature, since this enzyme is capable of depleting useful levels of anti-HIVactive dideoxyinosine. Chemically synthesized 2'-ß-fluorodideoxyinosine

(25) was shown to be completely resistant to purine nucleoside phosphorylase upon incubation with the enzyme (Marquez et al., unpublished). In

addition, the anti-HIV activity of 2'-ß-fluorodideoxyinosine (25) was identical to that of its progenitor compound 24 and dideoxyadenosine (figure 12).

In this system, a 10-μM concentration of the drug was able to provide com71
plete protection against HIV without significant cytotoxicity, as judged by

the equal height of the bars representing infected vs. noninfected cells,

respectively (figure 12).

FIGURE 12. Inhibition of the cytopathic effect of HIV by 2’- -flurorodideoxyinosine (25) in ATH8 cells

Compounds 23 and 24 (figure 9) were also independently synthesized and

tested in the MT-4 cell system (Herdewijn et al. 1987). In this system, the

same difference was observed: the 2'-ß-fluoro isomer 24 showed potent

anti-HIV activity, whereas the 2'- -fluoro isomer 23 was completely inert.

The fact that in MT-4 cells dideoxyadenosine appeared to be slightly more

potent than the active fluoro isomer 24 indicates that there are differences

in sensitivity between the ATH8 and MT-4 test systems (Baba et al. 1987).

In the case of the two possible 3'-fluoro isomers, 26 and 27 (figure 9), the

results in the MT-4 assay were consistent with the premise that the 3'-

fluoro substituent appears to be a good isostere for the hydroxyl oxygen of

2'-deoxyadenosine (Herdewijn et al. 1987). This compound (26), although

72
approximately one-eighth as potent as the parent dideoxyadenosine, coofirmed the previously observed trend in the pyrimidine system for a preferred -fluoro stereochemistry at the 3'-position. The 3'-ß-fluoro isomer 27

was totally devoid of activity (Herdewijn et al. 1987).

Aglycon-Modified Dideoxynucleosides

Pyrimidines. The most obvious and chemically accessible position for substitution on a pyrimidine base is at carbon 5. In the uracil series, however,

fluorine substitution was considered unwise, since cleavage of the glycosylic

bond in the resulting compound, 5-fluorodideoxyuridine, would have been a

source of highly cytotoxic 5-fluorouracil. However, because dideoxycytidine, which is one of the most potent known anti-HIV agents, was not a

substrate for cytidine deaminase (Kelley et al. 1987), it was decided to synthesize and investigate the anti-HIV activity of analogs in the cytidine series

(Kim et al. 1987). In an effort to increase the lipophilic character of dideoxycytidine, the 5-fluorodideoxycytidine analog 28 (figure 13) was synthesized along with other members of a small series that included the more

lipophilic analogs, methyl- and bromo-substituted derivatives (29 and 30,

figure 13).

Dose (μM) % Protection % Cytotoxicity

12, X=H 5 100 0

28, X=F 0.5 100 0

29, X=Br >200 <10 10

30, X=Me >100 <20 2 0

FIGURE 13. Anti-HIV activity (ATH-8 ceils) of 5-substituted cylosine

nucleosides

Active compounds with increased lipophilic character appeared important

enough, since it is known that HIV has a selective tropism for certain

cells of the central nervous system (CNS), and a wide variety of CNS

73
complications, including dementia, can result from this CNS infection (Price

et al. 1981; McArthur 1987). With the parent compound, dideoxycytidine,

CSF:plasma ratios of only 3 to 5 percent are achieved in rhesus monkeys,

and its poor CNS penetration is suspected to be related to its strong hydrophilic character, as indicated by its negative log P value (-1.26) in the

octanol/water system (Kelley et al. 1987; Cheung 1988). Among the 13

AIDS-related nucleosides recently measured for partition coefficient values

by the National Cancer Institute, AZT was the single compound that was

lipophilic, but only barely so (log P=+0.04) (Cheung 1988). Unfortunately,

the more lipophilic methyl- and bromo-substituted dideoxycytidines proved

to be inactive against HIV in the ATH8 assay system (Kim et al. 1987).

On the contrary, the 5-fluorodideoxycytidine analog was just as potent as

the parent dideoxycytidine and afforded complete protection against HIV at

a dose of 0.5 μM (figure 13) (Kim et al. 1987). However, the modest increase in lipophilicity afforded by this compound (log P=-1.05) did not

change the CSF:plasma ratio, which was virtually the same as for dideoxycytidine (Kelley et al., unpublished data). In this instance, therefore, the

fluorine atom in the pyrimidine ring behaves as a simple isostere of hydrogen, in which its stronger electronegativity appears not to have any effect,

positive or negative, on the resulting anti-HIV activity of dideoxycytidine.

Purines. Only one published report addresses halogen substitution in the

aglycon of a didcoxy purine (Haertle et al. 1988). The main thrust behind

this project was provided by the finding that 2-chloro-2'-deoxyadenosine was

not significantly deaminated in human cells, while at the same time being

efficiently phosphorylated in T lymphocytes. As discussed previously, dideoxyadenosine is catabolized first by adenosine deaminase, and the resulting product dideoxyinosine is further cleaved and rendered inactive through

the action of purine nucleoside phosphorylase. The 2-haloderivatives (compounds 31 through 33, figure 14) were found to be extremely poor substrates for adenosine deaminase, and the corresponding 2-halodideoxyinosine

analogs were generated only after prolonged incubation with the enzyme

(Haertle et al. 1988). Prevention of the deamination reaction was critical in

2-halodideoxyadenosine series because the corresponding 2-halodideoxyinosine congeners had significantly less anti-HIV activity (Haertle et al.

1988). This is in sharp contrast with the 2-unsubstituted didcoxyadcnosine

and dideoxyinosine nucleosides, which are virtually equal in their anti-HIV

activity (Ahluwalia et al. 1987; Johnson et al. 1988). Inhibition of HIV

infection, measured in cultured MT-2 lymphoblasts by the reduction in formation of syncytia, was achieved by 2-fluoro-, chloro- and bromodideoxyadenosine nucleosides at 10-μM concentration (figure 14) (Haertle et al.

1988). At the same concentration, these drugs also blocked HIV replication

in CEM T lymphocytes, as measured by a p24 (gag) antigen capture assay.

Cytotoxicity was observed generally at concentrations twofold higher than

those required to inhibit HIV-induced syncytia formation (Haertle et al.

1988). However, the observed trend was that with increasing size of the

halogen substituent, cytotoxicity was reduced without a concomitant

74
reduction of the anti-HIV activity. Another important difference, which was

independent of the type of halogen substituent, was the failure of the 2-

halodideoxyadenosine derivatives to inhibit HIV replication in deoxycytidine

kinase-deficient CEM mutants (Haertle et al. 1988). By contrast, dideoxyadenosine exerted equivalent anti-HIV activity in wild-type CEM cells as

well as in the deoxycytidine kinase-deficient variant. This would indicate

that dideoxyadenosine is also a substrate for adenosine kinase as well as for

deoxycytidine kinase (figure 14). In summary, introduction of a 2-halo substituent into the purine aglycon prevents catabolism by adenosine deaminase

but limits the activation of the drugs by eliminating substrate recognition

from adenosine kinase. The 2-halodideoxyadenosines are also more toxic

than the parent dideoxyadenosine against T cells. The order of toxicity is

F>Cl>Br (Haertle et al. 1988). These differences could be due in part to

the release of toxic 2-halo-adenine bases from the very unstable

dideoxynucleosides.

HIV-induced

Syncytia Dose dCK- dCK+

21, X=H 0 10 μm + +

31,

32,

X=F 0 10 μM

10 μM

- +

X=CI 0 - +

33, X=Br 0 10 μM - +

FIGURE 14. Anti-HIV activity (MT-2T cells) of 2-substituted adenine

nucleosides

CONCLUSION

The most salient feature of fluorine as a substituent on the sugar moiety of

active anti-HIV dideoxynucleosides is that it can effectively function as an

isostere of oxygen or hydrogen, depending on its position and stereochemical orientation on the dideoxyribose moiety. When the aglycon base is

75
uracil or thymine, fluorine is an excellent isostere of the oxygen atom of

the 3'- -hydroxyl function. This hydroxyl group appears to be essential for

enzymatic recognition at some critical stage towards the activation of these

compounds by the various kinases. This effect of fluorine is significantly

less, and in some cases detrimental to anti-HIV activity, when the aglycon

bases are cytosine or adenine. In these instances, it appears that the

3'- -hydroxyl function has a less critical role during the enzymatic activation that occurs by way of different kinases. When the fluorine is substituted at the 2'-position, only the ß configuration appears to be compatible

with anti-HIV activity. This effect appears to be more pronounced in the

dideoxypurine case, where the fluorine atom brings an additional stability to

the glycosylic bond towards chemical or enzymatic cleavage. An a-fluorine

at the 2'-position appears less desirable, since it will make these molecules

look more like ribosides. As a result, all 2'- -fluoro-substituted dideoxynuclecsides are inactive against HIV. Substitution by fluorine at various

positions of the aglycon moiety of dideoxycytidine and didcoxyadenosine

nucleusides does not appear to destroy activity, and only minimal changes

are observed with respect to the parent compounds. Clearly, in these situations, fluorine appears to be functioning as an isostere of hydrogen.

In summary, the most important effect of fluorine in anti-HIV-active dideoxypurine occurs when the fluorine is substituted at the C-2'-ß position.

Particularly for the purine case, it increases the stability of these drugs

without compromising biological effectiveness. This increase in chemical

and enzymatic stability offers many practical advantages for the development of effective long-lasting oral forms of this important class of antiviral

agents.

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78
AUTHOR

Victor E. Marquez, Ph.D.

Laboratory of Medicinal Chemistry

National Cancer Institute, NIH

Bethesda. MD 20892

79
Opiate Antagonists and Anti-HIV

Agents

Elliot F. Hahn

INTRODUCTION

It is unfortunate but true that most drugs of abuse were originally introduced as therapeutically useful agents that were intended to relieve and not

to add to an individual’s ills. In fact, clinical practice still regards many of

these abused agents as valuable and essential tools. With the diversion of

drugs to illicit use, it was recognized that opiates such as morphine impact

on the immune system, since immunological deficits were commonly seen

in opiate addicts.

The effects and some of the beneficial properties of the opium poppy were

known to man long before medical records were established. Egyptian findings indicate that opium was used to calm children, and other discoveries

described the early use of opium in some form as a source of relief from

many types of aches, pains, and mental disorders. Aside from its medical

applications, the poppy was also used (abused) in social settings, where it

was either eaten or smoked.

Although the pharmacological properties of the poppy were recognized for

centuries, an understanding of the nature of opium activity did not begin

until the early 19th century, when the principal constituents of the resin of

the poppy were isolated. These included the clinically useful compounds

morphine and its 3-methyl ether, codeine. Another important component

was identified as thebaine, which was shown to be medically useless because of its extreme toxicity. However, the value of thebaine resided in its

transformation products, which include both opiate agonists, such as oxymorphone and oxycodone, and the pure opiate antagonists. The discovery

of this latter type of agent, of which there are few, represents one of the

most interesting developments in research associated with the opiates. By

definition, opiate antagonists are compounds that are able to block or

reverse the pharmacological effects of the opiate analgesics. The first

observation of this type of drug was made in 1915. when N-allylnorcodeine

80
was shown to antagonize the respiratory depressant effects of morphine

(Pohl 1915). It was 30 years later that N-allylnormorphine (nalorphine,

Nalline) was made and its antagonistic action confirmed (Hart 1943). It

was not until 1951 that this property was clinically utilized, when Eckenhoff

demonstrated that nalorphine was an antidote for morphine poisoning in

man (Eckenhoff et al. 1951). The discovery of these properties of nalorphine stimulated an intensive search for other such agents. The key to

opiate antagonist activity appeared to reside with the substituent on the

piperidine ring nitrogen (Winter 1957). Pharmacological testing showed that

compounds containing a three-carbon chain, such as allyl or cyclopropylmethyl, displayed antagonist properties (Jacobson et al. 1970; Jacobson

1977). Naloxone was the first pure opiate antagonist to be synthesized

(Lewenstein and Fishman 1966; Jasinski et al. 1967). More recently, naltrexone has become clinically available (Martin et al. 1973). The rest of

the vast majority of other derivatives that were synthesized showed varying

degrees of agonist activity.

Approximately 15 years ago, the author was involved in a research program

to synthesize pure opiate antagonists that would exhibit greater potency and

a longer duration of action than naloxone. He was also interested in a

compound that would be orally active, since the possible use of pure opiate

antagonists as therapeutic agents in the treatment of opiate addiction was

being advocated. Initial studies (Linder and Fishman 1973) focused on the

preparation of various esters at C-3 and C-6 of the molecule (table 1).

Although an increase in potency was observed for a number of derivatives,

no significant oral bioavailability was noted.

TABLE 1. Biological activity of esters of naloxone

Compound

Relative

Intravenous (IV)

Potency

Naloxone 1.0

3-Acetate 2.0

14-Acetate 1.5

3,14-Diacetate 0.66

3-Sulfate 0.22

14-Sulfate 0.10

3,14-Disulfate 0.09

3-Sulfate,l4-Acetate 0.25

3-Cinnimate 1.8

3-Nicotinate 1.3

3-Phosphate 0.53

81
The C-6 carbonyl group in the naloxone molecule lends itself readily to

modification, and it appeared reasonable that structure-activity correlations

derived at this position for opiate agonists might also translate to the

antagonists. Replacement of the C-6 carbonyl by methylene resulted in

morphine and codeine analogs up to 75 times more potent than the parent

compound (Chadha and Rapoport 1957; Okun and Elliott 1958). Interestingly, this increase in potency was accompanied by a decrease in duration

of action except in the 14-hydroxy series of compounds. Both naloxone

and naltrexone underwent a Wittig reaction with triphenyl phosphonium

methylide to yield the respective methylene analogs in greater than 90 percent yield (Hahn et al. 1975). Table 2 (Hahn et al. 1975) shows the relative potencies of a series of C-6 derivatives of naloxone and naltrexone as

determined in the hot plate and tail clip tests. The methylene derivatives of

both naloxone and naltrexone (nalmefene) exhibited significant increases in

potency relative to the parent compounds.

TABLE 2. Mouse hot plate and tail clip assay

C-3

Substitution Oral

Antagonist

C-6 C-14 N Potency

OH =O OH -CH2CH=CH2 1.0

OH =O OH -CH2-C3H5 10.0

OAc =O OH -CH2CH=CH2 2.0

OH =O OAc -CH2CH=CH2 0.5

OAc =O OAc -CH2CH-CH2 1.0

OH CH2 OH -CH2CH=CH2 1.0

OH CH3···OH OH -CH2CH=CH2 5.0

OAc XH3 OH OH -CH2CH=CH2 2.0

OH CH3

···OH OH -CH2

-C3H5 12.0

OH =CH2 OH -CH2CH=CH2 1.5

OAc =CH2 OH -CH2CH=CH2 3.0

OH =CH2 OH -CH2

-C3H5 100

OAc =CH2 OH -CH2

-C3H5 50

82
The effect of spatial orientation of the C-6 oxygen substituent as a determinant of agonist and antagonist potency was examined using the guinea

pig ileum longitudinal muscle assay (Ronai et al. 1977). Table 3 indicates

that, if the substituent is alpha to the plane of the molecule, an enhancement of agonistic properties is observed. In contrast, when the substituent

at C-6 is beta, no agonist component is introduced. The 6-methylene derivatives that exhibit similar stereochemistry to that of the parent keto compound also are devoid of agonist activity and have increased potency

(table 3).

TABLE 3. Kinetic parameters of naloxone, naltrexone, and their 6-alphaand 6-beta-hydroxy derivatives on the longitudinal muscle

strip of guinea pig ileum

Compound ID50 (nM) Ke (nM)

Naloxone 3.9±0.5

(n=7)

6-Beta-Hydroxy

naloxone

6.3±0.9

(n=7) (n=3)

6-Alpha-Hydroxy

naloxone

346.5±46.1 9.9±1.6

(n=4) (n=3)

Naltrexone 71,433 0.8±0.1

(n=3) (n=4)

6-Beta-Naltrexol

(n=4)

1.6±0.1

(n=4)

68-Alpha-Naltrexol 33.7±3.8 1.2±0.2

(n=4) (n=4)

Further evaluation of nalmefene was carried out to establish its pure opiate

antagonist character. Results from both in vivo and in vitro studies indicated that nalmcfene was without agonist properties. These results included

the following:

(1) No signs characteristic of morphine were produced on injection into

drug-naive animals (Heilman et al. 1976).

(2) It was inactive in the mouse acetic acid writhing test at doses of

100 mg/kg subcutaneously (SC) (Heilman et al. 1976).

83
(3) It did not depress the contraction of coaxially stimulated guinea pig

ileum muscle (Nash et al. 1984).

(4) Nalmefcne had a sodium index of less than 1.0 at the opiate receptor

in rat brain membranes (Michel et al. 1984).

The binding of nalmefene to opiate receptors in rat brain membranes was

also tested (Michel et al. 1984). Nalmefene displaced the radiolabeled

prototype ligands dihydromorphine (mu), D-Ala-D-Leu enkephalin (delta),

and ethylketocyclazocine (kappa) with IC50s in the low nanomolar range.

As can be seen in table 4, nalmefene was more effective at inhibiting

agonist binding at central kappa sites than either naloxone or naltrexone.

TABLE 4. Relative affinities to the mu, delta, and kappa

mu delta kappa

Naloxone 0.85 0.06 0.09

Naltrexone 0.77 0.13 0.10

Nalmefene 0.67 0.09 0.24

A comparison of the potency of nalmefene and naloxone after either parenteral or oral administration is shown in table 5 (Baker Cummins Pharmaceuticals’ nalmefene information for investigators). Nalmcfene was more

effective in reversing the effects of an ED80 dose of morphine after both

SC and IV administration. Oral nalmefene (0.4 mg/kg) antagonized this

dose of morphine 3 hours after administration. In contrast, oral naloxone

(up to 5 mg/kg) was inactive.

TABLE 5. Narcotic antagonist activity of nalmefene and naloxone

Compound Procedure AD50* (mg/kg) Time**

Nalmefene Mouse tail flick 0.004 SC

Naloxone Mouse tail flick 0.03 SC

Nalmefene Mouse tail flick 0.2 PO

Nalmefene Rat tail flick 0.008 IV

Naloxone Rat tail flick 0.016 IV

Nalmefene Rat tail flick 0.4 PO

Naloxone Rat tail flick Inactive at 5.0 PO

*Dose to get 50 percent antagonism of an ED80 dose of morphine

**Time from administration of narcotic antagonist.

20 min

20 min

20 min

1 5 min

15 min

3 hr

3 hr

84
In clinical studies, nalmefene was shown to prevent respiratory depression

induced by fentanyl in a dose-dependent manner (figure 1) (Gal and Difazio

1986). In another study, nalmefene was compared to naloxone and placebo

in reversing the effects of meperidine (figure 2) (Barsan et al., 1989).

Nalmefene was shown to be effective through the course of the study

(4 hours), while naloxone, after about 45 minutes, was no longer significantly different from placebo.

FIGURE 1. Nalmefene prevents respiratory depression by fentanyl

*p<.05, significant difference from placebo treatment.

**p<.01, significant difference from placebo treatment.

NOTE: Control (C) values for VEKI (Mean ± SEM) after placebo or nalmefene (NF) pretreatment

and fentanyl challenge (2 μg/kg) 1, 2, 3, 6, and 8 hours later.

The bioavailability of nalmefene after oral administration was compared

using tablets relative to an oral solution (figure 3) (Baker Cummins Pharmaceuticals’ nalmefene information for investigators). Single 50-mg doses of

nalmefene HCI were administered to six normal subjects as five 10-mg tablets or as a 50-mg oral solution with a l-week washout period between

treatments. Blood samples were collected periodically for 48 hours, and the

plasma concentrations of intact nalmefene were quantitated. Statistical

analysis of biopharmaceutical parameters, such as maximum plasma

85
FIGURE 2. Opiate antagonism of meperidine by naloxone and naltnefene

a

p<.05 nalmefene vs. placebo.

b

p<.05 nalmefene vs. naloxone.

concentration (Cmax), time to reach maximum plasma concentration (Tmax),

area under the plasma concentration-time curve (AUC), and elimination halflife (t 1/2), indicated bioequivalency of the tablets and solution.

The harmonic mean half-life of nalmefene following administration of the

tablet was 8.3 hours; the corresponding value following the solution was

8.8 hours. These values are in close agreement with the terminal elimination half-life of nalmefene observed following IV administration. The absolute bioavailability of orally administered nalmefene has been estimated at

about 40 to 50 percent by comparison of the AUCs following both IV and

oral administration of the drug to four subjects.

With the discovery of endogenous opioids (Hughes 1975; Pastemak et al.

1975; Terenius and Wahlstrom 1975; Cox et al. 1975), the potential indications for the use of pure opiate antagonists has expanded dramatically.

Listed below are some of the clinical uses that have been investigated and

reported. A number of reviews describing these studies have been published (Olson et al. 1986; Zadina et al. 1986).

86
Parenteral:

(1) Anesthesia and opiate-overdose reversal

(2) Central nervous system (CNS) trauma

(3) Hypotension

(4) Cardiovascular

FIGURE 3. Mean nalmefene plasma concentrations after administration of

solution or tablet to healthy humans (n=6)

87
Oral:

(1) Puritus

(2) Gastrointestinal—reversal of constipation

(3) Compulsive behaviors (anorexia nervosa, eating disorders)

(4) Premenstrual syndrome (PMS)

(5) Fatigue

(6) Antineoplastic activity

(7) Endocrine disorders

(8) AIDS—immune modulator

The interest in pure opiate antagonists as immune modulators stems from

the numerous reports that show an impact of opioid peptides and exogenous

opiates on the immune system. Table 6 lists some of the reports that

support an interaction between the endogenous opioid and immune systems.

Table 7 shows that at least some of these effects involve mediation by

opioid receptors, since they are blocked or reversed by opioid antagonists.

It should be noted that neither table 6 nor table 7 is meant to be all

inclusive.

The recent interest in the use of opiate antagonists as agents for the treatment of AIDS stems from the report by Bihari et al. (1987) in which naltrexone (1.75 mg, four times a day) was administered to AIDS patients in a

double-blind placebo-controlled study. After 3 months, the naltrexone group

showed a significant drop in pathologically elevated serum alpha-interferon

levels compared to controls, and this correlated with a lesser incidence of

opportunistic infections. At this time, all patients including controls were

given naltrexone and monitored for 12 months. Approximately 60 percent

of the patients responded and showed decreases in serum alpha-interferon

levels. Of the nonresponders, 87 percent died within 9 months, while only

17 percent of the responders died within 12 months, and 26 percent died

within 18 months. No additional clinical studies using opiate antagonists

as anti-HIV agents have been reported since the report by Bihari and

coworkers (1987).

In conjunction with the National Cancer Institute Developmental Therapeutics Program, the author has evaluated the anti-HIV activity of nalmefene

using in vitro assays. Nalmefene was inactive in both the MT-2 and

CEM-V cell lines at concentrations up to 1.25X102 μg/ml.

In studies carried out at Dupont Corporation (Dr. Ii. Tabor, personal communication), the effect of naltrexone on various immune responses was

examined in both in vitro and in vivo assays. Nallrexone (0.012 to

100 μg/ml) had no effect in vitro on the proliferation of splenocytes in

response to phytohemagglutin (PHA). Similarly, activation of tumoricidal

88
TABLE 6. Evidence for an interaction between the endogenous opioid and

immune systems

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

Normal human T lymphocytes possess receptors for met-enkephalin and

beta-endorphin (Wybran et al. 1979; Hazum et al. 1979).

Human phagocytic leukocytes contain opiate receptors (Lopker et al.

1980).

Enkephalins prolong survival time of mice exposed to tumor cells

(Plotnikoff 1982).

Met-enkephalin increases active T-cell rosette-forming cells in

peripheral blood lymphocytes (Miller et al. 1983).

Enkephalins inhibit T-dependent antibody responses In Vitro (Johnson

et al. 1982).

Enkephalins or beta-endotphin enhance human peripheral blood

lymphocyte (PBL) natural killer (NK) cell activity (Faith et al. 1984;

Kay et al. 1984; Kay et al. 1987).

Met-enkephalin enhances T cells in patients with Kaposi’s sarcoma

(Plotnikoff et al. 1986).

Met-enkephalin temporarily enhances selected immune responses in

HIV-infected patients (Zunich and Kirkpatrick 1988).

TABLE 7. Impact of opioid antagonists on immune system functions

(1) Naloxone reverses effect of enkephalins and beta-endorphin on PBL

NK cell activity (Kay et al. 1984).

(2) Naloxone blocks beta-endorphin and met-enkephalin enhancement of

cytotoxic T-cell generation (Carr and Klimpel 1986).

(3) Naloxone blocks alpha-interferon-induced changes in hypothalamic

neuronal activity (Nakashima et al. 1987).

(4) Naltrexone has a dose-dependent stimulatory or inhibitory effect on

neuroblastoma growth (Zagon and McLaughlin 1983).

(5) Naloxone binds to human T lymphocytes (Madden et al. 1987).

89
macrophages was unaffected by naltrexone (0.01 to 10 μg/ml). The generation of lymphokine-activated killer (LAK) cells in the presence of IL2(1U/ml) was unchanged by naltrexone (0.01 to 10 μg/ml). In vivo studies

in mice showed a lack of effect of naltrexone (25 and 100 mg/kg) on

NKcell activity. In the tumor cell-clearance assay, naltrexone (0.01 to

10 mg/kg) was ineffective in changing the ratio in treated or control

animals. Finally, no change in ear swelling was observed after naltrexone

(0.01 to 10 mg/kg) in the assay that measures delayed-type hypersensitivity.

Taken together, the data are not suggestive of a beneficial effect resulting

from the use of opiate antagonists in the treatment of AIDS. Although intriguing, the study by Bihari et al. (1987) needs to be repeated under more

controlled conditions, since it is not clear that decreases in serum alphainterferon levels are indeed beneficial to the patient. In fact, studies are

currently ongoing in which alpha-interferon is administered in a regimen

with AZT to reduce the toxic side effects of the latter drug.

Recently the author has prepared a series of compounds that are either

homo- or heterodimers of specific dideoxynucleosides and elevated their

activity against HIV. The phosphate-linked dimers of the dideoxynucleosides are of the general formula shown in figure 4, where R

1

 and R

2 may

be AZT, ddA, or ddI, and R

3

 may be hydrogen, cyanoethyl, or either a

metal anion or organic anion salt. The substituents R

1

 and R

2

 may either

be the same or be derived from different dideoxynucleosidcs to yield phosphate dimers that are either homo or hetero analogs. The scheme for the

synthesis of the dimers is illustrated in figure 5, which describes the

preparation of AZT-P-ddA from AZT-5'-cyanoethylphosphate (Tener 1961)

and ddA using various coupling agents generated in situ.

FIGURE 4. Structure of dimers

When the dideoxynucleosides are linked by a phosphate bridge, the dimerized compounds might be more effective agents because of the following:

90
(1) Two nucleosides are being delivered to the cell simultaneously.

(2) The compound contains a masked phosphate, with the masking unit

also being active.

(3) The dimer could function as a prodrug.

(4) The intact dimer might also be active.

FIGURE 5. Synthetic scheme for synthesis of dimers

Table 8 lists the results of an assay in which the inhibition of syncytium

formation is examined at various concentrations of drug. Over the concentration range examined, both AZT-P-ddA and AZT-P-ddI are at least as

potent as AZT. Table 9 (Busso et al. 1988) shows the results of studies in

which the cytotoxic effect (ID50) of specific compounds is measured so that

a cytotherapeutic index (CTI=ID50/ED50) may be generated. Greater CTI

values than that of AZT are indicative of drugs that are either less toxic to

cells or more potent. The data (table 9) show that both AZT-P-ddI and

9 1
AZT-P-ddA have larger CTI values than AZT, a result that is due to their

being considerably less toxic to the cells.

TABLE 8. Primary screen results

Drug

Percent Inhibition of Syncytia Formation

100 μg/ml 10 μg/ml 5 μg/ml 1 μg/ml 0.5 μg/ml 0.1 μg/ml 0.01 μg/ml

AZT-P-ddA 100 100 100 96 96 6 4 0

AZT 100 100 100 100 100 57 0

AZT-P(CyE)ddA 100 100 100 72 58 0 0

AZT-P-ddI 100 100 100 100 98 76 0

ddA 100 100 100 9 3 87 4 2 0

NOTE: MT-2 cells were incubated with HIV (TM) and then resuspended to a final concentration of

3X105 cells/ml (96-well microtiter plate) with an MOI of 0.001. The drug was added after

the cells were transferred to the wells. On day 4, the number of syncytia per well was

compared to controls. Results are the mean of quadruplicate cultures (one experiment).

TABLE 9. Comparison of the cytotherapeutic index of specific dimers

ID50* ED50

Drug (mg/ml) (mg/ml) CTI

AZT-P-ddA** >100 0.08 >1,250

AZT 75 0.06 1,250

AZT-P(CyE)ddA** >100 0.6 >150

AZT-P-ddI** >100 0.05 >2,000

ddA 75 0.1 750

*MT-2 cells (2X105 cells/ml) were seeded with 100 μg/ml or 50 μg/ml of drug. Every 3 to 4 days,

media changes were performed and fresh drug added. On day 14, cell counts were performed (trypan

blue exclusion method). Results are the mean of two replicates per concentration.

**No evidence of cytotoxicity at 100 μg/ml.

Reverse transcription (RT) has been shown to be involved in the replication

of HIV (Mitsuya et al. 1987). The inhibition of RT by various dimers is

shown in table 10. AZT-P-ddI at a concentration of 1 μg/ml inhibited the

enzyme through at least 11 days, whereas AZX at 1 μg/ml was effective at

7 days but not at 11 days. In this assay, the activity of AZT-P-ddA was

comparable to that of AZT.

In other studies, the author examined the uptake of radiolabeled AZTP(CyE)ddA into H-9 cells. After incubation for 24 hours at 37 °C, the

cells were separated from the medium by the centrifugation. and the super92
natant was decanted. After washing and extraction with 70 percent methanol, the evaporated cellular extract was purified on silica gel, and bands that

coeluted with authentic standards of AZT-P-(CyE)ddA and AZT-P-ddA were

removed, extracted, and counted in a scintillation counter. The results

showed that 7 percent of the incorporated radioactivity corresponded to

AZT-P-(CyE)ddA, and 20 percent corresponded to AZT-P-ddA. The data

suggest that at least some of the dimer enters the cell intact and that AZTP(CyE)ddA is acting as a prodrug for AZT-P-ddA.

TABLE 10. Inhibition of reverse transcriptase activity*

Drug

Evaluation Period (Days)

7 1 1 14

Control (No Drug) 11,000 38,000 34,000

ddA (10 μM) 500 3,000 4,500

ddA (1 μM) 5,500 45,000 39,500

AZT-P-ddA (5 μg/ml) 500 500 500

AZT-P-ddA (1 μg/ml) 1,500 12,000 26,500

AZT (1 μg/ml) 1,500 12,000 23,500

AZT (0.5 μg/ml) 2,000 16,500 28,000

AZT-P(CyE)-ddA (5 μg/ml) 1,000 4,500 12,000

AZT-P(CyE)-ddA (1 μg/ml) 6,000 43,000 36,000

AZT-P-ddI (5 μg/ml) 1,000 1,500 25,000

AZT-P-ddI (1 μg/ml) 1,000 3,000 10,000

*Target cells=H9 cells. MOI=1.0. Media changes with the addition of fresh drug were performed at 3-

to 4-day intervals. Results are expressed as CPM-RT activity per ml in culture supernatant.

CPM>5,000 signifies abnormal elevations in RT activity. The results represent the mean of duplicate

studies in one experiment.

The stability of AZT-P-ddA as a function of temperature was examined in

human plasma (figure 6) (Hahn et al. 1989). At 37 °C, approximately

10 percent of drug was metabolized per hour. This metabolism was found

to be species dependent (figure 7) (Hahn et al. 1989). Analysis of the

nature of the metabolites by HPLC showed that only AZT and ddI were

formed.

93
FIGURE 6. Stability of AZT-ddA in human plasma (in vitro) at various

temperatures (4 μg/ml)

SOURCE: Hahn et al. 1989, copyright 1989, American Chemical Society

Figure 8 shows the distribution of radioactivity in serum and cells at various times following IV injection of [

3H]-AZT-P-ddA (5 mg/kg) into rats.

After 1 hour, about 7.8 percent of the injected dose is found in red blood

cells compared to 4.2 percent in the serum. This disproportionate uptake

into cells is no longer seen at 2 and 3 hours after injection, at which time

the serum contains approximately twice as much radiolabel when compared

to cells. The nature of the radioactivity found in the kidney at 3 hours

94
FIGURE 7. Stability of AZT-ddA in plasma of various species at 37 °C

(4 μg/ml)

SOURCE: Hahn et al 1989, copyright 1989, American Chemical Society.

after administration was examined. After sacrifice, kidneys were removed,

homogenized, and extracted. The residue after evaporation of the solvent

was analyzed by radioscans of thin-layer chromatography plates developed

in two different systems. The scans showed that the principal compound

present was AZT-P-ddA, indicating that intact dimer was still found in

tissue 3 hours after administration of the drug.

95
HOURS

FIGURE 8. [

3H]-Labeled material in serum and cells following IV

injection of [

3H]-AZT-P-ddA (5 mg/kg) into rats

In conclusion, a series of dimers of dideoxynucleosides linked by a phosphorous bridge has been synthesized. The results that have been obtained

show the following:

(1) The cytotherapeutic index of some of the dimers is better than the corresponding value for AZT.

(2) The intact dimeric compounds exhibit cellular uptake.(3)The dimers are

bioavailable after in vivo administration to animals.

(4) Since the dimers are not metabolized in a manner comparable to the

individual nucleoside components, it is conceivable that additional advantages may be associated with their in vivo administration. Additionally, as an intact compound, both components are present in the

cell simultaneously and may provide an added therapeutic effect.

96
(5) Since at least some of the administered drug enters the cell intact, a

possible new mechanism of action is suggested.

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ACKNOWLEDGMENTS

The following individuals collaborated in various phases of this research:

J. Fishman, Ph.D. (IVAX Corp.); L. Resnick, M.D., and M. Busso, M.D.

(Mt. Sinai Medical Center); and C.C. Lin, Ph.D., (Schering Corp.).

AUTHOR

Elliot F. Hahn, Ph.D.

IVAX Corporation

8800 Northwest 36th Street

Miami. FL 33178

99
Classical and Nonclassical

Cannabinoids: Mechanism of

Action—Brain Binding

Allyn C. Howlett, M. Ross Johnson, and

Lawrence S. Melvin

INTRODUCTION

Following characterization and publication by Gaoni and Mechoulam (1964)

of the structure of delta-9-tetrahydrocannabinol (delta-9-THC), researchers

enthusiastically pursued new therapeutants for 17 years, based on this active

principle from marijuana. These research efforts culminated with the marketing of delta-9-THC (Marinol) by Unimed and nabilone (Cesamet) by

Lilly and clinical investigation of levonantradol from Pfizer and nabitan

from SISA (figure 1). Delta-9-THC and nabilone are approved as an antiemetic for cancer patients being treated with chemotherapy. Levonantradol

was studied both as an antiemetic and an analgetic, and nabitan was studied

as an analgetic. This chapter will briefly review the research at Pfizer, Inc.

related to discovery of the synthetic cannabinoid CP-55,940 as an analgetic

in rodents and continuing research at St. Louis University Medical School

responsible for the discovery of a mechanism of action and brain-binding

site for this compound.

DISCUSSION

At the outset of these research efforts, two key points of information in the

cannabinoid literature provided a basis for the discovery of a unique class

of nonclassical cannabinoids. First, 9-nor-9-beta-hydroxyhexahydrocannabinol (HHC) was prepared (Wilson and May 1975) and found to exhibit analgetic activity in rodents, with equal potency to morphine (Wilson et al.

1976; Bloom et al. 1977). These results provided a novel chemical target

from which potent. nonopiate analgetics could be discovered. Second, published and presented research supported a contention that significant biological activity was present in naturally occurring and synthetic cannabinoids

only if they contained the rigid benzopyran ring system present in

100
Delta-9-THC Nabilone

Levonantradol Nabitan

FIGURE 1. Delta-9-tetrahydrocannabinol and derived drugs

delta-9-THC (Mechoulam and Edery 1973; Razdan 1987; Mechoulam et al.

1987). This second point was inconsistent with several hypotheses that we

had formulated to pursue a mechanism-based approach to the development

of drugs based on HHC. Therefore, an early research objective was to

directly challenge the need for incorporation of a benzopyran ring in

cannabinoid-based drugs. An additional benefit from this structure-activity

relationship (SAR) test was that it allowed synthesis of a greater diversity

of structurally novel compounds than may have been anticipated from previous studies. This point was also necessarily important for the successful

discovery and development of a drug based on HHC. Delta-9-THC and

structurally related biologically active cannabinoids present a broad spectrum

of activities in animals and, where tested, in man. Such a plethora of

effects by a compound would usually contraindicate its use as a drug. The

ultimate objective of this research was to structurally isolate the various

pharmacophores present in delta-9-THC and HHC and then rebuild compounds with high specificity and potency of action. To achieve progress on

this major objective, it was not fortuitous that we hypothesized that the cannabinoids must produce their biological effects on subtypes of an as-yetunknown specific receptor system.

Initial research leading to the discovery of levonantradol provided substantial

understanding of the SAR in the pyran ring portion of HHC (Johnson et al.

1981; Johnson and Melvin 1986). Using analgesia as an endpoint, it was

shown that the pyran oxygen atom in HHC could be replaced by carbon,

101
nitrogen, or alkyl-substituted nitrogen. Equatorial substitution at C-6 was

favored, but varied substitution was also found to be allowable and included

just hydrogen. These results indicated the gemdimethyl pyran ring of HHC

was not, in itself, a pharmacophore for analgetic activity in HHC. Nonetheless, it was still possible that the rigidity of the benzopyran ring was

responsible for a favorable three-dimensional interaction of more distal functionality (alcohol, phenol, C-3 chain) with a receptor. This possibility was

tested with the synthesis of structure 1, lacking the pyran ring of HHC

(Melvin et al. 1984a) (figure 2). Compound 1 also incorporated the 1,1-

dimethylheptyl C-3 chain, previously used in nabilone (Archer et al. 1977;

Archer et al. 1986) and developed in early work (Adams et al. 1949; Loev

et al. 1973), which was expected to provide optimal potency. The phenylpentyloxy C-3 chain of levonantradol and similar side chains were equally

suitable for use in structures such as 1. Analgetically, 1 was more potent

than HHC and equipotent with morphine. In fact, molecular modeling had

predicted a low-energy conformation of 1 in which the spatial arrangement

of the alcohol, phenol, and C-3 chain of HHC and 1 were superimposable.

This initial result confirmed the original proposal that the pyran ring of

HHC was not a necessary pharmacophore in cannabinoids and that any molecular rigidifying effects it provided were surmountable.

FIGURE 2. Comparison of HHC and compound 1

Broad SAR studies of 1 guided in part by an earlier and now less attractive

proposal of a prostaglandin mechanism of action (Milne and Johnson 1981),

led to the synthesis and extensive study of 2 (CP-55,940), a single enantiomer (Johnson and Melvin 1986) (figure 3). Structure 2 differs from 1 and

HHC by having a hydroxypropyl residue in the space previously occupied

by the pyran ring of HHC. Nuclear magnetic resonance (NMR) data confirmed that both the hydroxypropyl group and aryl-cyclohexyl bond are

freely rotating. Compound 2 was shown to have significantly enhanced analgetic potency with respect to morphine (6 to 37 times as potent) and

to be equipotent with levonantradol. Similar to levonantradol and desacctyllevonantradol, the analgetic activity of 2 was not blocked by naloxone

102
(McIlhenny et al. 1981; Yaksh 1981). The nonclassical cannabinoids, including 2, exhibit varying degrees of cannabimimetic activity, in addition to

analgetic and antiemetic effects (Weissman et al. 1982; Koe et al. 1985;

Little et al. 1988). The enhanced analgetic effect of 2 over 1 was attributed to the introduction of a potential new receptor binding element (hydroxypropyl), which interacted with a previously unutilized site at the proposed

cannabinoid analgetic receptor. To gain support for the identification of a

new binding element, compound 2 was modified in various ways to induce

stable conformations of the hydroxypropyl unit. It was argued that such a

conformationally restricted binding element would provide maximum biological potency when matched to its binding site at the receptor.

FIGURE 3. Compound 2 (CP-55,940) and the rigid derivative 3

(CP-55,244)

Ultimately, a conformationally restricted derivative of 2 was synthesized

with a hydroxymethyl-containing decalin ring (Johnson and Melvin 1986).

Compound 3 (CP-55,244), a single enantiomer, showed enhanced analgetic

potency (2 to 13 times) over 2 and a strong stereochemical preference for

an axial vs. equatorial hydroxymethyl group (31 to 53 times) (figure 3).

The enantiospecificity of analgetic activity for 3 was dramatically increased

to greater than 5000 times vs. 209 times for 2 and 3 times for 1, implying

a very specific receptor fit. These findings supported the conclusion that

the hydroxypropyl functionality on 2 was a significant new contributor to

receptor binding.

The less rigid bicyclic cannabinoid prototype illustrated by compound 1 was

extended to two other interesting modifications. SP-1, the active form of

the prodrug nabitan (Razdan et al 1976; Razdan and Howes 1983), possesses a unique N-propargyl group in place of the alcohol in 6a, 10a-dehydro

HHC and utilizes a 1,2-dimethylheptyl C-3 chain. Removal of the pyran

ring in SP-1 provided compound 4, which was still equipotent with HHC as

an analgetic but lost potency on some endpoints vs. SP-1 (1 to 8 times)

(figure 4). Optimum timing for drug administration was not taken into

103
consideration. A similar relationship also held true for the fully saturated

piperidine ring derivative of 4 (Melvin et al. 1984b).

FIGURE 4. Relationship of SP-1 and compound 4

Cannabidiol (CBD) is the natural pyran-ring-opened derivative of delta-9-

THC (Mechoulam and Shvo 1963). The pharmacology of CBD has been

broadly studied, showing that it lacks analgetic activity and is a relatively

weak cannabimimetic (Mechoulam et al. 1970; Dewey 1986; Razdan 1986;

Consroe and Snider 1986). We speculated that the inactivity of cannabidiol

was a result of an unfavorable steric interaction between the bulky isopropenyl group and a phenolic hydroxyl. Unlike 1, 2, or 3. cannabidiol cannot

readily adopt a conformation that would overlap its vinyl methyl (possibly

oxidized to hydroxymethyl), phenol, and C-3 chain with those of delta-9-

THC. Since it was already shown that the pyran ring was not necessary for

biological activity, it seemed unlikely that the simple presence of a second

phenol in cannabidiol would be so deleterious. Syntheses of compound 5

would test this idea, since 5 contains both phenols of cannabidiol but lacks

the isopropenyl group (figure 5). As predicted, compound 5 and its axial

alcohol isomer exhibited analgetic activity comparable to that of 1 and its

isomer (Melvin et al. 1984a).

With support and direction from accumulating research data, biological

studies were initiated to investigate the mechanism of action for the analgetic synthetic cannabinoids. Earlier studies had demonstrated that active

cannabinoids, in particular delta-9-THC and desacetyllevonantradol, were

reversible inhibitors of adenylate cyclase activity in membranes from neuroblastoma cells (N18TG2) (Howlett 1984; Howlett 1985; Howlett 1987).

104
Cannabidiol 5

FIGURE 5. Relationship of cannanidiol and diphenol 5

Table 1 demonstrates the potent inhibition of adenylate cyclase and the high

degree of enantiospecificity of the nonclassical cannabinoids and levonantradol (Howlett et al. 1988). The inhibition of neuroblastoma adenylate

cyclase is exhibited by all analgetically active cannabinoids with a significant degree of correlation for potency, enantiospecificity and stereospecificity. Further work supports a conclusion that actual enzyme inhibition by

cannabinoids occurs via interaction with a Gi protein complex (Howlett et

al. 1986). The receptor mechanism whereby cannabinoids inhibit adenylate

cyclase is distinct from that by which the opiates act (Devane et al. 1986).

It is tentatively concluded that the mechanism whereby cannabinoids exhibit

in vivo analgetic activity is identical to their in vitro inhibition of adenylate

cyclase (Howlett et al. 1988).

TABLE 1. Inhibition of adenylate cyclase activity in vitro

Kinh (nM)*

Compound (-) Enantiomer**

1 79

2 25

3 5

Levonantradol 100

Desacetyllevonantradol 7

Delta-9-THC 430

(+) Enantiomer**

135

>5,000

>10,000

>5,000

*Inhibition of secretin-stimulated activity. Data is fmm a mean of at least three determinations.

**In all cases studied to date, the (-) rotation has been exhibited by the enantiomer with absolute configuration 6aR,10aR (using HHC as an example) and this is the absolute configuration in which all

structures in this manuscript are drawn.

105
The original premise driving research to segregate the various biological

activities of cannabinoids, i.e. that the effects of cannabinoids are the result

of direct interaction with a discrete receptor(s), was initially pursued in rat

brain (Devane et al. 1988). A potent, stable cannabinoid radioligand was

needed for this research. Compound 2 (CP-55,940) was chosen and tritium

labeled in the C-3 chain by catalytic reduction of the olefinic precursor 6

(figure 6) (Devane et al. 1987).

6 [

3H2]-2

FIGURE 6. Precursor 6 and tritium-labeled 2

Specific binding in rat brain to cortical P2 membranes and synaptosomes

from hippocampus plus prefrontal cortex was characterized with tritiated 2.

Compound 2 binds to the rate P2 membrane preparation with a Ki=133±11

pM and a Bmax of 1.85 pmol/mg of protein. Desacetyllevonantradol exhibits a binding Ki=123±34 pM, while delta-9-THC is approximately tenfold

less potent as a ligand with Ki=1.6±0.56 nM. Conversely, the analgetically

inactive cannabidiol gave less than 50 percent displacement of tritiated 2 at

1 μM.

The analgetically less active enantiomer of 2 showed a fiftyfold weaker

affinity with Ki=3.4±1.9 nM. Together with unreported data and the continuing accumulation of new data, a significant correlation between analgetic

potency in vivo and binding to the rat brain receptor in vitro defined by

tritiated 2 has been shown. As was the case for inhibition of neuroblastoma adenylate cyclase, evidence has been obtained suggesting that the brainreceptor site is linked to a Gi protein complex (Devane et al. 1988). Table

2 summarizes the data that corroborate our conclusion that a specific brain

receptor has been identified that is responsible for at least the analgetic

activity of cannabinoids.

CANNABINOIDS AND THE IMMUNE SYSTEM

With respect to the possible effects of cannabinoids on the immune system,

these lipophilic substances can now be evaluated more specifically for their

cell receptor effects. This is important because the literature has not clearly

106
defined an effect of cannabinoids, particularly delta-9-THC, on the immune

system of mammals. Several caveats should be considered when reviewing

the reported immunological effects of marijuana or delta-9-THC. First,

those effects ascribed to the inhalation route of delivery of delta-9-THC

from marijuana are not usually controlled for effects resulting from the

simple act of smoking a marijuana or generic cigarette. Second, when evaluating a user population of marijuana, it is difficult to control for the use of

other potentially immuno-affecting substanas such as tobacco, alcohol, and

other drugs of abuse. Finally in vitro effects on cells are difficult to assess

because measurements are frequently conducted under nonphysiological conditions of drug and protein concentrations. Several recent reviews summarize research on the potential effects of marijuana on the immune system

(Friedman et al. 1988; Yahya and Watson 1987; Kawakami et al. 1988;

Murison et al. 1987; Mishkin and Cabral 1987; Press et al. 1987).

TABLE 2. Fulfilled requirements for a cannabinoid drug-receptor

site interaction

Receptor Binding of 2 (CP-55,940) to Rat Brain*

High Affinity K=133 pM

Rapid Equilibrium <45 min

Reversible/Saturable 100%/100%

Enantioselective (-)/(+)=0.02

Functional Activity and Correlation with Analgesia

High Potency μg/kg

Enantioselective (-)/(+)=0.004

SAR Correlatable With Binding R=0.9

*Assay uses [

3H]-2 (CP-55,940) of specific activity 50Ci/mmole.

CONCLUSION

Utilizing a nonclassical cannabinoid analgetic as a radioligand, a specific receptor was identified in rat brain. In vitro binding of cannabinoids to this

receptor and in vivo rodent analgetic potency correlate significantly. Additionally, analgetic cannabinoids inhibit neuroblastoma adenylate cyclase

activity in vitro with a significant correlation to in vivo potency. Preliminary data support a further conclusion that inhibition of adenylate cyclase and

brain receptor binding both occur via interaction with a single Gi protein

complex.

Biological and chemical probes are now available to allow an indepth

exploration of the pharmacology and mechanisms of action of the cannabi107
noids. Physiologically relevant receptor subtypes can be searched for,

including perhaps peripheral receptors that are associated with pain. Antagonists and mixed agonists-antagonists for these receptors can be pursued,

and endogenous binding substances can now be looked for. It is anticipated

that worthwhile exploitation of these results will follow in the form of

action-specific drugs based on cannabinoids.

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AUTHORS

Allyn C. Howlett, Ph.D

Department of Pharmacology

St. Louis University School of Medicine

1402 South Grand Boulevard

St. Louis, MO 63104

M. Ross Johnson, Ph.D

Glaxo Inc.

5 Moore Drive

Research Triangle Park, NC 27709

Lawrence S. Melvin, Ph.D

Central Research

Pfizer Inc.

Groton, CT 06340

111
Cocaine Receptor-Design

of Ligands

F.I. Carroll, Abraham Philip, and Anita H. Lewin

INTRODUCTION

Abuse of cocaine, an alkaloid obtained from the leaves of Erythroxylan

coca, has grown from a relatively minor problem a few years ago to a

major U.S. health problem today (Grabowski 1984; Clouet et al. 1988).

Concern is rising, especially over the much more potent effects obtained by

the growing fashion of smoking the alkaloid itself as free base (crack).

Since cocaine may affect immune system function (Watson et al. 1983;

Van Dyke et al. 1986; Donahoe et al. 1986; Havas et al. 1987; Klein et al.

1988) and can be toxic to the point of being lethal (Clouet et al. 1988),

studies directed toward gaining information about the mechanism of cocaine

action and toxicity are of importance.

POSSIBLE BASIS FOR REINFORCING PROPERTIES OF COCAINE

Cocaine is a psychomotor stimulant with rewarding or reinforcing properties.

Since cocaine has been shown to inhibit the transport of dopamine, norepinephrine, and serotonin (Coyle and Snyder 1969; Javitch et al. 1984; Ross

and Renyi 1969; Blackburn et al. 1967; Horn et al. 1974), any one or a

combination of these binding sites could presumably mediate the effects that

are related to the abuse of cocaine. However, the pharmacologically relevant receptor must possess a significant correlation between potency of

drugs in producing a response and in binding to the recognition site. Recently Kuhar et al. (1988) and Ritz et al. (1987) reported that ligand binding affinity to the 3H-mazindol binding site on the dopamine transporter

correlated with the reinforcing properties of cocaine and related drugs. In

contrast. no correlation was found for binding sites on other monoamine

transporters. In general, these workers found that compounds potent in selfadministration studies were also potent inhibitors of binding at the transport

site for dopamine. Conversely, compounds that were weak in selfadministration studies were also weak inhibitors at the dopamine binding

site. The study included compounds structurally similar to cocaine (table 1)

112
as well as other ester-linked local anesthetics and closely related psychostimulants (table 2). This information led Kuhar et al. to conclude that the

dopamine transporter may be the cocaine receptor that is responsible for

cocaine stimulant and reinforcing properties. Thus, cocaine euphoria would

be explained on the basis of dopamine uptake blockade causing increased

dopamine concentration in the synaptic cleft, leading to increased neurotransmission in the brain reward system.

TABLE 1. Inhibition of binding at dompamine transporter: Cocaine and

structurally similar compounds

SOURCE: Kuhar et al. 1988.

113
TABLE 2. Inhibition of binding at the dopamine transporter: Local

anesthetics and psychostimulants related to cocaine

SOURCE: Kuhar et al. 1988

The data in tables 1 and 2, while limited, do suggest some structural requirements for the receptor. An aromatic ring and an amino group are

apparently required but are not sufficient for good affinity to the receptor.

All the tropane structures as well as methylphenidate possess a carbomethoxy group. However, the fact that (+)-pseudococaine, (+)-cocaine, and

WIN-35,065-3 show low affinity for the receptor reveals that the stereochemistry of the carbomethoxy group is important. Nevertheless, the high

affinity of mazindol demonstrates that a carbomethoxy function is not a

114
strict requirement for high affinity to the receptor. The high affinity of the

2-aryl substituted tropanes (WIN-35,428; WIN-35,065-2; and WIN-35,981)

and methylphenidate shows that the receptor can accommodate aryl groups

located in different positions relative to the amino group. The high affinity

of methylphenidate and the comparable affinities of not-cocaine to cocaine

and WIN-35,981 to WIN-35,065-2 demonstrate that a tertiary amine is not

necessary for high affinity.

CONSTKUCTION OF COCAINE ANALOGS

In collaboration with the Addiction Research Center (ADR) of the National

Institute on Drug Abuse, the authors are conducting an SAR study to learn

more about the cocaine receptor. The goal of this research is to gain information about the structural and stereochemical features required for good

binding to the cocaine receptor. The scope of the study includes the design, synthesis, and receptor binding evaluation of cocaine analogs. The

design and synthesis of several cocaine analogs are presented in this chapter. The receptor binding data are not available at this date and will be

published later.

Structurally, natural cocaine is (lR,2R,3S,5R)-3-(benzoyloxy)-8-methyl-8-

azabicyclo[3·2·1]octane-2-carboxylic acid methyl ester. There are three other

possible stereoisomers of cocaine, each existing as an enantiomeric pair; all

seven compounds have been prepared in the authors’ laboratory (figure 1).

Cocaine and two of the isomers (table 1) have been evaluated in the dopamine transport receptor binding assay (Kuhar et al. 1988). Evaluation of

the other five isomers will answer the question of stereoselective binding.

The cocaine analogs evaluated by Kuhar et al. (1988) have all been

2-carbomethoxytropane derivatives (table 1). In order to gain information

concerning the effect of the size, volume, and oxidation state of this group

on binding, compounds 1-9 listed in figure 2 have been prepared.

In order to obtain information about the location of the ionic/hydrogen

bonding site of the cocaine receptor, it was decided to compare the binding

affinities of tropacocaine (figure 3) which showed moderate binding to the

dopamine transporter labeled with 3H-cocaine (Reith et al. 1986) and its

isomer 6-methyl-6-azabicyclo[3·2·1]octan-3 -ol benzoate (10). Molecular

modeling studies were used to compare the structural features of 10 to

tropacocaine. The molecular models of 10 and tropacocaine were constructed by using x-ray fragments and normal computer-supplied connections

available from the SYBYL computer graphics system (Tripos Associates,

St. Louis, MO 63117). The geometry was optimized with the MAXMIN2

module of SYBYL. The results, shown as an overlay of compound 10 and

tropacocaine, both with endomethyl conformations (figure 4) show a high

degree of similarity between the two structures, so that the effect of the

position of the nitrogen on binding can be assessed.

115
(-)-Cocaine

(-)-Allococaine

(+)-Cocaine

(+)-Allococaine

(+)-Pseudococaine

(-)-Allopseudococaine

(-)-Pseudococaine

(+)-Allopseudococaine

FIGURE 1. Stereoisomers of cocaine

116
FIGURE 2. Cocaine analogs modified at the 2 position of the

tropane ring

Cocaine and WIN-35,065-2 have good affinity for the dopamine transporter

(table 1). even though the phenyl ring is closer to the tropane ring in

WIN-35,065-2. Structures for cocaine and WIN-35,065-2 were constructed

using the SYBYL software system. Systematic conformational searches

using the SEARCH module of SYBYL were used to locate the global and

other low-energy conformations of both compounds. Figure 5 shows an

overlay of the global energy minimum conformation of WIN-35,065-2 and a

low energy conformation of cocaine. Inspection of figure 5 suggests that

the 3-naphthyl group of compound 11 (figure 3) would possess aryl groups

in the space occupied by the electron-rich portions in both cocaine and

WIN-35,065-2.

The p- and m-isothiocyanate derivatives of cocaine, 12 and 13 respectively,

have been prepared as potential irreversible binding ligands for the dopamine transporter. In addition, compound 14, the tritium-labeled analog of

WIN-35,065-2, was prepared as a new radioligand for the cocaine receptor.

117
FIGURE 3. Cocaine and WIN-35,065-2 analogs

CONCLUSION

The potency of cocaine and cocainelike drugs in self-administration studies

correlates with their potencies in inhibiting 3H-mazindol binding to the dopamine uptake site in rat striatum. Since there is no correlation for binding to

a large number of other pre- and postsynaptic binding sites, the cocaine

receptor related to substance abuse appears to be the one associated with

dopamine uptake inhibition.

118
FIGURE 4. Computer-generated molecular overlay of compound 10

and tropacocaine

NOTE: Hydrogens have been removed for clarity

FIGURE 5. Computer-generated molecular overlay of WIN-35,065-2

(global minimum conformation) and cocaine (one lowenergy conformation)

NOTE: All hydrogen atoms have been removed for clarity.

119
Several rigid azabicyclo analogs of cocaine. in which the size, stereochemistry, and/or the relative location of the amine functionality, carbomethoxy group, and aromatic ring were varied, have been prepared. The examination of these compounds in the dopamine transporter receptor binding

assay will provide information about the cocaine pharmacophore. In addition, other cocaine analogs were prepared that may serve as new biochemical tools for the study of the cocaine receptor.

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Blackburn, K.J.; French, P.C.; and Merrills, R.J. 5-Hydroxytryptamine

uptake by rat brain in vitro. Life Sci 6:1653, 1967.

Clouet, D.; Asghar, K.; and Brown, R. Mechanism of Cocaine Abuse and

Toxicity. National Imtitute on Drug Abuse Research Monograph 88.

DHHS Pub. No. (ADM)88-1585. Washington, DC: Supt. of Docs., U.S.

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Coyle, J.T., and Snyder, S.H. Antiparkinsonian drugs: Inhibition of dopamine uptake in the corpus striatum as a possible mechanism of action.

Science 166:889-901, 1969.

Donahoe, R.M.; Nicholson, J.K.A.; Madden, J.J.; Donahoe, F.; Shafer, D.A.;

Gordon, D.; Bokos, P.; and Falck, A. Coordinate and independent effects

of heroin, cocaine, and alcohol abuse on T-cell E-rosette formation and

antigenic marker expression. Clin Immunol Immunopathol 41:254-264,

1986.

Grabowski, J., ed. Cocaine: Pharmacology, Effects, and Treatment of

Abuse. National Institute on Drug Abuse Research Monograph 50.

DHHS Pub. No. (ADM)83-1326. Washington, DC: Supt. of Docs., U.S.

Govt. Print. Off., 1984.

Havas, H.F.; Dellaria, M.; Schiffman, G.; Gcller, E.B.; and Adler, M.W.

Effects of cocaine on the immune response and host resistance in BALB/c

mice. Int Arch Allergy Appl Immunol 83:377-383, 1987.

Horn. A.S.; Cuello. C.; and Miller, R.J. Dopamine in the mesolimbic system of the rat brain: Endogenous levels and the effects of drugs on the

uptake mechanism and stimulation of adenylate cyclase activity.

J Neurochem 22:265-270, 1974.

Javitch, J.A.; Blaustein, R.O.; and Snyder, S.H. [

3H] Mazindol binding

associated with neuronal dopamine and norepinephrine uptake sites.

Mol Pharmacol 26:35-44, 1984.

Klein, T.W.; Newton, C.A.; and Friedman, H. Suppression of human and

mouse lymphocyte proliferation by cocaine. In: Bridge, T.P.; Mirsky,

A.F.; and Goodwin, F.K., eds. Psychological, Neuropsychiatric, and Substance Abuse Aspects of AIDS. New York: Raven Press, 1988.

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Kuhar, M.J.; Ritz, M.C; and Sharkey, J. Cocaine receptors on dopaminc

transporters mediate cocaine-reenforced behavior. In: Clouet, D.;

Asghar, K.; and Brown, R., eds. Mechanism of Cocaine Abuse and

Toxicity. National Institute on Drug Abuse Research Monograph 88.

DHHS Pub. No. (ADM)88-1585. Washington, DC: Supt. of Docs., U.S.

Govt. Print. Off., 1988. pp. 14-22.

Reith, M.E.A.; Meisler, B.E.; Sershen, H.; and Lajtha, A. Structural requirements for cocaine congeners to interact with dopamine and serotonin

uptake sites in mouse brain and to induce stereotyped behavior. Biochem

Pharmacol 35:1123-1129, 1986.

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on dopamine transporters are related to self-administration of cocaine.

Science 237: 1219-1223, 1987.

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Van Dyke, C.; Stesin, A.; Jones, R.; Chuntharapai, A.; and Seaman, W.

Cocaine increases natural killer cell activity. J Clin Invest 77:1387-1390,

1986.

Watson, E.S.; Murphy, J.C.; Elsohly, H.N.; Elsohly, M.A.; and Turner, C.E.

Effects of the administration of coca alkaloids on the primary immune

responses of mice: Interaction with -tetrahydrocannabinol and ethanol.

Toxicol Appl Pharmacol 71:1-13, 1983.

ACKNOWLEDGMENT

This research was supported by U.S. Public Health Service grant

DA 05477-01 from the National Institute on Drug Abuse.

AUTHORS

F. I. Carroll, Ph.D.

Abraham Philip, Ph.D.

Anita H. Lewin, Ph.D.

Research Triangle Institute

P.O. Box 12194

Research Triangle Park, NC 27709

121
Structural and Functional

Similarities of Opiate Receptors of

Neural and Immune Origin

Daniel J. J. Carr and J. Edwin Blalock

INTRODUCTION

Opioid peptides, originally described as interacting with specific receptors

found in neural tissue (Lord et al. 1977; Cuello 1983), have been reported

to modulate various immune system parameters (Weigent and Blalock

1987). The immune modulatory capacity of opioids has, in many cases,

been shown to be reversible or inhibited by opioid receptor antagonists,

such as naloxone, leading investigators into hypothesizing the existence of

opioid receptors on cells of the immune system. In addition, the recent

observations describing the production of opioid peptides by cells of the

immune system (Blalock et al. 1985) has led to the hypothesis of bidirectional pathways shared between the immune and neuroendocrine systems,

based on the commonality of ligands and receptors (Blalock 1984). In an

effort to further characterize the interaction between the immune and neuroendocrine systems, a comparison of the physicochemical properties of opioid

receptors on cells of the two systems was initiated.

CHARACTERISTICS OF OPIOID RECEPTORS OF

NEUROENDOCRINE ORIGIN

Classically, opioid receptors are defined according to their selectivity for

various opioid ligands. Thus far, three predominate species of receptor

classes have been defined, including μ, , and (Barnard and DemoliouMason 1983). Of these, the μ and subtypes have been studied structurally as well as functionally at the cellular and molecular levels (Simonds

1988). Structural analysis reveals the S-class opioid receptor, isolated from

a neuroblastoma x glioma hybrid cell line, to have a molecular weight of

58,000 daltons (Klee et al. 1982; Simonds et al. 1985; Carr et al. 1987).

122
The μ-class opioid receptor reportedly possesses a molecular weight of 58

to 65 kilodaltons, depending on the type of neural tissue used (Simon 1987;

Cho et al. 1986). These observations, along with previous investigations

(Bowen et al. 1981), suggest the neural opioid receptor may be a single

glycoprotein species with specific regions associated with ligand binding

influenced by ionic strength.

Molecular mechanisms by which neural opioid receptors transduce messages

appear to be linked to G protein-adenylate cyclase pathways and ionic conductance channels (Simonds 1988). Opioids that interact with both μ- and

b-class receptors have been shown to inhibit adenylate cyclase activity

through Gi proteins. In addition, electrophysiologic analysis of opioid

receptor-ligand interactions shows a modulatory capacity on both Ca2+ and

K

+

 ion conductance. For example, opioids reportedly activate inwardly rectifying potassium currents in various neuronal systems. They also reduce

voltage-dependent calcium channels (Simonds 1988). Thus, much information as to the characteristics of opioid receptors on neural tissue has been

obtained.

PIIARMACOLOGIC AND PHYSICOCHEMICAL CHARACTERISTICS OF IMMUNE-CELL OPIOID RECEPTORS

As previously mentioned, early evidence suggesting opioid receptors on cells

of the immune system (immunocytes) came predominately from functional

assays on immune responsiveness. Human T-cell rosetting was shown to be

modulated by [Met]-enkephalin and morphine (Wybran et al. 1979). Moreover, this effect was reversed with the addition of naloxone, an opioid receptor antagonist. Other naloxone-reversible effects of opioids have been

well documented for both the humoral and cellular arms of the immune system (Sibinga and Goldstein 1988). Furthermore, biological responses arc by

no means the only methods that have been employed to show opioidlike receptors on immunocytes. By pharmacologic techniques, early work demonstrated opioidlike binding sites on immunocytes that were, in some cases,

saturable and of relatively high affinity (Lopker et al. 1980; Mehrishi and

Mills 1983; Ausiello and Roda 1984). Recently, specific binding sites for

the ligand bremazocine were demonstrated on EL-4 thymoma cells with a

Kd of 60 nM and Bmax of 2,700 fmols/106 cells (Fiorica and Spector 1988).

Similarly, using the K-selective ligand U69,593, the authors have determined

that the P388d1-macrophage cell line possesses opioid receptors with a Kd

of 17 nM and Bmax of 54 fmol/106

 cells (Carr et al. 1989). Thus, it seems

apparent that cells of the immune system (as determined by pharmacologic

techniques) possess opioid receptors that appear to have some selectivity for

specific classes of ligands and that possess high-affinity binding sites. In an

effort to further define immunocyte-receptor characteristics, immunologic

and biochemical techniques were employed. Documentation of the mass of

the opioid receptor(s) from neural tissue was originally determined using

detergent-solubilization procedures followed by molecular sieve

123
chromatography and radioreceptor assays of the various fractions (Simon

1987). With the advent of more sophisticated peptide ligands and classspecific alkaloids as well as the introduction of chemical crosslinkers with

short linker arms, it became possible to selectively label opioid receptors

in situ and determine their molecular weight via polyacrylamide gel electrophoresis. In an effort to determine if an opioid receptor from immunocytes

could be specifically labeled in this manner, 125I- -endorphin was incubated

with immunocytes in the presence or absence of unlabeled naloxone.

Bound ligand was then covalently attached to the receptor and subsequently

analyzed by gel electrophoresis and autoradiography. In the presence of a

reducing agent (2-mercaptoethanol), a -endorphin-labeled protein with an

apparent molecular weight of 46 kilodaltons was observed (Carr et al.

1988b). The labeling was specific, since in the presence of unlabeled

naloxone, no band was observed. Interestingly, a 46-kilodalton protein has

reportedly been observed to be associated with opioid receptors of neuroendocrine origin as well (Yeung 1987: Roy et al. 1988). Since -endorphin

binds with near equal affinity to both μ- and -class binding sites (Barnard

and Demoliou-Mason 1983), a more selective ligand was sought. To this

end, cis-(3)-methylfentanylisothiocyanate (SUPERFIT), a -class opioid receptor ligand was employed. In situ labeling of T-cell-, B-cell-, and

macrophage-enriched populations revealed the specific labeling of a protein

that migrated with an apparent molecular weight of 58,000 daltons under

nonreducing conditions on polyacrylamide gels (Carr et al. 1988c). Under

reducing conditions, the molecular weight shifted to 70,000 daltons, indicative of intradisulfide chains, which have previously been reported in neural

opioid receptors (Gioannini et al. 1985; Kamikubo et al. 1988). Interestingly, the same procedures when applied to the neuroblastoma x glioma hybrid

cell line NG108-15 yielded identical results (figure 1). Moreover, the labeling of the protein species on immunocytes was nearly identical, as one

might predict using neuroendocrine receptors. That is, the -selective

ligand [D-Ala2,D-Leu5]-enkephalin (DADLE) was able to block SUPERFIT

labeling, whereas the p-selective ligand [D-Ala2,N-Me-Phe4,Gly5-ol] enkephalin (DAGO) did not block binding of SUPERFIT (Carr et al. 1988c). In

addition, naloxone was able to block greater than 70 percent of the binding

of SUPERFIT, suggesting a classical opioid receptor-ligand interaction.

Taken together, these results indicate an opioid binding site on cells of the

immune system that demonstrates selectivity in ligand preference as well as

biochemical characteristics similar to neuroendocrine-derived receptors.

FUNCTIONALITY OF IMMUNOCYTE OPIOID RECEPTORS

Mechanistically, the immunocyte opioid receptor appears to be coupled to

the same second-messenger pathways. Investigations into the immunocyte

CAMP pathway have determined that opioid peptides such as -endorphin

and [Met]-enkephalin are able to suppress adenylate cyclase activity in a

naloxone-reversible fashion at 10-7 to 10-9 M (Fulop et al. 1987; Carr et al.

1988a). In addition, prostaglandins, which are known to elevate CAMP in

124
FIGURE 1. Part A. Autoradiogram of electrophoresed [

3H]-SUPERFITlabeled opioid receptors from NG108-15 cells under

reducing (lanes 3 and 4) and nonreducing (lanes and 2)

conditions. Part B. Autoradiogram of murine peripheral

blood lymphocytes labeled with [

3H]-SUPERFIT.

NOTE: Part A. NG108-15 cells were incubated (45 minutes, 4 °C) with [3H]-SUPERFIT in the

presence (lanes 2 and 4) or absence (lanes 1 and 3) of unlabeled SUPERFIT. Cells were

washed once and lysed in denaturing buffer (in the presence or absence of 2-mercaptoethanol

and 6 M urea. Cell lysates were electrophoresed on a 10-percent polyacrylamide gel, and the

resultant gel was dried and subjected to autoradiography for 5 days at -70 °C. Intensifying

screens were employed to amplify the signal. Part B. Approximately 5x106 murine peripheral blood lymphocytes were incubated (45 minutes, 4 °C) with [

3H]-SUPERFIT in the presence (lane 2) or absence (lane 1) of unlabeled SUPERFIT. Cells were washed and lysed in

denaturing buffer (2-percent SDS, 0.062 M Tris-HCI, lo-percent glycerol, 5-percent 2-

mercaptoethanol). Cell lysates were electrophoresed on a 10-percent polyacrylamide gel, and

the resultant gel was dried and subjected to autoradiography for 5 days. Enhancing screens

were used to amplify the image of the bands.

SOURCE: Carr et al. 1988c, copyright 1988, Academic Press, Inc.

neural tissue, are reportedly antagonized by opioids in immunocytes (Farrar

1984). It is interesting to note that an increase in lymphocyte CAMP has a

general inhibitory effect on the immune response (Boume et al. 1974).

Thus, observations that show opioids to inhibit adenylate cyclase activity in

lymphocytes are in agreement with their stimulatory effects on immune

functions, such as enhancement of natural killer activity (Mathews et al.

1983) or enhancement in the generation of cytotoxic T cells (Carr and

Klimpel 1986).

125
Another pathway of intracellular signaling is ionic conductance channels.

Two predominate ion channels, the K

+

 and Ca++ channels, have been identified and defined not only in neuronal cells but in cells of the immune system as well (Chandy et al. 1985). In addition, similarities in channel

profiles have been found in cells of both systems. It is well known that

opioids modulate both K

+

 and Ca++ currents in association with hyperpolarization of neurons (Simonds 1988). Recently it has been shown that the

endogenous opioid peptide -endorphin suppresses outward K

+

 currents in

T cells. This effect is partially reversed by naloxone (Carr et al. 1988b).

Whether the effects opioids have on K

+

 or other ionic conductances in

immunocytes are associated with biological. responses remains to be determined. However, leukocyte potassium channels appear to be associated

with cell proliferation (Chandy et al. 1984) and natural killer activity (Sidell

et al. 1986). Thus, future investigations may reveal or identify associations

between the modulation of potassium fluxes and biological responsivity to

opioid peptides.

As mentioned earlier, calcium conductance in neurons is influenced by opiates. Investigations into calcium fluxes in leukocytes and the ability of

opioids to modulate such function have recently begun using the highly

fluorescent (Ca2+ indicator FURA-2AM. The introduction of the -class

opioid receptor ligand U-50,488H to lymphocytes loaded with FURA-2AM

causes a rapid influx of Ca2+ ions into the cell, as measured by spectrofluorometry (Carr and Blalock 1989). Interestingly, Concanavalin A also

induces a rise in intracellular Ca2+ over nearly the same time interval. This

relationship between a T-cell mitogen and the ligand’s ability to increase

Ca2+ suggests Ca2+ flux as a common and early event associated with proliferation and/or potentiating immune responses.

These results have led us to define other features of the lymphocyte opioid

receptor(s). In an attempt to purify the receptor(s), an anlibody to the

opioid receptor(s) was produced by a procedure based on the molecular recognition theory (Bost et al. 1985). Antibody to the opioid receptor was

able to block specific binding of -endorphin to the -class opioid receptor

as well as to compete with -endorphin and naloxone for binding to receptors on the neuroblastoma x glioma cell line NG108-15 cells (Carr et al.

1986). Moreover, this antibody was used to purify the b-class opioid receptor from NG108-15 cells, which was then shown to retain its binding

specificity and capacity (Carr et al. 1987). Likewise, the antibody was

shown to block specific binding of dihydromorphine to leukocytes. In addition the antibody possessed agonist activity, similar to -endorphin in reducing cAMP content in leukocytes in a naloxone-reversible fashion (Carr

et al. 1988a). Recently the antibody was used as an affinity absorbant for

the purification of the opioid receptor from leukocytes. The protein species

purified by affinity chromatography was analyzed by polyacrylamide gel

electrophoresis and was determined (similar to the in situ data) to have a

molecular weight of 58,000 daltons under ronreducing conditions and

126
70,000 daltons under reducing conditions (Carr et al., in press). In addition,

purified receptor was shown to possess the capacity to specifically bind the

class ligand SUPERFIT (Carr et al., in press). Future endeavors are

directed to the purification of the opioid receptor from both neuronal and

immunocyte sources for the eventual comparison of amino acid sequence.

It is anticipated that these sequences will be very similar if not identical.

CONCLUSION

The authors have investigated immunocyte opioid receptors at both the cellular and molecular levels and found them to possess many traits similar to

their neuroendocrine counterparts. For example, saturable, high-affinity

receptors appear on leukocytes, which in the case of the -class receptor

exhibit specificity in their ligand preference like neuronal receptors. In

addition, the molecular weight of the -class receptor isolated from leukocytes is identical to receptors purified from neuronal and neuronal-derived

cell lines. Furthermore, the delta class receptor possesses intrachain

disulfide bridges that cause a migratory shift on polyacrylamide gels under

reducing versus nonreducing conditions. Moreover, the molecular

mechanisms that appear to be affected upon receptor activation are parallel

between the two systems, suggesting them to be composites of one another.

Future work will undoubtedly revolve around in vivo manipulations of these

receptors in an attempt to understand their physiologic function (Shavit

et al. 1986; Weber et al. 1986). It is anticipated that this will lead

ultimately to a better understanding of the dynamic relationship between the

immune and neuroendocrine systems and how such a relationship impinges

on diseases such as AIDS.

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ACKNOWLEDGMENTS

Synthesis of cis-(+)-3-methylfentanylisothiccyanate was provided by

Drs. B. DeCosta, C.-H. Kim, A.E. Jacobson, and K.C. Rice, National

Institute of Diabetes and Digestive and Kidney Diseases. This work was

supported in part by grant #R01 DK38024 from the National Institutes of

Health Cancer Center Core grant CA13148, and Council for Tobacco

Research (#2222).

AUTHORS

Daniel J.J. Carr, Ph.D.

Research Assistant Professor

J. Edwin Blalock, Ph.D.

Professor

Department of Physiology and Biophysics

University of Alabama at Birmingham

UAB Station

Birmingham, AL 35294

130
lmmunomodulatory Effects of

Chronic Morphine Treatment:

Pharmacologic and Mechanistic

Studies

Henry U. Bryant, Edward W. Bernton, and

John W. Holaday

INTRODUCTION

Morphine and its related alkaloids produce a myriad of pharmacological

activities, including the antinociceptive, antidiarrheal, euphoric, and addictive

properties that have been well characterized. However, a number of other

pharmacologic responses following opiate administration have been described, including effects on endocrine function (Holaday and Loh 1979),

thermoregulation (Clark 1979), behavior (Lowy and Yim 1983), metabolism

(Lamb and Dewey 1981), respiration (Holaday and Malcom 1987) urine

output (Huidobro-Toro and Huidobro 1981), and the cardiovascular system

(Holaday 1983). Data from a number of laboratories indicate that immunomodulatory effects should be added to the list of activities attributed to

morphine and its related congeners.

The concept that opiates might affect immune function is not entirely a

recent development. In reports as early as 1967, an increased incidence of

bacterial and viral infection was demonstrated in heroin abusers (Louria

et al. 1967). While this phenomenon was at first attributed to the sharing

of infected drug paraphernalia and contaminated needles, there is now a

considerable volume of evidence suggesting that the opiate itself is involved,

via suppression of a variety of immunologic endpoints. Lymphocyte proliferative responses to mitogens such as concanavalin A (Con A), phytohemagglutinin, and pokeweed mitogen are suppressed in heroin addicts (Brown

et al. 1974). T-cell rosette formation (Wybran et al. 1979) and the total

number of circulating lymphocytes are also reduced in this population

(McDonough et al. 1980). In animal models, opiates can be shown to

affect a number of important immunologic correlates. Lymphocyte

131
proliferative responses (Bryant et al. 1987a), natural killer-cell cytotoxic

activity (Shavit et al. 1986), and the phagocytic and killing properties of

polymorphonuclear leukocytes (Tubaro et al. 1985) are all attenuated following morphine exposure. Morphine also inhibits antibody (Lefkowitz and

Chiang 1975) and serum hemolysin formation (Gungor et al. 1980) in mice

inoculated with sheep etythrocytes. Poly I:C-induced interferon production

is suppressed in morphine-treated animals (Hung et al. 1973), as is the development of delayed hypersensitivity in response to mycobacterium bovis

(BCG).

Immunomodulatory effects of morphine have been described following injection of a single dose (Weber and Pert 1989) and after daily injections with

escalating doses (Gungor et al. 1980). The issue of chronic exposure to

morphine with regard to its effect on immune function was of particular

interest. One of the most common methods of continuous exposure to morphine in rodents is the subcutaneous (SC) implantation of a pellet containing

morphine base. This technique allows for the induction of tolerance and

dependence within 12 to 24 hours. This chapter focuses on this work and

the findings of other laboratories that have utilized this model to better

understand the effects of chronic exposure to morphine on immune function.

The generation of an “immunologic profile.” in morphine-pelleted mice,

pharmacologic studies, and possible mechanisms for the phenomenon are

presented in this chapter.

EFFECTS OF MORPHINE-PELLET IMPLANTS ON VARIOUS

IMMUNE ENDPOINTS

The initial, and perhaps most striking, observation with regard to the effects

of morphine-pellet (NIDA) implantation on the immune system was the

marked atrophy of the spleen and thymus that occurs within 72 hours of SC

implantation of a 75-mg morphine pellet (figure 1). This atrophy of the

spleen and thymus was observed following correction for the loss in overall

body weight associated with chronic morphine treatment and was accompanied by reduced cellularity of these organs (Bryant et al. 1987a).

In addition to the reduction of immune organ weight and cellularity, a

suppression of mitogen-induced lymphocyte proliferation was observed in

morphine-pelleted mice. Blastogenic responses to Con A, a T-cell mitogen,

or to lipopolysaccharide (LPS), a B-cell mitogen, were measured by assessment of tritiated thymidine incorporation into the DNA of the proliferating

lymphocytes. As with spleen and thymus size, 72 hours following implantation of the morphine pellet, blastogenic responses to both Con A and to

LPS were significantly suppressed (Bryant et al. 1987a). Lymphocytes

stimulated to proliferate by cells carrying foreign histocompatibility antigens

in a mixed lymphocyte response were less responsive when taken from

morphine-pelleted mice (figure 2). It was also found that, in mice immunized with sheep erythrocytes, morphine-pellet implants suppressed the

132
FIGURE 1. Spleens and thymi removed from C3H/HeN mice 72 hours

following implantation of an inert placebo pellet or a pellet

containing 75-mg morphine

NOTE: Pellets supplied by the National Institute on Drug Abuse.

ability of B-lymphocytes to produce antibody in response to sheep erythrocytes in a plaque-forming assay (figure 2). This latter finding is consistent

with the results of Weber et al. (1987), who utilized a similar morphinepellet model and assessed antibody formation in vivo. Interestingly, TNPovalbumin-induced antibody production was preferentially suppressed in

morphine-pelleted mice, suggesting a T-cell dependent effect (Weber et al.

1987). Flow cytometric analysis has shown that this morphine-pellet regimen leads to a significant reduction in the relative abundance of B cells

without significantly affecting the total T-cell population, although there is a

133
slight elevation in the relative abundance of the T-suppressor subpopulation

(Peritt et al. 1988). Therefore, evidence from a variety of immunologic

endpoints, such as blastogenic responses, antibody production, and relative

abundance of lymphocyte subpopulations, idicates that chronic morphine

administration affects both T- and B-cell mediated immunity.

MLR PFA

FIGURE 2. Effect of morphine-pellet implants (72 hours) on a mixed

lymphocyte response (MLR) and on antibody production as

assessed by a plaque-forming assay (PFA)

*p<.05 vs. appropriate placebo control

The effects of this morphine regimen on another major group of cells in the

immune system, the macrophage, have also been examined. Agents such as

BCG, when injected into the peritoneum, elicit macrophage migration into

the peritoneal cavity. These macrophages are activated as evidenced by

their tumoricidal activity vs. tumor cell targets in vitro. Morphine-pellet

implantation attenuates the tumoricidal activity of BCG-elicited peritoneal

macrophages (Bryant et al. 1988d). Interestingly, exogenous administration

of gamma-interferon in vitro to peritoneal macrophages obtained from

morphine-pelleted mice restores tumoricidal activity. This suggests that

134
suppressed gamma-interferon production, as has been reported by others

(Hung et al. 1973), is a likely contributor to the suppression of macrophage

activation. These studies also showed that Ia expression on peritoneal

macrophages induced by BCG is reduced in morphine-pelleted mice.

These effects of morphine-pellet exposure on in vitro indices have some

relevance in vivo as evidenced by the finding that morphine-pelleted animals

also show increased risk to bacterial infection. A sublethal dose of Listeria

monocytogenes in nonpelleted mice produced a 75-percent mortality rate in

morphine-pelleted mice vs. 12-percent mortality in noninfected, morphinepelleted mice (Bryant et al. 1988d). The timecourse of the toxicity suggested that the increased lethality was related to the bacterial infection, as

opposed to a morphine-related toxicity. This finding is in agreement with

the work of Tubaro et al. (1983), who found that continuous morphine administration via an implanted osmotic minipump increased lethality due to

infection with Candida albicans.

PHARMACOLOGIC ANALYSIS OF MORPHINE-PELLET-INDUCEID

IMMUNOSUPPRESSION

An essential pharmacological issue to establish with any opiate-induced

effect is reversibility or blockade by an opiate-receptor antagonist. This is a

particularly critical issue when discussing effects of opiates on the immune

system, since, in certain instances, opiate modulation of immune function is

not naloxone reversible (Schweigerer et al. 1982). In order to determine

sensitivity of the immunologic deficits induced by morphine-pellet implantation to reversal by an opiate antagonist, the morphine-pellet was coimplanted with a 6-mg naloxone pellet in one series of experiments. As would be

expected in a typical opiate-receptor-mediated phenomenon, the splenic and

thymic atrophy and suppression of Con A- and LPS-induced blastogenesis

caused by morphine were prevented by chronic cotreatment with naloxone

(Bryant et al. 1987b) (table 1). A 48-hour exposure to the naloxone pellet

alone was largely ineffective, although a slight reduction in spleen size was

noted in these animals. Weber et al. (1987) demonstrated that morphinepellet-induced suppression of antibody production in vivo also is prevented

by naloxone and that the effect is stereospecific, offering further evidence

that this effect is mediated by an interaction at an opiate receptor.

Another important concern, particularly when dealing with chronic treatment

regimens, is the timecourse of the effect. Again, this issue is of particular

relevance when examining immune endpoints, as changes in immune function are often dependent upon the time at which the response is assessed

following challenge. Macrophage activation by endotoxin is a good example of this point. While endotoxin is an excellent stimulus for activation

of macrophages following acute exposure, continuous stimulation results in a

suppression of the response (Friedman 1972). Furthermore, physiologic

responses following acute exposure to opiates arc often quite different from

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TABLE 1. Effect of naloxone and tolerance on morphine-pellet-induced

immunomodulation

Parameter Morphinea

Morphine +

Naloxoneb Naloxone

Morphine +

Morphine PRE-TXc

Spleen Weight 55±3*

Thymus Weight 36±2*

Con A-Induced Proliferation

0.25 μg/ml 32±21*

0.5 μg/ml 47±7*

1.0 μg/ml 52±10*

2.0 μg/ml 60±16

LPS-Induced Proliferation

0.1 μg/ml 51±27*

0.5 μg/ml 44±20*

5.0 μg/ml 74±17

94±1

82±3

121±23

102±9

102±9

94±10

103±17

89±10

102±7

85±4*

90±7

101±13

100±8

96±9

90±2

99±8

82±6

94±4

72±5*

32±4*

72±12

100±14

97±19

85±24

117±11

108±26

96±23

*p<.05 vs. placebo group; values represent percent of mean placebo response ± SEM.

a

48-hour morphine-pellet (75 mg) exposure period.

b

6-mg naloxone pellet coimplanted with morphine or placebo pellet.

c

72-hour pre-exposure period to 75mg morphine-pellet, followed by a second

pellet for 48 hours.

those observed in tolerant animals (Morley et al. 1982). Therefore, extensive temporal analyses were conducted of the effects of morphine-pellet

implantation on immune function. T-lymphocyte proliferative responses

were largely unaffected at 6 or 24 hours following morphine-pellet implantation, but were reduced at 48 and 72 hours. Interestingly, the response

was essentially normal by 96 hours and a rebound stimulation was observed

at 120 hours (Bryant et al. 1988a), a time when antinoiceptive tolerance to

a morphine challenge is still increasing (figure 3). B-lymphocyte proliferative responses to LPS and the thymic and splenic atrophy followed a similar

pattern of suppression, followed by a return toward normal at the later time

points. The suppression of B-cell blastogenesis and thymic atrophy

appeared to be the most sensitive endpoints.

The loss of immunosuppression at the later time points is most easily explained by either (1) exhaustion or encapsulation of the morphine pellet

leading to a decreased bioavailability or (2) to the development of tolerance

to the effect. Blood morphine levels were assessed in morphine-pelleted

mice in a collaborative effort with Dr. Byron Yobum of St. John’s University. Following an early surge at 6 and 24 hours, serum morphine levels

136
FIGURE 3. Timecourse for the development of tolerance to antinociception following morphine challenge in morphine-pelleted

mice (panel A) compared to the timecourse for the effect

on concanavalin A (Con A)-induced lymphocyte proliferation (panel B)

SOURCE: Bryant et al. 1988a, copyright 1988, American Society for Pharmacology and

Experimental Therapeutics

137
plateaued and remained fairly constant (approximately 600 ng/ml) over the

following 4 days (Bryant et al. 1988b). Therefore, the difference in

immunomodulatory effects at 72 and 120 hours postpelleting are not due to

an altered bioavailability of morphine from the morphine pellet. This is

consistent with the early findings of Patrick et al. (1975) who demonstrated

that the analgetic ED-50 and morphine levels in the brain are elevated for

as long as 144 hours following morphine-pellet implantation.

Since altered release of morphine was not a satisfactory explanation for the

altered immunomodulatory effects, additional studies were conducted to

evaluate the development of tolerance to these effects. In these experiments, animals were pre-exposed to a 75-mg morphine-pellet for 72 hours,

then given an additional pellet for a 48-hours period and the immune variables evaluated. In the morphine-pretreated animals, the degree of thymic

atrophy was comparable to nonpretreated mice, but the severity of splenic

atrophy was not as great as in 48-hour morphine-implanted animals not preexposed to morphine (table 1) (Bryant et al. 1987b). The suppression of

Con A- and LPS-induced blastogenesis was completely lost in the

morphine-pretreated group. Therefore, it is apparent that tolerance does

develop to the immunomodulatory effects of chronic morphine treatment and

that the loss of splenic and thymic atrophy and mitogen-induced lymphocyte

proliferation at later time points in the model reflects the development of

tolerance to the morphine pellet.

The investigators have only scratched the surface of important pharmacological questions regarding morphine-induced immunomodulation. Receptorsubtype involvement, a central vs. peripheral site of action, and effects of

other opiate agonists in a similar model are all important questions yet to

be addressed. The role of withdrawal in the immunomodulatory effects has

been addressed in some preliminary investigations. ‘Temporal factors again

appear involved, as at early time points (e.g., 48 hours), precipitated withdrawal has no effect on the suppressive effects of morphine-pellet implantation (Bryant et al. 1987b). However, at later time points (e.g., 144 hours),

increases in Con A-induced blastogenesis may be observed in withdrawing

animals (Kenner et al. 1988a).

MECHANISTIC STUDIES

Studies into possible mechanisms for the immunosuppressive effects observed 48 hours following morphine-pellet implantation have focused on

three areas: (1) direct effects of morphine on lymphocyte proliferative

responses; (2) the level of the morphine-induced deficit in the blastogenic

cascade; and (3) possible mediation via alterations in neuroendocrine

systems, particularly of prolactin and corticosterone.

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Direct Effects

Several reports have suggested the presence of opiate receptors on lymphocytes (Carr 1988). Therefore, morphine interaction with these receptors is

one possible hypothesis that might explain the immunomodulatory effects in

the model. However, exposure of lymphocytes obtained from normal mice

to a wide range of morphine concentrations in vitro did not suppress

Con A-induced blastogenesis. In fact, concentrations in the micromolar

range (comparable concentration to serum levels recorded in the morphinepelleted mice at the 48-hour time point (Bryant et al. 1988b) produced an

elevation in lymphocyte proliferation in response to Con A (figure 4).

Furthermore, the opiate antagonist naloxone produced a similar profile in

vitro (i.e., stimulation at micromolar concentrations). These observations

argue against a direct mode of action of morphine at the lymphocyte level

as an explanation for the immunosuppressive effects.

FIGURE 4. Effect of morphine addition in vitro to normal mouse

(C3H/HeN) lymphocyte cultures stimulated by 1.0 mcg/ml

Con A

139
Level of Morphine-Induced Immunosuppression

The blastogenic response of lymphocytes depends upon the interplay of

events, which include the recognition and processing of foreign antigen by

antigen-presenting cells, production of monokine and lymphokine mediators

for amplification of the response, and. ultimately, the proliferation and differentiation of antigen-responsive lymphocytes capable of cell-mediated cytotoxicity, antibody production, and macrophage activation. The lymphocyte

product interleukin-2 (IL-2) plays a central role in the proliferation of

activated T cells.

In work done with Dr. Thomas Jerrells (University of Texas Medical

Center, Galveston, TX), it has been found that the attenuated lymphocyte

proliferative responses to Con A in the morphine-pelleted mice are directly

linked to a deficiency in production of IL-2 by the lymphocytes. A cell

line (CTLL) that is highly sensitive to IL-2 was used as a bioassay for IL-2

levels in the supernatant of proliferating cells from either placebo or

morphine-pelleted mice. A marked reduction in the IL-2 level in these

supernatants was observed in cell cultures taken from morphine-pelleted

mice as compared to those taken from placebo-pelleted mice (Peritt et al.

1988). As observed with the proliferation data, IL-2 levels were not

reduced in cell suspensions taken from morphine-pelleted mice that were

also given a naloxone pellet.

Further evidence was provided by experiments that demonstrated that addition of exogenous IL-2 to the media elevated the proliferative responses of

lymphocytes obtained from morphine-pelleted mice to mitogen, although not

to the same magnitude as was observed with lymphocytes from placebopelleted mice. Addition of supernatants from normal Con A-stimulated

lymphocytes (which contain IL-2 in addition to other important growth factors) also reversed the attenuation of the response in lymphocytes obtained

from morphine-pelleted mice. Possible changes in lymphocyte responsiveness to IL-2 were ruled out with an assay developed by Dr. Jerrells and

David Peritt. In this procedure, lymphocytes from morphine- and placebopelleted mice were bulk stimulated with Con A. Following a sufficient

period of time to allow for blastogenesis, those lymphocytes that had responded to the Con A stimuli to form blastocytes were separated from the

remaining cells on a Ficoll gradient. As might be expected based on the

proliferative data, the number of blastocytes was significantly reduced in

cultures obtained from morphine-pelleted mice. Equal numbers of blast

cells from morphine and placebo mice were then exposed to IL-2 in culture.

Following incubation, proliferation of the blast cells was then determined.

The blast-cell procedure is a very useful tool to allow comparison of cells

with fully expressed IL-2 receptor. Interestingly, blast cells from morphinepelleted mice showed no reduction in IL-2-elicited proliferation when compared to blast cells originating from placebo-pelleted mice (Peritt et al.

1988). These studies indicate that the morphine-induced “defect” most

140
likely occurs prior to receptor recognition of IL-2 and that it can be

accounted for at least in part by attenuated production of lymphokine mediators of the mitogen-induced growth response.

These studies suggested that an earlier event in the proliferation cascade

might be responsible for the reduced IL-2 production and ultimate inhibition

of proliferative responses. Therefore, a separate series of experiments was

aimed at determining possible effects of chronic morphine treatment on the

macrophage. Macrophages play an important role in the processing of antigen and the production of monokines that eventually lead to the production

of IL-2 by lymphocytes. A possible effect of morphine-pellet implantation

on the macrophage was suggested by two preliminary observations. The

first was that, as previously mentioned, morphine-pellet implantation greatly

increased the lethality of a sublethal injection of L. monocytogenes in mice.

The second observation was that, in addition to inhibiting Con A-induced

lymphocyte proliferation, chronic morphine treatment also inhibited the

mixed lymphocyte response in mice. The macrophage seemed to be a possible common denominator in this case based on the knowledge that

L. monocytogenes is primarily a macrophage pathogen and that the mixed

lymphocyte response requires antigen processing (i.e., by macrophages)

since lymphocytes from an allogeneic animal serve as the proliferative

stimuli in this assay.

To assess possible effects of morphine treatment on macrophage involvement in these proliferative defects, macrophages and T-lymphocytes were

isolated from spleens obtained from placebo- or morphine-pelleted mice and

then combined in a crossover design (figure 5). With this approach, it was

demonstrated that when lymphocytes from chronically morphine-treated mice

are incubated with macrophages from placebo-pelleted mice, only a mild reduction in Con A-induced proliferation is observed. However, when lymphocytes from placebo-pelleted mice are incubated with macrophages from

morphine-pelleted mice, the more familiar marked reduction of Con Ainduced proliferation is observed (Bryant et al. 1988d). These findings

suggest a critical role for the macrophage in morphine-induced immunosuppression. The possibility has been ruled out that prostaglandins originating from the macrophage are involved in the immunosuppressive effect,

since the suppression cannot be blocked by indomethacin added in vitro.

Therefore, future investigations will examine the effects of chronic morphine

treatment on the production of macrophage activation factors (e.g., gammainterferon) and on monokine production (e.g., interleukin-1) by the macrophage itself.

Since a direct effect of morphine appeared unlikely, possible indirect effects

were assessed. Cold exposure is known to suppress the immune system

(Hardy et al. 1986), and morphine produces hypothermia. However, this

possible indirect mechanism was ruled out by the timecourse studies since,

141
at the later time points, morphine-pelleted mice were hypothermic but were

not immunosuppressed (Bryant et al. 1988a).

FIGURE 5. Design for macrophage/T-cell crossover study

NOTE: Numeric values represent tritiated thymidine uptake induced by 0.5 mcg/ml Con A as a

percentage of the placebo macrophage/T-cell control combination.

Neuroendocrine-Based Mechanisms

Possible effects of morphine on neuroendocrine correlates have also been

examined as a possible mechanism for the immunosuppressive effect.

Based on the germinal research efforts by Dr. Bernton, investigation of the

immunopermissive role of prolactin has taken place (Bemton et al. 1988).

Since morphine is known to affect prolactin release (Holaday and Loh

1979), this link was first examined as a possible mechanism for the

immunosuppressive effect of chronic morphine treatment. Implantation of

pellets containing ovine prolactin or injection of prolactin-releasing drugs

such as the dopamine antagonist metoclopramide prevented the suppression

of mitogen-induced proliferative responses induced by morphine. Interestingly, the splenic and thymic atrophy subsequent to morphine-pellet

142
implantation were not reversed by these prolactin-modifying regimens. Prolactin levels were assessed in the morphine-pelleted mice, and, surprisingly,

no significant differences were noted when compared to placebo-pelleted

mice at 6, 24, 48, 72, 96, or 120 hours postimplantation, indicating an

apparent difference in the effects of morphine on prolactin release between

mice and rats (Bryant et al. 1988b). Recent findings by Julie Kenner

(Kenner et al. 1988) and by others (Montgomery et al. 1987) have demonstrated the presence of a lymphocyte prolactin or prolactinlike protein that is

elevated following mitogenic stimulation. Levels of lymphocytic prolactin

are also unaltered by morphine-pellet implantation. When considered with

the rest of the data, these findings suggest that, while prolactin-modifying

regimens can reverse certain aspects of morphine-pellet-induced immunosuppression, a role for alterations in prolactin as a possible mechanism for the

effect of morphine is unlikely. These findings also indicate that this

morphine-pellet model may be useful in identifying immunopotentiating

substances.

Glucocorticoid mobilization subsequent to stress is known to be one mediator of stress-induced immunosuppression (Solomon and Amkraut 1981),

since glucocorticoids are well-recognized immunosuppressants (Parrillo and

Fauci 1979). Morphine is also known to activate the hypothalamo-pituitaryadrenal (HPA) axis (George and Way 1955; Holaday and Loh 1979). Indeed, several lines of evidence suggested involvement of glucocorticoids in

the immunosuppressive-type effects following chronic morphine treatment:

(1) timecourse studies indicated a significant degree of adrenal hypertrophy

in the morphine-pelleted mice (Bryant et al. 1988a); (2) the thymic atrophy

produced by morphine-pellet implantation is primarily of the thymic cortex,

a particularly sensitive area to the lytic effects of glucocorticoids (Bryant

et al. 1988b); (3) the profile of events blocked by morphine in the lymphocyte proliferative cascade closely parallels steps blocked by glucocorticoids

(figure 6); and (4) corticosterone levels are elevated in morphine-pelleted

mice, with a timecourse that roughly resembles the suppression of mitogendriven proliferative responses in these animals (Bryant et al. 1988b).

Therefore, the immunosuppressive effects of 48-hour morphine-pellet implants were evaluated in adrenalectomized mice and in mice treated with the

glucocorticoid receptor antagonist RU-486 in order to determine the contribution of adrenal corticosteroids to chronic morphine-induced immunosuppression. As reviewed in table 2, the magnitude of splenic and thymic atrophy was less pronounced in the adrenalectomized, morphine-pelleted mice.

The relative degree of the attenuation of LPS-induced lymphocyte proliferation was also smaller in the adrenalectomized morphine-pelleted mice and

morphine-induced reduction of Con A-stimulated proliferation was absent in

the adrenalectomized animals (Bryant et al. 1988b).

Adrenalectomy represents a rather severe manipulation to the organism.

Complete adrenalectomy also removes adrenergic outputs from the adrenal

medulla, which may have some impact on immune function (Johnson et al

143
FIGURE 6. Comparison of steps in the lymphocyte proliferative cascade

that are sensitive to glucocorticoid administration (bars)

and to chronic morphine treatment (asterisks)

1981). Therefore, as a corollary to the adrenalectomy studies, the effects of

RU-486, a steroid congener known to act as an antagonist at the glucocorticoid receptor were also examined (Moguilewsky and Philibert et al. 1984).

Overall, effects similar to adrenalectomy were observed in morphine-pelleted

mice given 20 mg/kg RU-486 twice per day of morphine exposure (table 2)

(Bryant et al. 1988b). As with adrenalectomy, splenic atrophy was less

pronounced with RU-486 administration. RU-486 had only marginal effects

144
on thymic atrophy and the suppressed proliferative responses of splenocytes

from morphine-pelleted mice to Con A or LPS. The suppressed mixedlymphocyte response in morphine-pelleted mice was also less severe when

RU-486 was administered. These studies indicate that morphine-induced

immunosuppression is at least in part due to morphine-induced activation of

the HPA axis. In addition to direct activation of the HPA axis, chronic

morphine treatment may lead to inflated circulating corticosterone levels

indirectly, since morphine and corticosterone compete for the same metabolic enzymes for degradation and ultimate clearance (Holaday and Loh

1979).

TABLE 2. Comparison of immunosuppressive effects in adrenalectomized

(ADX) and RU-486 treated mice

Parameter

Morphine-pellet* Morphine-pellet

Sham ADX Saline RU-486

Spleen Weight

Thymus Weight

Con A-Induced Proliferation

(0.25 μg/ml)

LPS-Induced Proliferation

(0.5 μg/ml)

Mixed Lymphocyte

Response

6.5±2 83±5 60±1 82±5

44±2 78±5 45±3 52±5

44±4 123±12 52±8 70±7

62±6 74±10 68±14 92±8

56±1 84±7

*48-hour morphine-pellet (75 mg) exposure period

NOTE: Values represent percent of mean placebo response ± SEM.

SOURCE: Bryant et al. 1988b.

Several lines of evidence suggest that the immunosuppressive effect of

chronic morphine administration is related to its stimulatory effect on the

HPA axis and subsequent mobilization of glucocorticoids. Traditional stress

models are known to result in analgesia, adrenal hypertrophy, thymic atrophy, increased levels of circulating corticosterone and cholesterol, and

hyperphagia (Neill et al. 1970; Stewart and Eikelboom 1981; Hulse et al.

1982; Morley et al. 1983; Rothfeld et al. 1983; Terman et al. 1984). Stress

also results in attenuated blastogenic responses to mitogenic stimulation

(Solomon and Amkraut 1981). Chronic morphine treatment produces a remarkably parallel pattern of responses (Pang et al. 1977; Holaday and Loh

1979; Lowy and Yim 1983; Bryant et al. 1987c). Morphine-induced

145
immunosuppression represents another effect of the opiate that is shared

with chronic stress. This similarity has prompted the suggestion that

chronic morphine treatment may be considered as a pharmacologic mimic of

more traditional stress models. Finally, the immunosuppressive effects of

morphine may have potentially important clinical relevance, particularly in

individuals at a hign risk for infection or in immunocompromised patients.

It has been speculated that the increased incidence of acquired immunodeficiency syndrome in narcotic drug abusers may have some basis in the

immunomodulatory effects of the opiate. Indeed, research efforts reported

in this monograph support this conjecture (Watson et al., this volume).

Effects on immunocompetence should represent an additional area of

concern regarding untoward effects in the development of novel opiaterelated pharmaceuticals.

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AUTHORS

Henry U. Bryant, Ph.D.

Department of Immunology, Pulmonary and

Leukotriene Research

Eli Lilly and Co.

Indianapolis, IN 46285

Edward W. Bernton, M.D.

John W. Holaday, Ph.D.

Department of Medical Neuroscience

Division of Neuropsychiatry

Walter Reed Army Institute of Research

Washington, D.C. 20307

149
Morphine-Induced Immune

Modulation: Does It Predispose to

HIV Infection?

Prince Kumar Arora

INTRODUCTION

While the prevalence of HIV seropositivity in the United States is highest

for homosexual males (ranging from 20 percent to 70 percent), the incidence of seropositivity among intravenous drug abusers can be even greater,

ranging from about 5 percent in certain areas to 75 percent in others

(Centers for Disease Control 1984). Of all seropositives, intravenous drug

abusers are generally regarded as posing the most substantial risk of spreading the disease to heterosexuals, with the risk being greater in urban areas

(Centers for Disease Control 1984). Also at risk are infants born to parents

who are intravenous drug users. Current statistics suggest that one out of

two children born to seropositive women develop and almost certainly die

of AIDS before the age of 5. There is growing evidence that drug abuse

leads to an increased susceptibility to infections (Chetubin and Millian 1968;

Sapira et al. 1970) and widely abused drugs such as the opiates have been

shown to produce profound effects on the immune system (Cherubin and

Millian 1968; Sapira et al. 1970; McDonough et al. 1980; Donahoe

et al. 1985; Donahoe et al. 1986; Shavit et al. 1986; Yahya and Watson

1987; Bryant et al. 1988; Arora et al., in press). These findings prompted

the study of the relationship(s) between drug abuse and increased

susceptibility to infection.

This chapter will report the finding that opiate addiction causes marked

atrophy of immune organs and a robust and sustained elevation of

CD4+:CD8+ (T helpet/inducer:T suppressor/cytotoxic) and CD5+:CD8+

(T helper:T suppressor/cytotoxic) cell ratios in rodents implanted subcutaneously (SC) with a single morphine pellet. These observations demonstrate that morphine-induced changes in immune competence may predispose

for the higher incidence of HIV infection in opiate abusers.

150
MATERIALS AND METHODS

Morphine Pellet Implantation

Male B10.BR mice (Jackson Laboratory, Bar Harbor, ME) were implanted

SC with pellets containing either morphine (75 mg, free base) (Gibson and

Tingstad 1970) or microcrystalline cellulose (placebo). Morphine and placebo pellets were prepared by K. Sones and G. Grimes of the National

Institutes of Health (NIH) pharmacy. This regimen of morphine produced a

mortality rate of 10 to 30 percent. These deaths occurred usually within

the first 24 hours of pellet implantation and occasionally as late as

48 hours. The appearance of an abstinence syndrome following naloxone

challenge (2 mg/kg, intraperitoneally (IP)) was used to assess the efficacy of

pellet implantation. Both morphine- and placebo-treated mice were challenged with naloxone HCl 24 to 96 hours after implantation, and their behavior was assessed for an additional 15 minutes. An abstinence syndrome,

including hyperactivity, vocalization, jumping, and wet-dog shakes, was

apparent less than 3 minutes after naloxone treatment in morphine-treated

mice. In contrast, naloxone produced only a transient hyperactivity in

placebo-treated mice. Morphine treatment also resulted in a slight weight

loss (less than 10 percent of the total body weight) at 72 hours compared to

placebo-treated animals.

Organ Weight and Viable Cell Count

Mice were killed by cervical dislocation at 24-hour intervals following pellet

implantation. Immune organs were removed, weighed, and placed in sterile

Hanks balanced salt solution (HBSS) (GIBCO, Grand Island, NY) containing 10 percent heat-inactivated fetal bovine serum (FBS) (Hazleton Laboratories Inc.) (HBSS+FBS). Single-cell suspensions from individual organs

were prepared as previously described (Arora and Shearer 1981). Cells

were treated with ACK-lysing buffer (NIH media unit) to remove erythrocytes and washed twice in HBSS+FBS. Viable cell counts of the spleen

and thymus were determined by trypan blue exclusion. Each data point is

the mean plus or minus the standard error of the mean of five animals.

These results represent three to four separate experiments.

Histopathology of Immune Organs

Histopathology of immune organs was done by Dr. Kim Waggie (Comparative Pathology Section, Division of Research Services, NIH). At 72 hours,

both the spleen and thymus were removed from mice implanted with either

morphine or placebo pellet. The immune organs were fixed in 10 percent

formalin, embedded in wax, and slices cut by microtome. Single slices of

the immune organs were stained with hematoxylin and eosin and examined

under microscope.

151
Flow Cytofluorometric Analysis

At various time intervals following morphine-pellet implantation, single-cell

suspensions from individual organs were prepared and stained with monoclonal antibodies (mAb) specific for cell-surface markers (sIg, Thy 1, CD4,

CDS, and CD8) as described previously (Arora et al. 1987b) (table 1). In

brief, 2x106 cells were incubated on ice (in the dark) with 5 μl of appropriate fluorescent isothiocyanate (FITC)-conjugated mAb for 45 minutes. After

two washes with HBSS+FBS (2 percent), the cell pellet (2x106 cells) was

resuspended and analyzed by flow cytofluorometry. In some cases, cells

were incubated with mAb, washed, and then incubated with a 5 μl of

FITC-conjugated goat F(ab’)-anti-rat IgG antibody specific for k-chain on

ice for 30 minutes, washed, and analyzed. FlTC-conjugated monoclonal

antibodies specific for sIg+ cells were obtained from Organon-Teknika Corporation, West Chester, PA. FITC-conjugated antibodies specific for Thy

1

+

lyt l

+

 (CD5+). and lyt 2

+

 (CD8+) cells were obtained from Becton Dickinson, Inc., Mountain View, CA. L3T4+

 (CD4+)-specific antibody was obtained from Becton Dickinson, Mountain View, CA). Goat anti-rat k-chain

mAb was obtained from Organon-Teknika Corporation, West Chester, PA.

TABLE 1. Surface markers used to define cell types

Cell Surface Marker Cell Type

sIg+

Thyl+

CD4+

CD5+

CD8+

B lymphocytes

T lymphocytes

Helper/Inducer T lymphocytes (H/I TL)

All T lymphocytes

Suppressor T lymphocytes (STL)

Cytotoxic T lymphocytes (CTL)

RESULTS

Time Kinetics of Morphine Effect on Immune Organ Weights

As shown in figure 1, implantation of a single morphine pellet had a

marked effect on the weight of immune organs. A 40-percent reduction in

splenic weight was observed within 24 hours of implantation of morphine

pellet (figure 1, top panel). The magnitude of spleen atrophy became greater, with maximum suppression at 72 hours (65 percent reduction relative to

placebo). This effect waned and then was undetectable after 6 to 8 days.

Atrophy of the thymus in morphine-treated mice (figure 1, bottom panel)

displayed a similar pattern. Thymic atrophy was most pronounced during

48 to 72 hours postimplantation (70 to 80 percent reduction relative

152
FIGURE 1. Time course of morphine-induced effects on spleen and

thymus wet weights in mice

NOTE: Mice were sacrificed at various time intervals after implantation of a 75-mg morphine pellet

(----) or a placebo pellet (—). The spleen and thymus were removed and weighed, and the

weights corrected for the individual animal’s body weight. Each data point represents the

mean organ weight in g/kg of body weight ±SEM for five animals.

SOURCE: Arora et al., in press.

153
to placebo), but, unlike spleen, thymus size remained significantly smaller

for a longer period (17 to 21 days postimplantation of the morphine pellet).

A 10- to 30-percent rate of lethality was observed in mice given morphinepellet implants. These deaths occurred usually within the first 24 hours of

pellet implantation and occasionally as late as 48 hours.

Time Kinetics of Morphine Effect on Viable Cell Count in Spleen

and Thymus

The reduction in spleen weight was associated with a decrease in spleencell number as shown in figure 2, top panel. As with splenic weight, the

greatest reduction of spleen-cell number was observed at 48 to 72 hours

postimplantation intervals (90 percent at 48 hours to 62.5 percent at

72 hours relative to placebo). At subsequent time points (17 to 21 days

later), the spleen-cell number of morphine-pelleted mice returned toward that

of the placebo group. Thymic atrophy was also associated with a decrease

in cell number (90 percent at 72 hours to more than 9.5 percent at 96 hours

relative to placebo group) in the thymus (figure 2, bottom panel), the magnitude of which paralleled the reduction in thymus weight. The recovery of

cell numbers in immune organs occurred at different rates, but the reduced

cell number persisted after normalization of organ weights had occurred

(compare figure 1 with figure 2). Cell viability as assessed by trypan blue

exclusion was unaffected by morphine treatment. Histopathological analyses

of immune organs (spleen and thymus) demonstrated a significant depletion

of the number of periarteriolar lymphoid sheaths (PALS) in the spleen of

morphine-implanted (figure 3B) compared with placebo-implanted mice (figure 3A). In addition, a significant depletion of lymphocytes in the white

pulp area was observed in spleens of morphine-implanted mice (figure 3D)

compared to controls (figure 3C). Morphine treatment also caused a significant depletion of cells in the thymus (figure 4B compared to figure 4A).

Thc localization pattern of cells in the thymus revealed depletion of thymocytes in the cortex region (figure 4).

Flow Cytofluorometric Analysis

Spleen. Figure 5 summarizes data on the cytofluorometric analysis of

spleen cells. At various time periods following implantation of morphine

pellet, cells were stained with FITC-conjugated monoclonal antibodies specific for cell surface markers. Morphine did not cause any significant

change in the number of B (sIg+) cells (figure 5B to figure 5E) when compared with the placebo group (figure SA). Within 24 hours following

morphine-pellet implantation, there was a sharp increase in the number of T

(Thy 1

+

) cells in the spleen (figure 5F compared with figure 5G). The

number of Thy 1

+

 cells remained high for an additional 3 days (figure 5H

to figure 5J). Figure 6 shows data on the number of CD4+, CD5+, and

CD8+ cells in the spleen of both placebo-pellet implanted animals (figure

6A, 6F, and 6K) and morphine-pellet implanted animals (figure 6B to 6E,

6G to 6J, and 6L to 60) at different time periods. The occurrence and

154
FIGURE 2. Time coarse of morphine-induced changes in viable cell number in spleen and thymus at various time intervals after

implantation of a 7.5-mg morphine pellet (----) or a placebo

pellet (—)

NOTE: Each data point represents the mean variable cell count ± SEM of five animals. these results

are representative of 3 to 4 separate experiments.

SOURCE: Arora et al., in press.

155
FIGURE 3. Hermatoxylin and eosin staining of spleen 72 hours after

implantation of mice with placebo pellet (A and C) or morphine pellet (B and D)

NOTE: Note depletion of the number of periarteriolar lymphoid sheaths (white pulp areas) and loss of

lymphocytes in the white pulp area. Magnification X 50 (A and B); X 200 (C and D).

SOURCE: Arora et al., in press.

significant increase in the number of CD4+ and CD5+ cells are readily evident in figures 6B to 6E and 6G to 6J, respectively. There was little or no

change in the number of CD8+ cells in the morphine-pelleted group (figure

6L to 60) when compared to the placebo group (figure 6K). The observed

increases in the number of CD4+ and CD5+ cells occurred in 100 percent of

the animals in the morphine-pelleted group.

Thymus. The cytofluorometric data on thymocytes shown in figure 7 indicatc that morphine caused a significant drop in the number of Thy 1

+

 cells

(figure 7B to 7E) when compared to the placebo group (figure 7A). Morphine also had an effect on the number of CD4+

 (figure 7G to 7J) and

CD8+ (figure 7L, to 7O). but the drop in the number of CD8+ cells was

156
FIGURE 4. Hematoxylin and eosin staining of thymus 72 hours following

implantation of mice with placebo pellet (A and C) or morphine pellet (B and D)

NOTE: Note the depletion of thymocytes in both the cortical and medullary regions. Magnification X

80 (A and B); X 310 (C and D).

SOURCE: Arora et al., in press.

more marked when compared to the drop in the number of CD4+ cells at all

time points examined.

Morphine Effect on Cell to Cell Ratios

As shown in table 2, morphine markedly increased the ratios of T:B,

CD4+:CD8+, and CD5+:CD8+ cells in the spleen and CD4+:CD8+ cells in the

thymus. The increased CD4+:CDS+ and CD5+:CD8+ ratios were due to increases in the number of CD4+

 and CDS’ cells respectively, compared with

placebo-implanted animals.

157
FIGURE 5. Flow cytofluorometric analysis of spleen cells obtained from

mice implanted with placebo or morphine, stained with

FITC-conjugated goat anti-mouse or anti- Thy 1.2 antibodies

NOTE: Cells obtained on day 1 (1B and 1G). day 2 (1C and 1H). day 3 (1D and 1I), and day 4 (1E

and 1J) from mice implanted with placebo (1A and 1F) or morphine (1B to 1E and 1G to 1J)

pellets were stained with FITC-conjugated goat anti-mouse IgG (1A to 1E) or anti-Thy 1.2

(1F to 1J) antibodies. Each cytofluorogram shows the fluorescence of stained (shaded area)

vs. unstained (solid line) cells.

158
FIGURE 6. Flow cytofluorometric analysis of spleen cells obtained from

mice implanted with placebo or morphine, stained with

CD4, CD5, or CD8 monoclonal antibodies

NOTE: Cells obtained on day 1 (2B, 2G. and 2L), day 2 (2C, 2H. and 2M), day 3 (2D. 2I, and 2N),

and day 4 (2E, 2J, and 2O) from mice implanted with placebo (2A, 2F, and 2K) or morphine

(2B to 2E, 2G to 2J, and 2L to 2O) pellets were stained with CD4+ (2A to 2E), CD5+ (2F to

2J), or CD8+ (2K to 2O) monoclonal antibodies. Each cytofluorogram shows the fluorescence

of stained (shaded area) vs. unstained (solid line) cells.

159
FIGURE 7. Flow cytofluorometric analysis of thymocytes obtained from

mice implanted with placebo or morphine

NOTE: Ihymocytes obtained on day 1 (3B, 3G, and 3L). day 2 (3C. 3H, and 3M). day 3 (3D, 3I,

and 3N), and day 4 (3E, 3J, and 3O) from mice implanted with placebo (3A, 3F, and 3K) or

morphine (3B to 3E, 3G to 3J, and 3L to 3O) pellets were stained with Thy 1+ (3A to 3E),

CD4+ (3F to 3J), or CD8+ (3K to 3O) monoclonal antibodies. Each cytofluorogram shows the

fluorescence of stained (shaded area) vs. unstained (solid line) cells.

160
TABLE 2. Immunofluorescent analysis of spleen cells and thymocytes

Cell:Cell Ratio

Spleen Thymus

Groups Thy 1

+

:sIg+ CD4+:CD8+ CD5+:CD8+ CD4+:CD8+

Placebo 0.44 1.52 3.28 1.01

Morphine

24 hr 0.82

48 hr 0.86

72 hr 0.84

96 hr 0.89

120 hr 0.34

SOURCE: Arora et al., in press

2.56 5.21 2.54

2.56 4.37 2.22

2.16 4.24 2.11

2.70 4.65 2.03

2.66 3.39 N.D.

DISCUSSION

While the mechanisms responsible for these morphine-induced changes in

immune function are unknown, they may be mediated directly through

opiate receptors present on lymphocytes (Sibinga and Goldstein 1988), indirectly via opiate receptors in the central nervous system, or by activating

the hypothalamic-pituitary-adrenal (HPA) axis (George and Way 1955) to

release immunosuppressive glucocorticoids (Parrillo and Fauci 1979).

Morphine-induced changes in immune function have also been postulated to

mimic the effects of stress (Bryant et al. 1987). However, while chemically

induced stress (Arora et al. 1987a) and behaviorally induced stress

(Laudenslager et al. 1983; Stein et al. 1985) can affect T-cell function, these

procedures do not produce the rapid and profound changes in cell number

or organ weight reported here.

The reduction in spleen and thymus size may suggest possible morphineinduced changes in lymphocyte trafficking because a decrease in organ

weight was associated with a decrease in cell number. However, the observation that cell numbers in immune organs recovered at rates different from

the normalization of organ weights would argue against the above possibility. Since opiate receptors are found on the surface of lymphocytes (Sibinga

and Goldstein 1988), a direct action is also possible. In vitro studies, however, have generally found opioids to be stimulatory in nature (Wybran

et al. 1979; Johnson et al. 1982; Gilman et al. 1982; Miller et al. 1984).

Cold stress has long been known to suppress immune function (Hardy et al.

1986). The hypothermia often observed in opiate-treated animals therefore

161
might act as a “cold stress” to suppress immunity. In the present study,

morphine-treated mice did demonstrate a significant reduction of the body

temperature at each time interval. However, at later time points, when body

temperature of morphine-treated mice was back to normal, the cell numbers

in the spleen and thymus were still reduced. These results would suggest

that morphine-induced hypothermia may not be responsible for the immunomodulatory effects.

Chronic morphine treatment in itself may represent a “stressful” stimulus to

the organism. Stressful stimuli have been known to have marked effects on

immune function (Kelley et al. 1985; Arora et al. 1987a), and many of the

effects observed in stressed animals, such as analgesia, hypcrcholesterolemia,

hyperphagia, changes in thermoregulation and a variety of endocrine parameters, and prevention of the preovulatory leutinizing hormone surge are also

observed in morphine-treated animals (Pang et al. 1977; Holaday and Loh

1979; Lowy and Yim 1983; Bryant et al. 1987). Morphine-induced immune

modulation may thus represent another effect of this drug shared with

chronic stress.

Tne morphine-induced changes in B cells; T cells; and CD4+, CD5+, and

CD8+ cells are temporally dissociated from the alterations in both cell number (figure 2) and organ weight (figure l), which suggests that these phenomena are regulated by separate mechanisms. Determination of B-cell; Tcell; and CD4+

-, CD5+-, and CD8+-cell numbers involves several steps, e.g.,

white-blood-cell count, differential counts to determine the percentage of

lymphocytes, monoclonal antibody staining and fluorescent analysis to measure the percentage of B- and T-cell subpopulations. and then appropriate

calculations. As a consequence, there is more variability in measured Tsubset numbers than occurs with a single measurement. The difference

between control populations and those just described in morphine-pelleted

mice was nevertheless significant and was confirmed in several sequential

periods of observations. Moreover, these changes in T-cell populations did

not correspond to the development of either tolerance or dependence to

morphine (Bryant et al. 1987). That these changes may have clinical relevance is supported by recent findings of opiate-induced increases in the

OKT4+ population that were positively correlated with the period of heroin

use (Donahoe and Falek 1988).

The infection of CD4+ (T helper/inducer) cells by HIV has been well

described (Popovic et al. 1983; Chen et al. 1983; Klatzman et al. 1984;

Kalish and Schlossman 1985; Fauci 1988), resulting in modulation of CD4

antigen, virus production, cytopathologic changes, and cell death (Klatzman

et al. 1984; Kalish and Schlossman 1985). The CD4 antigen has been reported to be the HIV viral receptor (Dalgleish et al. 1984) and viral infection can be blocked by antibody to the CD4 antigen (Klatzman et al. 1984;

Dalgleish et al. 1984). This unique viral tropism may explain many of the

immunologic manifestations of AIDS. Moreover, HIV infectivity is

162
probably enhanced in proliferating CD4+

 cells (McDougal et al. 1985), suggesting that susceptibility to HIV may be increased under circumstances in

which CD4+ cells are elevated relative to other cellular elements. The

presence of elevated levels of CD4+ cells in the immune organs of animals

chronically exposed to morphine suggests that this environment may facilitate access for HIV infection of these cells in opiate abusers.

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ACKNOWLEDGMENTS

Dr. Phil Skolnick, Chief, Laboratory of Neuroscience, National Institute of

Diabetes and Digestive and Kidney Diseases, NIH, provided constructive

criticism and suggestions regarding this manuscript. Portions of this work

were supported by grant N00014-87-G-0178 from the Office of Naval

Research, Department of the Navy.

AUTHOR

Prince Kumar Arora, Ph.D.

Laboratory of Neuroscience

National Institute of Diabetes and

Digestive and Kidney Diseases

National Institutes of Health

Building 8, Room 11

Bethesda, MD 20892

165
A Murine Retrovirus Model for

Studies of the Role of Cofactors

and Ethanol in AIDS Development

Ronald R. Watson

INTRODUCTION

Promising opportunities for the study of immunological changes, cofactor

involvement in disease, and immunostimulatory drug testing involve a

murine retrovirus model that causes many immunopathological functional

changes similar to those seen in acquired immunodeficiency syndrome

(AIDS). The effects of the LP-BMS murine leukemia virus infection on

immune systems will be reviewed in this chapter, with special emphasis on

T and B cells, and macrophages.

LP-BM5 infection suppresses cell functions while causing polyclonal lymphocyte activation. Murine immunological characterization, availability of

inbred mouse strains, economy of using mouse vs. primate or human models, and similarity of the immune changes caused by murine retroviruses

compared to those seen in AIDS caused by human immunodeficiency virus

(HIV) encourage rapid development of the LP-BMS murine leukemia model.

Ethanol consumption suppresses, while dietary vitamin A stimulates, immune functions during murine retroviral infection. High dietary vitamin A

(retinyl palmitate) caused increased numbers of activated macrophages. It

also increased the percentage of cells with markers for Ia+ cells and macrophages in the retrovirally infected mice compared to infected controls. In

uninfected mice, retinyl palmitate stimulated the percentage of cells with

activated lymphocytes bearing IL-2R, and T cytotoxic cells. These were

associated with a retarded death rate during infection with LP-BM5 murine

leukemia in C57/BL6 mice. Thus, vitamin A stimulation may play a role

in the slower death rate for retrovirally infected mice. Morphine treatment

also increased the percentage of cells with markers for macrophages and

activated macrophages in virally infected mice, while suppressing them in

uninfected mice. Thus, these drugs of abuse can modulate the peripheral

blood lymphoid subsets, which are suppressed by retroviral infection.

166
MURINE RETROVIRAL MODEL FOR STUDIES OF IMMUNOMODULATORY COFACTORS IN AIDS

To understand AIDS and develop treatments, a range of animal models have

been proposed (Mosier 1986). Models that simulate many aspects of AIDS,

yet have the benefits of large numbers of subjects, low cost, and a welldefined immunological system, are critically needed for studies of the effects of cofactors such as alcohol, nutrition, and drugs of abuse. An animal

system that meets the criteria of well-developed immunological reagents,

well-characterized immune systems, and a variety of genetically inbred

strains that can be infected with retroviruses, is the mouse. Just as individual retroviruses may transform different cell types and infect different organisms, it is likely that they impair the immune systems to some extent by

differing routes. Understanding the precise cellular and subcellular mechanisms that lead to immunodeficiency in murine AIDS would represent a

considerable step forward in the understanding of retrovirus-induced immunodeficiency in man and the role of cofactors, and a step forward in the

development of appropriate therapeutic approaches. At least 60 percent of

the research literature on retroviruses has dealt with a murine model, improving its value as a model system.

The family Retroviridae classifies all RNA viruses known to replicate by

way of a DNA intermediate integrated in the host cell genome, with oncogenic ones grouped in the subfamily Oncovirinae. Many animal retroviruses

are not associated with cancer, while some are agents of chronic degenerative diseases of animals, and others are entirely apathogenic. Immunodeficiency is among the major, nonneoplastic disorders associated with murinc

retroviral infection. The ability to immunosuppress the host is shared by so

many exogenous retroviruses infecting such widely divergent species that the

property might be regarded as having significant survival value for such viruses. Viruses, including murine and human retrovituses, can potentially

cause disease by at least four general methods involving immunomodulation:

(1) as a result of viral replication, perhaps lysing or functionally impairing

the lymphoid cells they infect; (2) immunosuppression from the activity of

soluble factors of viral or host origin released from infected cells; (3) damage due to infection of cells involved in phagocytosis, antigen presentation,

and nonspecific effector aspects of cell-mediated immunity; and (4) suppression of immunity from viral triggering of an imbalance in immune regulation with overactivity of T-suppressor or helper cells.

There are clearly some characteristics shared by murine and human retroviruses. For example, both virions are enveloped by a lipid bilayer derived

from the host-cell membrane and contain a surface glycoprotein spike as

well as an internal spherical core. They contain the genome and several

enzymes, including the reverse transcriptase. The genomes are RNA resembling eukaryotic mRNA and present in two copies. Finally, all major

events in replication are unique to retrovituses: synthesis of double-stranded

167
linear viral DNA in the cytoplasm of infected cells by a process that leads

to the formation of long terminal repeats; integration of viral DNA into cellular DNA to form a provirus; and transcription of the provirus using cellular machinery to form new RNA genomes and mRNAs. The genome contains genes for the group-specific antigen gag, for the reverse transcriptase

pol, and for envelope proteins env.

LP-BM5 MURINE RETROVIRUS INFECTION OF MICE

Mosier, Yetter, and Morse have characterized a murine retroviral disease

model, LP-BM5 murine retrovirus, that approaches the immunological damage of AIDS in man (Mosier 1986; Salzman 1986; Buller et al. 1987;

Mosier et al. 1987; More et al. 1988; Klinken et al. 1988; Yetter et al.

1985). Its characteristics form a syndrome similar to AIDS and will be described in some detail as a superb murine model. Quantitative loss of

T-cell function occurs far in advance of reduction in the numbers of L3T4+

(CD4+) cells. Natural killer (NK) cell function is abolished early, but

lymphocyte-activated killer function remains until 8 to 10 weeks after initiation of the infection (table 1). H-2-restricted cytotoxic T-lymphocyte (CTL)

killing of allogeneic cells is depressed starting 3 to 4 weeks after infection.

In the early stages of disease (through about 6 to 8 weeks after infection),

addition of interleukin-2 (IL-2) restores CTL activity. After that time, addition of exogenous IL-2 has only a very small restorative effect, if it has any

effect at all (Mosier et al. 1985; R. Yetter, personal communication). Therc

are few early changes in other hematopoietic elements in the mouse model.

LP-BM5 MuLV infection does not result in a general depression of bone

marrow function, as seems to occur in other animal models for AIDS.

The virus mixture LP-BM5 was originally isolated by Latarjet and Duplan

(1962) and produces nonneoplastic lymphoproliferative disease in adult mice.

The ecotropic component of the LP-BM5 mixture seems to function to promote replication of the poorly replicating, pathogenic mink cell focusforming component. LP-BM5 was obtained from a nonthymic lymphoma

that had been induced by irradiation of mice. Then bone marrow stromal

cells were isolated from the infected mice and, grown as cell lines, yielded

the agent. It is a cluster of viruses that includes a B-tropic ecotropic virus

and a B-tropic mink cell focus-inducing virus (Watson 1989). The latter

virus alone does not cause immunological disease, but mice infected with a

nonpathogenic ecotropic virus and then infected with the cell focus-inducing

virus do develop disease. Thus, this virus or one that passages with it is

the etiologic agent in the disease, with the ecotropic virus functioning to

spread it in mouse tissues. The characteristics of the infection are that the

animals develop hypergammaglobulinemia (IgM, IgE, and IgG), lymphadenopathy, T-cell functional immunodeficiency, and later neurological signs,

including paralysis as well as opportunistic infections (Buller et al. 1987;

Mosier et al. 1987; Salzman 1986). The virus infects macrophages, B cells,

and, to a lesser extent, T cells. Lymphomas of monoclonal B cell origin

168
TABLE 1. Comparison of changes in immune cells and functions induced

by HIV-1 infection in man and LP-BM5 murine leukemia

infection in mice

Immunological Parameter

Presence During Retroviral

Infection With

HIV LP-BMS MuLV

Abnormalities of B Cells

Increased lmmunoglobulin Production

Increased Spontaneous Proliferation

Reduced Responsiveness to Antigens

From Immunization and Infection

Abnormalities of T Lymphocytes

Reduced Resistance to Opportunistic

Infections and Neoplasms

Reduced Numbers of CD4+

 Cells (T Helper)

Decreased Cytotoxic Lymphocyte Functions

for Nonrettovitus, Vitally Infected Cells

Increased Spontaneous Proliferation

Diminished T-Helper Functions

Abnormalities of Mactophages

Decreased Numbers of Activated

Mactophages

Reduced Numbers of Mactophages

Diminished In Vitro Pathogen Killing

Spontaneously Increased PGE and IL-1

Secretion

Abnormalities of NK Cells

Reduced Tumor Cell Cytotoxicity

Increased Activity With IL-2 Incubation

In Vitro

+ +

+ +

+ +

+ +

+

+ +

+ +

+ +

variable

+

+

+

+

+ +

+ +

+ +

SOURCE: Modified from Watson 1989d.

169
have occurred in a small percentage of infected mice, and a few mice

develop neurologic symptoms near the end of the clinical course of disease

(Mosier 1986). Molecular and genetic analysis of spleen cells showed that

early in LP-BM5 polyclonal, there was proliferation of both B and T

lymphocytes (Klinken et al. 1988). These cells infiltrate various organs including the brain, and they may be associated with the retrovirus induced,

late-developing, neurological changes (Klinken et al. 1988). Derived cell

lines continued to produce LP-BM5 and were B cell in origin. This is evidence of transformed B cells. The demonstration of terminal B-cell lymphomas caused by LP-BM5 MuLV infection extends the similarities of that

infection to human AIDS (Klinken et al. 1988). Although lentiviruses were

associated with human AIDS, and C-type retroviruses appear causative in

murine AIDS, both are very similar in causing reduced resistance to pathogens, more neoplasias, and profound immunosuppression.

EFFECTS OF MURINE LP-BM5 RETROVIRUS INFECTION ON

LONGEVITY AND DISEASE RESISTANCE

Infected C57/BL6 mice live 5 to 6 months before succumbing to disease or

constriction of airways by expanding lymphoid tissues. They frequently die

from respiratory failure due to mediastinal lymph node enlargement (Buller

et al. 1987), while AIDS patients die from a variety of opportunistic infections and tumor-associated conditions. Exposure to an environment with

many potential pathogens and normal flora in the animal rooms results in

death much earlier from LP-BM5-induced immunosuppression by infectious

disease (R. Yetter, personal communication). On the other hand, mice without functional T cells, infected with the retrovirus, survived past 11 months

when the experiment was terminated. Infection of adult C56/BL6 mice with

LP-BM5 MuLV leads to the rapid induction of clinical symptomatology

with virtually no latent phase. Elevations in serum IgM levels can be detected 1 week postinfection, and lymphadenopathy and splenomegaly are

apparent within 3 weeks. Although AIDS patients are susceptible to a variety of pathogens of the immunosuppressed, including Cryptosporidium and

Cryptococcus, laboratory mice live in significant isolation from exposure to

many pathogens, so the information on disease resistance is limited. Nonetheless, there is evidence of increased colonization by Candida albicans

(R. Yetter, personal communication), with spontaneous secondary infections

mainly at 3 to 4 months postinfection. These have involved primarily

mouse hepatitis virus, and, more rarely, Pneumocystis carinii (Mosier 1986).

Natural horizontal transmission of LP-BM5 MuLV infection has not been

observed (Mosier 1986). The infection is experimentally transmitted by

intraperitoneal (IP) injection of virus into either neonatal or adult animals.

Initially uninfected mice cohoused with LP-BM5 MuLV-infected mice for

up to six months show no signs of disease or viral infection (Mosier 1986).

Ectromelia virus (mousepox) is a naturally occurring agent that is transmitted via skin abrasions. While some mouse strains succumb to mousepox

170
readily, B6 replicates it at low levels and survives. Adult B6 mice have resistance to mousepox infection, which was significantly suppressed by the

retrovirus LP-BM5. LP-BM5 MuLV in vitro and in vivo suppressed responses to antigens, so that no antibodies developed to subsequent infection

with Ectromelia (Buller et al. 1988). Combined Ectromelia and MuLV infection resulted in increased numbers of deaths when mousepox infection

occurred for a prolonged time after exposure to the retrovirus. Cytotoxic

T-lymphocyte defenses did not develop to mousepox when its infection followed LP-BM5 MuLV infection by several months. This suggests that the

retrovirus significantly suppresses directly or indirectly both B- and T-cell

specific responses to this pathogen. Lethality due to mousepox was likely

induced by immunosuppression due to the mink focus-forming cell component of the LP-BM5 virus mixture (Buller et al. 1988). Some mice resisted

Ectromelia challenge when infected with the retrovirus, which may have

been due to the continuing functioning of macrophages in some animals.

Necrosis of the spleen similar to that in susceptible mice suggested that

death was due to Ectromelia.

T CELLS DURING LP-BM5 MULV RETROVIRAL INFECTION

AIDS in man progresses to significant immunosuppression, as does the

LP-BM5 MuLV infection in mice (table 1). The role of T cells is critical

to AIDS development in man. HIV causes abnormalities in the immune

system by depleting the CD4+ T-cells and helper cells and by changing their

functions. In addition, HIV activates B cells and infects macrophages,

which can be critical to antigen presentation (Fauci 1988). LP-BM5 MuLV

does not increase number or activity of T cells, nor are viral components or

the various mixtures directly immunosuppressive (Mosier 1986).

While T cells are not the main source of viral replication, they are required

for early pathogenesis and disease. LP-BM5 MuLV decreases T-helper cell

functions. Although the number of T-helper cells stays relatively constant

during early stages of infection, there is some suppression of T-suppressor

cells, yielding an increased T-helper:T-suppressor ratio in mice (table 1).

However, there is rapid T-cell activation (1 month postinfection) with reduction in response to viral antigens, in vitro mitogens, and MHC self-restricted

antigens (Salzman 1986; Mosier et al. 1986). This is similar to humans infected with HIV who gradually lose T-cell functions (Fauci 1988). Loss of

some of these functions may be due to reduced ability of cells from MuLVinfected mice to produce IL-2. However, mitogenesis is not restored by

exogenous addition of IL-2, while responsiveness to MHC self-restricted

antigens occurs early in the retrovirus infection (Morse et al. 1988). The

mouse model diverges also from AIDS in humans in the extent of B-cell

lymphoproliferation and in the absence of a long latent phase of infection

(Fauci 1988).

171
The model has recently been further developed by studies with T-cell deficient C57B1/10 (mu/mu) nude mice (Mosier et al. 1987). In the absence of

helper T cells, the mice were not killed by the effects of the retroviral infection during the 5 to 6 months as is usual and survived for 11 months,

the length of the experiment. They did not develop many of the characteristics of the disease. The murine system makes possible studies requiring

special strains and inbred mice for such immune reconstitution studies.

Since viral replication of components of the LP-BM5 MuLV occurred to a

similar extent (Mosier et al. 1987), the reduced development of the disease

was not due to reduction in virus production. Rather, the development of

murine AIDS in the mice required the virus mixture and functional T cells.

The requirement for other cells such as B cells or macrophages was not determined. Since the virus was produced in similar levels, however, by the

nude mice without mature T cells, it appears that lymphoid cells such as B

cells, null cells, and macrophages/monocytes are the major sites of infection

by the retrovirus (Mosier 1986). This is logical, as the polyclonal B-cell

activation seen in human and murine AIDS requires several lymphokines, at

least in normal B-cell activation in uninfected animals. The model was further extended by depleting the L3T4+

 helper T cells with the appropriate

antisera, which also prevented development of murine AIDS (Mosier et al.

1988). Immunodeficiency in this murine model seems to significantly involve modification of B-cell activation via participation of T cells.

B-CELL FUNCTIONS DURING MURINE LP-BM5 MULV

RETROVIRAL INFECTION

B cells and their production of immunoglobulins show a significant number

of alterations due to retroviral infection in man (Fauci 1988). As mentioned

above, there are substantial changes in B-cell functions due to the murine

retroviral infection, in part due to changes in T- and accessory-cell functions

(table 1). Activation of both T and B lymphocytes can be detected within

1 week of infection, as judged by flow cytometric analysis of the size of T

and B cells and measurements of the percentage of B cells secreting IgM.

There is an absolute increase in the number of B cells in the spleen and

lymph nodes, and a threefold increase in the fraction of B cells in cell

cycle (Mosier 1986). The proliferative response to both T- and B-cell mitogens is depressed by more than 80 percent; L3T4+

 helper function for

T-dependent antibody responses in vitro and in vivo are virtually absent; and

responses to T-independent antigens are vastly diminished. While T cells

from LP-BM5 MuLV-infected mice will not provide helper activity to normal B cells for a specific antibody response in vitro, they do stimulate elevated polyclonal IgM secretion by normal B cells (Mosier 1986). During

the early part of the MuLV infection, the B cells produce large amounts of

immunoglobulin, with an increased number of plasma- or immunoglobulinproducing cells. There is widespread polyclonal activation of B cells

(Salzman 1986; Mosier et al. 1985). The cells can become unresponsive to

new antigens, either due to B- or T-cell defects. As the disease progresses,

172
there is a shift to production of only a few antibodies, even monoclona1

proliferation of B cells with development of B-cell lymphomas. Interestingly, responsiveness to mitogens (Mosier et al. 1985) in vitro and in vivo is

suppressed and cannot be restored by T cells from uninfected mice. The

later stages of the murine AIDS-like disease involve continued expansion of

members of the B lymphocyte lineage, including all stages from pre-B cells

to plasma cells, increased numbers of the monocyte- macrophage lineage,

and appearance of immature T cells in the periphery (Mosier 1986). Although there is functional evidence of an early depletion of L3T4+

 cells, T

cells bearing the L3T4 marker continue to be present throughout the course

of the disease. The similarities between this murine model for AlDS and

early stages of AIDS are striking in many respects. There is an early phase

of B-cell hyperactivity and polyclonal activation in both, and the order of

deletion of T-cell function is similar, with helper T-cell function lost well

before cytotoxic/suppressor T-cell function (Watson 1989d).

The LP-BM5 mouse model diverges from AIDS in humans in the extent of

B-cell lymphoproliferation and in the absence of a long latent phase of infection (Mosier 1986). This model also diverges in genomic composition,

which is still incompletely known, LP-BM5 does produce an envelope protein env, gag antigen, and RNA-dependent DNA polymerase pol, with some

similarities in function to similar proteins produced by HIV-l. It apparently

lacks, however, the other genes of HIV-l. Information on whether the infection increases thymosin alpha-l, produces circulating immune complexes,

alters delayed hypersensitivity responses, or reduces serum thymosin levels

is lacking for LP-BM5. In addition, LP-BM5 does not stimulate production

of antilymphocyte antibodies or suppressor substances. As knowledge of

the cellular and subcellular mechanisms of immunosuppression retroviruses

increases through use of murine models, even though they diverge from

HIV-l genetically and functionally to some extent, general understanding

will be expanded, with opportunities for more rapid application to human

retroviral infections and treatment. Mice infected with LP-BMS do not pose

a known risk of transmission of the agent to man (Mosier 1985; Mosier

et al. 1985; Mosier et al. 1988).

Although murine retroviral infections are not complete models for human

retroviral disease, the LP-BM5 MuLV model has many benefits for immunological studies (table 1). The viral infection simulates well key aspects of

AIDS-related complex (ARC) and AIDS. LP-BM5 virus infects inbred

mice, yet does so differently with the few strains tested to date, allowing

mechanistic studies with the many immunological parameters in mice that

are well defined. Although the virus has a rapid clinical course with no

latent phase, it produces lymphadenopathy, hypergammaglobulinemia, and

late neurological changes. Even though the murine virus is not very closely

related to HIV, it does cause T-cell defects and polyclonal B-cell activation

and infects and acts on macrophages, whereas in mice without T cells, there

is no clinical disease. In both human and murine retroviral infection, there

173
are multiple alterations of lymphoid cells, and impairment of macrophages

seems to be critical. Alteration of these cells, especially of their accessory

functions, represents a unifying determinant in the genesis of many manifestations of retroviral-induced immunodeficiency. Mosier et al. (1985; Mosier

et al. 1987) concluded that the LP-BM5 MuLV-induced disease is a good

mouse model for profound immunosuppression by a retrovirus and that the

model has considerable relevance for similar retrovirus-induced syndromes

in humans.

CHANGES IN LYMPHOCYTE AND MACROPHAGE SUBSETS DUE

TO MORPHINE AND ETHANOL TREATMENT DURING A

RETROVIRUS INFECTION CAUSING MURINE AIDS

Dietary materials that suppress immune systems may also be critical in the

rate of progression to AIDS after retroviral infection (Watson 1989a;

Watson 1989b; Watson 1989c; Watson and Wallace 1989). Possible cofactors that are immunosuppressive include alcohol (Watson 1988b; Mufti

et al. 1988; Watson et al. 1988b; Watson and Darban 1988; Watson 1988b)

and morphine (Yahya and Watson 1987). Ethanol use has major effects on

disease resistance depending upon the amount consumed and the duration.

Alcohol-induced cirrhosis suppresses cellular immune functions as well as

resistance to viral pathogens. There is evidence that ethanol acts as an

immunosuppressive agent in vitro and in long-term exposure without liver

disease (Watson 1988b). Morphine (Yahya and Watson 1987) also routinely

suppresses cellular immune functions and reduces resistance to various pathogens. Retroviral infection in mice is an exciting system to study the

effects of such cofactors on immune functions. It has many characteristics

of AIDS, such that it has been suggested as an excellent murine model for

infection with HIV in man. Therefore, the present investigation examines

the effects of ethanol and morphine treatments on changes in numbers of

regulatory lymphocytes and monocytes/macrophages during murine retroviral

infection.

C57/BL6 female mice, 3 to 4 weeks old, weighing approximately 15 g,

were obtained from Simonsen Laboratories, Inc., Gilroy, CA. They were

routinely provided with Purina laboratory chow and water ad libitum.

Thirty mice were injected with morphine sulfate IP every day for 7 days

during the same weeks others were exposed to ethanol. The morphine was

given at a daily dose of 75 mg/kg body weight IP. For the initial ethanol

exposure, 30 mice were subjected to a Carnation Slender liquid diet containing 7 percent (v/v) ethanol (36 percent of calories) for the initial 7 days of

the study as described previously (Watson et al. 1988b). Then all mice fed

ethanol were again provided the chow diet for 3 weeks, before being fed a

5-percent ethanol diet for 1 week, which was 3 weeks postviral infection.

Some mice were sacrificed; the remainder received the chow diet for 3 additional weeks, followed by 1 week of 5 percent ethanol diet. At this time,

174
8 weeks postinfection, more were sacrificed. Uninfected mice in groups of

20 animals were treated identically with each drug of abuse.

C57/BL6 mice were chosen because they had previously been observed to

consume sufficient amounts of ethanol in a liquid diet to develop functional

tolerance and physical dependence. Physical dependence on ethanol was

achieved as evidenced by the high incidence of convulsions and tremors

4 to 8 hours after the removal of dietary ethanol. Mice used developed

signs of ataxia several days before sacrifice. After the first week of exposure to drugs of abuse, each animal (30 mice per group) was injected IP

with 0.1 ml of LP-BMS murine leukemia inoculum, which was donated by

Dr. R.A. Yetter.

Identification of T cells isolated from the peripheral blood was carried out

as described elsewhere (Watson et al. 1988b, Watson et al. 1988a). Ethanol

increased the percentage of T-helper cells in virally infected mice compared

to infected controls (table 2). However, ethanol consumption did not affect

the percentage of any lymphocyte subsets except those with the activation

markers, IL-2R, in uninfected mice, while morphine suppressed them.

TABLE 2. Effects of morphine or dietary ethanol on frequency of

lymphocytes identified by surface markers 8 weeks postLP-BM5 murine leukemia virus infection1

Control Ethanol Morphine

Virus Virus Virus

Uninfected Infected Uninfected Infected Uninfected Infected

T Helper 33.5±4.9 24.3±1.1* 38.0±2.5# 41.0±2.6*# 25.6±1.5* 20.0±1.0*

T Suppressor/

Cytotoxic 20.5±0.7 11.3±0.5* 20.6±1.1# 19.0±2.6*# 9.3±0.6* 8.3±0.5*

Total

T Cell 68.0±2.8 73.0±2.6 67.3±0.5 64.3±9.6# 73.6±1.5 59.0± 1.0#

B Cell

(Mu ±) 17.5±10.6 8.3±0.5* 26.0±1.0# 2.3±1.1# 11.0±2.0# 12.0±1.0#

1Mean ± SD (n=4).

*Significantly different from uninfected control.

#Significantly different from virus-infected control.

Similar effects were seen with the T-suppressor cells with both compounds.

Neither drug of abuse significantly affected total T cells in uninfected mice,

while both tended to suppress total T cells in infected mice. Both morphine and ethanol suppressed the percentage of activated lymphocytes identified by IL-2R on their surfaces in uninfected mice. Morphine had a

175
tendency to increase IL-2R bearing cells during infection as compared to

infected controls (table 2).

As shown in table 3, LP-BM5 MuLV infection reduced significantly the

percentage of peripheral blood cells with markers for macrophages and activated macrophages. Both ethanol in the diet and morphine injection reduced the percentage of these cells. Ethanol consumption by retrovirally

infected mice had a trend toward a lower percentage of each of these cell

types compared to ethanol-fed or virus-infected controls (table 3). However,

morphine injection during infection increased the percentage compared to

morphine-treated or virally infected controls (table 3). Extremely similar

results were seen at 4 weeks postinfection with both compounds.

TABLE 3. Effects of morphine and dietary ethanol on frequency of

macrophages identified by surface markers 8 weeks postLP-BM5 murine leukemia virus infection1

Control Ethanol Morphine

Virus Virus Virus

Uninfected Infected Uninfected Infected Uninfected Infected

Macrophage

(MAC1+) 21.5±14.8 8.0±1.0* 10.6±1.1* 3.0±2.0*# 5.3±1.5* 17.3±2.0#

Activated or

Stimulated 23.0±18.3 5.6±2.0* 17.0±2.6# 3.3±1.1* 7.3±2.0* 11.3±3.0#

Macrophages

(MAC2+)

Ia+15.0±7.0 5.6±0.5* 9.0±1.0*# 2.3±1.1*# 15.6±2.0 12.0±1.7#

1Mean ± SD (n=4).

*Significantly different from uninfected control (p<0.05).

#Significantly different from virus-infected control.

After 25 weeks of infection, there had been mortality in the infected mice,

with 45.0 percent surviving. Among the controls not treated with a drug of

abuse or infected, none died by 25 weeks. In the ethanol-fed, infected

mice, however, there was greater mortality, with 8.6 percent surviving.

There were deaths among ethanol-fed uninfected mice, with 41.7 percent

surviving. In these mice, deaths occurred at the first two ethanol exposures

when mice were small; in the ethanol-fed infected mice, deaths continued

throughout the experiment, particularly from 20 to 25 weeks after infection.

Among the morphine-injected, infected animals at 25 weeks postinfection,

44.8 percent survived. There were deaths after morphine exposure, and

38.4 percent of morphine-injected uninfected mice survived at 25 weeks

postinfection, which was not significantly different. Survival was less in thc

virus-infected, ethanol-fed mice than was expected, if deaths due to

176
exposure to ethanol and virus were combined without interaction

(18 percent). On the other hand, survival in mice injected with morphine

and retrovirus infected was higher than would have been expected by

survival rates from morphine injection alone and virus infection alone

(17.3 percent). The altered survival may be in response to several

immunological changes. Even though the LP-BM5 MuLV-infected mice are

more susceptible to infectious diseases, in clean animal facilities they are

presumed to be relatively unexposed to pathogens. In addition,

virus-stimulated lymphoid cells yield increased lymphomas and expanded

lymphoid tissues, bringing premature death (Salzman 1986). Thus,

suppressed or enhanced stimulation of lymphoid cells, particularly B and T

cells, would be expected to change survival rates (Mosier et al. 1987).

Finally, recent in vitro work shows that immunological stimulation of cells

increases the likelihood that the retrovirus will be transmitted to other cells.

Lymphoid cell activation measured by the percentage with activation

markers were changed by these treatments. Thus, any of the above

mechanisms could have been significantly involved in death rate changes.

Others have shown that T cells are critical to development of the murine

disease with death, even in the presence of continued viral infection

(Klinken et al. 1988). Nude mice without mature T cells did not die as

early as the immunologically competent mice did. Thus, ethanol, which increased T-helper and T-suppressor cells and reduced the number of T cells

in virally infected mice compared to infected controls, may exacerbate

immunological changes caused by retroviral infection. Morphine may not

produce these effects, as it tended to reduce the number of T cells, while

relatively increasing macrophages in infected mice. The increase in activated macrophages due to morphine occurred concomitantly with an increase

in macrophages, so that the ratio was similar to that for infected mice and

lower than that for controls, particularly ethanol-fed mice. The effect of

morphine to increase the percentage of activated lymphocytes and macrophages could increase the cells that can be infected by retrovirus. LP-BMS

MuLV infects T cells, and such changes suggest that drugs of abuse modulate aspects of the cellular immune systems, particularly in retrovirally

infected mice (Watson et al. 1988b). They could be cofactors in modifying

the progression to AIDS by retroviruses.

CONCLUSION

The chronic, occasional exposure to dietary ethanol during retroviral infection significantly affected the numbers of various T subsets in the peripheral

blood, while not affecting uninfected mice. This may be the first demonstration of a dietary component acting to uniquely alter T cells during a

retroviral infection. Changes in numbers and activation of T cells and

macrophages indicate potentially decreased host defenses. The fact that

ethanol exposure similar to binges in man caused significant functional

changes during retroviral infection needs more detailed studies. The levels

177
of ethanol in the blood were similar to levels attainable in man and were

associated with withdrawal signs in the mice. Therefore, the existing

evidence indicates that high levels of ethanol and/or its metabolites have the

potential, during retrovirus infection, to alter the numbers of cells that can

participate in cellular immune responses.

LP-BM5 MuLV infection resulted in suppression in the numbers of T-helper

cells and macrophages, confirming previous results (Mosier et al. 1985;

Mosier et al. 1987). It was found, in addition, that activated macrophages

were reduced by the infection. The loss of both macrophages, as well as

activated macrophages (MAC-2) and lymphocytes (IL-2R positive cells),

may be critical in the eventual death and altered immune functions associated with the virus. It can explain changes in immunoregulation and reduction in disease resistance to pathogens caused by this retrovirus.

REFERENCES

Buller, R.M.L.; Yetter, R.A.; Fredrickson, T.N.; and Morse, H.C. Abrogation of resistance to severe mouse pox in C57/BL6 mice infected with

LP-BMS murine leukemia viruses. J Virol 61:383-387, 1987.

Fauci, A.S. The human immunodeficiency virus: Infectivity and mechanisms of pathogenesis. Science 239:617-622, 1988.

Klinken, S.P.; Fredrickson, T.N.; Hartley, J.W.; Yetter, R.A.; and

Morse, H.C., III. Evaluation of B cell lineage lymphomas in mice with a

retrovirus-induced immunodeficiency syndromes, MAIDS. J Immunol

140:1123-1131, 1988.

Latarjet, R., and Duplan, J.-F. Experiment and discussion on leukaemogenesis by cell-free extracts of radiation-induced leukemia in mice. Int J

Radiat Biol 5:339-344, 1962.

Mosier, D.E. Animal models for retrovirus-induced immunodeficiency

disease. Immunol Invest 15:233-261, 1986.

Mosier, D.E.; Yetter, R.A.; and Morse, H.C., III. Retroviral induction of

acute lymphoproliferative disease and profound immunosuppression in

adult C57BL/6 mice. J Exp Med 161:766-784, 1985.

Mosier, D.E.; Yetter, R.A.; and Morse, H.C. Functional T lymphocytes are

required for a murine retrovirus-induced immunodeficiency disease

(MAIDS). J Exp Med 165:1737-1742, 1987.

Mufti. S.I.; Prabhala, R.; Moriguchi, S.; Sipes, I.G.; and Watson, R.R.

Functional and numerical alterations induced by ethanol in the cellular

immune system. Immunopharmacology 15:85-94, 1988.

Salzman, L.A., ed. Animal Models of Retrovirus Infection and Their

Relationship to AIDS. Orlando, FL: Academic Press, 1986.

Watson, R.R., ed. Diagnosis of Alcohol Abuse. Boca Raton, FL: CRC

Press, 1988a.

Watson, R.R. Alcohol, immunomodulation, cancer. Prog Food Nutr Sci

12:189-209, 1988b.

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Watson, R.R., ed. Cofactors in Human Immune Deficiency Virus infection

and Progression to AIDS. Boca Raton, FL: CRC Press, 1989a.

Watson, R.R., ed. Drugs of Abuse, Immune Functions, and Resistance to

Diseases. Boca Raton, FL: CRC Press, 1989b.

Watson, R.R. Immunomodulation by alcohol, a cofactor in development of

acquired immune deficiency syndrome (AIDS) after retrovirus infection.

In: Watson, R.R., ed. Cofactors in HIV-1 Infection and AIDS. Boca

Raton, FL: CRC Press, 1989c.

Watson, R.R. Murine models for acquired immune deficiency syndrome.

Life Sci 44:i-xiii, 1989d.

Watson, R.R., and Darban, H. Alcohol and immunosuppression. Clin

Immunol Newsletter 9: 129-131, 1988.

Watson, R.R.; Prabhala, R.H.; Abril, E.; and Smith, T.L. Changes in

lymphocyte subsets and macrophage functions from high, short-term

dietary ethanol in C57/BL6 mice. Life Sci 43:865-870, 1988a.

Watson, R.R.; Prabhala, R.H.; Darban, H.R.; Yahya, M.D.; and Smith, T.L.

Changes in lymphocyte and macrophage subsets due to morphine and

ethanol treatment during a retrovirus infection causing murine AIDS. Life

Sci 43:v-xi, 1988b.

Watson, R.R.; Yahya, M.D.; Darban, M.R.; and Prabhala, R.H. Enhanced

survival by vitamin A supplementation during a retrovirus infection

causing murine AIDS. Life Sci 43:xii-xvii, 1988c.

Watson, R.R., and Wallace, C.L. Drugs of abuse as cofactors in the

progress of HIV infection to AIDS. In: Watson, R.R., ed. Cofactors in

Human Immune Deficiency Virus Infection and Progression to AIDS.

Boca Raton, FL: CRC Press, 1989.

Yahya, M.D.; and Watson, R.R. Minireview. Immunomodulation by morphine and marijuana. Life Sci 41:2503-2510, 1987.

Yetter, R.A.; Hartley, J.W.; Hoffman, P.M.; Mosier, D.E.; and

Morse, H.C., III. Non-neoplastic sequelae of infections with murine leukemia viruses. In: Skamene, E., ed. Genetic Control of Host Resistance

to Infection and Malignancy. New York: A.R. Liss, 1985. pp. 671-682.

ACKNOWLEDGMENT

R. Prabhala, Ph.D., D. Yayha, M.S., T. Smith, Ph.D., and H. Darban, M.S.,

all of the University of Arizona, assisted in some of the mouse experiments.

The work was supported by grants from Wallace Genetics, NIH P50

AA08037 and DA04827.

179
AUTHOR

Ronald Ross Watson, Ph.D.

Department of Family and Community Medicine

Co-Director of NIAAA-Funded Specialized

Alcohol Research Center

University of Arizona

Arizona Health Sciences Center

Tucson, AZ 85724

180
Drug Abuse and AIDS: Causes for

the Connection

Robert M. Donahoe

INTRODUCTION

The principal demographic factor connecting intravenous (IV) drug abuse

with blood-borne pathogens like HIV1 is the needle sharing habits of addicts. Addicts who contract AIDS, in turn, serve as the major reservoir for

its heterosexual and pediatric transmission. Because of these circumstances,

over 25 percent of current AIDS cases in the United States are either directly or indirectly connected to needle sharing by IV drug abusers. Since

public health measures have been largely unsuccessful in reducing the

spread of AIDS in addicts, as compared with other demographic groups at

high risk for AIDS, this percentage is on the rise.

Factors other than needle sharing are also relevant to the spread of AIDS

by addicts. Prominent among these is the influence of the drugs themselves. By altering host immunocompetence, drugs of abuse represent a

source of acquired immunodeficiency independent of HIV1, with the potential to alter susceptibility of addicts to HIV1, and the development of AIDS.

This chapter explores potential causes for immunomodulation by drugs of

abuse and their influence on the course of AIDS in addicts.

Opiates are the primary drug abused intravenously. However, because of

increased availability and reduced cost, IV administration of cocaine has recently increased dramatically, particularly among heroin users. Therefore,

most of the ensuing discussion focuses on the role of opiates and heroin

addiction in modulating host immunocompetence, although much of this discussion is relevant to the immunological effects of behaviorally active drugs

of abuse in general.

HISTORICAL PERSPECTIVE ON IMMUNOMODULATION

BY OPIATES

A chronological documentation of observations that support the linkage between opiates and altered immune function appears in table 1. These and

181
related findings have recently been reviewed by several authors (Yahya and

Watson 1987; Sabinga and Goldstein 1983). The evidence indicates that the

functions of most, if not all, circulating cells of the immunological system

are influenced by opiates, which clearly supports the notion that IV drug

abusers can suffer from generalized drug-induced immunodeficiency (DII),

which may influence the course of HIV1 infection. Importantly, the observations in table 1 also suggest that a specific target for opiate effects may

be the HIV1 viroreceptor itself, the CD4 molecule, and that opiates affect

immunological cells both directly and indirectly through effects on the

neuroendocrine system.

TABLE 1. Chronological history of key reports linking opiates with

immunomodulation and AIDS

Year Observation Authors

1909

1928

1970

1972

1974

1979

1980

1980

1980

1983

1983

1984

1985

1985

Morphine depresses phagocytosis in vivo and

in vitro.

Morphine addiction alters leukocyte

differentials in dogs.

Infection and cancer are elevated in heroin

addicts.

Cytogenetic damage is elevated in lymphocytes

of heroin addicts.

Heroin addicts exhibit depressed mitogenesis

and elevated immunoglobulin levels.

Morphine depresses T cell E-rosette formation

in vitro in naloxone-reversible fashion.

Heroin addicts exhibit naloxone-reversible

depression of T cell E-rosette formation.

Phagocytes have opiate receptors.

Morphine injected into mice alters immune

function.

Heroin addicts are a high-risk group for AIDS.

Rodents injected with morphine are more

susceptible to infection and cancer.

Opioid-mediated stress depresses NK function.

Inhibition of receptor microdisplacement defined

as cause for opiate effects on E-receptors.

Th/Ts ratios depressed in heroin addicts in the

absence of HIV1.

1986 Morphine modulates CD2, CD4, and CD8

in vitro.

1987 Purified human T cells have opiate-binding sites

Archard et al.

Pierce and Plant

Sapira et al.

Falek et al.

Brown et al.

Wybran et al.

McDonough et al.

Lopker et al.

Gungor et al.

Wormser et al.

Tubaro et al.

Shavit et al.

Donahoe et al.

Des Jarlais et al.

Donahoe et al.

Madden et al.

182
GENERALIZED IMMUNOLOGICAL EFFECTS OF OPIATES:

THEIR ROLE IN AIDS

There is no clear understanding of the role of immunity in protection of a

host against infection with HIV1 or containment of the infection thereafter.

The membrane-budding nature of the virus suggests that protection or containment will require both cell-mediated and humoral branches of the

immune response. However, the types of immune response that are most

pertinent are unknown. Therefore, despite evidence that opiates modulate

the function of T and B cells, macrophages, polymorphonucleocytes, and

natural killer (NK) cells, it is largely uncertain how such effects might influence susceptibility of addicts to HIV infection and development of AIDS.

A possible exception to this generalized uncertainty relates to the finding

that opiates modulate expression of the HIV1 viroreceptor, the CD4

molecule. This exception is detailed later in this discussion.

One approach to defining the relationship between generalized immunological effects of opiate addiction and AIDS is to use experimental animal

models. Investigations with rodent models (Tubaro et al. 1983) have shown

that morphine alters animal susceptibility to bacterial infection and tumor

transplantation. However, these findings are difficult to relate directly to

the AIDS situation, since the pathogens and hosts involved in the rodent

model systems are far removed from the circumstances inherent to AIDS.

Rodent models directed at opiate effects on AIDS-like viruses would of

course be more appropriate. However, the relevance of such models to

human AIDS is open to debate from at least three different aspects of experimental design: neuroendocrine/pharmacological relevance; immunological relevance; and virological relevance.

To maximize the relevance of an animal model to the circumstances of

human AIDS, the author has established a rhesus monkey model. Unquestionably, a monkey model is phylogenetically close to the human situation.

Especially relevant is the shared susceptibility of monkeys and man to very

closely related immunodeficiency viruses. Use of a monkey model is aimed

at establishing the effects of morphine on the immunological status of monkeys subjected to a standard addiction paradigm and at determining their

subsequent susceptibility to infection with the M-9 strain of simian immunodeficiency virus isolated from sooty mangabey monkeys (SIV/SMM). This

virus crossreacts serologically with HIVl, infects human lymphocytes, and

causes an AIDS-like syndrome in rhesus monkeys (Fultz et al. 1986). Two

separate experimental approaches are being taken: (1) direct examination of

the pathogenicity of SIV/SMM in animals simultaneously being addicted to

morphine; and (2) examination of the long-range immunological effects of

morphine addiction before testing animals for susceptibility to SIV/SMM.

Preliminary data from the first approach suggest that intercurrent morphine

addiction does not alter the ability to isolate SIV/SMM from the blood of

infected monkeys, unless the animal is stressed by withdrawal from opiates.

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Data from the second approach indicate that cell-mediated immunity is

altered in addicted monkeys in comparison with controls, within weeks of

injection of opiates. The main parameters affected are T-cell numbers and

percentages of various T-cell subtypes, blastogenic responsiveness, mixedlymphocyte responsiveness, and NK activity. Interestingly, the effect of

morphine, to some extent, appears to be one of aggravating stress-mediated

immunodeficiency inherent to the manipulation of the monkeys and their

subsequent adaptive response. Further study with this model will reveal

whether pathogenicity associated with SIV/SMM infection will be altered by

morphine addiction.

THE AIDS VIRORECEPTOR CD4 IS MODULATED IN VIVO AND

IN VITRO BY OPIATES

In 1980, McDonough et al. reported that heroin addicts typically have depressed levels of T cell E-rosettes, which are reversible in vitro by naloxone. This observation led to the demonstration that heroin addicts also

experience depressed ratios of their T-helper and T-suppressor lymphocytes

(Th/Ts) (Donahoe et al. 1987) in the absence of infection with HIV1, a

finding corroborating that of Des Jarlais et al. (1985). Notably, addicts who

do not exhibit depression in their Th/Ts, as a group, exhibit increasing

percentages of circulating Th cells corresponding to the duration of their addiction. While substantiating further the immunomodulatory potential of

addiction, such observations also suggest that addict susceptibility to HIV1

may be affected by alteration of the expression of Th cells, the principal

target cells for HIVl.

To better establish the relevance of these in vivo findings suggestive of opiate effects on T-cell functions and to identify possible mechanisms involved,

the in vitro effects of morphine on E-rosette formation and expression of

the antigenic markers of Th (CD4 molecules) and Ts (CD8 molecules) lymphocytes as well as their CD2 markers were investigated. Since the nature

of the E-rosetting phenomenon was incompletely understood at the time

these investigations started, experiments were devised to assess kinetics of

E-rosette formation to define the processes involved and the effects of opiates thereon. Multiphasic kinetics of E-rosette formation were observed

(Donahoe et al. 1985) under certain thermal conditions (reciprocally increasing and then decreasing). These kinetics were indicative of cyclical processes of E-rosette formation and loss. They suggested that the effect of

opiates on these processes was to inhibit conformational changes in the

E-receptor molecule, known as microdisplacement, that are responsible for

allowing dormant or cryptic E-receptor molecules within the plasma membrane to be up-regulated.

Adaptation of the kinetic procedures for assessment of E-rosette formation

to cytofluorometric types of analyses resulted in procedures capable of measuring the effects of morphine on CD2 expression at the molecular level.

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Initially, a single-color, antigenic-marker-staining paradigm was used to

detect effects of morphine on CD2. Later, assessment of CD2 was done simultaneously with either CD4 or CD8 by using standard double-staining

paradigms. These studies (Donahoe et al. 1987) showed that morphine

modulated all three T cell surface markers (CD2, CD4, and CD8). Their

expression was affected by cyclical processes of receptor modulation as had

been seen with the E-rosetting procedures. Transmodulation between receptor molecules and endocytic processing appears to be the primary process

involved in this modulation. Similar, longer range in vitro effects of endorphin on CD4 expression have also been reported by Puppo et al. 1985.

These observations indicate that the modulation of surface markers observed

with T cells from heroin addicts to some extent must be mediated through

their direct interaction with opiates. This conclusion is supported by numerous reports documenting the presence of opiate-binding sites on lymphocytes

and other cells of the immune system (Sabinga and Goldstein 1988), including a study done with the author’s colleague (Madden et al. 1987) incorporating rigorous pharmacological criteria and preparations of highly enriched

T cells.

In a separate study (Bueso-Ramos et al. 1988), it was also found that selected anti-CD2 and anti-CD4 monoclonal antibodies reciprocally down-regulate

the mutual expression of CD2 and CD4 molecules in both the presence and

absence of morphine. This mutual down-regulation was evident for the

Leu3a but not the OKT4 epitope of CD4. This is notable because the

Leu3a epitope is positioned near the NH, terminus of the CD4 molecule on

the “outermost” part as it protrudes from the cell membrane and is responsible for HIV1 binding (figure 1). Thus, such Leu3a modulation could be

projected to interfere with HIV1 infectivity by directly altering viroreceptor

expression. Since in vitro data suggest that a long-range (hours-days) effect

of opioids is depression of CD4 expression (Puppo et al. 1985), it seems

logical to project that such effect would reduce chances of HIV1 infection

of Th cells in vivo. However, studies using monoclonal antibodies to block

HIV1 infectivity for Th have shown that lack of a full complement of CD4

receptors is insufficient cause to reduce HIV1 infectivity (Weber and Weiss

1988). In lieu of stoichiomctry of CD4 as the deciding factor in determining susceptibility of Th for HIV1, it may be that generalized immunomodulation by opiates, including its effects on CD4 expression, is a more

pertinent factor. Another factor may be that modulation of CD4 expression

by morphine affects feedback mechanisms controlling circulating levels of

Th. Such effect would be harmonious with the previously discussed findings (Des Jarlais et al. 1985; Donahoe, et al. 1987) that heroin addicts frequently experience elevated circulating levels of Th cells. In this way,

chronic addiction leading to increases in Th could be expected to increase

the likelihood of HIV1 infection. Obviously, further study is needed to

separate strictly immunological effects from viroreceptor effects of opiates

as contributing factors in the pathogenesis of HIV1 infection in heroin

addicts.

185
FIGURE 1. Effects of opiates and HIV1 on the intersystemic network

connecting the neural and immune systems

NOTE: Opiates and other drugs of abuse are exogenous modulators of this network. These drugs,

their endogenous congeners, and the hormonal products influenced by them work together to

set the homeostatic tone of the various physiological response systems in the network. A

common focus of these effects is the plasma membrane of affected cells. Here, they

modulate expression of surface receptor molecules relevant to both behavioral and

immunological functions. As illustrated in the T cell, this receptor modulation occurs within

the membrane itself via transmodulation as well as via second-messenger effects (note that the

T cell is represented as a thymocytic precursor of CD4+ and CD8+ circulating T cells).

Since the CD4 molecule is both an HIV1 viroreceptor and a key regulatory component of

immunoresponsivity. its modulation in an addictive setting is bound to be critical to the

immunocompetence of addicts and their susceptibility to AIDS. According to this network

theme, any perturbation in the network including psychological stressors, may affect CD4

expression, immune function, and susceptibility to HIV1 and its complications.

THE EFFECTS OF OPIATES ON IMMUNOCOMPETENCE MAY BE

BOTH DIRECT AND INDIRECT

Opiates are also likely to affect susceptibility to HIV1 infection and AIDS

in more indirect ways than those outlined above. One way is to moderate

immunoresponsiveness indirectly through alterations in the neurocndocrine

system. Another way is to affect the production of, and response to, peptides and hormones secreted by leukocytes that are connected to endogenous

opioid circuitry, such as immunosecreted enkephalins and endorphins and

other analogs of classical neuroendocrine hormones produced by T cells and

other leukocytes (Weber, this volume).

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In regard to the latter consideration, there is presently no specific information to determine how heroin addiction might be influenced by such factors.

Nonetheless, it is inconceivable that secretion of, or response to, enkephalins

and endorphins secreted by T cells and other leukocytes (and, consequently,

immunoresponsiveness affected by these factors) would not be influenced by

heroin addiction. Regarding the possibility of indirect neuroendocrine influence, the study of Shavit et al. (1984) probably best illustrates that the

immunological effects of heroin addiction are also mediated to some extent

by opiate effects on the central neuroendocrine system. The study showed

that splenic NK activity in rats is depressed by a naloxone-reversible type

of footshock stress but not by a nonreversible type. Shavit et al. (1986)

also showed that NK activity is depressed after both systemic and intracerebral administration of morphine to rats but not with a systemically administered morphine analog that does not traverse the blood-brain barrier. Both

studies led to the conclusion that NK activity of rat spleen cells was centrally mediated by opiates, although a precise neuroanatomical site regulating immune function has proved elusive. Weber and Pert (in press) and

Weber (this volume) have extended these findings. Using the technique of

focal microinjection of morphine through guide cannulae aimed at various

brain structures, they show that opiates act in the periaqueductal gray matter

(PAG) of the mesencephalon, but not in other opiate-receptor-containing

neuroanatomical sites, to produce suppression of NK cell activity. The observed suppression is mediated through opiate receptors, since it can be

blocked by naltrexone, an opiate antagonist. These findings suggest that

opiate release and subsequent action in the PAG may be components of an

important neural pathway involved in stress-induced immunosuppression and

illness and neoplasia associated with it. A complexity is introduced into

these circumstances, however, when the data of Kay et al. (1984) that show

that morphine and endorphin enhance NK activity directly in vitro are considered. This finding suggests that systemic regulation of NK activity

differs from the in vitro circumstance.

Interestingly, studies with rhesus monkeys indicate that NK activity is enhanced 2 weeks after initiation of four-times-daily injections of morphine.

The enhancement observed is counter to depressed effects in a number of

T cell functional parameters. Since the data from these experiments indicate that control levels of T cell responsivity are rebounding to historical

norms, while levels for morphine-exposed animals are not, it appears that

the control animals are rebounding from the effects of an undetermined

stressor common to the experimental conditions, but that the animals exposed to morphine are not. This suggests that the NK enhancement observed is related to effects of opiates on some stress-mediated phenomenon

and may, therefore, be centrally mediated. It is, however, unclear how

these data relate to those of Shavit et al. (1986), since their findings indicated depression of NK activity by morphine, not enhancement. Kraut and

Greenberg (1986) have also shown enhanced NK activity in response of

mice to morphine injection, so the reasons for such differences are unclear.

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Clearly, differences in species and experimental protocols used must be considered when assessing the immunological effects of opiates.

AIDS AND OPIATES: CAUSES FOR THE CONNECTION

The foregoing discussion suggests that opiates govern immunocompetence in

human addiction in a variety of ways. The possibilities in this regard are

represented schematically in figure 1, which illustrates the interdependencies

of the immune system with other organ systems as they are influenced by

opiates and HIV1. Since both opiates and HIV1 directly influence both the

immunoendocrine and neuroendocrine systems, and these systems are networked between themselves and other organ systems, there is an understandable uncertainty about the exact role of opiates in AIDS.

The complexities of this issue are extended by the uncertain relevance of

in vitro and animal-model studies to the situation of human addiction and

by the uniqueness of the HIV1 pathogen, which is the final determinant in

host susceptibility to AIDS. Unquestionably, innate interindividual variation

is also relevant to determination of host response to pathogens and drugs, as

are other environmental factors that interdict in the intersystemic network

impacted by addiction, as in figure 1. Yet, despite the complexities of the

intersystemic interdependencies illustrated in figure 1, there are some comforting commonalities that allow better understanding of the AIDS-drug connection and provide direction for future research. It is the cells of the

immune system themselves that are most pertinent in this regard. In the

final analysis, it is the response of these cells to the addictive milieu that

decides immunocompetence. Thus, we need to know how drugs affect

these cells directly and indirectly through the pathways delineated in

figure 1 so that this knowledge can be integrated with that concerning infectivity and pathogenicity of human immunodeficiency viruses.

We have noted that opiates and other drugs of abuse (cocaine and alcohol)

have a common ability to modulate surface molecules on T cells (Donahoe

et al. 1986). Though this effect is common, the causes arc diverse. With

opiates, the effects are presumably mediated through receptor-ligand interaction, whether it is the T cell acting directly with the opiate or some hormonal product resulting from other systemic and centrally mediated points

of opiate-receptor stimulation. With alcohol, as another example. the cause

for surface molecular modulation appears to principally involve alterations in

membrane fluidity, which influence receptor activity within the membrane of

both the T cell and the cells with which it interacts within the intersystemic

network, as outlined in figure 1 (Madden et al. 1984; Donahoe et al. 1986).

With cocaine, a key effect may relate to its anesthetic properties of

Na-channel blockade, which could alter surface-receptor expression within

the network in various ways (Donahoe et al. 1986). Importantly, it is

equally likely that environmental stressors can influence this situation in the

same way as drugs, because the intermediaries of the stressors are the

188
endogenous congeners of the drugs or products of endogenous responses to

them. The main point is that by altering receptor expression, which all

drugs of abuse do as mimics of endogenous peptides and hormones, behavior of the cells affected will be changed. This is the principal commonality

of importance that is to be derived from figure 1, receptor change will lead

to behavioral change. Since AIDS viruses and opiates mediate these

changes to some extent at the same immunoregulatory molecule, CD4, these

factors are inextricably linked in the puzzle that is AIDS.

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ACKNOWLEDGMENTS

Grant support is acknowledged from the following sources: (1) National

Institute on Drug Abuse, DA 01451, DA 04400, DA 0498; (2) State of

Georgia, Department of Human Resources; and (3) grant RR-00165 from

the Division of Research Resources to the Yerkes Regional Primate

Research Center.

AUTHOR

Robert M. Donahoe, Ph.D.

Department of Psychiatry

Laboratory of Psychoimmunology

Emory University School of Medicine

Georgia Mental Health Institute

1256 Briarcliff Road, N.E.

Atlanta, GA 30306

191
Immune Function in Heroin Addicts

and Former Heroin Addicts in

Treatment: Pre- and Post-AIDS

Epidemic

Mary Jeanne Kreek

INTRODUCTION

According to the various surveys of the National Institute on Drug Abuse

particularly the DAWN survey, and also the New York State Division of

Substance Abuse Services, there are now over 2 million people in the

United States who abuse heroin, of whom 500,000 are classified as “hardcore” heroin addicts. “Hardcore” is defined as repeated use of heroin three

to six times a day for over 1 year, with development of tolerance, dependence, and drug-seeking behavior (Kreek 1987b; National Institute on Drug

Abuse 1983; National Institute on Drug Abuse 1987). There are probably

another 500,000 who use heroin regularly, but not necessarily for the length

of time or to the extent defined above. Most of this heroin use is by

needle. Also, the DAWN survey suggests 22 million persons in the United

States have used cocaine at some time, and approximately 6 million have

used it recently, suggesting that use may be regular (Kreek 1987b; National

Institute on Drug Abuse 1983; National Institute on Drug Abuse 1987).

Alcohol is still the most commonly misused agent, with problem drinking

occurring in approximately 11 million and alcoholism in approximately

6 million persons in the United States. In New York City, there are

approximately 250,000 hardcore heroin addicts at this time. In the DAWN

surveys, which are carried out at regular intervals, a change was found

between 1985 and 1987 with respect to the top five drugs (excluding alcohol use alone) mentioned in emergency room visits: cocaine now heads the

list, alcohol in combination with other drugs is now second. and heroin has

dropped to third position. However, in the medical examiners’ surveys,

alcohol in combination with other drugs still leads the list of deaths due to

drug abuse. Heroin is second; cocaine is third; and two other opiates, propoxyphene and codeine, are in fourth place.

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In this chapter, a historical survey of what my research group and others

found to be immunological abnormalities in street heroin addicts prior to the

advent of human immunodeficiency virus (HIV) infection, the acquired

immunodeficiency syndrome (AIDS) epidemic, and the rapid increase in the

use of cocaine is presented. Although recent retrospective studies of the

HIV epidemic suggest that the cocaine epidemic may have occurred even

earlier, cocaine abuse escalation was recognized in 1981 when the price

dropped, availability increased, and the numbers of regular users rose

dramatically over the next 3 years (Novick 1989b). An even greater escalation in the magnitude of cocaine abuse occurred around 1985 when the very

potent, free-base form of cocaine, “crack,” which is usually selfadministered by smoking with inhalation of the drug, became available.

AIDS was first diagnosed in 1981 and was recognized regularly in intravenous (IV) drug abusers in New York City by 1982. Retrospective studies

have documented that HIV infection reached the parenteral drug-abusing

population of New York City around 1977 to 1978 (Des Jarlais et al. 1989;

Des Jarlais et al. 1984; Novick et al. 1986b; and Novick et al. 1986c).

PRE-AIDS IMMUNE FUNCTION IN HEROIN ADDICTS

The first historical era for this review of immunological information is 1966

to 1978, prior to the HIV epidemic, prior to common cocaine use, and in

fact prior to any large-scale pharmacological treatment of narcotic addiction,

although methadone-maintenance treatment had already started as an experimental treatment. From 1966 to 1968, there were a few reports appearing

from New York City and also from Lexington, KY, of immunological abnormalities in heroin addicts not in methadone-maintenance treatment either

entering detoxification treatment or being seen at post mortem (Cherubin

and Milliam 1968; Helpern and Rho 1966; Louria et al. 1967; Sapira 1968).

In the late 1960s the medical examiner of the city of New York reported

diffuse lymphadenopathy at time of post mortem in essentially all deaths of

what was called “narcotism,” which was heroin overdose (Helpern and Rho

1966). This is a very important early finding to be reconsidered, because

this common physical finding in IV drug abusers prior to the advent of HIV

infection means that clinicians and scientists now cannot identify HIV infection in street drug abusers by using lymphadenopathy as one of the physical

findings. Next, diffuse abnormalities in levels of serum immunoglobulins

were found in heroin addicts, along with a very high prevalence of biologically false-positive tests for syphilis, a test that depends on abnormal

amounts of IgM antibodies, in these prepharmacological-treatment study

groups; these findings were reported by several workers (Cherubin and

Milliam 1968; Louria et al. 1967; Sapira 1968).

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METHADONE PHARMACOKINETICS AS USED IN

MAINTENANCE TREATMENT: IMPLICATlONS FOR IMMUNE

FUNCTION

In 1964, Professor Vincent Dole, the late Marie Nyswander, and I initiated

studies of possible long-term pharmacological treatment of narcotic addiction. At the time, it was decided that a drug selected for treatment should

have characteristics very different from heroin or morphine. The theoretically desirable attributes for such a drug were that it would be orally effective, have a very long duration of action with little peak effect, and yet

would sustain a person in an essentially normal state physiologically (Dole

et al. 1966; Kreek 1972). In 1964, analytical technology was not yet available to measure the very low plasma levels of many basic amine drugs,

such as heroin, morphine, and methadone; thus, we had to rely primarily on

clinical observations to estimate the pharmacokinetic profiles of each agent.

Methadone had already been used in the management of addiction, primarily

on the street by drug addicts who would use it to self-detoxify or selfmaintain in settings of heroin shortages or desire for treatment. It was also

used effectively in a few established inpatient treatment resources for shortterm (10 to 14 days) detoxification of heroin addicts. Methadone was

known to be an orally effective drug. In early clinical studies of methadone used for treatment of pain, apparent cumulative effects, including

undesired respiratory depression after repeated doses, were observed despite

its relatively short duration of action in pain relief. These findings

suggested that methadone might have a much longer duration of action than

initially had been reported. At that time (1964) methadone was reported to

have the same duration of action as morphine with respect to pain relief.

We now know that heroin (and its major metabolite, morphine) and methadone in tolerant subjects have very different characteristics. Methadone has

approximately a 24- to 36-hour duration of action, with withdrawal symptoms initially appearing around 24 hours following dosing (Dole and Kreek

1973; Kreek 1973c; Kreek et al. 1976). This is sharply contrasted with

heroin used on a chronic basis; withdrawal symptoms will begin within 3 to

4 hours after the last dose of heroin. The pharmacokinetic profiles are now

known to be strikingly different for these two agents. By giving a drug

orally, as opposed to parenterally (using hypodermic needles and syringes),

one can get away from both the lure and the psychological ramifications of

needle use and also from the potential hazards that accompany “dirty”

needle usage, such as infection with a variety of viral and bacterial agents

that may contaminate shared or unsterilized needles and other injection

paraphernalia.

When gas-liquid-chromatographic techniques were developed for measuring

plasma levels of methadone, there was found analytically what had been

seen clinically much earlier, that is, following oral dosing, there is a very

low peak plasma level of methadone, barely a doubling of the nadir or

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steady-state level, during chronic high-dose methadone administration to tolerant and dependent former heroin addicts; this small peak is followed by a

slow decline, with sustained plasma levels and thus perfusion at critical

opiate receptor sites of action for a 24-hour dosing interval (Kreek 1973c;

Kreek et al. 1976; Kreek et al. 1978). If the dose of methadone is selected

to be less than the degree of tolerance developed by the individual in

treatment, there is no narcotic effect perceived, no euphoria, no sleepiness,

no “high,” nor any other narcotic effect. However, the dose must be selected with the strategy of staying below the degree of tolerance developed by

that individual, in sharp contrast to the strategy for the treatment of pain,

for which, to achieve analgesia, one must always exceed the degree of tolerance developed by the individual requiring treatment. The steady-state,

methadone-maintained patient receiving a steady dose of methadone has no

analgesic effects from the methadone administration and thus, in the setting

of pain stimulus, will experience pain and will respond as any other person

to appropriate usual, or slightly higher than usual, doses of a short-acting

narcotic coadministered with methadone; this is the pain management of

choice in such patients.

Further studies were performed of the pharmacokinetics of methadone in

former addicts in steady-state, methadone-maintenance treatment (Kreek

et al. 1976; Kreek et al. 1979; Kreek et al. 1978; Nakamura et al. 1982).

Because of the extensive distribution and the resultant very low plasma

levels of this drug, it was not possible to use classical radioisotope tracer

techniques; the amounts of isotope that would be required exceed the ethical

limits in human research (Kreek et al. 1979; Nakamura et al. 1982). Therefore, stable isotope-labeled species of methadone were used to do these

studies (Kreek et al. 1979; Nakamura et al. 1982). In collaboration with

Dr. Peter Klein and his group, formerly at Argonne National Laboratories

and now at Baylor Medical School, three different species of stable isotopelabeled methadone, tri-, penta-, and octadeuteromethadone, were synthesized

with one specific label on each of the enantiomers and also on the racemic

compound used in therapeutics, to determine the metabolic fate of the two

enantiomers simultaneously and measure quantitatively the entire methadone

mass using gas chromatography, chemical ionization—mass spectrometry

with selected ion monitoring (Kreek et al. 1979; Nakamura et al. 1982).

Using these techniques, we have been able to determine that in humans the

active R(1;-)enantiomer of methadone has a slower elimination than does the

inactive S(d;+)enantiomer (Kreek et al. 1979; Nakamura et al. 1982). Based

on studies both with and without use of stable isotope techniques, we have

found that the half-life of methadone in humans is 24 hours for the racemic

compound and approximately 48 hours for the active enantiomer. This

sharply contrasts with the 1- to 4-hour half-life of heroin in humans. Also,

methadone has greater than 90 percent systemic bioavailability after oral

administration, contrasted to less than a 30-percent bioavailability for heroin.

These pharmacokinetic differences arc probably the primary explanation of

why one cm transform a formerly behaviorally abnormal person into a

195
normal well-functioning one during chronic methadone-maintenance treatment and also why physiological indices, profoundly perturbed by the

chronic use of the short-acting heroin, may become increasingly normal with

time in stabilized methadone maintenance treatment (Kreek 1973b; Kreek

1978).

There are currently approximately 100,000 former heroin addicts now in

methadone-maintenance treatment in the United States and abroad. In good,

well-staffed programs, usually those that provide counseling, rehabilitation

efforts, and access to medical care and behavioral care as needed, the voluntary retention rates range from 55 to 80 percent. If adequate doses of

methadone are used, illicit heroin use persists in less than 10 percent of

patients (Dole 1988; Kreek 1973c; Kreek 1987b). However, in several followup studies it has been shown that more than 80 percent of former

methadone-maintained patients, irrespective of the degree of the rehabilitation, relapse to heroin or other narcotic use within 2 years of leaving

methadone treatment (Kreek 1987b). These data are superimposable on outcome data of former heroin addicts treated in drug-free programs; less than

30 percent are able to stay narcotic free after leaving residential drug-free

treatment. These findings may teach us something about the biology of

addictive disease and emphasize the need to look more deeply into what

may be some of the mechanisms of addiction. Also. some of those mechanisms may be very important for an understanding of the immune phenomena associated with drug abuse. Methadone-maintenance treatment has a

general 55- to 80-percent long-term success rate; treatment with an opioid

antagonist, such as naltrexone, has a 15- to 20-percent success rate in

unselected heroin addicts; drug-free treatment is successful in 20 to 30 percent of such patients in a variety of different kinds of programs in which

long-term followup studies were performed.

PRE-AIDS IMMUNE FUNCTION IN METHADONE-MAINTAINED

FORMER ADDICTS

Starting with the first patient, prospective studies were begun to examine

the medical status of heroin addicts at time of entry to experimental methadone treatment in 1964 and during 3 or more years of continuous maintenance treatment (Kreek 1973a; Kreek et al. 1972; Kreek et al. 1979).

Within 3 or 4 years, other groups also were beginning to study heroin

addicts entering or during methadone-maintenance treatment, either prospectively or at “one point in time” (Adham et al. 1978; Brown et al. 1974;

Cushman 1973a; Cushman 1973b; Cushman and Grieco 1973; Cushman et

al. 1977; Cushman and Sherman 1974; Dunk et al. 1987; Falek et al. 1972;

Falek 1980; Geller and Stimmel 1973; Grieco and Chuang 1973; Madden

et al. 1979; McDonogh et al. 1980; Sherwood et at. 1972). This availability of a treatment that had a very high rate of retention permitted study

of some of the physiological effects of pharmacological treatment, in

addition to study of the medical status before and during treatment (Kreek

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1973a; Kreek et al. 1972; Kreek et al. 1986). Some of the very earliest

observations included absolute lymphocytosis in 20 to 30 percent of

subjects; elevated levels of serum IgM and IgG in 50 to 70 percent of

subjects; and diverse serum protein abnormalities (Kreek 1973a; Kreek

1973b; Kreek et al. 1972). Abnormalities in T-cell rosette formation and

impaired lymphocyte responsiveness to mitogens were also reported (Brown

et al. 1974; Cushman et al. 1977). Abnormal natural killer (NK) cell

cytotoxicity in patients who recently entered methadone treatment was found

in early studies (Lavie et al., unpublished).

Three sets of workers looked at changes in immunological function with

time in methadone treatment: our group at The Rockefeller University, the

groups of Stimmel at Mount Sinai Medical Center, and Cushman at

St. Luke’s-Columbia Medical Center (Brown et al. 1974; Cushman 1973a;

Cushman 1973b; Cushman and Grieco 1973; Cushman et al. 1977; Cushman

and Sherman 1974; Geller and Stimmel 1973; Grieco and Chuang 1973;

Kreek 1973a; Kreek et al. 1972). We all found that lymphocytosis was

present in over 30 percent of the patients both at the time of entry and

during 3 years or more of high-dose methadone-maintenance treatment

(Kreek 1973a; Kreek et al. 1972). We found that even after 3 years or

more of methadone-maintenance treatment, 76 percent of patients had elevated levels of IgM, 48 percent had elevated levels of IgG, and 7 percent

had elevated levels of IgA. Of those patients with elevated levels of IgM,

52 percent had biological false-positive tests for syphilis (Kreek 1973a;

Kreek et al. 1972). The persistent antigenic stimulus for the elevations in

immunoglobulin levels remains unknown. Dr. Deborah Doniach in England

performed extensive studies searching for autoantibodies in sera of these

patients, in collaboration with our group. It was found that the prevalence

of autoantibodies was actually less than would have been anticipated, given

the prevalence of chronic liver disease in this population. Over 60 percent

of all these methadone-maintained patients had evidence of chronic liver

disease at lime of entry (Kreek et al. 1972).

Stimmel and coworkers, also studying heroin addicts prospectively from

time of entry into methadone-maintenance treatment, but following them for

a shorter term in treatment than our studies, found elevated levels of IgM in

87 percent and IgG in 63 percent of subjects entering methadonemaintenance treatment (Brown et al. 1974). After 1 to 18 months of treatment, these abnormalities had lessened. Percentages of patients having

abnormalities decreased from 87 to 76 percent for IgM and from 63 to

48 percent for IgG. Possibly more important, the levels of elevations of

immunoglobulins observed also decreased significantly (Brown et al. 1974).

Cushman similarly found that there was a reduction both in numbers of

patients with elevations in levels of immunoglobulins and in the actual

degree of those elevations for IgM and IgG during long-term methadonemaintenance treatment (Cushman 1973a; Cushman 1973b; Cushman and

Grieco 1973; Grieco and Chuang 1973). Cushman also showed that the

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numbers of patients with biological false-positive tests for syphilis decreased

with time in methadone-maintenance treatment, from 23 percent of the

entire study group of heroin addicts at time of entry to 6 percent after a

mean of 23 months in treatment (Cushman and Sherman 1974). Cushman,

Grieco, and colleagues showed that abnormal T rosetting was found in most

heroin addicts but that the numbers of methadone-maintained patients showing T-cell and B-cell rosetting abnormalities seemed to decrease with time

in treatment (Cushman et al. 1977). Stimmel and colleagues showed that

lymphocytes from heroin addicts had a significantly reduced ability to

respond to mitogenic stimulation in vitro using phytohemagglutinin and concanavalin A as mitogens (Brown et al. 1974). Followup studies of these

subjects during methadone treatment showed significant improvement in

some, but not all, subjects (Brown et al. 1974). In all these studies, those

of our group, of Stimmel, and of Cushman, appropriate adequate treatment

doses of methadone (60 to 100 mg per day) were administered (Dole 1988).

Therefore, all study subjects in long-term methadone-maintenance treatment

were being exposed to a higher opioid load than they had been when they

were self-administering heroin on the street. Also, since methadone has a

plasma half-life of around 24 hours, but heroin only 1 to 4 hours, the area

under the plasma concentration-time curve for methadone is significantly

greater than that for heroin (Kreek 1973c; Kreek 1989; Kreek et al. 1976).

Following heroin self-administration, plasma-level peaks occur three to six

times a day, depending on the number of self-administrations, followed by

rapid decline to a nadir with resultant low areas under the plasma

concentration-time curves.

Also, in each of these three sets of studies, it was found that only 20 to

30 percent of heroin addicts entering treatment had a history of clinically

apparent hepatitis, but more than 50 to 70 percent had laboratory evidence

of chronic liver disease. When markers for hepatitis B were measured, it

was found in each of these studies that 5 to 15 percent of heroin addicts

were, and still are, chronic carriers of hepatitis B antigen (Kreek 1973a;

Kreek 1973b; Kreek 1978; Kreek 1987b; Kreek et al. 1972; Kreek et al.

1986; Novick 1988b; Novick ct al. 1986b). Hepatitis B is still the major

infectious disease of easy transmissibility within the population of IV drug

abusers in most regions of our Nation (Novick et al. 1986b). Alcohol

abuse also was then and is now a major problem (Kreek 1973a; Kreek

1978; Kreek 1981; Kreek et al. 1972). It was found, in the prospective

studies beginning in 1964, that approximately 30 percent of street heroin

addicts in New York were abusing alcohol to excess and that this problem

continued concomitantly with their heroin addiction (Kreek 1973a; Kreek

1978; Kreek 1981; Kreek 1987b; Kreek et al. 1972). Thus, alcohol abuse,

along with hepatitis B and hepatitis delta (identified in the late 1970s and

shown by retrospective analysis of our banked sera collection appeared in

the New York City drug-abuse population in the mid-1970s), is the major

cause of liver disease in this population (Kreek et al. 1987; Kreek

et al. 1986; Novick et al. 1988b; Novick et al. 1985; Novick et al. 1986b).

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Delta hepatitis virus is an incomplete RNA virus that is dependent upon the

hepatitis B DNA virus for its replication, expression, and infectivity; it

causes a potentially lethal viral liver disease. Serological evidence of

infection with delta virus is now present in between 10 and 50 percent of

New York City street heroin addicts (Kreek et al. 1987; Kreek et al. 1986;

Novick et al. 1986b; Novick et al. 1985; Novick et al. 1986b).

In all immunological studies from 1964 onward, the question was asked of

whether or not the presence of liver disease, which is known to cause many

kinds of immune abnormalities, and alcohol abuse, which also causes a

variety of immune abnormalities, could account for the abnormalities in

immunoglobulins and also in T-cell function observed, including the

abnormal responses to mitogens and T-cell rosetting. Cushman, Stimmel,

and our own group found that there was no predominant correlation

between the immunological abnormalities and the presence of liver disease.

Indeed, although liver disease and alcohol abuse may contribute to the

abnormalities observed, they are not the primary or sole factors causing

these abnormalities.

In progress is a prospective study of young adult heroin addicts who

became hardcore addicts before the age of 18, some of whom now have

been in methadone-maintenance treatment for up to 20 years (Kreek et al.

1986). Whereas more than 15 percent of these youngsters had hepatitis B

antigenemia, and most of these were chronic carriers of hepatitis B virus at

time of entry to treatment, when stabilized for 10 years or more in

methadone-maintenance treatment, none (0 percent) still had hepatitis B antigenemia (Kreek et al. 1986). The number of patients in this followup study

is too small to do meaningful statistical determinations for significance, but

the rate of conversion from a chronic carrier state to a “protected“ immunized state (with hepatitis B surface antibodies) in this group is at least

brisk. This again raises the question: Is immune competency improved

during chronic methadone-maintenance treatment?

NEUROENDOCRINE EFFECTS OF HEROIN AND OF

METHADONE AS USED IN MAINTENANCE TREATMENT:

IMPLICATIONS FOR IMMUNE FUNCTION

In 1973 and 1975, some very exciting and relevant scientific findings were

made, that is, the discovery of specific opioid receptors and the endogenous

ligands of three distinct classes that bind to these receptors. The distinct

groups of endogenous opioids remain three, and each has been defined by

both biochemical and molecular biological techniques: the enkephalins, the

dynorphins, and beta-endorphin from proopiomelanocortin (POMC). Although all of the opiate-receptor-active endogenous opioids contain the four

amino acids characteristic of met- or leu-enkephalin, nevertheless only

enkephalins and dynorphins are known to be processed to met- or leucnkephalin. Of the three endogenous opioid classes, the one that has

199
attracted the most attention is the POMC precursor. In humans, ACTH and

beta-endorphin are released from POMC in equimolar amounts from the anterior pituitary into blood; POMC also is processed in the hypothalamus in

humans to yield beta-endorphin and ACTH.

Much research work has been reported recently, documenting both the

expression of the message for genes of the endogencus opioids and synthesis of the peptides of three classes in peripheral sites outside the brain,

hypothalamus, pituitary, and spinal cord. The mRNA for the enkephaiin

series clearly is expressed in many other sites. For instance, my group,

interested in the possible role of the brain-gut axis in the addictive diseases,

has been studying the endogenous opioid system of the gastrointestinal tract.

We have recently shown that the message for the preproenkephalin gene is

extensively expressed in the gastrointestinal tract of the guinea pig,

especially in the colon (Zhang et al. 1988). Zurawski and colleagues have

recently reported that the message for preproenkephalin is expressed by

mouse T-helper cells that have been activated by concanavalin A and also

that enkephalin peptidc synthesis by these activated T cells occurs

(Zurawski et al. 1986). The message for the dynorphin gene also may be

expressed in several different peripheral sites, along with synthesis of

dynorphin peptides; work continues on this question.

The precursor peptide POMC is particularly of interest, because it yields

one of the major stress hormones, ACTH, which in turn drives the adrenal

cortex to produce corticosteronc in rodents and cortisol in humans. Two

independent groups of investigators, Westly and colleagucs (1986) and

Lolait and colleagues, have reported expression of the message for the

POMC gene in Newcastle disease virus-infected mouse splenocytes and in

unstimulated mouse splenic macrophages, respectively (Lolait et al. 1986;

Lolait et al. 1984).

The endocrine effects for drugs of abuse, such as heroin, and also treatment

drugs, such as methadone, arc potentially very important for several reasons

(Kreek 1978; Kreek 1987a; Kreek 1987b). One is that endocrine and

neuroendocrine function modulate many other important physiological functions, including immune function. Cortisol (corticosterone in rodents),

ACTH, and also possibly beta-endorphin may affect specific cellular or

humoral elements of the immune system. Our research group is interested

in the possible relationship between neuroendocrinc function and the behavior of addictive disease per se and is also interested in the relationship

between neuroendocrine function and immune function (Kreek 1987a; Kreek

1989). In man, in sharp contrast to most of the recently reported findings

in the rodent model, an acute injection of a short-acting or long-acting

opiate, such as heroin, morphine, or methadone, will cause inhibition of

release of ACTH and beta-endorphin. with resultant blunted release of cortisol from the adrenal cortex and a flattened circadian rhythm of that release

(Kreek 1972; Kreek 1973a; Kreek 1978; Kreek 1987a). During subacute

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and chronic injections of short-acting narcotics, intermittent multiple doses

of drug must be used to achieve desired effects, with opiate peaks followed

by rapidly declining levels of opiate; these declining levels are accompanied

by mild or severe withdrawal symptoms several times each day in the

addict. In heroin addiction, inhibition of the neuroendocrine peptides at

time of peak plasma levels of short-acting opiate continues, but if narcotic

withdrawal is allowed to proceed, then the opposite response begins, which

is the stress response of increased release of beta-endorphin, ACTH, and

cortisol (Kreek 1972; Kreek 1973a; Kreek 1978; Kreek 1987a). Other

neuroendocrine effects of exogenous short-acting opioids in man that have

been well described include inhibition of release of LH and concomitant

lowering of levels of testosterone, along with increased release of vasopressin and prolactin (Kreek 1978; Kreek 1987a).

In a very simplified scheme of the hypothalamic-pituitary-adrenal (HPA)

axis in humans, normally a negative feedback control mechanism is operative, with suppression of corticotropin-releasing-factor at hypothalamic sites

and also suppression of processing and release of POMC peptides, including

beta-endorphin and ACTH, at the level of the anterior pituitary and probably also at the level of the hypothalamus by cortisol released from the

adrenal cortex in response to ACTH. This negative feedback loop is altered

during long-term heroin addiction, with suppression of release of the POMC

peptides and resultant abnormal levels of cortisol. In withdrawal from narcotics, an opposite, different alteration in this loop, with the increased peptide hormone and cortisol release. In the first 2 months of methadonemaintenance treatment, the findings are similar to those during heroin addiction, that is, a suppression of release of ACTH and beta-endorphin, but after

3 months or more of steady dose treatment, when the full treatment dose of

methadone has been achieved by slowly increasing the daily dose and then

stabilizing the daily dose, the circadian rhythm of release and plasma levels

of ACTH and beta-endorphin become normalized, and the release of cortisol

and the circadian rhythm of its release also become normalized (Kreek

1972; Kreek 1973a; Kreek 1973b; Kreek 1978; Kreek et al. 1981). In longterm methadone-maintained patients, as in normal control subjects, the

plasma levels of both beta-endorphin and ACTH are highest in the morning;

then the levels begin to drop, with lowest levels in the late afternoon or

early evening, according to the known, normal circadian rhythm (Kreek et

al. 1981; Kreek et al. 1983).

We have been using a provocative test, the administration of metyrapone, a

compound that selectively blocks 11-beta-hydroxylation in the adrenal cortex, thus blocking the last step in the metabolic biotransformation pathway

to cortisol production (Kreek 1972; Kreek 1973a; Kreek 1978; Kreek et al.

1984). Since cortisol is the only steroid operative in the negative feedback

loop in humans for the control of the HPA axis, the negative feedback loop

is therefore disrupted, which in turn causes hypothalamic and pituitary sites

to produce more of the POMC peptide hormones ACTH and beta-endorphin.

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This provocative test is used in neuroendocrine evaluation and is called a

test of hypothalamic-pituitary reserve. In normal subjects, additional

amounts of ACTH and beta-endorphin, and thus increased amounts of the

precursors of cortisol, should be produced in response to this cutoff of the

negative feedback control loop. In certain endocrine deficiency diseases, in

cycles of heroin addiction, and during the first 3 months of methadone

treatment, there is an apparent inadequate hypothalamic-pituitary reserve;

that is, in response to the cutoff of this negative feedback loop, the pituitary

does not put out additional amounts of ACTH and beta-endorphin, and the

adrenal cortex does not produce increased amounts of the immediate precursors of cortisol (Kreek 1972; Kreek 1973a; Kreek et al. 1984). However,

we have shown that during long-term methadone-maintenance treatment, this

abnormality in response to metyrapone disappears, and the responses

become normal (Kreek 1973a; Kreek 1978; Kreek 1987a; Kreek

et al. 1984).

For the past few years, since sensitive and specific laboratory tests for the

quantitative measurements of low plasma levels of the peptides ACTH and

beta-endorphin have been developed and validated, the metyrapone test is

now carried out by a single-dose administration of metyrapone, which

abruptly lowers cortisol levels (Kreek et al. 1984). Direct measurements of

peptide hormones released into plasma in response to this abrupt lowering

are then carried out (Kreek et al. 1984). In the earlier metyrapone test,

multiple doses of mctyrapone were administered over a l-day period, and

during that day and the next 2 days, the precursors of cortisol were measured as urinary excretory products (Kreek 1972; Kreek 1973a; Kreek

1978). Using the single-dose test in methadone-maintained patients, a twofold to fourfold increase in plasma levels of both ACTH and beta-endorphin

is observed over the 4 hours following metyrapone administration in

response to an abrupt decrease in cortisol production; this result is identical

to that defined as a normal response to metyrapone testing in otherwise

healthy subjects (Kreek 1987a; Kreek et al. 1984). Thus it has been shown

that neuroendorine functions, both baseline levels and responses during various kinds of provocative tests, are normal in stable-dose, long-term,

methadone-maintained former heroin addicts.

In studies still in progress of drug-free former heroin addicts and former

methadone-maintained patients no longer receiving methadone, preliminary

observations indicate a hyperresponsivity of the drug-free former-narcoticdependent person to metyrapone testing. This contrasts sharply with the

hyporesponsivity seen during cycles of heroin addiction (Kreek 1987a;

Kreek et al. 1984). Whcthcr or not this excess production of stress-related

peptides is related to behaviors observed in drug-free heroin addicts or zerodose methadone-maintained patients, that is, the relapse to use of illicit

narcotic drugs, which would convert this hyperresponsive state into a hyporesponsive state, is not yet known. However, this observed hyperresponsivity of the HPA axis during metyrapone testing, a chemically induced stress,

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must now be considered in the context of possible relationships to other

kinds of stress in former heroin addicts not receiving opioid medication.

This excessive release of HPA-axis peptide and steroid hormones is also

similar to that seen when narcotic use is abruptly stopped in narcoticdependent persons and also, to a lesser extent, between doses of selfadministered short-acting heroin. The relationship of this hyperresponsivity

of neuroendocrine function to immune function in the setting of relative or

absolute narcotic withdrawal is not known.

COCAINE, ETHANOL, AND POLYDRUG ABUSE DURING

METHADONE TREATMENT: IMPLICATIONS FOR IMMUNE

FUNCTION

Methadone-maintenance treatment per se does not specifically or directly

treat alcoholism, cocaine dependency, or any other nonopiate drug dependencies. Howevcr, methadone-maintenance treatment may help engage a

patient in drug-free treatments for alcoholism and cocaine dependency,

which ideally would be available in all treatment programs for these common, concomitant addiction problems. At this time, even during effective

methadone-maintenance treatment, with, essentially, cessation of illicit heroin

use, there is continued heavy use of alcohol, a use which usually began

before or during heroin addiction, by 25 to 50 percent of patients. Since

1978 to 1983, there is also extensive use of cocaine by 25 to 50 percent of

methadone-maintained patients, just as there is by street heroin addicts

(Kreek 1973a; Kreek 1978; Kreek 1981; Kreek 1987b). From a treatment

standpoint, these findings make it imperative to attempt to combine drugfree treatment for these problems with methadone-maintenance treatment for

narcotic addiction (Gordis 1988). In addition, there is an urgent need for

the development of pharmacological treatments for cocaine dependency and

alcoholism, since drug-free and psychotherapeutic treatments for these addictions have had very limited success rates over the long term (Gordis 1988;

Kreek 1987b). Chronic abuse of alcohol, cocaine, and other agents also

may have profound implications with respect to the potential normalization

of neuroendocrine function, which is hypothesized to be closely linked to

the normalization of immune function observed in former heroin addicts in

methadone-maintenance treatment (Kreek 1989; Van Dyke et al. 1986;

Watson et al. 1985). Preliminary studies by our group and others suggest

that both cocaine abuse and alcohol abuse may significantly alter neuroendocrine function as well as immune function in these patients (Kreek

1989; Van Dyke et al. 1986; Watson et al. 1985). Many groups are now

studying both the acute and chronic and direct and indirect effects of

alcohol and cocaine on specific indices of immune function, primarily

because of the possible relationship of any such effects to HIV infection

and AIDS (Kreek 1989; Van Dyke et al. 1986; Watson et al. 1985).

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NEUKOENDOCRINE EFFECTS OF NALTREXONE AND RELATED

OPIOID ANTAGONISTS: IMPLICATIONS FOR IMMUNE

FUNCTION

To explore further the role of the endogenous opioid in addictive disease

and to determine the effects of any drug used to treat drug abuse, specifically in this case heroin addiction, on both neuroendocrine function and

immune function. an additional sequence of studies has been carried out on

the effects of chronic treatment with the specific opioid antagonist naltrexone on neuroendocrine function. Former heroin addicts in naltrexone

treatment for a mean of 5 months, with at least 5 weeks of naltrexone treatment, were studied in collaboration with Drs. Kostcn and Kleber; it was not

possible to study patients in treatment for a longer period of time (Kosten

et al. 1986a; Kosten et al. 1986b). It was found that the morning basal

levels of beta-endorphin were significantly increased in the naltrexonetreated patients as compared with otherwise healthy control subjects (Kosten

et al. 1986b). These findings were not surprising, since acutely, naloxone

when given in an IV bolus or naltrexone when given either IV or orally in

a single dose will cause a brisk rise in ACTH and beta-endorphin and, in

response, a rise in plasma levels of cortisol levels, all of which then fall off

rapidly following the pharmacokinetic profile of each antagonist. Cortisol

levels were also increased during naltrexone treatment in many patients.

We had the opportunity to restudy seven patients after they stopped naltrexone treatment and had been naltrcxone free for a period of time; basal

morning levels of cortisol returned to normal range after stopping naltrexone

treatment (Kosten et al. 1986a). Naltrcxone-treated former heroin addicts

received the drug three or even as infrequently as two times a week for

feasibility and compliance reasons; therefore the doses administered were

very large because of the relatively short half-life of naltrcxone (4 to

6 hours). A high peak plasma level occurred after dosing, followed by a

steady decline, rather than a stabilized, steady-state plasma level of the

drug. It was not surprising that no full normalization of neuroendoctine

function occurred during chronic naltrexone treatment (Kosten et al. 1986a;

Kosten et al. 1986b).

Real levels of beta-endorphin, ACTH, and cortisol became normal in drugfree, former-methadone-maintained patients, but subjects have not responded

normally to provocative tests of neuroendocrine function. We have not

been able to do provocative tests in chronic naltrexone-treat patients yet.

However, abnormal results of provocative testing might be anticipated, since

during chronic naltrcxonc treatment, there is no normalization in baseline

neuroendocrine function. The three hormones of potential interest with

respect to immune modulation, ACTH, beta-endorphin, and cortisol, are at

least intermittently elevated during naltrexone treatment (Kosten et al.

1986a; Kosten et al. 1986b).

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POST-AIDS IMMUNE FUNCTION IN HEROIN ADDICTS AND IN

METHADONE-MAINTAINED FORMER ADDICTS

AIDS has changed many things, and for those of us working in research

related to addiction, it has become an additional confounder, beyond hepatitis B, hepatitis delta, alcohol abuse, cocaine abuse, and polydrug abuse, all

of which commonly occur both in street addicts and in patients in treatment

and also confound or complicate research design, patient selection, and data

interpretation. AIDS also has become a confounder for treatment of narcotic addiction, with the former hopes of full recovery now often not possible

to realize. As of September 5, 1988, there were 72,645 cases of AIDS in

the United States; 23 percent of these were in New York City. Since January 1, 1988, IV drug use has been the primary risk factor for new cases of

AIDS in New York City, now accounting for 43 percent of cases. In the

Nation as a whole, parenteral drug abuse is the primary risk factor in

approximately 2.5 percent of AIDS cases; parenteral drug abusers are the

group at second highest risk for the development of AIDS. Both male and

female IV drug users arc affected. Children with AIDS are primarily the

children of IV drug users at this time (Des Jarlais et al. 1985; Des Jarlais

et al. 1989; Kreek 1989). IV drug users are probably the largest

contributors to the spread of AIDS to the heterosexual community (Des

Jarlais et al. 1985; Des Jarlais et al. 1989; Kreek 1989).

Identifying Initial HIV Infection in New York City

In 1984, studies were carried out on unlabeled banked sera, which had been

collected from 1969 onward, to determine when HIV seemed to attack the

New York City population of heroin users (who were examined on entry to

and during methadone-maintenance treatment) (Des Jarlais et al. 1989; Des

Jarlais et al. 1984; Novick et al. 1986b; and Novick et al. 1986c). At the

same time, Des Jarlais carried out various studies both in street addicts and

in persons entering various kinds of treatment programs for parenteral drug

abuse (Des Jarlais et al. 1985; Des Jarlais et al. 1984). It was found that

50 to 60 percent of parenteral drug abusers on the streets of New York

were positive for the antibody test, indicating infection with HIV in 1984

(Des Jarlais et al. 1989; Des Jarlais et al. 1984; Novick et al. 1986b; and

Novick et al. 1986c). Around 50 percent of former drug abusers who had

entered methadone-maintenance treatment after 1982 were also positive for

the HIV antibody (Novick et al. 1986b; Novick et al. 1986c). We found,

howcvcr, that less than 10 percent of former parenteral drug abusers who

had entered and remained in effective methadone-maintenance treatment

before the epidemic reachcd New York City in 1978 were HIV-antibody

positive (Des Jarlais et al. 1984; Novick et al. 1986b; Novick et al. 1986c).

This 10 percent of patients with positive anti-HIV tests could be the

cocaine-abusing group of patients in methadone-maintenance treatment; at

least 10 to 20 percent of cocaine abusers who arc active heroin addicts or

former heroin addicts in treatment use cocaine by the IV route, placing

205
these persons at continuing risk for HIV infection. The finding that less

than 10 percent of patients in effective methadone-maintenance treatment

were anti-HIV positive at a time when greater than 50 percent of street

addicts were anti-HIV positive was highly significant in 1984 and still has

important public health implications in 1988. It is imperative to get hardcore heroin addicts into effective treatment at this time. Data presented by

Dr. Olof Blix of the University of Uppsala at the Fourth International AIDS

meeting held in Sweden in June 1988 showed that those heroin addicts who

had entered Swedish methadone programs prior to 1979 and were still in

treatment in 1988 had less than a 5-percent anti-HIV positivity prevalence

(Blix et al. 1988). New entries into methadone treatment were not allowed

in Sweden between 1979 and 1983, but the government did allow those

already in treatment to remain. New patients again were allowed to enter

methadone-maintenance treatment programs in 1983; this study showed that

more than 50 percent of those who entered treatment from 1983 to 1988

were anti-HIV positive at the time of entry (Blix et al. 1988). These data

are therefore essentially superimposable on our data from New York City.

Post-AIDS Problems in Studying Immunological Abnormalities in

Active and Former Heroin Addicts in Treatment

AIDS has altered our ability to do large-scale immunological studies in

populations of street IV drug abusers in many locations. It has also posed

some new problems related to immunological abnormalities in drug abusers

(Novick et al. 1988a). Our group has recently described false positive tests

for anti-HIV using the standard ELISA tests in 7 percent of unselected

parenteral drug abusers entering hospital primarily for treatment of liver

disease. Although these patients repeatedly had positive ELISA tests for

anti-HIV, on further testing they repeatedly had negative Western blot tests.

The question was, who were these false-positive testing subjects. In our

study, 5 out of 70 in a group of parenteral drug abusers had false-positive

test results; analysis of data showed that the patients with false-positive test

results were parenteral drug abusers who also had a very long duration of

alcohol abuse and, as a result of that alcohol abuse, had profoundly abnormal laboratory tests reflecting liver function and protein synthesis, with

elevated levels of serum globulins and diverse immunoglobulin abnormalities. The abnormal levels of immunoglobulins, in part due to chronic

alcohol abuse in these narcotic addicts, may have caused a false-positive

anti-HIV test (Novick et al. 1988a).

Another recent study related to immune dysfunction in drug-using populations has been performed by our group in collaboration with Des Jarlais and

Trepo (Kreek et al. 1987). We studied a group of unselected parenteral

drug abusers, without AIDS but with about a 50-percent positive HIVantibody test result, and a group of parenteral drug abusers with active

AIDS requiring treatment (Kreek et al. 1987). In the non-AIDS group,

5 percent had hepatitis B antigenemia, and 21 percent had positive

206
antihepatitis delta serological tests, indicating prior delta hepatitis infection

(Kreek et al. 1987). Of the 374 subjects without AIDS, only three had

hepatitis delta antigencmia. In the study group with AIDS, however,

15 percent had hepatitis B antigenemia, and 6 percent also had hepatitis

delta antigenemia (Kreek et al. 1987). Hepatitis delta antigen marker is

normally cleared within the first 2 to 4 weeks after hepatitis delta infection,

and the nonprotective antibody appears. In the group with AIDS, no subject was found with hepatitis delta antibodies, a highly significant difference

from the street drug abusers without AIDS, with or without anti-HIV positivity, suggesting that production of the delta antibody cannot occur or is

suppressed in the setting of the immunosuppression of AIDS (Kreek et al.

1987). The infectivity of the antigenemic state for delta hepatitis is much

greater than when the antigen, reflecting active viral replication, has cleared.

However, the infectivity of the delta hepatitis virus is possible only in those

persons who have actively replicating hepatitis B virus, as reflected by

hepatitis B antigencmia, since hepatitis delta is a defective RNA virus

requiring replication of the DNA hepatitis B virus for its own expression

and infectivity. Hepatitis delta virus may still be infectious after the

antigenemia has cleared and the nonprotective antidelta antibodies have

appeared, but only as long as there is replicating hepatitis B virus. These

findings suggest that the natural history of both hepatitis B and hepatitis

delta may be significantly altered in the setting of AIDS. They also

suggest that the infectiousness of patients with both hepatitis B and

combined hepatitis B and delta may be increased in the setting of active

AIDS. These findings make imperative a serious consideration of vaccination of all persons in the risk groups for HIV infection and also all health

care and laboratory workers working with patients with HIV infection and

AIDS.

Important issues to be clearly delineated when considering the possible

effects of drugs of abuse and drugs used to treat drug abuse with respect to

the immune system are: What are the direct drug effects, and what are the

indirect drug effects? When one performs studies in which drugs are added

to the experimental system in vitro, any effects observed are, by definition,

direct drug effects on specific components of the immune system. Such

studies of potential direct drug effects can be carried out using cells from

otherwise healthy normal subjects or cells from abnormal populations.

When a drug is administered to an intact animal or to a human, and then

the effects of the drug are studied either in vivo or in cells from such a

subject in vitro, potentially both direct effects and indirect effects are

observed. It is impossible in such an experimental design to completely

separate the indirect from the direct effects. They may be equally contributory, or one may be primarily contributory. It is therefore essential to consider this also before attempting to interpret data obtained from patients

either abusing drugs or receiving a drug in treatment.

207
NEED FOR CLINICAL STUDIES OF IMMUNE FUNCTION

RELATIVE TO DRUG ABUSE AND ITS TREATMENT

Clinical studies of immune dysfunction in humans with drug abuse or addictions of a variety of types, with and without AIDS, and also in humans in

treatment for drug abuse or addiction and receiving a variety of therapeutic

pharmacological agents, need to be carried out. One of the most important

things to be remembered in study design and in data interpretation is that

one must define precisely who the study subjects are and what drug is

being abused or being administered in treatment. The length of time of

abuse or treatment and the doses of drug received as well as the schedule

upon which drugs are received are also important factors to be evaluated.

We need to conduct studies in humans, because humans arc significantly

different from even the closest primates and certainly profoundly different

from rodents with respect to neuroendocrinc function, certain specific

immune indices, and the pharmacokinetics of many of the drugs which

should be studied. In vitro studies using both human and animal material

and also studies using animal models, preferably in many species, must be

carried out. Clinical studies, however, must ultimately be performed.

These studies must be as well controlled as possible. When possible, it is

very important to have healthy control subjects or contrast groups to answer

specific questions. If active street addicts or abusers of specific drugs are

to be studied, certain questions should be asked repeatedly, so that they

may be answered very carefully and as honestly and correctly as possible,

including the length of time of drug abuse or addiction prior to treatment;

the types of drugs abused; the route of administration and the, approximate

doses of the drug used; the length of time in treatment; the type of treatment being receivcd, including the drug or drugs being used in treatment;

the doses of drugs being administered; and the dosing schedule by which

these treatment agents arc administered. Also this verbal data. especially

with regard to any possible continuing drug abuse, should be confirmed by

urine monitoring. Thc medical status and the laboratory status of the potential study subject must be well defined. with a careful definition of liver

disease, including the type and relative severity thereof, and the status with

respect to HIV infection as well as other indices. The available clinical

literature of immune function in drug abusers since the onset of the AIDS

epidemic is extraordinarily deficient in this regard. Therefore. rather than

presenting the diffuse, often uninterpretable literature, studies will be

discussed in which a few of these variables have been controlled.

POST-AIDS IMMUNE FUNCTION IN HEROIN ADDICTS:

RELATIONSHIP TO CHRONIC LIVER DISEASE

In a study reported in 1985, Des Jarlais examined both street drug abusers

who were HIV negative and those who were HIV positive by ELISA testing and Western blot confirmation (Des Jarlais et al. 1985). He found that

there were highly significant differences between the anti-HIV-negative and

208
anti-HIV-positive subjects with respect to the absolute numbers of cells of

the immune system. Total lymphocytes, total numbers of T cells, and total

numbers of B cells were all significantly reduced in the anti-HIV-positive

group as compared with the anti-HIV-negative parenteral drug abusers; Tcell subsets were also altered, with a significant reduction in T-helper cells

in the anti-HIV-positive group, coupled with an increase in T-suppressor

cells and a resultant reduced T4:T8 ratio in the anti-HIV-positive group; this

reduction in T4:T8 ratio was greater than the minimal reduction in T4:T8

ratio in the anti-HIV-negative group (lower limit of normal T4:T8 ratio for

the laboratory used was 1.5) (Des Jarlais et al. 1985). Similarly, Des

Jarlais found that IgG was increased in the anti-HIV-positive group to an

extent significantly greater than the already significantly elevated and

abnormal levels observed in the parenteral drug abusers who were anti-HIV

negative (Des Jarlais et al. 1985).

NK cells are the first line of defense in humans against many viral infections and tumor cells. NK cells can be studied immediately in the laboratory, that is, on the same day they are obtained from study subjects, as

opposed to studying them after several days of incubation and manipulations

that may alter the activity or responsivity from time of acquisition of the

cells. We concentrated on studies of NK-cell cytotoxicity activity after

drugs had been administered in vivo and in vitro. At least two other groups

reported that NK-cell cytotoxicity activity is significantly reduced in street

heroin addicts; we have recently confirmed this (Nair et al. 1986; Novick

et al. 1989a; Poli et al. 1985). There is a question whether significant

lowering of NK-cell activity in heroin addicts is a direct drug effect, an

indirect effect, or an epiphenomenon related to diverse injected foreign substances or to exposure to a variety of diseases, such as hepatitis B, hepatitis

delta, undetected HIV infection, or other infections to which heroin addicts

are exposed by use of unsterile needles and self-injection paraphernalia.

The sexual preference of the heroin addict also must be taken into account.

We have been performing studies in a variety of different populations to

address these questions. The first was carried out by Novick and colleagues

in England (Novick et al. 1986a). This was a study of 53 heterosexual and

homosexual males, all of whom had hepatitis B infection and all of whom

were otherwise healthy except for their chronic liver disease; presumably

none had HIV infection, given the time when the study was performed

(Novick et al. 1986a). NK-cell activity was significantly lower in the

homosexual population, independent of hepatitis B virus infection (Novick

et al. 1986a).

In a second study, also carried out in England, NK-cell activity was determined in patients with liver disease of a variety of types and degrees of

severity (Dunk et al. 1987). NK-cell activity was found to be reduced in

patients with cirrhosis and even more reduced in patients with hepatocellular

carcinoma (Dunk et al. 1987). However, NK cytotoxicity activity was not

209
reduced in patients with mild chronic liver disease (Dunk et al. 1987). This

is of considerable importance for immunological studies of NK-cell activity

in parenteral drug abusers or former addicts in treatment; more than 60 percent of New York addicts in treatment have liver disease of a postviral or

postalcoholic type, yet a very low percentage, less than 10 percent, have

actual cirrhosis (Kreek 1972; Kreek 1973a; Kreek 1978; Kreek 1981; Kreek

et al. 1972; Kreek et al. 1986; Novick et al. 1985; Novick et al. 1986b).

Therefore, NK-cell activity is not apt to be compromised by the mild

chronic liver disease that is present in most study patients and indeed most

former heroin addicts in treatment or active heroin addicts.

POST-AIDS STATUS OF IMMUNE FUNCTION IN METHADONEMAINTAINED FORMER ADDICTS: RELATIONSHIP TO TIME

AND STABILIZATION IN TREATMENT

We hypothesized that whereas lowered NK-cell cytotoxic activity has been

documented in heroin addicts and may be due to one or more of the causes

enumerated above, in former heroin addicts receiving steady-state, stabledose, long-term methadone-maintenance treatment, in the absence of or in

the presence of otherwise mild chronic liver disease and even possibly in

the presence of sporadic use but not regular abuse of another drug or

alcohol, normalization of NK-cell activity may occur (Nair et al. 1986;

Novick et al. 1989a; Ochshom et al. 1989; Poli et al. 1985).

Standard techniques are used to measure NK-cell cytotoxic activity (Novick

et al. 1986a; Novick et al. 1989a; Ochshorn et al. 1989). Initial titrations

were done to determine the optimal effector:target ratio with this group; a

ratio of 100:1 was selected for many of these studies (Novick et al. 1989a;

Ochshom ct al. 1988; Ochshom et al. 1989). The NK-cell cytotoxicity

assay has been modified and standardized over the past few years; there is

now an intra-assay coefficient of variation of 6.9 percent and intraindividual

interassay coefficient of variation of 14.5 percent (Ochshom et al. 1988;

Ochshom et al. 1989). We have controlled many factors, including the time

of day at which the bloods are drawn. Although it is true that possible

circadian rhythm of NK-cell activity in humans has not been well studied;

however, because of the well-established neuroendocrine circadian rhythmicity, especially of the HPA axis, all our bloods for NK-cell activity are

drawn in the early morning hours, when levels of beta-endorphin, ACTH,

and cortisol are the highest. Patients are placed at rest for at least 30 minutes before blood is drawn, because exercise may also change NK-cell

activity or actual killer cell number, by redistributing cells into the central

circulating pool.

In an initial clinical study, 34 unselected patients in one methadonemaintenance treatment program, which also has abstinence base treatment

for other chemical dependencies, were studied (Ochshom et al. 1989). They

were unselected with respect to the presence or absence of cocaine abuse,

210
alcohol abuse, or any other kind of drug abuse, and they were unselected

with respect to the length of time in treatment or dose of methadone being

administered. The only patients excluded from the study were patients

documented to be hepatitis B antigen carriers, because immune dysfunction

may be pronounced in such patients. However, patients with all other types

and degrees of severe chronic liver disease were included. We found that

in the unselected methadone-maintained clinic population studied, 18 patients

(53 percent) had normal NK-cell activity; 7 had modestly lowered NK-cell

activity, 2 to 3 standard deviations below normal; and 9 had a more pronounced lowering of NK-cell activity greater than 3 standard deviations

below normal (Ochshorn et al. 1989). Thus, more than one-half of the unselected methadone-maintenance patients had normal NK activity, and by

extrapolation from these studies in heroin addicts, normalized NK-cell

activity occurred. In this particular clinic population, where anti-HIV testing has not and cannot be carried out routinely because of staff and patient

fears of the possibility of unintentional breach of confidentiality and also

because of a lack of sufficient numbers of well-qualified staff to do the

counseling needed to prevent any adverse behavioral consequences of routine or mass testing of anti-HIV testing, it is not known whether or not

some of the patients with very low NK-cell activity may be anti-HIV positive. Some patients have been tested for anti-HIV; although this is only a

small percentage of the total study group, all of these patients showed

abnormal lowered NK-cell-activity levels. Therefore, in this particular

study, possible HIV infection, which is known to reduce NK-cell activity,

cannot be ruled out as a cause for the observed significant reduction in NK

activity. This underscores the problem of carrying out immunological

studies in parenteral drug abusers at this time. Certainly for this particular

study, a reanalysis of data after anti-HIV testing, if and when that is done,

will be of great interest. We are also obtaining absolute cell numbers of

total lymphocytes, B cells, monocytes, NK cells, T cells, and T-cell subsets

in these patients in collaboration with Dr. Neal Flomenberg of the Memorial

Hospital-Sloan Kettering Medical Center, New York. In analysis of these

data of cell numbers determined to date, it is intriguing that more than

50 percent of the unselected methadone-maintained subjects have normal

T4:T8 ratios. Further studies and analyses of data are currently in progress.

A second clinical study performed by Novick, Ochshom, and me has been

completed very recently (Novick et al. 1989a). This study was performed

using carefully selected study subjects, as contrasted to the clinical study

previously described (Novick et al. 1989a; Ochshom et al. 1989). In this

study, there were three groups of subjects: (1) active heroin addicts, without cocaine abuse and without significant alcohol abuse and with negative

tests for HIV infection; (2) former heroin addicts who are now very longterm methadone-maintained patients, all in treatment for 11 years or more

and receiving a methadone dose of more than 60 mg per day; and

(3) otherwise healthy, nondrug-abusing, control subjects. NK-cell cytotoxic

activity was significantly reduced in the active heroin addict group.

211
Howevcr, in the long-term methadone-maintained patients, NK-cell cytotoxicity activity was normal, indistinguishable from the normal control

subjects (Novick et al. 1989a). Analyses of additional data collected from

the three groups show that several other specific indices of immune function

have normalized in the long-term methadone-maintained group, as contrasted

with the heroin addict group, in which many abnormalities were observed

(Novick et al. 1989a).

IN VITRO STUDIES OF EFFECTS OF OPIOID AGONISTS AND

ANTAGONISTS ON IMMUNE FUNCTION

The literature on the direct opiate drug effects in vitro on NK activity and

on the direct effects of endogenous opioid ligands on NK-cell activity is full

of conflicting and sometimes contradictory reports, as is, in fact, all of the

immune literature with respect to the endogenous and exogenous opioids on

specific indices of immune function (Kreek 1989). There are reports suggesting that peptide ligands from each of the three classes of endogenous

opioids may increase, have no effect on, or occasionally decrease NK-cell

activity.

In a series of laboratory studies, we investigated whether NK cells are normally controlled or significantly modulated by the cndogenous opioids and

if so, whether this modulation would be reversed or altered by adding a

specific opioid antagonist in vitro, thus removing any endogenous opioids

bound to specific or nonspecific classical or nonclassical opiate receptors on

NK cells. In the first set of in vitro studies, the commercially available and

clinically used active (1;-)-enantiomer of naloxone was added in increasing

concentrations to the buffers used in assays for NK-cell activity (Ochshorn

et al. 1988). Cells from otherwise normal healthy subjects and cells from

long-term methadone-maintained patients were used in these studies. There

was no significant effect of the active naloxone enantiomer on NK-cell

activity until a concentration of greater than 10-4 M was reached (Ochshorn

et al. 1988). These concentrations were greater than any achieved in a

therapeutic setting of use of an opiate antagonist and could be considered to

be in “toxicological“ ranges. They also were greater than the amounts required to remove cndogenous opioids bound to mu, delta, and kappa opiate

receptor subtypes. We then carried out precisely the same in vitro studies

using the inactive (d;+)enantiomcr of naloxone and made exactly the same

findings, that is, there were no effects on NK-cell activity until concentrations of greater than 10-4 M were reached, and at that point the inactive

enantiomer, like the active enantiomer, significantly reduced NK-cell activity

(Ochshorn et al. 1988). Examination of the cells at the end of these studies

showed that integrity of the cell membranes had been maintained. These

findings suggest that although there may be a physical chemical event related to binding or attachment of either the active or inactive enantiomer of

naloxone to NK cells, thus altering their activity when very high concentrations of antagonists are added, this, by definition, cannot be due to

212
“classical“ opiate receptor binding, since the altered activity, that is,

reduction of NK-cell activity, occurred only at extremely high concentrations, beyond those required for naloxone to act at kappa and delta receptor

subtypes as well as at the receptor subtype of its preference, the mu

receptor subtype. The same effects were observed when high concentrations

of inactive enantiomer were used as when the opiate receptor active enantiomer of naloxone was used. Thus any residual endogenous opioids, bound

to any specific subtype of opiate receptors, would have been removed by

much lower concentrations of active antagonist. Using cells obtained from

methadone-maintained patients, we made exactly the same findings; any

specifically bound exogenous opioid would have been removed by the low

concentrations of active naloxone, which had no effect on NK-cell activity

in vitro.

In a second set of in vitro studies, we now are studying the effects of the

active (R;l;-) and the inactive (S;d;+)enantiomers of methadone NK-cell

activity (Kreek 1989). Methadone as it is delivered clinically is the racemic

mixture of equal amounts of these two enantiomers. To date, the data

resulting from these studies, in which methadone has been added in vitro to

cells from otherwise healthy persons as well as from methadone-maintained

patients, have been essentially superimposable on the data obtained using

the opioid antagonist naloxone in similar in vitro studies (Kreek 1989). No

changes of NK-cell activity, neithcr enhancement nor reduction of activity,

are found until methadone concentrations of 10-4 M or greater are reached;

at concentrations of 10-4 M and greater, a significant reduction of NK-cell

activity is seen with both enantiomers of methadone. These studies suggest

that neither endogenous opioids nor exogenous opioid antagonists or agonists cause direct effects on NK-cell activity until very high concentrations of

drugs are used. These studies further suggest that these effects, observed at

high concentrations. of potential opioid receptor ligands are not directly

linked with any binding to any opioid receptors that may or may not exist

on NK cells. Although these very high concentrations of drugs could be

considered to cause a toxic effect, such as altering cell-membrane integrity,

trypan-blue exclusion by cells remained normal to the end of the study.

Therefore the integrity of membranes was Preserved throughout all of these

agonist as well as antagonist studies. More subtle alterations in membranes

may be altering NK-cell activity at the high concentrations used in vitro.

SUMMARY

These studies suggest that specific opiate receptors are not involved significantly in modulating NK-cell activity by any direct effect. The role normally played by the endogenous opioids in directly modulating NK-cell

cytotoxic activity may be minimal and certainly is not a clinically relevant

controlling factor. These findings also suggest that the repeatedly observed

lowering of NK-cell activity in untreated heroin addicts is not due to a

direct drug effect. It may be due, however, to an indirect drug effect,

213
possibly by way of altering neuroendocrine function, which we and others

have shown predictably occurs during cycles of heroin addiction, and which,

as discussed above, has been shown to become normalized during

steady-dose, long-term, methadone-maintenance treatment. The lowering of

NK-cell activity in heroin addicts may also be due primarily to the use of

unsterile needles, with exposure to and infection with multiple diseases as

well as injection of many foreign substances. Clinical studies of all the

important immunological indices will have to be carried out in wellcharacterized human populations, including normal healthy control subjects,

drug abusers, drug addicts, and former drug addicts in defined treatment

status, before the actual roles of drugs of abuse or drugs used to treat drug

abuse in immune function in humans will be fully understood.

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AUTHOR

Mary Jeanne Kreek, M.D.

Associate Professor and Physician

Laboratory of Biology of Addictive Diseases

The Rockefeller University

1230 York Avenue

New York, NY 10021-6399

219
Opioid Peptides, Receptors, and

Immune Function

Hemendra N. Bhargava

INTRODUCTION

Thc abuse of drugs in the United States has reached epidemic proportion.

Associated with it are the problems of bacterial, fungal, and viral infections.

It is believed that patients consuming drugs of abuse are at a much higher

risk of contracting AIDS, because drugs of abuse are immunosuppressants

(Coleman and Curtis 1988; Farci et al. 1989; Kaplan 1989; Martin and

Schoub 1989; Stimson et a1.1988). In this chapter, the effects of chronic

administration of opioid agents, particularly morphine and heroin, on clinical

complications and biochemical markers of immune function are described.

Evidence is provided for the presence of opioids in peripheral circulation

(blood and its components) and the existence of opioid receptors on lymphocytes and phagocytes. The effects of opioid peptides, -endorphin, and

methionine-enkephalin, on several in vitro parameters of immune functions

are described. Finally, preliminary findings on the effects of methionine-enkephalin on biochemical markers of immune function and on clinical symptoms in the acquired immunodeficiency syndrome (AIDS)-related complex

(ARC) and AIDS patients are described.

DRUG ADDICTION AND IMMUNE FUNCTION

The modification of immune function by subjects taking drugs of abuse has

received considerable attention in recent years. Although it has been recognized for some time that patients using drugs of abuse like morphine or

heroin. alcohol, and marijuana have depressed immune function (Watson

ct al. 1985; Lundy et al. 1975; Patel et al. 1985) leading to increased risk

of infection, the possible mechanisms by which these effects are produced

have been studied only recently. Since opiates like morphine and heroin

arc self-administered by addicts using intravenous injections, sharing unsterilized needles might be one of the ways the micro-organisms enter the

blood stream and produce infections. The other possibility is that drugs of

abuse like heroin may have a direct action on the immune function of the

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host. For almost half a century, clinical observations of heroin addicts have

shown that these subjects have increased incidence of bacterial, protozoal,

and viral infections. They include bacterial endocarditis (Luttgens 1949;

Hussey et al. 1944; Olsson and Romansky 1962), septicemia (Briggs et al.

1967; Hussey et al. 1944; Cherubin and Brown 1968), pulmonary infarction,

tetanus, hepatitis, malaria, thrombophlebitis, and skin sepsis (Hussey and

Katz 1950; Louria et al. 1967).

Both clinical and preclinical studies suggest that chronic morphine treatment

depresses the immune function, which leads to a higher incidence of infections. Various types of infections in 82 opiate addicts were summarized in

a recent study by Lazzarin et al. (1984). The major infections included

viral hepatitis, recurrent dental abscesses, subcutaneous abscesses, phlebitis,

lues, and respiratory infections.

The factors that can contribute to the immunological abnormalities of heroin

addicts are: (1) drugs and/or drug-contaminant-induced defects in both cellular and/or humoral immunity; (2) needle contamination causing viral infections, e.g., hepatitis disturbs cellular and humoral immune mechanisms; and

(3) the abuse of other drugs, such as alcohol and marijuana, which are also

known to affect the immune function (Lundy et al. 1975; Patel et al. 1985).

THE NORMAL IMMUNE RESPONSE

A brief description of the role of blood cells in the regulation of immune

function will be given here. Cellular immunity, which is controlled or

regulated by white blood cells, falls into two classes: T cells, which

originate in the bone marrow but complete their maturation process in the

thymus gland, and monocytes or macrophages. B cells are the source of

antibodies, the proteins that bind to antigens and help in their removal or

destruction. T cells are more complex in classification and function. In

general, T cells cannot recognize free antigen circulating in blood or lymph,

and can respond to antigen on a cell surface only under certain conditions.

After recognizing antigen, the T lymphocytes, the cytotoxic T cells, actively

defend the body by killing infected, foreign, or malignant cells by destroying their cell membrane. There are other kinds of T cells that modulate the

immune response by secreting messenger proteins. They include: inducer

T cells, which facilitate the maturation of T lymphocytes from precursor

forms and the action of other T cells; helper T cells, which facilitate

B cells in secreting antibody; and suppressor T cells, which dampen the

immune response of B and T cells. Thus, helper and suppressor cells act

in opposite directions. Based on the biochemical markers on their surface,

the T cells have been classified primarily into two groups: CD4 cells,

which function as helpers and inducers, and CD8 cells, which have suppressor and cytotoxic function. The other contributors in the immune response

are the macrophages and natural killer cells. These are scavenger cells that

develop from monocytes found in the skin and other tissues and act by

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engulfing an organism, breaking down its proteins and displaying the antigenic protein fragment on the cell membrane. Macrophages prepare the

antigen for recognition by CD4 cells. Natural killer cells destroy virusinfected cells and tumor cells spontaneously without the involvement of

lymphocytes or recognizing antigens.

In response to an infection, such as a viral infection, virus-infected cells

secrete proteins known as interferons which stimulate the natural killerkiller cells. Macrophages also secrete soluble proteins called monokines,

such as gamma-interferon and interleukin-1. The latter activates T cells that

have recognized the viral antigen on a cell surface, preparing them to diffcrentiate and divide. Activated T cells produce soluble factors, which are

called lymphokines. CD4 and CD8 cells secrete interleukin-2, and stimulate

proliferation into cytotoxic suppressor and helper T cells. Cytotoxic T cells

lyse virus-infected cells, which are then removed from the body.

MORPHINE OR HEROIN ADDICTION AND IMMUNE FUNCTION

Returning to the effects of chronic morphine treatment on the immune function in addicts, cellular immunity was found to be compromised by functional deficits of polymorphonuclear leukocytes and T lymphocytes

(Lazzarin et al. 1984). Immunological function in heroin addicts was

studied in 38 patients. The immunological abnormality was evidenced by

hypergammaglobulinemia, with higher than normal levels of IgM and IgG,

and false positive test for syphilis. Cellular immunity was also found to be

impaired, as evidenced by decreased responsiveness of lymphocytes to

milogen-induced blastogenesis (Brown et al. 1974). McDonough et al.

(1980) also reported a decrease in absolute number of total T lymphocytes

in peripheral blood of opioid addicts as measured by the ability of lymphocyte to rosette sheep red blood cells (and an increase in null lymphocyte),

but no significant change in B lymphocytes or total white blood cell count

was noted. Naloxone was able to reverse these changes. Naloxone not

only increased T cell percentage, it also improved the appearance of the

E-rosettes. The lymphocytes of addicted patients also had decreased the

ability of phytohemagglutin to induce stimulation of 3H-thymidine incorporation into the DNA. Morphine addicts were also shown to exhibit a severe

depression of phagocytosis and killing properties of polymorphonuclear

leukocytes and monocytes and their ability to generate superoxide anion

(Tubaro et al. 1985).

IN VITRO EFFECTS OF MORPHINE

In Vitro, morphine inhibits T cell E-rosetting (Donahoe et al. 1985). Some

controversial results have been obtained on the in vitro effects of morphine

on T, B, and null cells. Thus, Bocchini et al. (1983) reported that at

107 M concentration of morphine and naloxone, the percentage of T cells

was increased, that of null cells decreased, and there was no change in B

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cells. This was accompanied by an enhanced effect of phytohemagglutininstimulated lymphocyte response. It is possible that these effects are dose

related, since at high concentration (10-3 M), morphine inhibits the lymphocyte response to phytohemagglutinin.

EFFECT OF MORPHINE ADDICTION ON IMMUNE FUNCTION IN

ANIMALS

Several studies have been carried out in animals in an effort to understand

more precisely the mechanisms by which opioids, particularly morphine,

modulate the immune response. Chronic administration of morphine to

mice decreased the responsiveness of lymphocytes to concanavalin A stimulation when compared to lymphocytes of nonaddicted mice. This suppressive effect was reversed partially by concurrent administration of naloxonc

(Ho and Leung 1979). In morphine-addicted mice, the primary immune

response was inhibited, as evidenced by decreases in the spleen to body

weight ratio and serum hemolysin production against sheep red blood cells.

These effects were naloxone reversible (Gungor et al. 1980). Chronic treatment of mice for 3 days with morphine (75 mg/kg/day) produced immunosuppression, as shown by using a plaque-forming cell assay to evaluate

antibody-forming efficiency (Lefkowitz and Chiang 1975) and a rosetteforming cell test (Lefkowitz and Nemeth 1976). In guinea pigs, chronic

treatment with morphine affected the lymphocyte metabolism of cyclic

nucleotides and lymphocytic responsiveness to concanavalin A stimulation

(Law et al. 1978). Many of the above morphine-induced immunosupprcssive effects were either not reversed or partially reversed by naloxone.

Chronic treatment of mice with morphine also resulted in decreased resistance to bacterial and fungal infections. Infections due to Klebsiella pneumoniae and Candida albicans were enhanced in chronic morphine-treated

mice (Tubaro et al. 1983). Morphine decreased reticuloendothelial system

activity, phagocyte count, natural cell activity killer and superoxide anion

formation in polymorphonuclear leukocytes and macrophages. Additionally,

morphine induced a reduction of lymphoid organ weight (Tubaro et al.

1983).

EVIDENCE FOR THE PRESENCE OF OPIOID BINDING SITES ON

CELLS CONTROLLING IMMUNE FUNCTION

As discussed above, since morphine causes immunosuppression in both animals and humans, and these effects are at least partially reversed by the

opioid antagonist naloxone, it is suggested that opioid receptors may be

involved in the immunomodulation. Since the discovery of opioid receptors

in 1975, to date at least five major subtypes of opioid receptors have been

postulated. They include μ-(preferring morphine), -(preferring enkephalins), -(preferring ethylketocyclazocine or dynorphins), -(preferring

N-allylnormetazocine, SKF 10,047), and -(preferring -endorphin). With

223
the discovery of opioid receptors in mammalian brain and other tissue, a

search was started to isolate the endogenous ligands for the receptors, which

resulted in the isolation of a family of peptides having opioidlike activity.

Three major classes of compounds were enkephalins (methionine and leucine), dynorphin(1-13), and -endorphin. Endogenous opioids have been

implicated in a number of physiological and pathophysiological states

involving neural and hormonal actions (Morley et al. 1987). It is thus possible that the endogenous opioids are also involved in the modulation of the

immune function via the autonomic nervous system, the endocrine system,

and the central nervous system. As noted earlier, T lymphocytes are the

major cells in immune function. The first indirect evidence for the

presence of opioid receptors on human T lymphocytes was provided by the

studies of Wybran et al. (1979) who showed that morphine inhibited and

methionine-enkephalin increased the percentage of active T rosettes. This

effect was antagonized by naloxone but not by the inactive levomoramide,

suggesting the presence of specific opioid receptors on T lymphocytes.

Binding sites for 3H-leucine enkephalin on cultured human T lymphocytes

were present, but the binding of the ligand could not be displaced or

inhibited by naloxone (Ausiello and Roda 1984). The binding sites for

3H-naloxone were deteted on lymphocytes and platelets from peripheral

blood of healthy human volunteers. The binding was displaced by unlabeled naloxone and morphine (Mehrishi and Mills 1983). Cultured human

lymphocytes were shown to have specific binding sites for 125I-h-[D-Ala2]-

ß-endorphin. The binding was not inhibited by opiate agonists and antagonists or by enkephalin analogs, but was inhibited by ß-endorphin or its

analogs (Hazum et al. 1979). These studies demonstrated the presence of

binding sites for ß-endorphin on lymphocytes that were specific for

ß-endorphin but were nonopioid in nature. Similar binding sites have been

found on the surface of mouse thymoma cells (Schweigerer et al. 1985).

In vitro studies using phytohemagglutinin-stimulated human peripheral blood

lymphocytes demonstrated the binding of 3H-methionine-enkephalin. and this

could be displaced by unlabeled methionine-enkephalin but not with

200 μM naloxone (Plotnikoff et al., personal communication).

The -opiate receptor ligand, cis(+)-3-methylfentanyl-isothiocyanate

(SUPERFIT), was found to label a protein from both B- and T-cell enriched

murine splenocytes peripheral blood lymphocytes and human peripheral

blood lymphocytes (Carr et al. 1988).

EVIDENCE OF THE PRESENCE OF INTACT OPIOID PEPTIDES

IN PERIPHERAL BLOOD

ß-Endorphinlike material is present in the serum of rats and, upon stress, it

is apparently released from the pituitary into the blood (Guillemin et al.

1977). Although ß-endorphin is present in low concentration in human

serum, appreciable concentrations have been found in plasma of patients

with certain endocrine disorders (Suda et al. 1978). Methionine-enkephalin

224
is also present in the human plasma in the intact form in the amount of

55 pg/ml and is possibly secreted by the adrenal medulla (Clement-Jones

et al. 1980). Furthermore, the plasma levels of methionine-enkephalin are

unrelated to the levels of endogenous adrenocorticotropic hormone (ACTH),

-lipotropin (ß-LPH), and ß-endorphin, which suggests that methionineenkephalin is derived from its own separate precursor distinct from ACTH,

-LPH, and ß-endorphin (Clement-Jones et al. 1980). Finally, human leukocyte interferon (hIFN- ), which is a group of proteins with antiviral

activity, contains recognizable endorphin and ACTH-like activities (Smith

and Blalock 1981).

EFFECT OF SOME OPIOID PEPTIDES ON IMMUNE FUNCTION

Several studies have been carried out with opioid peptides, particularly

ß-endorphin and methionine-enkephalin in animals, humans, and in vitro, to

understand their possible role in immune function. Although the concentration of circulating ß-endorphin is very low in the rat, it is increased

severalfold following acute stress, producing plasma ß-endorphin concentration to almost 10 ng/ml (Guillemin et al. 1977). The possible relationship

between stress, immune function, and endorphins has been reviewed (Amir

et al. 1980). The effect of -endorphin, ß-endorphin, and D-Ala2-Met5-

enkephalin to modulate the proliferative responses of splenic lymphocytes to

mitogenic stimulation was measured. The results indicated that of the three

peptides, only ß-endorphin potentiated concanavalin A, and phytohemagglutinin-induced proliferation of T lymphocytes, but had no effect on the

response to the B cell mitogen lipopolysaccharide/dextran sulfate (Gilman

et al. 1982). The potentiating effect of ß-endorphin was not reversed by

naloxone, which suggests that the effect may be mediated by a nonopioid

but ß-endorphin-specific mechanism. On the other hand, McCain et al.

(1982) reported that ß-endorphin is a potent and efficacious suppressor of

phytohemagglutinin-induced blastogenesis in cultured human T lymphocytes

that could not be antagonized by naloxone. The reduction in lymphocyte

reactivity induced by ß-endorphin did not appear to be due to cytotoxicity,

since the lactic dehydrogenase activity of the supernatant fluid, an indicator

of cell lysis, was not affected. The differences in the studies of Gilman

et al. (1982) and McCain et al. (1982) were that in the former spleenocytes

were used, whereas in the latter cultured human leukocytes were used. The

influence of ß-endorphin on lymphocyte function has been demonstrated to

be dependent on the donor used. The ß-endorphin-induced inhibition of

lymphocyte proliferation appeared to be due to fragment ß-endorphin10-16

amino acid sequence, and the effect is probably mediated by interference in

the mobilization of intracellular calcium. ß-Endorphin and methionineenkephalin stimulate chemotaxis of human blood mononuclear cells, an

effect which is antagonized by opiate antagonist naloxone (van Epps and

Saland 1984), indicating that such an effect is mediated via endogenous

opioids. Similar response has also been shown for neutrophils. ß-Endorphin at very low concentration (10-14 M) and methionine-enkephalin (10-9 M)

225
have been shown to enhance the activity of natural killer cells from peripheral human blood, and this effect was blocked by naloxone (Matthews et al.

1983). Thus endogenous opioids modify immune function via both opioid

and nonopioid receptors. The opioid receptor-mediated effects include

increased natural killer cell activity, increased a-interferon and interleukin-2

production, release of histamine from mast cells. enhancement of chemotaxis, and enhancement of T-cell subsets. The effects, which do not appear

to involve opioid receptors, include modification of phytohemagglutininstimulated proliferation, superoxide production, and binding to terminal complexes of complement (SCSB-9 and CSB-9).

EVIDENCE FOR THE PRESENCE OF METHIONINE-ENKEPHALIN

IN PERIPHERAL BLOOD COMPARTMENTS

Methionine-enkephalin is a peptide (Tyr-Gly-Gly-Phe-Met) that has been

found in a variety of tissues, including brain, spinal cord, and gastrointestinal tract of several species (Simantov et al. 1977; Yang et al. 1977;

Wesche et al. 1977; Gros et al. 1977; Hughes et al. 1977; Miller et al.

1978). It has also been detected in human gut, brain, and cerebrospinal

fluid (Polak et al. 1977; Gramsch et al. 1979; Akil et al. 1978).

It has been indicated earlier that methionine-enkephalin. an endogenous

opioid peptide, circulates as intact pentapeptide in human plasma (ClementJones et al. 1980). Although methionine-enkephalin is degraded rapidly by

tissue enkephalinases and aminopeptideases, making its half-life extremely

short (Roda et al. 1986), the presence of intact peptides in the circulation

raises the question of whether there are protective mechanisms present in

the peripheral blood. If so, what are their distribution patterns and what arc

their physiological roles? Picogram quantities of immunoreactive

methionine-enkephalin were detected in human, rat, and rabbit platelets.

The platelets’ methionine-enkephalin concentration in the Sprague-Dawlcy

rats was not affected by either adrenalectomy or by hypophysectomy

(DiGiulio et al. 1982). However, plasma concentrations of methioninecnkephalin were increased after adrenalectomy in both normal and

hypophysectomized rats (Panerai 1988). Therefore, the origin of

methionine-enkephalin in blood is still not certain. The distribution and uptake of methionine-enkephalin into human blood cells have also been

studied. Blood obtained from normal volunteers was separated into red

blood cells (RBCs), white blood cells (WBCs), and platelets, and

methionine-enkephalin content of cells was determined by radioimmunoassay. On a per cell basis, WBCs were found to contain about 100-fold

greater amounts of methionine-enkephalin than either RBCs or platelets.

Similarly, the uptake of 3H-methionine-enkephalin was far greater in WBCs

than in RBCs or platelets. These studies suggest that WBCs may act as a

compartment of distribution for methionine-enkephalin in blood (Valentine

et al. 1988).

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Effects of Methionine-Enkephalin on Immune Function: In Vitro and

Animal Studies

Wybran et al. (1979) provided suggestive evidence for the presence of

methionine-enkephalin receptors on human blood T lymphocytes. Human

T lymphocytes are easily recognized by their specific ability to form

rosettes with sheep red blood cells (SRBC). In vitro, methionine-enkephalin

increased the percentage of active T rosettes, an effect that was inhibited by

naloxone (Wybran et al. 1979). Subsequently, the presence of opioid receptors on human phagocytic leukocytes was reported (Lopker et al. 1980).

The distribution and presence of methionine-enkephalin receptors on cells

involved in immune function prompted further studies on the possible role

of methionine-enkephalin in the host defense mechanisms. Plotnikoff’s

group has shown that the enkephalins increase phytohemagglutinin-induced

lymphocyte blastogenesis (Plotnikoff and Miller 1983) and prolong survival

of BDF, mice inoculated with an attenuated L1210 strain of tumor cells

(Plotnikoff and Miller 1983; Plotnikoff 1982). Methionine-enkephalin significantly increases the active T cell rosette-forming cells in peripheral

blood lymphocytes from lymphoma patients (Miller et al. 1983). EnkephaLins also stimulate at low doses and inhibit at high doses T-dependent antibody responses in vitro (Johnson et al. 1982). Both methionine- and

leucine-enkephalin significantly increased natural killer (NK) cell activity in

isolated human peripheral blood lymphocytes (Faith et al. 1984). NK cells

belong to the subpopulation of lymphocytes that play an important role in

host defense mechanisms against neoplastic disease.

Further evidence for the possible role of methionine-enkephalin in the

immune function was provided by the studies of Zurawski et al. (1986),

who demonstrated that mitogenic activation of mouse T-helper cells induces

preproenkephalin mRNA synthesis. A complementary DNA library prepared

from a cloned concanavalin A-activated mouse T-helper cell line was found

to encode preproenkephalin mRNA. The supematants from induced

T-helper cell cultures were found to have methionine-enkephalin immunoreactivity. These studies suggest that enkephalins like methionineenkephalin serve a function as neuroimmunomodulators via T-dependent

immune functions. With this background information, the effects of

methionine-enkephalin in patients with AIDS, ARC, and cancer were determined (Wybran et al. 1987).

Effects of Methionine-Enkephalin on the Immune Function of ARC and

AIDS Patients

AIDS is characterized by opportunistic infections and malignant diseases.

AIDS is associated with persistent lymphadenopathy and characteristic

immunological abnormalities with selective T-cell deficiency, persistent

decreased CD4/CD8 ratio, elevated serum immunoglobulins, decreased in

vitro lymphocyte proliferative responses, decreased NK cell activity and

227
cell-mediated cytotoxicity, and elevated serum levels of immune complexes.

The earlier and less severe form of AIDS is called ARC. Clinical symptoms include fever, fatigue, malaise, night sweats, weight loss, anorexia, oral

candidiasis, lymphadenopathy, depression, and diarrhea (Seligmann et al.

1984). The effect of methionine-enkephalin on the immunologic markers

and clinical symptoms of ARC patients was determined. Studies were conducted in seven patients with ARC. The patients received methionineenkephalin three times a week intravenously for a minimum of 21 days.

The doses ranged from 20 to 100 μg/kg at each injection. The immunologic results have been summarized in table 1.

TABLE 1. Immunological changes after 21 days of treatment in patients

with ARC and AIDS

Day 0 Day 21 P

Lymphocyte Count 1207±186 1129 ± 223 NS

OKT3 Percentage 68±3 77 ± 2 < 0.02

OKT4 Count 174 ± 4.5 271± 71 < 0.05

NK Activity (%) 19 ± 3 24 ± 2 < 0.10

IL-2 Production (U) 0.11 ± 0.05 0.8 ± 0.3 < 0.05

PHA Response (cpm 10”) 162 ± 30 261 ± 60 < 0.01

NOTE: The results are expressed as mean ±SEM. The p value is based on Student’s t test comparing

paired data on day 0 and day 21.

SOURCE :Wybran et al. 1987. copyright 1987, New York Academy of Sciences.

Following the treatment with methionine-enkephalin, there was a significant

increase in the number of blood OKT3 (CD3) T lymphocytes and OKT4

(CD4) (T helper cells) lymphocytes without an increase in the absolute

number of lymphocytes, significant increase in NK cell activity and

interleukin-2 (IL-2) production, and enhancement of the phytohemagglutinin

response. The symptoms like weight loss, night sweats, recurrent scrotal

infection, depression, malaise, fatigue, and swelling of the lymph nodes

regressed. The data clearly indicated that methionine-enkephalin can enhance some immunologic functions in ARC patients, which was also associated with some therapeutic benefits. Obviously, these studies need to be

extended to a larger population of ARC patients.

One AIDS patient, who received a single injection of 20 ug/kg of

methionine-enkephalin, had his phytohemagglutinin response increase from

4300 cpm to 14000 cpm. Another AIDS patient with Kaposi’s sarcoma,

who received chronic treatment with methionine-enkephalin, had his lesions

228
remain stable for 4 months. Thus, methionine-enkephalin administration

was associated with the regression of the Kaposi’s sarcoma.

SUMMARY AND CONCLUSIONS

The studies have clearly demonstrated that binding sites for opioid peptides,

-endorphin, and methionine-enkephalin exist on T lymphocytes. -Endorphin appears to be immunodepressant, whereas methionine-enkephalin is

immunostimulant. Both in vitro and in vivo studies have shown that

methionine-enkephalin can influence some immune functions. Since in vitro

modification of immune function requires very low concentrations, it is reasonable to believe that methionine-enkephalin plays a physiological role in

the immune system. Although not well established, methionine-enkephalin

appears to activate T lymphocytes via opioid receptors and triggers a series

of intracellular signals leading to the activation of receptors for interleukin-2

(IL-2), OKT10, and active sheep T red blood cell receptors. Methioninecnkephalin enhances the activity of NK cells and induces the production of

IL-2, which in turn may recruit and activate other T-cell subsets like CD3

and CD4. Methionine-enkephalin also enhances mitogen-induced proliferation of lymphocytes. Since preliminary studies with methionine-enkephalin

in ARC patients have provided beneficial effects by the improvements in

their symptoms, it will be worthwhile to extend these observations to a

larger number of patients with ARC and AIDS. Finally, it appears that

some endogenous opioid peptides and their analogs, in addition to

methionine-enkephalin, may provide therapeutic benefits not only in ARC

and AIDS but also in other immunodeficient states.

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AUTHOR

Hemendra N. Bhargava, Ph.D.

Professor of Pharmacology

Department of Pharmacodynamics

College of Pharmacy

The University of Illinois at Chicago

Health Sciences Center

Chicago, IL 60612

233
Summary of Recommendations for

Future Research

Louis Harris

The National Institute on Drug Abuse was established by law in 1974 as

one of three Institutes in the Alcohol, Drug Abuse, and Mental Health

Administration, a Public Health Service Agency within the Department of

Health and Human Services. Since its inception, the Institute has been the

lead Federal agency responsible for reducing the demand for illicit drugs.

Through the congressional reauthorization process, the Institute has been

directed to carry out its mission utilizing various mechanisms, such as

categorical service grants, block grant services, and a wide range of

intramural research projects and extramural research grants and contracts.

Currently, the National Institute on Drug Abuse is authorized as a research

institute, and I emphasize research, to study the causes and consequences of

drug abuse and ways of improving our ability to treat and prevent drug

abuse and to collect information on the incidence and prevalence of drug

abuse.

In recent years, the emergence of widespread abuse of cocaine and its

public health consequences have lent a greater immediacy to the mission of

the Institute and resulted in significant budgetary increases. However, as

Dr. Schuster pointed out. the realization that intravenous or parenteral abuse

of drugs is associated with nearly 30 percent of the reported cases of AIDS

has created an overwhelming need to attack the problem of drug abuse with

all the tools available to society. The main function of the Institute in this

regard is to provide the research base on which public health decisions and

policies can be made intelligently.

During the course of this technical review meeting, a number of distinguished scientists discussed the “Current Chemical and Pharmacological

Advances on Drugs of Abuse Which Alter Immune Function and Their

Impact Upon AIDS.” Many elegant synthetic and medicinal chemistry

studies of the opiates, cocaine, and the cannabinoids were presented. Those

present at the meeting also heard about the beginning of systematic studies

to determine whether and how the immune system may be affected by the

234
opioids and other drugs of abuse. Some common themes have emerged,

which have led to recommendations for future research and development.

1. There is a need for continued and expanded support for basic chemical.

biochemical, and pharmacological research. General suggestions include the following:

a. the need for highly selective agonists and antagonists for the

variety of opioid and other postulated receptors;

b. the preparation of labeled ligands and affinity labels for positron

emission tomography (PET) scanning and the binding and

isolation of receptors;

C. the determination of the physiological role of the purported

receptors;

d. the isolation and determination of the structure of the opioid receptor or receptors, which should obviously be expanded to include other postulated receptor types; and

e. expansion of our efforts to determine whether there are specific

receptors or binding sites for other drugs of abuse, such as the

cannabinoids, cocaine. the sedative hypnotics, and so on. Of particular need is the development of specific antagonists and other

molecular probes.

2. The exciting discovery that cells of the immune system have specific

opioid binding sites calls for expanded research in this area. A number of general recommendations have emerged:

a. The use of immunologic tools in the study and isolation of receptors for drugs of abuse should be expanded.

b. Careful and systematic studies of the effect of the opioids and

other classes of drugs of abuse on the immune system should be

expanded. This is particularly true for cocaine, the cannabinoids,

and the inhalants, such as the nitrites, which are associated with

populations particularly at risk for HIV infection.

c. There is a real need for careful studies to determine whether and

how the observed effects on the immune system of drugs of

abuse in animals and man are related to the spread of the HIV

virus and the occurrence of opportunistic diseases. This means

that drugs of abuse and the pharmacotherapeutic agents used for

treatment of dependence on these drugs should be tested for their

235
effects on the immune system in the disease state as welt as in

the normal.

. . 3 Finally, there are a number of specific recommendations. These include the following:

a. There is a great need for the pharmaceutical industry to devote

more attention to the development of new drugs for the treatment

of the addictive disorders. This should not be confined to tne

opioids but should be expanded to include the stimulants such as

cocaine, the hallucinogens such as PCP, and the abused central

depressants.

b. As a corollary to this, the current development of known possible

pharmacotherapeutic agents should be expedited. This includes

making (-) -acetylmethadol (LAAM) rapidly available; possibly

developing nor-LAAM as a replacement or backup; developing

buprenorphine; and producing new long-lasting antagonists or

depot preparations of known antagonists. In addition, pharmacotherapies for cocaine dependence should be rapidly developed. A

number of current “leads,” such as desipramine, calcium channel

blockers, and others, exist, and studies should be expanded to

determine their efficacy and safety so that they can be made

available to the therapeutic community as rapidly as possible.

C. There is a need for expanded animal and human testing facilities

to provide standardized data for feedback to the chemists to aid

in the design of new molecules. This should include immunological studies as well as dependence studies.

d. Efforts should be continued and expanded to make available to

interested investigators supplies of new substances that have

emerged from chemical and biochemical research. The availability of these new tools will accelerate research and lead to the

more rapid development of new and more effective treatments for

substance abuse.

e. A uniform data base should be developed by the National

Institute on Drug Abuse to relate chemical structure to

pharmacological and immunological findings. The data base

should be interactive and easily accessible.

AUTHOR

Louis S. Harris, Ph.D.

Research Technology Branch

National Institute on Drug Abuse

Parklawn Building, Room 10A-13

5600 Fishers Lane

Rockville, MD 20857

236
While limited supplies last, single copies of the monographs may be

obtained free of charge from the National Clearinghouse for Alcohol and

Drug Information (NCADI). Please contact NCADI also for information

about availability of coming issues and other publications of the National

Institute on Drug Abuse relevant to drug abuse research.

Additional copies may be purchased from the U.S. Government Printing

Office (GPO) and/or the National Technical Information Service (NTIS) as

indicated. NTIS prices arc for paper copy; add $3.00 handling charge for

each order. Microfiche copies arc also available from NTIS. Prices from

either source are subject to change.

Addresses are:

NCADI

National Clearinghouse for Alcohol and Drug Information

P.O. Box 2345

Rockville, MD 20852

GPO

Superintendent of Documents

U.S. Government Printing Office

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NTIS

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U.S. Department of Commerce

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(703) 487-450

For information on availability of National Institute on Drug Abuse

Research Monographs 1 through 24 (1975-1979) and others not listed

write to NIDA, Community and Professional Education Branch,

Room 10A-54, 5600 Fishers Lane, Rockville, MD 20857.

237
25 BEHAVIORAL ANALYSIS AND TREATMENT OF SUBSTANCE

ABUSE. Norman A. Krasnegor. Ph.D., ed.

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26 THE BEHAVIORAL ASPECTS OF SMOKING. Norman A. Krasnegor,

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Health.)

GPO out of stock NTIS PB #80-118755/AS $23

30 THEORIES ON DRUG ABUSE: SELECTED CONTEMPORARY

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31 MARIJUANA RESEARCH FINDINGS: 1980. Robert C Petersen, Ph.D.,

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32 GC/MS ASSAYS FOR ABUSED DRUGS IN BODY FLUIDS. Rodger L.

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36 NEW APPROACHES TO TREATMENT OF CHRONIC PAIN: A

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Lorenz K.Y. Ng, M.D., ed.

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37 BEHAVIORAL PHARMACOLOGY OF HUMAN DRUG DEPENDENCE.

Travis Thompson, Ph.D., and Chris E. Johanson, Ph.D., eds.

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38 DRUG ABUSE AND THE AMERICAN ADOLESCENT. Dan J. Lettieri,

Ph.D., and Jacqueline P. Ludford, M.S., eds. A RAUS Review Report.

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40 ADOLESCENT MARIJUANA ABUSERS AND THEIR FAMILIES.

Herbcrt Hendin, M.D.; Ann Pollinger, Ph.D.; Richard Ulman, Ph.D.; and

Arthur Carr, Ph.D., eds.

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42 THE ANALYSIS OF CANNABINOIDS IN BIOLOGICAL FLUIDS.

Richard L. Hawks, Ph.D., ed.

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238
44 MARIJUANA EFFECTS ON THE ENDOCRINE AND REPRODUCTIVE

SYSTEMS. Monique C. Braude, Ph.D., and Jacqueline P. Ludford, M.S., eds.

A RAUS Review Report.

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45 CONTEMPORARY RESEARCH IN PAIN AND ANALGESIA, 1983.

Roger M. Brown, Ph.D.; Theodore M. Pinkert, M.D., J.D.; and Jacqueline P.

Ludford, M.S., eds. A RAUS Review Report.

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46 BEHAVIORAL INTERVENTION TECHNIQUES IN DRUG ABUSE

TREATMENT. John Grabowski, Ph.D.; Maxine L. Stitzer, Ph.D.; and Jack E.

Henningfield, Ph.D., eds.

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47 PREVENTING ADOLESCENT DRUG ABUSE: INTERVENTION

STRATEGIES. Thomas J. Glynn, Ph.D.; Carl G. Leukefeld, D.S.W.; and

Jacqueline P. Ludford, M.S., eds. A RAUS Review Report.

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48 MEASUREMENT IN THE ANALYSIS AND TREATMENT OF

SMOKING BEHAVIOR. John Grabowski, Ph.D., and Catherine Bell, M.S.,

eds.

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50 COCAINE: PHARMACOLOGY, EFFECTS, AND TREATMENT OF

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51 DRUG ABUSE TREATMENT EVALUATION: STRATEGIES,

PROGRESS, AND PROSPECTS. Frank M. Tims, Ph.D., ed.

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52 TESTING DRUGS FOR PHYSICAL DEPENDENCE POTENTIAL AND

ABUSE LIABILITY. Joseph V. Brady, Ph.D., and Scott E. Lukas, Ph.D., eds.

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53 PHARMACOLOGICAL ADJUNCTS IN SMOKING CESSATION.

John Grabowski, Ph.D., and Sharon M. Hall, Ph.D., eds.

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54 MECHANISMS OF TOLERANCE AND DEPENDENCE. Charles Wm.

Sharp, Ph.D., ed.

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55 PROBLEMS OF DRUG DEPENDENCE, 1984: PROCEEDINGS OF

THE 46TH ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON

PROBLEMS OF DRUG DEPENDENCE, INC. Louis. S. Harris, Ph. D., ed.

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56 ETIOLOGY OF DRUG ABUSE: IMPLICATIONS FOR PREVENTION.

Coryl LaRue Jones, Ph.D., and Robert J. Battjes, D.S.W., eds.

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57 SELF-REPORT METHODS OF ESTIMATING DRUG USE: MEETING

CURRENT CHALLENGES TO VALIDITY. Beatrice A. Rouse, Ph.D.;

Nicholas J. Kozel, M.S.; and Louise G. Richards, Ph.D., eds.

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58 PROGRESS IN THE DEVELOPMENT OF COST-EFFECTIVE TREATMENT FOR DRUG ABUSERS. Rebecca S. Ashery, D.S.W., ed.

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59 CURRENT RESEARCH ON THE CONSEQUENCES OF MATERNAL

DRUG ABUSE. Theodore M. Pinkert. M.D., J.D., ed.

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60 PRENATAL DRUG EXPOSURE: KINETICS AND DYNAMICS.

C. Nora Chiang, Ph.D., and Charles C. Lee, Ph.D., eds.

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61 COCAINE USE IN AMERICA: EPIDEMIOLOGIC AND CLINICAL

PERSPECI’IVES. Nicholas J. Kozel, M.S., and Edgar H. Adams, M.S., eds.

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62 NEUROSCIENCE METHODS IN DRUG ABUSE RESEARCH. Roger M.

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63 PREVENTION RESEARCH: DETERRING DRUG ABUSE AMONG

CHILDREN AND ADOLESCENTS. Catherine S. Bell, M.S., and Robert J.

Battjes, D.S.W., eds.

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64 PHENCYCLIDINE: AN UPDATE. Doris H. Clouet, Ph.D., ed.

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65 WOMEN AND DRUGS: A NEW ERA FOR RESEARCH. Barbara A.

Ray, Ph.D., and Monique C. Braude, Ph.D., eds.

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66 GENETIC AND BIOLOGICAL MARKERS IN DRUG ABUSE AND

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68 STRATEGIES FOR RESEARCH ON THE INTERACTIONS OF DRUGS

OF ABUSE. Monique C. Braude, Ph.D., and Harold M. Ginzburg, M.D., J.D.,

eds.

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69 OPIOID PEPTIDES: MEDICINAL CHEMISTRY. Rao S. Rapaka, Ph.D.;

Gene Barnett, Ph.D.; and Richard L. Hawks, Ph.D., eds.

GPO Stock #017-024-1297-1 $11 NTIS PB #89-158422/AS $39

70 OPIOID PEPTIDES: MOLECULAR PHARMACOLOGY, BIOSYNTHESIS, AND ANALYSIS. Rao S. Rapaka, Ph.D., and Richard L.. Hawks, Ph.D.,

eds.

GPO Stock #017-024-1298-0 $12 NTIS PB #89-158430/AS $45

71 OPIATE RECEPTOR SUBTYPES AND BRAIN FUNCTION. Roger M.

Brown, Ph.D.; Doris H. Clouet, Ph.D.; and David P. Friedman, Ph.D., eds.

GPO Stock #017-024-01303-0 $6 NTIS PB #89-151955/AS $31

72 RELAPSE AND RECOVERY IN DRUG ABUSE. Frank M. Tims, Ph.D.,

and Carl G. Leukefeld, D.S.W., eds.

GPO Stock #017-024-01302-1 $6 NTIS PB #89-151963/AS $31

73 URINE TESTING FOR DRUGS OF ABUSE. Richard L. Hawks, Ph.D.,

and C. Nora Chiang, Ph.D., eds.

GPO Stock #017-024-01313-7 $3.75 NTIS PB #89-151971/AS $23

74 NEUROBIOLOGY OF BEHAVIORAL CONTROL IN DRUG ABUSE.

Stephen I. Szara, M.D., D.Sc., ed.

GPO Stock #017-024-1314-5 $3.75 NTIS PB #89-151989/AS $23

75 PROGRESS IN OPIOID RESEARCH. PROCEEDINGS OF THE 1986

INTERNATIONAL NARCOTICS RESEARCH CONFERENCE. John W.

Holaday, Ph.D.; Ping-Yee Law, Ph.D.; and Albert Herz, M.D., eds.

GPO Stock #017-024-01315-3 $21 NCADI out of stock

Not available from NTIS

241
76 PROBLEMS OF DRUG DEPENDENCE, 1986. PROCEEDINGS OF

THE 48TH ANNUAL SCIENTIFIC MEETING, THE COMMITEE ON

PROBLEMS OF DRUG DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01316-1 $16 NCADI out of stock

NTIS PB #88-208111/AS $53

77 ADOLESCENT DRUG ABUSE: ANALYSES OF TREATMENT

RESEARCH. Elizabeth R. Rahdert, Ph.D., and John Grabowski, Ph.D., eds.

GPO Stock #017-024-01348-0 $4 NTIS PB #89-125488/AS $23

78 THE ROLE OF NEUROPLASTICITY IN THE RESPONSE TO DRUGS.

David P. Friedman, Ph.D., and Doris H. Clouet, Ph.D., eds.

GPO Stock #017-024-01330-7 $6 NTIS PB #88-245683/AS $31

79 STRUCTURE-ACTIVITY RELATIONSHIPS OF THE CANNABINOIDS.

Rao S. Rapaka, Ph.D., and Alexandra Makriyannis, Ph.D., eds.

GPO Stock #017-024-01331-5 $6 NTIS PB #89-109201/AS $31

80 NEEDLE SHARING AMONG INTRAVENOUS DRUG ABUSERS:

NATIONAL AND INTERNATIONAL PERSPECTIVES. Robert J. Battjes,

D.S.W., and Roy W. Pickens, Ph.D., eds.

GPO Stock #017-024-01345-5 $5.50 NTIS PB #88-236138/AS $31

81 PROBLEMS OF DRUG DEPENDENCE, 1987. PROCEEDINGS OF

THE 49TH ANNUAL SCIENTIFIC MEETING, THE COMMITEE ON

PROBLEMS OF DRUG DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01354-4 $17 NTIS PB #89-109227/AS

Contact NTIS for price

82 OPIOIDS IN THE HIPPOCAMPUS. Jacqueline F. McGinty, Ph.D., and

David P. Friedman, Ph.D., eds.

GPO Stock #017-024-01344-7 $4.25 NTIS PB #88-245691/AS $23

83 HEALTH HAZARDS OF NITRITE INHALANTS. Harry W. Haverkos,

M.D., and John A. Dougherty, Ph.D., eds.

GPO Stock #017-024-01351-0 $3.25 NTIS PB #89-125496/AS $23

84 LEARNING FACTORS IN SUBSTANCE ABUSE. Barbara A. Ray,

Ph.D., ed.

GPO Stock #017-024-01353-6 $6 NTIS PB #89-125504/AS $31

85 EPIDEMIOLOGY OF INHALANT ABUSE: AN UPDATE. Raquel A.

Crider, Ph.D., and Beatrice A. Rouse, Ph.D., eds.

GPO Stock #017-024-01360-9 $5.50 NTIS PB #89-123178/AS $31

86 COMPULSORY TREATMENT OF DRUG ABUSE: RESEARCH AND

CLINICAL PRACTICE. Carl G. Leukefeld, D.S.W., and Frank M. Tims,

Ph.D., eds.

GPO Stock #017-024-01352-8 $7.50 NTIS PB #89-151997/AS $31

242
87 OPIOID PEPTIDES: AN UPDATE. Rao S. Rapaka, Ph.D., and Bhola N.

Dhawan, M.D., eds.

GPO Stock #017-024-01366-8 $7 NTIS PB #89-158430/AS $45

88 MECHANISMS OF COCAINE ABUSE AND TOXICITY. Doris H.

Clouet, Ph.D.; Khursheed Asghar, Ph.D.; and Roger M. Brown, Ph.D., eds.

GPO Stock #017-024-01359-5 $11 NTIS PB #89-125512/AS $39

89 BIOLOGICAL VULNERABILITY TO DRUG ABUSE. Roy W. Pickens,

Ph.D., and Dace S. Svikis, B.A., eds.

GPO Stock #017-022-01054-2 $5 NTIS PB #89-125520/AS $23

90 PROBLEMS OF DRUG DEPENDENCE, 1988. PROCEEDINGS OF

THE 50TH ANNUAL SCIENTIFIC MEETING. THE COMMITTEE ON

PROBLEMS OF DRUG DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01362-5 $17

91 DRUGS IN THE WORKPLACE: RESEARCH AND EVALUATION

DATA. Steven W. Gost, Ph.D., and J. Michael Walsh, Ph.D., eds.

GPO Stock #017-024-01384-6 $10 NTIS PB #90-147257/AS $39

92 TESTING FOR ABUSE LIABILITY OF DRUGS IN HUMANS.

Marian W. Fischman, Ph.D., and Nancy K. Mello, Ph.D., eds.

GPO Stock #017-024-01379-0 $12 NTIS PB #90-148933/AS $45

93 AIDS AND INTRAVENOUS DRUG USE: FUTURE DIRECTIONS FOR

COMMUNITY-BASED PREVENTION RESEARCH. C.G. Leukefeld, D.S.W.;

R.J. Battjes, D.S.W.; and Z. Amsel, Ph.D., eds.

GPO Stock #017-024-01388-9 $10 NTIS PB #90-148941/AS $39

94 PHARMACOLOGY AND TOXICOLOGY OF AMPHETAMINE AND

RELATED DESIGNER DRUGS. Khursheed Asghar, Ph.D.; Errol De Souza,

Ph.D., eds.

GPO Stock #017-024-01386-2 $11 NTIS PB #90-148958/AS $39

IN PRESS:

95 PROBLEMS OF DRUG DEPENDENCE, 1989. PROCEEDINGS OF

THE 51ST ANNUAL SCIENTIFIC MEETING. THE COMMITTEE ON

PROBLEMS OF DRUG DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

96 DRUGS OF ABUSE: CHEMISTRY, PHARMACOLOGY, IMMUNOLOGY, AND AIDS. Phuong Thi Kim Pham, Ph.D., and Kenner Rice, Ph.D.,

eds.

97 NEUROBIOLOGY OF DRUG ABUSE: LEARNING AND MEMORY.

Lynda Erinoff, ed.

98 THE COLLECTION AND INTERPRETATION OF DATA FROM

HIDDEN POPULATIONS. Elizabeth Y. Lambert, M.S., ed.

243

U.S GOVERNMENT PRINTING OFFICE: 1990 -0- 270-468
DHH Publication No. (ADM) 90-1676

Alcohol, Drug Abuse, and Mental Health Administration

Printed 1990