Narcotic Antagonists:

NALTREXONE

PHARMACOCHEMISTRY

AND SUSTAINED-RELEASE

PREPARATIONS

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service • Alcohol. Drug Abuse • and Mental Health Administration
Narcotic Antagonists:

Naltrexone Pharmacochemistry and

Sustained-Release Preparations

Editors:

Robert E. Willette, Ph. D.

Gene Barnett, Ph. D.

NIDA Research Monograph 28

1981

DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration

National Institute on Drug Abuse

Division of Research

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Rockville, Maryland 20657

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

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The NIDA Research Monograph series is prepared by the Division of Research

of the National Institute on Drug Abuse. Its primary objective is to provide

critical reviews of research problem areas and techniques, the content of

state-of-the-art conferences, integrative research reviews and significant

original research. Its dual publication emphasis is rapid and targeted

dissemination to the scientific and professional community

Editorial Advisory Board

Avram Goldstein, M.D.

Addiction Research Foundation

Palo Alto, California

Jerome Jaffe, M,D.

College of Physicians ond Surgeons

Columbia University, New York

Reese T. Jones, M.D.

Langley Porter Neuropsychiatric Institute

University of California

Scn Fransico, California

William McGlothlin, Ph.D.

Department of Psychology, UCLA

Los Angeles, California

Jack Mendelson, M.D.

Alcohol and Drug Abuse Research Center

Harvard MedicaI School

McLean Hospital

Belmont, Massachusetts

Helen Nowlis, Ph.D.

Office of Drug Education DHHS

Washington, D.C.

Lee Robins, Ph.D.

Washington University School of Medicine

St. Louis, Missouri

NIDA Research Monograph series

William Pollin, M.D.

DIRECTOR, NIDA

Marvin Snyder, Ph.D.

DIRECTOR, DIVISION OF RESEARCH, NIDA

Robert C. Petersen, Ph.D.

EDITOR-IN-CHIEF

Eleanor W. Waldrop

MANAGING EDITOR

Parklawn Building 5600 Fishers Lane, RockviIle, Maryland 20857
Narcotic Antagonists:

Naltrexone Pharmacochemistry and

Sustained-Release Preparations
ACKNOWLEDGMENTS

This monograph is based on papers presented at a technical

review on the clinical pharmacochemistry of naltrexone, held

at Parklawn Building, Rockville, Maryland, on May 30 and 31,

1919.

The National Institute on Drug Abuse has obtained permission

from the Pharmaceutical Society of Great Britain to reprint a

portion of the data in An Improved Long-Acting Delivery

System for Narcotic Antagonists, by Anand L. Misra and

Ronald B. Pontani, as noted in the text. Further reproduction

of this material without specific permission of the copyright

holder is prohibited. All other material, except short quoted

passages from copyrighted sources, is in the public domain and

may be used and reproduced without permission. Citation as to

source is appreciated.

The United States Government does not endorse or favor any

specific commercial product or commodity. Trade names or sup

pliers’ names appearing in this publication are used only be

cause they are considered essential in the context of the studies

reported herein.

Library of Congress catalog card number 81-600021

DHHS publication number (ADM)81-902

Printed 1981

NIDA Research Monographs are indexed in the Index Medicus.

They are selectively included in the coverage of BioSciences Information Service, Chemical Abstracts, Current Contents, Psychological Abstracts, and Psychopharmacology Abstracts.

i v
FOREWORD

With increasing frequency, Federal agencies are being called

upon to evaluate and develop new drugs and treatments for a wide

variety of diseases and related conditions. The so-called “orphan”

drugs, or drugs of little or limited commercial value, are being

shunned by the pharmaceutical industry, due primarily to the

ever-increasing developmental costs and risks associated with new

drugs. Thus, within the Public Health Service, a drug development

effort has emerged to fill this void.

The United States Congress has charged the National Institute

on Drug Abuse with the responsibility to develop and provide drugdependent people with adequate and, if necessary, new treatment

methods. Amongst the many approaches undertaken have been efforts to develop drug therapies to serve as adjuncts to psychologic

and sociologic efforts. The two principal initiatives have focused on

the maintenance drug, levo-alpha-acetylmethadol (LAAM), and the

narcotic antagonist, naltrexone. The continuing accounts of milestones in their development have been presented in numerous scientific and medical articles and summarized in several volumes of

this Research Monograph series. We consider this volume to represent volume 5 in our series on Drug Development.

The first of this series, Monograph 4, Narcotic Antagonists: The

Search for Long-Acting Preparations, represented a status report on

early efforts to develop such a preparation for naltrexone. Monograph 8, Rx

:3x/Week LAAM, Alternative to Methadone, and Monograph 9, Narcotic Antagonists: Naltrexone Progress Report, described progress on LAAM and naltrexone, respectively, as the Institute’s newly established drug development program was fully underway. In a departure from these advanced developmental programs, basic concepts in the design of new agents was the subject

of Monograph 22, Quantitative Structure Activity Relationships of

Analgesics, Narcotic Antagonists, and Hallucinogens.

Previous volumes on naltrexone have detailed the conceptual approach to the use of a narcotic antagonist or agent that blocks the

effects of heroin or other opioids. Of the early drugs tested, naltrexone emerged as the most promising. It is currently under clinical

v
evaluation, with a strong likelihood of being marketed in oral form

within the next year or so. Although this will provide a blocking

drug with three-times-a-week dosage, it is felt that a longer-acting

drug or dosage form would be more desirable in the treatment of

narcotic addiction. It has been nearly five years since we published

Monograph 4. At that time we summarized many promising approaches and interesting leads. Some of these encouraged us to feel

that at least one of the preparations would be ready for clinical

trials within the near future. That day has continued to elude us,

but we are now much wiser and more experienced and presently

have a preparation in hand that awaits a clinical trial.

In this volume, we summarize the most promising of the approaches pursued over the past several years. General overviews of

the background for chemical and pharmacological decisions are

given. In addition, details of the analytical methods for measuring

naltrexone levels, the pharmacokinetics of naltrexone in test animals and man, and the development and manufacture of clinical

materials are presented. We expect this volume to help serve as a

guide to other efforts in the area of drug development for longacting drug preparations.

The Editors

vi
CONTENTS

Foreword

The Editors

PART I INTRODUCTION AND OVERVIEW

Historical Perspective on the Chemistry and Development of

Naltrexone

Sydney Archer

Treatment of Heroin-Dependent Persons With Antagonists:

Current Status

Pierre Renault

PART II ANALYTICAL METHODS

An Electron-Capture Gas Chromatographic Assay for

Naltrexone in Biological Fluids

R. H. Reuning, S. B. Ashcraft, and B. E. Morrison

Quantitative Determination of Naltrexone, 6 ß-Naltrexol and

2-Hydroxy-3-methoxy-6ß-naltrexol (HMN) in Human Plasma,

Red Blood Cells, Saliva and Urine by Gas Liquid

Chromatography

Karl Verebey

Analytical Methods for Quantitative and Qualitative Analysis

of Naltrexone and Metabolites in Biological Fluids

Monroe E. Wall and Dolores R. Brine

PART III PHARMACOLOGY

Background on Animal Testing in the Drug Delivery Systems

Program

William L. Dewey

Pharmacological Evaluation of Narcotic Antagonist Delivery

Systems in Rhesus Monkeys

Stephen E. Harrigan and David A. Dawns

v

3

11

25

36

52

69

77

vii
A Comparative Study of the Oral, Intravenous, and

Subcutaneous Administration of 3H Naltrexone to Normal

Male Volunteers

Mario Perez-Reyes and Monroe E. Wall 93

PART IV METABOLISM

The Metabolism of Naltrexone in Man

Monroe E. Wall, Dolores R. Brine, and Mario Perez-Reyes 105

Current Status of Preclinical Research on Disposition,

Pharmacokinetics, and Metabolism of Naltrexone

Anand L. Misra 132

The Clinical Pharmacology of Naltrexone: Pharmacology and

Pharmacodynamics

Karl Verebey 147

PART V PHARMACOKINETICS

Overview of Human Pharmacokinetics of Naltrexone

Vojtech Licko 161

Pharmacokinetic Quantitation of Naltrexone Release From

Several Sustained-Release Delivery Systems

R. H. Reuning, S.H.T. Liao, and A.E. Staubus 172

PART VI SUSTAINED-RELEASE SYSTEMS

A Review of Parenteral Sustained-Release Naltrexone

Systems

James L. Olsen and Fred A. Kincl 187

Development of Drug Delivery Systems for use in Treatment

of Narcotic Addiction

A. Carl Sharon and Donald L. Wise 194

Use of Synthetic Polypeptides in the Preparation of

Biodegradable Delivery Systems for Narcotic Antagonists

Kenneth R. Sidman, Arthur D. Schwope, William D.

Steber, and Stephen E. Rudolph 214

Biodegradable Drug Delivery Systems Based on Aliphatic

Polyesters: Application to Contraceptives and Narcotic

Antagonists

Colin G. Pitt, Thomas A. Marks, and Anton Schindler 232

An Improved Long-Acting Delivery System for Narcotic

Antagonists

Anand L. Misra and Ronald B. Pontani 254

viii
Solid Solutions as Long-Acting Naltrexone Delivery Systems

James L. Olsen 265

Participants in Technical Review 272

List of Monographs 274
Part I

Introduction and

Overview

Naltrexone: Research Monograph 28

R. E. Willette and G. Barnett, eds.

National Institute on Drug Abuse, 1980

Historical Perspective on

the Chemistry and

Development of

Naltrexone

Sydney Archer

The first clinically useful narcotic antagonist was nalorphine. This compound was relatively weak, short-acting,

and produced a number of side effects, the most prominent

of which were psychotomimetic in nature. For these reasons

nalorphine did not qualify as a possible modality for the

treatment of heroin addiction. Cyclazocine is a totally synthetic narcotic antagonist which is much more potent and

longer acting than nalorphine. It was the first compound

used by Martin in clinical trials in postadicts. However,

its dysphoric effects necessitated long induction periods and

these CNS effects precluded its use in long-acting delivery

systems. Naloxone was a “pure” antagonist which did not

produce the psychotomimetic effects o either nalorphine or

cyclazocine. Although it is a potent antagonist when given

parenterally, it is shorter acting than cyclazocine. Replacement of the N-allyl substituent of naloxone with the cyclopropylmethyl radical of cyclazocine led to naltrexone,

which is even more potent than either naloxone or cyclazocine and has a longer duration than naloxone. Because of

this favorable combination of properties naltrexone proved

to be the drug of choice for inclusion in long-acting delivery

systems.

Although the first narcotic antagonist, N-allylnorcodeine (I), was

described by Pohl in 1915, the first important member of the class

of narcotic antagonists was nalorphine (II), an authentic sample of

which was prepared by Weijlard and Erickson in 1942 (1). Nalorphine showed no analgesic activity in the rodent laboratory assays

3
4 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

in vogue at that time, such as the D’Amour-Smith test in rats and

the Eddy-Leimbach test in mice, but instead was found to antagonize the agonist effect of morphine (2,3). Until recently, it was used

as a clinical antidote for acute narcotism but was effectively replaced by naloxone as the drug of choice for this condition.

In clinical studies designed to determine whether combinations

of nalorphine and morphine could produce analgesia with a reduced incidence of respiratory depression, Lasagna and Beecher (4)

reported that nalorphine when given alone was a potential analgesic in man. This clinical finding was confirmed by Keats and Telford who extended these studies to other narcotic antagonists and

found other examples which produced significant clinical analgesia

(5,6). Early studies on the addiction liability of nalorphine showed

that it produced neither physical nor psychic dependence after prolonged administration to postaddicts (7), although a later study

demonstrated that a mild physical dependence unlike that of morphine could develop (8). The major drawback of nalorphine and its

other analgesically active congeners was that intense dysphoric

side effects were observed at or very near clinically effective doses

in a large fraction of patients. Thus these compounds were judged

to be clinically unacceptable as analgesics.

The importance of these observations lay in the fact that for the

first time it was found possible to divorce potent analgesia from

morphine-like addiction liability. In retrospect it is curious that

what proved to be such a seminal observation attracted so little attention of medicinal chemists engaged in the synthesis of strong

analgesics. Two groups, one at the Sterling-Winthrop Research Institute (9,10), and the other at the University of Rochester (11), independently embarked on the synthesis of narcotic antagonists

with the intention of finding a nonaddicting strong analgesic.

The Rochester group explored the tetracyclic morphinan series

while the Sterling-Winthrop group concentrated on the tricyclic

benzomorphan series, the chemistry of which was the subject of a

series of papers by E. L. May (12). The major obstacle to this ap-
HISTORICAL PERSPECTIVE ON NALTREXONE 5

proach was the lack of a laboratory model which could detect agonist activity in putative mixed agonist-antagonists. As a matter of

fact, very few laboratories were testing for narcotic antagonist

action and the chemists at Sterling-Winthrop were fortunate that

Harris and Pierson had developed a quantitative test for this type

of pharmacological activity (13). Quite fortuitously, Gates and

Archer discovered that they were working in the same area, and

the former accepted an invitation to have his compounds tested by

Harris and Pierson. The results of this collaborative effort were

published simultaneously in the Journal of Medicinal Chemistry

(10,11).

The most interesting member of the Gates series was cyclorphan

(III). This compound proved to be a potent antagonist in laboratory

animals (14) and man. Although psychotomimetic effects were observed, they did not occur as frequently as with nalorphine (15). Cyclorphan was about forty times as potent as morphine on a milligram basis, i.e., 0.25 mg of III provided analgesia equivalent to 10

mg of morphine.

The compounds which attracted the greatest interest in the benzomorphan series were the weak antagonist pentazocine (IV), and

the powerful antagonist cyclazocine (V). Pentazocine was shown to

be about 1/3-1/6 as potent as morphine as an analgesic (16,17) with

reduced abuse potential (18) and with a reduced incidence of psychotomimetic effects (16). Cyclazocine was a highly effective analgesic in man when given either orally or parenterally (15). It is a

potent antagonist with a long duration of action, a result that

could not be predicted from animal studies (19). Dependence studies

in postaddicts showed that after escalating doses of the drug over a

prolonged period of time, abrupt withdrawal produced a withdrawal syndrome different from that of morphine in that it was quite

mild and no drug-seeking behavior on the part of the addict was
6 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

observed (20). Cyclazocine would have been an almost ideal analgesic except for the fact that a small but significant percentage of the

patients exhibited psychotomimetic effects at or near analgesic

doses. Tolerance to these effects appeared to develop on prolonged

administration.

Wikler (21) suggested that drug dependence evolves through a

series of several phases. At the beginning an individual may use

opiates to relieve daily tensions and discomforts. With each episode

of self-administration the habit is reinforced. As tolerance to the

drug develops, the effects of the opiate are reduced so that higher

doses are required to afford the same degree of relief. If the addict

fails to maintain an adequate drug intake his dependence is further reinforced. He may then seek assistance in becoming detoxified or may attempt a self-induced withdrawal. In either case

marked physiological changes take place during the withdrawal

phase which may last for several months even when detoxification

occurs slowly. This will lead to drug-seeking behavior or recidivism.

Wikler feels that an extinction of drug-seeking behavior could be a

means of reversing the conditioned abstinence. This could be accomplished by blocking the relief obtained from use of opioids

during this critical period. One way to accomplish this goal is to

administer a substance with antagonist action which in itself does

not induce tolerance or dependence but which blocks the pharmacological effects of the opiates.

On the basis of these considerations Martin considered the use of

narcotic antagonists as a method for treating heroin dependence.

Nalorphine was the first candidate, but its short duration of action

and high incidence of dysphoria prompted Martin to select cyclazocine for such a study (19). The rationale for the use of cyclazocine

was given by Martin as follows: “On the basis of our studies, 4 mg

per day of cyclazocine will provide protection against the euphorogenic actions of large doses of narcotics, prevent the development

of physical dependence, and will thereby control the pharmacological actions which are held responsible for narcotic

addiction....There may be other benefits....Wikler stated that two of

the most important reasons for relapse of the abstinent narcotic

addict are conditioned abstinence, which may be evoked by stimuli

that have been associated with the addict’s hustling activity to acquire drugs, and reinforcement of drug-seeking activity through repeated reductions of abstinence by drug. It is possible that in sub

jects who attempt to readdict themselves while receiving a narcotic

antagonist such as cyclazocine, there may be extinction of physical

dependence and drug-seeking behavior.”
HISTORICAL PERSPECTIVE ON NALTREXONE 7

Martin, Gorodetzky, and McClaine (19), and Jaffe and Brill (22),

were the first to study cyclazocine as a modality for treating opiate

addicts. These early studies were promising enough to warrant further clinical trials. Fink and his colleagues (23) succeeded in carrying out a more rapid induction of cyclazocine than Jaffe and Brill,

who required 10-40 days to reach the 4.0 mg/day dose which was

necessary for effective clinical antagonism of 25 mg heroin challenges. The slow induction period was necessary in order to permit

the subjects to become tolerant to the agonist effects of cyclazocine.

Fink and his colleagues (23) found that induction could be effected

over a period of 4 days by using naloxone as needed to antagonize

the unpleasant effects of cyclazocine. The dose of cyclazocine was

increased by 1.0 mg/day when 600 mg of naloxone was available

every 4 hours. At a stabilized dose of 4.0 mg/day of cyclazocine the

subjects reported no effect from a challenged dose of a $20 quantity

of “street” heroin, a transient “buzz” from twice that amount, and

a mild euphoria from a $60 amount taken 12-14 hours after oral

administration of cyclazocine.

Naloxone (VI) was synthesized by Lowenstein and Fishman in

1960, and in 1961 Blumberg reported that it was a powerful narcotic antagonist in animals (24). It was also reported that the compound was inactive as an analgesic. Foldes (25) found that naloxone

was an active narcotic antgonist in man with none of the dysphoric

effects of cyclazocine. Although a 1.0 mg intravenous dose of naloxone could block the effects of a 25 mg heroin challenge, when given

orally 3.0 g/day were required (26) to block a 50 mg challenge given

24 hours after the administration of VI.

The principal shortcoming of naloxone is its short duration of

action. Blockade of a heroin challenge rarely exceeded one day so

that the omission of a daily dose allowed the subject to become

high on heroin.

On the basis of these early experiences it became clear that what

was needed was 1) a long-acting, potent, “pure” narcotic antagonist

and 2) a delivery system which would keep the subjects under constant drug pressure.

347-877 0 - 81 - 2
8 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

The first of these requirements was met by naltrexone (VII) (27).

This compound is about twice as active as naloxone as an antagonist and inactive as an analgesic in the mouse. In the rat it showed

weak agonist action. Furthermore in man it was longer acting than

naloxone (28) and produced no unwanted side effects, thus eliminating the need for an induction phase.

The problem of a suitable delivery system remains. On the basis

of a simple pharmacokinetic analysis it was concluded that a

system with a release rate which followed zero-order was more desirable than one which manifested first-order kinetics (29). It would

also be desirable to have a delivery system whose matrix would biodegrade at approximately the same rate at which the drug was released so that when the administered dose of the drug was exhausted there would be no residual matrix left behind.

It was at this point in the history of the development of narcotic

antagonists that the National Institute on Drug Abuse stepped in.

Several contracts for the fabrication of long-acting biodegradable

naltrexone delivery systems were funded and long term toxicity

and pharmacokinetic studies on naltrexone were sponsored. As a

result of these efforts on the part of NIDA at least one system has

been brought to the point where a clinical trial may be initiated in

the foreseeable future.

REFERENCES

1. J. Weijlard and A.E. Erickson. N-allylnormorphine. J. Am. Chem. Soc., 64, 869-870

(1942).

2. E.R. Hart and E.L. McCawley. Pharmacology of N-allylnormorphine as compared

with morphine. J. Pharmacol. Exp. Ther., 82, 338-348 (1944).

3. K. Unna. Antagonistic effects of N-allylnormorphine upon morphine. J. Pharmacol. Exp. Ther., 79, 271 (1944).

4. L. Lasagna and H.K. Beecher. Analgesic effectiveness of nalorphine and nalorphine combinations in man. J. Pharmacol. Exp. Ther., 112, 356-363 (1954).

5. A.S. Keats and J. Telford. Nalorphine, a potent analgesic in man. J. Pharmacol.

Exp. Ther., 117, 190-196 (1956).

6. J. Telford, C.N. Papadopoulos, and A.S. Keats. Morphine antagonists as analgesics. J. Pharmacol. Exp. Ther., 133, 116 (1961).

7. H. Isbell. Attempted addiction to nalorphine. Fed. Proc., 15, 446 (1956).

8. W.R. Martin and C.W. Gorodetsky. Demonstration of tolerance and dependence

on nalorphine. J. Pharmacol. Exp. Ther., 150, 437 (1965).

9. S. Areher, N.F. Albertson, L.S. Harris, A.K. Pierson, J.G. Bird, A.S. Keats, J. Telford, and C.N. Papadopoulos. Narcotic antagonists as analgesics. Science, 137,

541-543 (1968).

10. S. Archer, N.F. Albertson, L.S. Harris, A.K. Pierson, and J.G. Bird. Strong analgesics and analgesic antagonists in the benzomorphan series. J. Med. Chem., 7,

123-127 (1964).
HISTORICAL PERSPECTIVE ON NALTREXONE 9

11. M.D. Gates and T. Montzka. Some potent morphine antagonists possessing high

analgesic activity. J. Med. Chem., 7, 127-131 (1964).

12. J. Hellerbach, O. Schnider, H. Besendorf, B. Pellmont, N.B. Eddy, and E.L. May.

Synthetic Analgesics. Pergamon Press, New York, 115-183 (1966).

13. L.S. Harris and A.K. Pierson. Some narcotic antagonists in the benzomorphan

series. J. Pharmacol. Exp. Ther., 143, 141-148 (1964).

14. L.S. Harris, A.K. Pierson, J.R. Demskinski and W.L. Dewey. The pharmacology

of (-)-3-hydroxy-N-cyclopropylmorphinan(cyclorphan). Arch. Int. Pharmacodyn.

Ther., 165, 112-126 (1967).

15. L. Lasagna, T.J. Dekornfeld, and J.W. Pearson. The analgesic efficacy and respiratory effects in man of a benzomorphan “narcotic antagonist.” J. Pharmacol.

Exp. Ther., 144, 12-16 (1964).

16. A.S. Keats and J. Telford. Studies of analgesic drugs. VIII. A narcotic antagonist

analgesic without psychotomimetic effects. J. Pharmacol. Exp. Ther., 143, 157-

164 (1964).

17. R. Janzen, W.D. Keidel, A. Herz, and C. Steichele (eds.). Pain. George Thieme,

Stuttgart, and Churchill Livingston, London, 339-430 (1972).

18. D.R. Jasinski, W.R. Martin, and R. Hoedtke. Effects of short- and long-term administration of pentazocine in man. Clin. Pharmacol. Ther., 11, 385-389 (1970).

19. W.R. Martin, C.W. Gorodetzky, and T.K. McClaine. An experimental study in

the treatment of narcotic addicts with cyclazocine. Clin. Pharmacol. Ther., 7,

455-458 (1966).

20. W.R. Martin, H.F. Fraser, C.W. Gorodetzky, and D.E. Rosenberg. Studies of the

dependence-producing potential of the narcotic antagonist 2-cyclopropylmethyl2-hydroxy-5,9-demethyl-6,7-benzomorphan (Cyclazocine, Win 20,740, ARC II-C,3).

J Pharmacol. Exp. Ther., 150, 426-429 (1965).

21. A. Wikler. Conditioning factors in opiate addiction and relapse. In Narcotics. D.I.

Wilner and G. G. Kassebaum (ed). McGraw-Hill, New York, 85-100 (1965).

22. J.H. Jaffe and L. Brill. Cyclazocine, a long-acting narcotic antagonist: Its voluntary acceptance as a treatment modality by narcotic abusers. Int. J Addiction,

1, 99-123 (1966).

23. M. Fink, A.M. Freedman, R. Resnick, and A. Zaks. Clinical status of the narcotic

antagonist in opiate dependence. In H.W. Kosterlitz, J.O.J. Collier, and J.E. Villareal (eds.), Agonist and Antagonist Actions of Narcotic Drugs, Macmillan

Press, Ltd., London, 266-277 (1972).

24. H. Blumberg, H.B. Dayton, M. George, and D. Rapaport. N-allylnoroxymorphone:

A potent narcotic antagonist. Fed. Proc., 20, 311 (1961).

25. F.F. Foldes, J.N. Lunn, J. Moore, and I.M. Brown. N-allylnoroxymorphone: A

new potent narcotic antagonist. Amer. J. Med. Sci., 245, 23-30 (1963).

26. A Zaks, T. Jones, M. Fink, and A.M. Freedman. Naloxone treatment of opiate

dependence. JAMA, 215, 2108-2110 (1971).

27. H. Blumberg and H.B. Dayton. Naloxone, naltrexone and related noroxymorphones. In M.C. Braude, L.S. Harris, E.L. May, J.P. Smith, and J.E. Villareal

(eds.). Narcotic Antagonists. Advances in Biochemical Psychopharmacology, Vol.

8. Raven Press, New York, 33-43 (1973).

28. W.R. Martin, D.R. Jasinski, and P.A. Mansky. Characteristics of the blocking effects of EN-1039A (N-cyclopropylmethyl-7,8-dihydro-14-hydroxynormorphinoneHCl). Quoted by Blumberg in reference 27.

29. S. Archer. Design consideration for long-acting antagonists, In M.C. Braude et al.

(eds.). Narcotic Antagonists. See reference 27.
10 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

AUTHOR

Sydney Archer

Department of Chemistry

Rensselaer Polytechnic Institute

Troy, New York 12181
Naltrexone: Research Monograph 28

R. E. Willette and G. Barnett, eds.

National Institute on Drug Abuse, 1980

Treatment of HeroinDependent Persons With

Antagonists: Current

Status

Pierre F. Renault

Naltrexone is an important new pharmacologic adjunct to

the treatment of heroin dependence. The development of

naltrexone has been nurtured in the mature recognition

that simple detoxification or simple opiate replacement

therapy is not appropriate for every heroin addict. Our current data indicate that naltrexone is safe and effective. Its

use may be limited to a minority of addicts, those who are

highly motivated and opiate free, because patient compliance has been a major problem with which clinicians using

naltrexone have had to contend. Patient compliance is a

problem, because there are no immediate consequences to

the patient for stopping his naltrexone regimen. Side effects

from naltrexone have been minimal and have occurred in a

minority of patients. They consist primarily of gastrointestinal symptoms, including nausea and occasionally abdominal pain.

INTRODUCTION

Like many chronic diseases, heroin addiction is a complex condition to treat and a therapist treating heroin dependence must often

satisfy nonmedical authorities that his treatment will have an

impact on the “problem.” The “problem” of heroin dependence,

however, is not simply an individual behavioral problem but one of

cultural, historical, and socioeconomic dimensions, Thus, it is not

11
12 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

uncommon for the effectiveness of a treatment to be measured

more in terms of its impact on crime rate or prevalence of heroin

addiction rather than the success in the treatment of afflicted individuals. This complexity has confused therapists and has left the

goals of individual treatment poorly defined. Conversely, the lack

of definition of treatment goals has encouraged unreasonable expectations of the treatment system and oversimplified conceptualizations of the problem of heroin addiction. In spite of this, effective

treatment programs have been established and a majority of

heroin-dependent individuals who come for treatment can expect to

be helped.

The ultimate goal of treatment for heroin-dependent persons

should be the patient’s control over the use of the drug; however,

in the case of heroin, society has demanded that control be defined

as abstinence. This demand has led Governments to support detoxification programs, because detoxification is generally applicable,

low in cost, and it is the most immediate and direct way of disentangling an individual from heroin. However, government emphasis on detoxification programs often betrays the oversimplified view

that heroin dependence is simply a pharmacologic problem. On the

contrary, heroin dependence involves every aspect of an individual

and his environment. Individual freedom of choice is undermined

by conditioned responses, pressures from peers, the social frustrations which often seem to form the substrate of heroin use, and

subtle pharmacologic factors such as the intense craving associated

with protracted abstinence.

An often more realistic definition of control over drug use is the

elimination of dysfunctional drug use. Dysfunctional drug use has

been defined as drug use that results in physical, psychological,

economic, legal, and/or social harm or discomfort to the individual

user or to others affected by that drug user’s behavior. It is the discomfort that drug use causes that brings an individual to treatment, and the elimination of that discomfort should be the most

immediate treatment goal. It was this conceptualization of treatment goal that nurtured the development of substitution or maintenance therapy of heroin addiction with methadone. Methadone

maintenance effectively returned the control of heroin to the individual by decreasing craving and eliminating abstinence symptoms.

It also promoted functioning in normal society by decreasing the

frequency of drug taking, the disruption by drug effects, and the

difficulty and costs of obtaining drugs. Although it is clear that

methadone or levo-alpha-acetylmethadol (LAAM) maintenance does

decrease dysfunctional drug use, it is not applicable to all addicts.

Maintenance or stabilization treatment has only been used in
CURRENT STATUS OF NALTREXONE TREATMENT 13

chronic addicts where the prospects of total abstinence are poor.

However, many narcotic-dependent individuals desire total abstinence and find the concept of methadone maintenance unacceptable. Other individuals are currently abstinent but are in danger of

relapsing. Among the latter are those recently detoxified from

heroin addiction, or those who are being released from prison, or

even others who, in spite of prolonged abstinence, have been suddenly tempted to take heroin again by the pressures of emotional

turmoil or the ready availability of heroin in their immediate environment. Finally, there are those individuals who have never been

dependent on heroin but are using the drug and, therefore, are at

risk of becoming dependent. Currently available treatments for

such individuals are limited and the pressures on the abstinent

addict to relapse are intense. These “abstinent addict” patients are

often strongly motivated to seek treatment and relapses often produce guilt and depression, but they are still unable to resist giving

in to the intense craving for heroin. They are strongly motivated,

but motivation simply is not enough. The recognition of the need to

broaden the spectrum of heroin dependence treatment to include

these patients has nurtured the development of narcotic antagonist

treatment.

THE CONCEPT OF NARCOTIC ANTAGONISTS

Narcotic antagonists are usually defined as chemical compounds

which block the effects of opiate drugs. Narcotic antagonists will

block the analgesia, the euphoria, and all the physiologic changes

such as pupilary constriction produced by agonist opiates. By blocking the effects of agonist opiates, narcotic antagonists also prevent

the development of physical dependence and tolerance to opiate

drugs.

Opiate drugs exist on a continuum with morphine, heroin, and

other agonists at one end and antagonists such as naloxone and

naltrexone at the other end. There are many well-known drugs

which fall between the pure agonists, such as heroin and morphine,

and the pure antagonists, such as naloxone and naltrexone. Drugs

such as pentazocine, cyclazocine, and nalorphine have mixed agonist and antagonist effects. For example, pentazocine, a commonly

used analgesic drug, will precipitate the abstinence syndrome in an

individual who is physically dependent on heroin. Cyclazocine is a

potent antagonist, but the induction period is characterized by

agonist effects of a dysphoric nature. During the induction onto cy-
14 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

clazocine, tolerance builds rapidly to the dysphoria, and not to the

antagonistic activity of the drug.

The blockade produced by narcotic antagonists is different from

that produced by disulfiram. Disulfiram (Antabuse) is a commonly

used drug for the treatment of alcoholism. It exerts its effect not by

blocking the effects of alcohol, but by interrupting the metabolism

of alcohol at the acetaldehyde stage. The build-up of acetaldehyde

produces severe symptoms of nausea, vomiting, and hypotension.

The knowledge that drinking will produce such symptoms may act

as a deterrent to further drinking for some alcoholics. Narcotic antagonists exert their activity by blocking the access of agonists to

the opiate receptor sites at the molecular level. Antagonists do not

produce symptoms when an agonist is administered. Rather, when

opiate receptor sites are occupied by an antagonist, opiate agonists

simply have no pharmacologic effect.

Although narcotic antagonists do not produce symptoms when

they are used in the treatment of heroin dependence, they will precipitate an abstinence syndrome in individuals who are physically

dependent on an opiate drug. By virtue of their greater affinity for

the opiate receptors, they will displace opiate agonists from the receptor sites. A heroin addict must be detoxified before he can be

treated with a narcotic antagonist. Once completely free of opiate

drugs, however, no symptoms will be produced by the administration of a narcotic antagonist.

The theory underlying narcotic antagonist treatment of heroin

dependence is based on the concept of extinction. This assumes

that the euphoriant effects of opiate drugs reinforce the self-administration of these drugs. If an ex-addict is given a narcotic antagonist and he takes an agonist drug, he will experience no effect, i.e.,

no euphoria. This lack of reinforcement will gradually result in the

extinction of heroin self-administration (1-3).

The concept of narcotic antagonist treatment has been criticized

on the basis that since these drugs do nothing “positive” for an individual (the only subjective effect is the absence of an effect when

an antagonist such as heroin is taken) and there are no pharmacologic consequences (such as withdrawal symptoms) for not taking

an antagonist, compliance with a treatment program employing a

narcotic antagonist drug will be quite low. Compliance is a problem

with medications for most chronic conditions where the medication

is given to prevent the progression of the disease rather than to alleviate a symptom which would recur if the medication were not

taken.

At least three solutions have been proposed to meet this objection. It has been proposed that naltrexone be administered in a
CURRENT STATUS OF NALTREXONE TREATMENT 15

long-acting form. Sustained action preparations lasting up to a

month or more could be developed. Another suggestion has been

that external coercion be used to force individuals to comply with a

narcotic antagonist regimen. For example, individuals released on

parole from the civil justice or civil commitment system could be

threatened with reincarceration for lack of compliance. Prisoners

in a work release program could be required to take a narcotic antagonist prior to leaving the prison to work each day. Finally, some

have suggested that an antagonist be sought which would have

some agonistic euphoriant properties, a mild euphoriant effect

which would not be disruptive or lead to dysfunctional drug use

but which would act as a reinforcer and encourage compliance.

THE DEVELOPMENT OF NALTREXONE

In the beginning of this decade, the decision was made to encourage the development of a narcotic antagonist to be used in the

treatment of heroin dependence. Available compounds included naloxone, cyclazocine, and naltrexone. Both naloxone (4-6) and cyclazocine (3,7-9) were tested in clinical trials. Both of these drugs

proved to be excellent antagonists but both had drawbacks. Cyclazocine was limited because of dysphoric side effects during the induction period. Naloxone is a pure antagonist, and thus, it is free

of side effects, but lacks potency when administered orally. Finally,

naltrexone was selected for development. Naltrexone is a potent

narcotic antagonist. It has a long duration of action, up to 3 days

following one oral dose. It is devoid of agonist activity and thus the

induction phase is virtually asymptomatic. Martin et al. (10) found

that a 50 mg oral dose of naltrexone will block the subjective effects of 25 mg of morphine for 24 hours and that it will attenuate

the development of physical dependence. O’Brien et al. (11) reported that the blockade produced by a 120-200 mg dose of naltrexone

at 48 hours was not absolute, but was sufficient that subjects discontinued self-injection of 1-4 hydromorphone (Dilaudid). Resnick et

al. (12) reported complete blockade of the agonist effects of 29 mg of

intravenous heroin 24 hours after a 50 mg or larger dose of naltrexone, and 48 hours after 120 mg or larger dose. They also found

some individual variability, i.e., one patient required 200 mg (their

maximum dose) to achieve 24-hour blockade. Finally, Martin et al.

(10) demonstrated that tolerance does not develop to the antagonistic action of naltrexone.

The drug development process in the United States requires extensive animal toxicity testing followed by phased human clinical
16 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

trials. Most of the required animal toxicity testing of naltrexone

has been completed. Phase I human trials involve the basic pharmacology of the drug in humans, such as the work reported by

Martin. Phase II involves the testing of the drug in otherwise

healthy heroin-dependent males. Phase II clinical testing of naltrexone was organized into two cooperative studies.

The first cooperative study was a fixed dose, randomly assigned,

prospective, double-blind comparison of naltrexone and placebo

conducted in five clinics following three protocols. In all, 192 patients participated; 94 were randomly assigned to naltrexone and

98 to placebo. The three protocols were determined by patient characteristics. In one protocol, street addicts were detoxified from

heroin and placed on naltrexone or placebo. In the second protocol,

individuals were placed on naltrexone or placebo immediately after

detoxifying from methadone maintenance; and in the third protocol, postaddicts from the criminal justice system were placed on

naltrexone or placebo. The other cooperative study involved a

loosely-knit group of 12 clinics, which followed a variety of protocols, but used a uniform data collection system. These studies were

largely open and uncontrolled. They were intended to gain clinical

experience with naltrexone and the data collected was useful primarily as an investigation of the safety of the drug in comparison

with the safety results reported in the double-blind study. In all,

1,005 patients participated in this study, 10 of whom were female.

Another important difference between the two studies was that

dropouts could reenter the open study.

Both cooperative studies have been completed, and the results

are available. Both show naltrexone to be safe. There were no drugrelated medical problems in the double-blind study. In the open

study, one individual developed idiopathic thrombocytopenic-purpura which may have been the result of sensitivity to naltrexone.

Fifty-three of the 1,005 cases were terminated for “medical reasons,” none of which were clearly drug-related. (In the double-blind

study, five of the 98 placebo-medicated patients were terminated

for “medical reasons.”) No subjects in either study were dropped

because of serious deviation in a clinical laboratory determination.

In the double-blind study, four laboratory parameters statistically

differentiated the naltrexone group from the placebo group. However, none of the differences in hemoglobin, total protein, bilirubin,

and urine red blood cells were of a clinically significant magnitude.

Only a slight increase in bilirubin was also found in the open

study. Again, this was not clinically significant. The naltrexone

group did have more side effects. These consisted primarily of gastrointestinal complaints, such as nausea and abdominal pain.
CURRENT STATUS OF NALTREXONE TREATMENT 17

There were also more complaints of headache and skin rash.

Again, these side effects were of low incidence and the safety data

have been judged to support the conclusion that naltrexone has a

wide margin of safety for clinical use.

The Phase II clinical trials did show the anticipated poor compliance with naltrexone. And, although retention rate tended to be

better and the use of narcotic drugs tended to be less in the naltrexone group compared to the placebo group, these differences,

when compared individually, were not statistically significant.

Since the theory behind narcotic antagonist treatment involves

extinction and the concept of extinction implies some use of narcotic drugs, a subsample of those individuals who had at least one

urine sample positive for morphine and/or methadone was studied.

Only 17 of the naltrexone and 18 of the placebo subjects actually

tested the blockade by using an opiate agonist (heroin or methadone). But, in this subsample, the naltrexone patients had significantly fewer subsequent urines positive for methadone or morphine

and this difference was statistically significant. Also, the pattern

between the two groups was different. The pattern in the naltrexone group was to test once or twice with heroin or methadone and

then to stop. The use of these drugs in the placebo group was sporadic during the entire course of treatment. Thus, it seems clear

that naltrexone can prevent impulsive heroin use in motivated patients from becoming a full-fledged relapse to chronic heroin dependence.

A very interesting finding from the double-blind study deserves a

special note. During the course of the study, subjects were administered a craving scale. This was an analogue scale on which subjects

rated the amount of craving they felt at the time. Response on this

instrument was variable, but the naltrexone patients reported statistically significantly less craving toward the end of their evaluation than did the placebo-treated patients.

CLINICAL USE OF NALTREXONE

Naltrexone is administered orally either in liquid or solid (capsule) form. The capsule is preferable, because naltrexone has an extremely bitter taste. However, there is some slight danger of diversion when capsules are used, and the liquid formulation was used

in most clinical trials to ensure that the subjects actually ingested

the administered dose. The frequency of administration is either

daily or three times a week. The daily dose is 50 mg and the dosage

for a three-times-a-week regimen is 100 mg on Monday and
18 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

Wednesday and 150 mg on Friday. Both of these regimens had been

found to provide an adequate blockade for the heroin used in the

United States.

Postaddicts who are not currently physically dependent have

generally been inducted onto naltrexone by 10 mg increments until

50 mg daily is reached. Then, they have been converted to a 100-

100-150 mg regimen. However, some clinicians feel that an induction period onto naltrexone is unnecessary in the postaddicted patient.

Patients who are currently physically dependent on an opioid

drug must first be detoxified before they can be put on naltrexone.

The selection of a method of detoxification is up to the individual

clinician. Once the patient has been detoxified, it has been customary to administer a small amount of naloxone intravenously to be

certain that the patient is no longer physically dependent. Thomas

et al. (13) suggest 0.8-1.2 mg intravenously. If there is no response

to this dose of naloxone, induction on a full dose of naltrexone

should be without discomfort.

The clinic setting in which naltrexone is administered is critical

to the success of the treatment. It should be recognized that naltrexone is not a treatment in itself. It is a pharmacologic adjunct to

treatment. The actual treatment consists of the efforts made by the

staff to help the individual patient alter his lifestyle so that he can

function successfully in normal society apart from the drug-abusing

subculture. It is not appropriate in this paper to enter into a

lengthy discussion of clinic milieu, psychotherapeutic techniques,

and rehabilitative measures that can be employed with heroin-dependent individuals. Suffice it to say that in the United States, the

tendency has been to create a supportive and permissive milieu

where individuals are strongly encouraged to remain on naltrexone

until they have significantly altered their lifestyle.

There are three types of patients for whom naltrexone seems

particularly well suited. These are adolescent heroin users, “abstinent” addicts at risk of recidivism, e.g., individuals recently released from prison, and patients recently detoxified from methadone maintenance. Adolescents will have had a relatively short experience with opiate drugs. Many may be experimenters with

heroin and not physically dependent. Those who are physically dependent will have been so for only a relatively short period of time.

All these individuals are at risk of becoming chronically heroin dependent. Currently, there is no available pharmacologic adjunct to

assist in the treatment of adolescent heroin users. Prisoners who

have been recently paroled have, presumably, been narcotic free

for the duration of their incarceration. However, it is well known
CURRENT STATUS OF NALTREXONE TREATMENT 19

that upon reentering their old environment, these postaddicts are

at risk of becoming readdicted. Often, the sequence of events begins

with an impulsive dose of heroin which leads precipitously to the

reestablishment of old behavioral patterns and compulsive heroin

use. Within the criminal justice system, naltrexone may also make

a significant contribution to work release programs. Naltrexone

may make it possible for addicts to participate in such programs

and thereby facilitate their rehabilitation (14). Patients recently detoxified from methadone maintenance are also known to be vulnerable to relapse. There is evidence that this vulnerability may have

a pharmacologic basis (15). In the future, naltrexone may form the

basis of a comprehensive after-care program for methadone maintenance patients which will carry them through this period of pharmacologic vulnerability.

Noticeably absent from this list are freshly detoxified heroin-dependent individuals. The experience in the United States during

Phase II clinical testing indicated that the motivation in this group

was less than that of the other groups tested as measured by their

compliance. Admittedly, this may not be true in other treatment

situations. The heroin-dependent individuals, who were treated

with naltrexone in the Phase II clinical trial, were free to leave at

any time, and had no incentive or social constraint to encourage

them to remain on naltrexone for sufficient time to participate

fully in the clinic’s treatment program. The problem of compliance

is not unique to naltrexone. It is common in all of medicine, but

especially with medications administered to asymptomatic individuals who have a latent form of a disease. A convenient analogy is

that of antihypertensive medication. It is a human characteristic to

forget the pain and the symptoms which led one to treatment, as

soon as those symptoms are eliminated by the treatment.

The problem of compliance with naltrexone treatment has led to

two basic philosophies underlying the clinical use of naltrexone.

Naltrexone has been conceived as a maintenance drug which an individual takes continuously for a year or two or perhaps indefinitely. Naltrexone has also been conceived as a crisis medication to be

used by individuals whenever they feel at risk of resuming heroin

use. Maintenance on naltrexone appears to be essential for the majority of patients. As a maintenance drug, naltrexone offers protection against the impulsive drug use which can easily lead to the

resumption of chronic heroin dependence. On the other hand, when

there are few incentives to continue naltrexone use, the problem of

compliance often interferes with maintenance. Thus, from a practical point of view, only a minority of patients will use naltrexone as

a maintenance drug. Most patients tend to drop out of treatment
20 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

prematurely, but many return requesting a resumption of naltrexone treatment when they experience themselves relapsing. The

problem of compliance has encouraged the free use of clinics and

naltrexone for support during crises. However, the recognition of

the danger of relapse requires a level of maturity, self-reliance, and

insight which is not characteristic of most patients.

THE FUTURE OF NALTREXONE

The immediate future of naltrexone will be the completion of

Phase III clinical trials and the eventual marketing of the drug for

general use. However, even with the marketing of naltrexone, several important research issues will remain.

We must improve our ability to select patients for naltrexone

treatment. This is part of a broader issue which includes all forms

of opiate addiction treatment. We are still unable to characterize

those individuals who do best with the currently available treatment modalities.

An exciting area of research will be on the effects of naltrexone

on the endorphin system. The fact that in the Phase II double-blind

study, naltrexone appeared to decrease craving suggests that it

may have a “healing” effect on the endorphin system. It may well

be that endorphins serve an important function in the regulation

of mood and sense of well-being. It also seems possible that prolonged use of heroin may atrophy this system and the subsequent

disruption in mood and sense of well-being may be the basis of prolonged craving in addition to the known pharmacologic phenomenon of prolonged abstinence. It would be of the utmost importance

if naltrexone proved to be not only a protection against impulsive

heroin use, but also directly beneficial by helping to reverse heroininduced atrophy in the endorphin system.

Along with the development of oral naltrexone, sustained action

preparations are also being developed. These are injectables which

can be removed if necessary. Their planned duration of action will

be approximately one month. However, none has been tested in

man as yet. Sustained action preparations have been regarded as a

solution to the problem of compliance; but, even monthly injections

will require patient compliance. Their most serious disadvantage is

that they will block narcotic analgesia and therefore must be removable or overridable in the event of severe pain (16).
CURRENT STATUS OF NALTREXONE TREATMENT 21

CONCLUSION

The immediate future of naltrexone will be the completion of

clinical testing and the marketing of naltrexone for general use.

However, broader research questions also remain. Who are those

patients likely to benefit from narcotic antagonist treatment? Is

naltrexone the “ideal” antagonist? Naltrexone was chosen for its

lack of agonistic properties which were experienced as dysphoric

side effects in the case of cyclazocine. But, would an antagonist

with some reinforcing euphoric agonistic properties be more acceptable to patients and, therefore, effective in a larger population of

patients? Does a pure antagonist such as naltrexone have a “healing” action on the disequilibrated endorphin system? What are the

effects of naltrexone on protracted abstinence? Is naltrexone best

viewed as a maintenance or a crisis drug? Answers to these questions will come as this important technical advance is used more

widely by thoughtful clinicians.

REFERENCES

1. A. Wikler. Recent progress in research on the neurophysiologic basis of morphine

addiction. Am. J. Psychiat., 105, 329-338 (1948).

2. A. Wikler. Conditioning factors in opiate addiciton and relapse. In D.I. Wilner

and G.G. Kassenbaum, (eds.), Narcotics. McGraw Hill, New York, 85100 (1965).

3. W.R. Martin, C.W. Gorodetxky, and T.K. McLane. An experimental study in the

treatment of narcotic addicts with cyclazocine. Clin. Pharm. Therap., 7, 455-465

(1966).

4. M. Fink, A. Zaks, R. Sharoff, A. Mora, A. Bruner, S. Levit, and A.M. Freedman.

Naloxone in heroin dependence. Clin. Pharm. Themp., 9, 568-577 (1968).

5. A. Zaks, T. Jones, M. Fink, and A.M. Freedman. Naloxone treatment of opiate

dependence: A progress report. JAMA, 215, 2108-2110 (1971).

6. P. Pierson, R. Rapkin, and H. Kleber. Naloxone in the treatment of the young

heroin abuser. Am. J. Drug Alcohol Abuse, 1, 243-252 (1974).

7. A. Freedman, M. Fink, R. Sharoff, and A. Zaks. Clinical studies of cyclaxocine in

the treatment of narcotic addiction. Am. J. Psychiat., 124, 1499-1504, (1968).

8. R. Resnick, M. Fink, and A.M. Freedman. Cyclaxocine therapy of opiate dependence: A progress report. Compr. Psychiat., 12, 491-502 (1971).

9. H. Kleber, K. Kinsella, C. Riordan, S. Greaves, and D. Sweeney. The use of cyclaxocine in treating narcotic addicts in a low-intervention setting. Arch. Gen. Psychiat., 30, 37-42 (1974).

10. W.R. Martin, D.R. Jasinski, and P.A. Mansky. Naltrexone, an antagonist for the

treatment of heroin dependence, effects in man. Arch. Gen. Psychiat., 28, 784-

791 (1973).

11. C.P. O’Brien, R.G. Greenstein, J. Mintz, and G.E. Woody. Clinical experience

with naltrexone. Am. J. Drug Alcohol Abuse, 2, 365-377 (1975).

12. R. Resnick, J. Volavka, A. Freedman, and M. Thomas. Studies of EN-1638A (naltrexone): A new narcotic antagonist. Am. J. Psychiat., 131, 646-650 (1974).
22 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

13. M. Thomas, F. Kauders, M. Harris, J. Cooperstein, G. Hough, and R. Resnick.

Clinical experience with naltrexone in 370 detoxified addicts. In D. Julius and

P. Renault (eds.), Narcotic Antagonists: Naltrexone. National Institute on Drug

Abuse Research Monograph 9. DHEW Pub. No. (ADM)76-387. Supt. of Docs.,

U.S. Govt. Print. Off., Washington, DC., 89 (1976).

14. L. Brahen, V. Wiechert, and T. Capone. Narcotic antagonist treatment of the

criminal justice patient. op. cit., p.93.

15. W.R. Martin and D.R. Jasinski. Physiological parameters of morphine dependence in man: tolerance, early abstinence, protracted abstinence. J. Psychiat.

Res., 7, 9-17 (1969).

ACKNOWLEDGMENT

This article was originally published in the Bulletin on Narcotics,

Volume 30(2), April-June 1978, pp. 21-29.

AUTHOR

Pierre F. Renault*

Chief, Clinical-Behavioral Branch

Division of Research

National Institute on Drug Abuse

Rockville, Md. 20857

*current address:

Associate Director

Division of Medical and Scientific Evaluations

National Center for Health Care Technology

Rockville, Maryland 20857
Part II

Analytical Methods

347-877 0 - 81 - 3

Naltrexone: Research Monograph 28

R.E. Willette and G. Barnett, eds.

National Institute on Drug Abuse, 1980

An Electron-Capture Gas

Chromatographic Assay

for Naltrexone in Biological

Fluids

R. H. Reuning, S. B. Ashcraft, and B. E. Morrison

The electron-capture gas chromatographic assay for naltrexone is an adaptation of the method published originally by

Sams and Malspeis (1). The current methodology, described

in detail, consists of an extraction procedure, derivatization

to form an electron-capturing triester, and gas chromatography using an OV-17 column. Extraction efficiencies indicate that either benzene or 0.25% butanol in cyclohexane

used as the organic phase yields maximal extraction of naltrexone and minimal extraction of most naltrexone metabolites. Methylene chloride, on the other hand, yields an optimal combination of clean chromatograms and high extraction efficiencies for both naltrexone and its metabolites.

Derivatization with either heptafluorobutyric anhydride or

pentafluoropropionic anhydride together with a basic catalyst yields a triester derivative. Use of an OV-17 chromatographic column with electron-capture detection permits

assay of naltrexone specifically with respect to known metabolites. One minor metabolite, 2-hydroxy-3-O-methyl-ß-naltrexol could interfere with naltrexone quantitation if present in sufficient quantities. This interference could readily

be detected if it were to occur. Data on the reproducibility

of this assay procedure indicate that it is sensitive to a concentration of 0.25 ng/ml plasma.

25
26 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

INTRODUCTION

The growth of knowledge concerning naltrexone metabolism and

pharmacokinetics has been dependent on the ability to assay naltrexone and its metabolites in biological fluids in a specific

manner. The relatively high potency and extensive degree of metabolism of this compound present a significant analytical challenge in terms of the sensitivity and specificity needed for unambiguous metabolic and pharmacokinetic studies. It was recognized

early that only a few techniques were likely to have the necessary

sensitivity and specificity; these being radiotracer assay, electroncapture gas chromatography (GLC-EC) and mass fragmentography.

The purpose of this report is to summarize several years of experience with a GLC-EC assay for naltrexone developed originally by

Sams and Malspeis (1). The current assay procedure will be described in detail in the Experimental section and research connected with each phase will then be reviewed.

EXPERIMENTAL

Reagents and Glassware

Naltrexone reference standard, naloxone reference standard and

naltrexone hydrochloride were obtained from Endo Laboratories

(Garden City, NY) and used without further treatment. Pentafluoropropionic anhydride (PFPA), obtained from PCR, Inc. (Gainesville,

FL), was redistilled if derivatization and GLC-EC analysis of naltrexone and related compounds yielded unsymmetrical peaks or

blank peaks which would interfere chromatographically. When

necessary, PFPA was redistilled into 0.5°C fractions between 70°

and 74°C. The purity of each fraction was checked by the above

procedure. The catalyst 4-dimethylaminopyridine (DMAP), from

Tridom/Fluka (Montreal, Canada), was recrystallized at least four

times from benzene.

High purity “Distilled-in-Glass” solvents (Burdick-Jackson Laboratories, Inc., Muskegon, MI) were used for extraction and stock solution preparation and all other chemicals were reagent grade.

Glassware was soaked overnight in sulfuric acid-nitric acid (4:1),

washed with Alconox and rinsed with distilled water. After drying

at 110° for several hours the glassware was dipped in a 1% solution

of Dri-film SC-87 (Pierce Chemical Co., Rockford, IL) in toluene for

1 minute and then dried again at 110° for several hours. The glassware was then washed again, rinsed with double distilled, demineralized water, and dried.
NALTREXONE ELECTRON-CAPTURE ASSAY 27

Extraction

Naltrexone assays are generally carried out in either a 10-100

ng/ml range or a 1-10 ng/ml range with differing standard curves

for each range. An appropriate amount of a solution of naloxone

internal standard (60 ng or 6 ng, respectively) in methanol is

placed in each screw-capped test tube, and the methanol is evaporated under mild heat and nitrogen. Samples of plasma or urine

containing naltrexone, generally 0.5 ml for the high concentration

range and 2.0 ml for the low range, are then added. When dilution

is needed for samples to be assayed within the range of the standard curve, plasma samples are diluted appropriately with blank

plasma and urine samples with distilled water. The sample is then

supplemented with 0.5 g sodium chloride (1.0 g if the 2.0 ml sample

volume is used), 2.5 ml phosphate buffer, pH 10.4 (2), and 5.5 ml

organic solvent (benzene, ethyl acetate, methylene chloride or

0.25% (v/v) butanol in cyclohexane). After shaking for 15 minutes

and centrifuging, the organic layer is transferred to a tube containing 1 ml 0.1 N sulfuric acid. After shaking and centrifuging, the

organic layer is discarded. The acid is then washed with 5.5 ml organic solvent and the organic phase is aspirated off. The acid layer

is supplemented with 0.5 g sodium chloride and 2.5 ml of the same

phosphate buffer and this mixture is then extracted with 5.5 ml organic solvent. The organic solvent is transferred to another tube

and evaporated to dryness. The uncapped tubes are then placed in

a vacuum desiccator until further analysis (at least 16 hr).

Derivatization and Gas Chromatography

The residue in each tube from the extraction procedure is dissolved in 50 μl benzene containing 1% DMAP and then treated

with 25 μl PFPA for 1 hr. at 68” in an oil bath heating block (LabLine Instruments, Inc., Melrose Park, IL). After cooling in an ice

bath the excess reagents are removed by adding 5 ml of a saturated

solution of sodium tetraborate, rotating on a Labquake mixer (Labindustries, Inc., Berkeley, CA) for 3 min and then centrifuging for

4 min. When a series of samples is assayed, each sample is washed

with the sodium tetraborate individually just prior to injection of a

1-3 μ1 aliquot of the separated benzene phase onto the gas chromatographic column.

The naltrexone derivatives are chromatographed on a HewlettPackard (Palo Alto, CA) 5713A gas chromatograph with a 63Ni-electron-capture detector. The coiled glass column, 2 mm internal diameter and 2.4 m in length, is packed with 3% OV-7 on 100-120

mesh Gas Chrom Q (Applied Science Laboratories, State College,
28 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

PA). The temperature settings are: oven, 203°; injection port, 250°;

and detector 300°. The argon-methane (95:5) carrier gas flow rate is

about 28 ml/min.

Standard Curve

A standard curve is run on each day of analysis. Prior to the initial addition of blank plasma or urine, appropriate known amounts

of naltrexone in methanol are added to five separate tubes so that

a standard curve over the desired concentration range is obtained.

These standards are then supplemented with plasma or urine and

assayed in the same run with the experimental plasma and urine

samples. Blank plasma or urine samples are also carried through

the assay procedure. Standard curves are constructed by plotting

the peak height ratio (naltrexone/naloxone) as a function of the

amount of naltrexone added.

Assay of ß-naltrexol and Conjugated Metabolites

The naltrexone metabolite, ß-naltrexol, can also be assayed in

plasma or urine by the procedure just described. One of the more

polar extracting solvents is necessary and the standard curves for

both naltrexone and ß-naltrexol are obtained from samples containing known amounts of both compounds.

Conjugates of naltrexone and ß-naltrexol can be determined by

assay of the sum of the levels of either compound plus its respective conjugates and then subtracting the appropriate assayed value

for unconjugated naltrexone or ß-naltrexol (3). When assaying for

the total of conjugated and unconjugated compound, urine or

plasma samples are incubated at 37° for 24 hrs with 2 volumes of

Gluculase (Endo Laboratories, Garden City, NY) that has been diluted 1:10 with 0.2 M acetate buffer, pH 5.0 (4). Experiments with

samples of plasma and urine (dog and monkey) containing naltrexone conjugates have shown that neither longer incubation time nor

higher enzyme concentration results in any further conjugate hydrolysis.

RESULTS AND DISCUSSION

Standard Curves

Linear standard curves with very high correlation coefficients

(r 0.998) and essentially zero intercepts are generally obtained

when naltrexone in either the 10-100 ng/ml or the 1-10 ng/ml

range is assayed in plasma or urine from monkey, dog and rabbit.
NALTREXONE ELECTRON-CAPTURE ASSAY 29

The sensitivity limit for naltrexone is less than 1 ng/ml. ß-naltrexol yields similar standard curves except that the correlation coefficient is often lower (r 0.980) and the sensitivity limit is about 2

ng/ml. A typical chromatogram (figure 1) illustrates that blank inFIGURE 1. Gas chromatogram of an extracted and PFPA-derivatized plasma sample

(monkey) to which 30 ng naloxone, 3.5 ng ß-naltrexol and 35 ng naltrexone were

added. A is the naloxone (internal standard) derivative, and B and C are the ßnaltrexol and naltrexone derivatives, respectively.
30 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

terference is generally negligible and that both naltrexone and ßnaltrexol can be quantitated from a single chromatogram.

Derivatization and Gas Chromatography

It has been shown previously (1,3) by mass and infrared spectrometry that derivatization of naltrexone with either PFPA or

heptafluorobutyric anhydride (HFBA) in the presence of a basic

catalyst yields the triester derivative, presumably at the 3, 6 and

14 positions (note scheme 1). This complete derivatization provides

maximal electron-capture sensitivity and avoids potential assay

error due to varying degrees of incomplete derivatization. In the

presence of catalyst, derivatization times longer than 1 hr do not

result in any further esterification (1). Available evidence would

suggest, however, that there is incomplete esterification if the catalyst is not added (1). Of those catalysts that have been tried (e.g.,

several concentrations of pyridine, triethylamine, and DMAP) the

1% DMAP appears to be optimal in that it yields both complete

derivatization and chromatograms with minimal blank interference. It is important, however, that the DMAP be recrystallized

several times in order to obtain chromatograms with low blank interference. Chromatograms from PFPA derivatization generally

have less blank interference than those from HFBA.

Once the ester derivatives of naltrexone are formed, the excess

anhydride and catalyst must be removed. Because of the presence

of catalyst, it is not possible to do this by evaporation. Thus, the

borate wash is used to remove both the anhydride and catalyst.

Sams and Malspeis (1) have shown that both the

pentafluoropropionate and the heptafluorobutyrate derivatives are

sufficiently unstable after the borate wash that samples should be

chromatographed as soon as possible. Thus, in the procedure described in the Experimental section each sample is individually
NALTREXONE ELECTRON-CAPTURE ASSAY 31

washed with the borate solution, separated and chromatographed

immediately. This procedure is necessary to obtain reproducible results with the naltrexone assay described herein. The instability of

the derivatives after the borate wash precludes automation of the

chromatography portion of the assay procedure.

Chromatographic conditions have been optimized for sensitivity

and for specificity with respect to known naltrexone metabolites.

Various percentages of the following column packing materials

have been tried; OV-1, OV-17 and SE-30 on Gas Chrom Q (Applied

Science Laboratories, State College, PA). Various temperature settings for the oven as well as temperature programming have also

been attempted (1). The best separation of derivatized naltrexone

and its known metabolites was obtained with a 2.4 m column

packed with 3% OV-17, together with the other conditions described in the Experimental section. An illustration of the results

obtained when known amounts of derivatized naltrexone and its

unconjugated metabolites are chromatographed is shown in figure

2. The retention times are as follows: naloxone (internal standard),

5.1 min; ß-naltrexol, 7.2 min; -naltrexol, 7.8 min; 2-hydroxy-3-Omethyl-ß-naltrexol, 7.9 min; naltrexone 8.3 min; 2-hydroxy-3-Omethylnaltrexone, 9.0 min; 3-O-methyl-ß-naltrexol, 12.1 min. The

derivatized a-naltrexol was omitted from figure 2 because the retention time was so close to that of derivatized 2-hydroxy-3-Omethyl-ß-naltrexol.

In monkey the only metabolite that has been found to be present

in appreciable quantities in blood and urine is ß-naltrexol. This is

fortunate since relatively high levels of 2-hydroxy-3-O-methyl-ß-naltrexol could potentially interfere with the naltrexone assay. Studies in which standards containing both of these compounds in varying ratios were derivatized and chromatographed indicate that

when the peak height ratio of naltrexone to 2-hydroxy-3-O-methylß-naltrexol is 5:1 the error in the assay of naltrexone is less than

3%. If the ratio is 2:1 the error is 9% and a ratio of 1:1 yields an

error of 21% in the naltrexone assay (5). However, this metabolite

appears to be a minor metabolite in man (6) and has not been observed in significant amounts in plasma samples from monkeys administered single intravenous or oral doses of naltrexone nor in

plasma or urine samples from dogs administered naltrexone intravenously. In an unusual application of this assay, a peak corresponding to the retention time of derivatized 2-hydroxy-3-O-methylß-naltrexol was observed in the assay of 1- and 24 hr plasma samples from monkeys dosed chronically with oral naltrexone, 1 to 20

mg/kg/day, for 52 weeks (7).
32 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

FIGURE 2. Gas chromatogram of a derivatized sample spiked with naltrexone,

naltrexone metabolites and internal standard. A = naloxone (internal standard), B

= ß-naltrexol, C = 2-hydroxy3-O-methyl-ß-naltrexol, D = naltrexone, E = 2-

hydroxy-3-O-methylnaltrexone, F = 3-O-methyl-ß-naltrexol. The dashed curve

represents derivatized blank solvent.

Extraction

Organic solvents that have been investigated for the extraction

procedure include benzene, methylene chloride, ethyl acetate, cyclohexane, 0.5%, 0.75%, 1%, 2%, 5%, 10% and 15% (v/v) isopropanol in cyclohexane, and 0.1%, 0.25% and 0.5% (v/v) n-butanol in

cyclohexane. Of these solvents, the four that yield an optimum

combination of high naltrexone extraction efficiency and a low

degree of blank interference are benzene, methylene chloride, ethyl

acetate and 0.25% (v/v) n-butanol in cyclohexane. Although ethyl

acetate has been used extensively in previous investigations, it has

been abandoned because of occasional blank interfence, variability.
TABLE 1. Extraction Efficiencies of Naltrexone and its Metabolites in the Naltrexone Assay Procedure
34 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

in duplicate assays, and low correlation coefficients for the standard curve.

Extraction efficiencies have been reported in detail elsewhere

(3,4,5) and only mean values for the final organic solvents chosen

are reported in table 1. It is evident from these results that ethyl

acetate and methylene chloride yield very high extraction efficiencies for both naltrexone and its metabolites. Thus, since methylene

chloride yields less day-to-day variability in assay performance, this

solvent appears to be optimal when assays for both naltrexone and

ß-naltrexol and perhaps other metabolites are desired. The solvent

that is most selective for unchanged naltrexone is 0.25% (v/v) butanol in cyclohexane. This solvent is particularly advantageous with

respect to the metabolite 2-hydroxy-3-O-methyl-ß-naltrexol which

could interfere with naltrexone quantitation if it were present in a

sufficiently high ratio to the naltrexone level. Morphine, which

may be present in biological samples from certain types of studies

on naltrexone (such as sustained release naltrexone evaluation in

self-administering monkeys), is not extracted by either benzene or

0.25% (v/v) butanol in cyclohexane (5).

General Aspects

Because of the requirement of sub-nanogram sensitivity for many

applications of this assay, several tedious and exacting procedures

are necessary. A troubleshooting list for the procedure could include the following: impure derivatizing agent, impure catalyst, solvents not of the highest quality available, deteriorated coating solution for the glassware, deterioration of organic extracting solvents

presumably due to absorption of water, insufficient drying of the

residue from the extraction procedure prior to derivatization, and

deterioration of chromatographic columns with repeated use. HowTABLE 2. Accuracy and Reproducibility of the

Naltrexone Assay Procedure

Concentration of Mean naltrexone Coefficient

Naltrexone added Concentration o f

to plasma (ng/ml) assayed (ng /ml)a

variation (%)

20° 20.0(10)

5

c

4.93(10)

0.5c

0.50(8)

0.25c

0.26(9)

aNumber of replicates in parentheses.

bOne-half ml monkey plasma was used.

c

Two ml monkey plasma was used.

4.6

9.0

9.1

21.5
NALTREXONE ELECTRONCAPTURE ASSAY 35

ever, patience and diligent attention to detail can result in the accuracy and reproducibility exemplified in table 2. Since most

assays are done in duplicate, the use of the assay on experimental

samples should be even better than the results in table 2 suggest.

Thus, for most applications the sensitivity limit of this assay is 0.25

ng/ml for naltrexone in plasma.

REFERENCES

1. R.A. Sams and L. Malspeis. Determination of naloxone and naltrexone as perfluoroalkyl ester derivatives by electron-capture gas-liquid chromatography. J.

Chromatogr., 125, 409-420 (1976).

2. S.J. Mule. Determination of narcotic analgesics in human biological materials.

Anal. Chem., 36, 1907-1914 (1964).

3. R.H. Reuning, V.K. Batra, T.M. Ludden, M.Y. Jao, B.E. Morrison, D.A. McCarthy,

SE. Harrigan, S.B. Ashcraft, R.A. Sams, MS. Bathala, A.E. Staubus and L.

Malspeis. Plasma naltrexone kinetics after intravenous bolus administration in

dogs and monkeys. J Pharm. Sci., 68, 411-416 (1979).

4. L. Malspeis, M.S. Bathala, T.M. Ludden, H.B. Bhat, S.G. Frank, T.D. Sokoloski,

B.E. Morrison and R.H. Reuning. Metabolic reduction of naltrexone I. Synthesis, separation and characterization of naloxone and naltrexone reduction products and qualitative assay of urine and bile following administration of naltrexone, a-naltrexol, or ß-naltrexol. Res. Commun. Chem. Pathol. Pharmacol.. 12,

43-65 (1975).

5. R.H. Reuning. Testing of drug delivery systems for use in the treatment of narcotic addiction. Quarterly report no. 21, NIDA contract HSM-42-73-182, pp. 4-13

(1978).

6. M. Wall. Personal communication.

‘7. R.H. Reuning. Testing of drug delivery systems for use in the treatment of narcotic addiction. Quarterly report no. 14, NIDA contract HSM-42-73-182, pp. 37-42

(1977).

ACKNOWLEDGMENT

Supported in part by contract HSM-42-73-182 from The National

Institute on Drug Abuse. We are indebted to Dr. M. Wall, Research

Triangle Institute, Research Triangle Park, NC, for providing samples of 2-hydroxy-3-O-methyl-ß-naltrexol, 2-hydroxy-3-O-methylnaltrexone and 3-O-methyl-ß-naltrexol. The authors wish to acknowledge the advice of Dr. Richard A. Sams, College of Veterinary

Medicine, The Ohio State University.

AUTHORS

R. H. Reuning, S. B. Ashcraft, B. E. Morrison

College of Pharmacy

Ohio State University, Columbus, Ohio 43210
Naltrexone: Research Monograph 28

R. E. Willette and G. Barnett, eds.

National Institute on Drug Abuse, 1980

Quantitative Determination

of Naltrexone, 6ß-Naltrexol

and 2-Hydroxy-3-Methoxy6ß-Naltrexol (HMN) in

Human Plasma, Red Blood

Cells, Saliva and Urine by

Gas Liquid

Chromatography

Karl Verebey

Two gas liquid chromatographic methods differing mainly

in sensitivity are described for the quantitative determination of naltrexone (NT) and its metabolites in human biofluids. Flame ionization detection of the N,O-bis-(trimethylsilyl) trifluoroacetamide (BSTFA) derivatives provided sufficient separation and sensitivity for quantitation of the

bases in urine. However, the thousand times lower levels in

serum, red blood cells (RBC) and saliva necessitated the use

of more sensitive electron capture detection methods of the

pentafluoro derivatives of NT and its metabolites. The 2-hydroxy-3-methoxy-6ß-naltrexol (HMN) and 6ß-naltrexol (ßOL) pentafluoro derivatives had nearly identical gas liquid

chromatographic retention times in a number of stationary

liquid phases. Thus, their se ration had to be achieved

prior to chromatography. Differential extraction was based

on the different partition characteristics of HMN and 6ßnaltrexol between aqueous and organic solvents. Applicabil36
QUANTITATIVE DETERMINATION OF NALTREXONE 37

ity of the methods was tested using the biofluids of four

subjects taking 2 x 200 mg naltrexone per day chronically.

Blood, saliva and urine samples were collected at the same

time (prior to drug administration), which was 16 and 24

hours after the doses. In the plasma the relative percentages of the bases were 73.5 ß-OL; 23.1 HMN and 3.4 NT.

The same in the urine were 76.6 ß-OL, 14.4 HMN and 9.0

NT The lipophilic nature of HMN and the hydrophilic

property of ß-OL may have influenced their distribution

into RBC and saliva. In the RBC 96.1% HMN and only

trace amounts of ß-OL distributed and in saliva 92.3% of

ß-SOL and no HMN was found; the difference in both cases

was made up by naltrexone to 100%.

INTRODUCTION

Naltrexone was already administered to men during preliminary

clinical trials (1,2) while methods were not yet available for the

monitoring of the drug and its metabolites in human biofluids. The

isolation and tentative identification of 6ß-naltrexol, the major urinary biotransformation product in humans, was first reported by

Cone in 1973 (3). Methodological advancement soon followed, resulting in the description of the urinary excretion profile of naltrexone

in man by Cone et al., Verebey et al., and Dayton and Inturrisi

(4,5,6). In 1975, Cone and Gorodetzky reported the presence of noroxymorphone, an N-dealkylated, strongly agonistic metabolite and

a methylated 2,3-catechol type metabolite of naltrexone in human

urine (7). Also in 1975, Verebey et al. described 2-hydroxy-3-methoxy-6ß-naltrexol (HMN), a minor metabolite of naltrexone isolated

from human urine and red blood cells (8). Determination of naltrexone and its metabolites in plasma was far more difficult because the concentration of the bases is approximately a thousandfold less than the same in urine. In 1976 Verebey et al. described a

quantitative method for the determination of naltrexone and 6ßnaltrexol in human plasma utilizing electron capture detection of

the pentafluoro derivatives of naltrexone and 6ß-naltrexol (9). Utilizing this technique the total disposition and most of the pharmacokinetics of naltrexone were determined in man by Verebey et al.,

following acute and chronic doses of naltrexone (10). Sams and Malspeis (11) and Sams (12) provided extensive data on the nature of

electrophore formation of naltrexone and naloxone for electron

capture detection. Naloxone is often used as an internal standard

for the quantitation of naltrexone. Later, naltrexone prodrugs were

synthesized for extended opiate receptor blockade through various

slow release drug delivery systems. Bruce et al., utilizing electron
38 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

capture gas chromatographic techniques, were able to quantitate

eleven ester and ether derivatives of the prodrugs (13).

More recently the structure of HMN was confirmed by synthesis

(14) and by nuclear magnetic resonance spectra (15). The availability of synthetic HMN has also encouraged methodological advances. In this communication methods are described for the quantitation of naltrexone, 6ß-naltrexol and HMN in plasma (or serum),

red blood cells, saliva and urine (16). It is the intention of this

report to concentrate on the difficulties and shortcomings of current methodology.

MATERIALS AND METHODS

Chemicals and Reagents

Analytical grade naltrexone, 6ß-naltrexol and 2-hydroxy-3-methoxy-6ß-naltrexol were provided by NIDA. Etorphine, the internal

standard for the urine method, was purchased from the American

Cyanamid Co., (Princeton, NJ) and nalorphine, the internal standard for the plasma method, was obtained from Merck & Co., Inc.

(Rahway, NJ). The derivatizing reagent, pentafluoropropionic anhydride (PFPA), glassware siliconizing agent, Dri-Film SC 87, and

column conditioner, Silyl 8, were purchased from Pierce Chemical

Co. (Rockford, IL) and N,O-bis-(trimethylsilyl) trifluoroacetamide

(BSTFA) was purchased from Regis Chemical Co. (Morton Grove,

IL). The organic solvents used in the procedure were all glass distilled, G.C. grade, purchased from Burdick and Jackson (Muskegon,

MI). Glusulase was purchased from Endo Laboratories (Garden

City, NY). All aqueous solutions, buffers and dilute acids were prepared in glass distilled water, and all glassware was siliconized

using 5% Dri-Film SC 87 in toluene.

Extraction Procedure (Urine)

After large doses of naltrexone, greater than 400 mg daily, 6ßnaltrexol levels were so much greater than naltrexone and HMN

levels that a separate extraction procedure was needed for the recovery and quantitation of naltrexone and HMN. This was accomplished by utilizing a pH (7.5) lower than the optimum for 6ß-naltrexol recovery and a nonpolar organic solvent (benzene). The

method is summarized in table 1. For the routine assay of naltrexone and HMN 1 to 2 ml of urine was used, while for the determination of 6ß-naltrexol 0.1 to 0.3 ml urine was adequate. Etorphine

was added to the urine samples: 2 μg for the naltrexone and HMN

assays and 20 μg for the 6ß-naltrexol assay.
QUANTITATIVE DETERMINATION OF NALTREXONE 39

TABLE 1

The urines were buffered to pH 8.5 using 0.5 ml of 0.2 M carbonate-bicarbonate buffer and the aqueous solution was extracted with

7.0 ml chloroform. The chloroform was back extracted into 5.0 ml

of 0.5 N HCl. The pH of the aqueous layer was adjusted to 8.5 and

re-extracted into 7.0 ml chloroform. The extractions at each step

consisted of 10 min shaking in a mechanical shaker and 5 min centrifugation. The chloroform extracts were evaporated to dryness in

a rotary evaporator under vacuum at 50” (Evapomix, Buchler, Fort

Lee, NJ).

Calibration Curves and Quantitation (Urine)

Standard solutions of NT (20 μg/ml), HMN (50 μg/ml) and etorphine (20 and 200 μg/ml) were prepared in water. Standard curves

for NT and HMN were prepared by adding 0.5, 1.0, 1.5 and 2.0 μg

347-877 0 - 81 - 4
40 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

of naltrexone, 5 to 30 μg of HMN and 2 μg of etorphine to each 2.0

ml of blank urine samples.

The standard curve for ß-OL was prepared separately, 3, 6, 9 and

12 μg ß-OL and 20 μg of etorphine was added to each 0.2 ml blank

urine. The standards were extracted, derivatized and chromatographed as the unknown samples.

Glusulase Hydrolysis

The concentration of urinary naltrexone and 6ß-naltrexol was

determined before (free) and after hydrolysis (total) while values of

HMN in both systems were equal, indicating that no conjugation of

HMN occurs (10). Thus HMN quantitation can be performed with

non-hydrolyzed samples. A typical hydrolysis and extraction system

is outlined in table 1.

Derivatization Procedure (Urine)

The tubes were sealed with rubber caps, flushed with dry nitrogen, BSTFA (20-30 μl) was added, mixed on a Vortex mixer for 5

sec and the reaction allowed to proceed in a dry heating block containing sea sand at 60°C for 15 min. Following the reaction 1 μl of

BSTFA containing silylated bases was injected into the gas chromatograph.

Gas-Liquid Chromatographic Conditions (Urine)

The gas chromatographic analysis was performed on a PerkinElmer 900 gas chromatograph, equipped with a hydrogen flame

ionization detector. The column was a 2 m x 2 mm I.D. glass

spiral. The packing consisted of 3% OV-17 on Gas-Chrom Q, 80-100

mesh. The temperature of the detector and flash heater was 285°.

The carrier was helium with a flow-rate of 60 ml/min. The flowrates of hydrogen and air were 30 ml/min and 300 ml/min, respectively. The column oven temperature was 270°.

Extraction Procedure (Plasma, Serum, RBC and Saliva)

An extraction outline of HMN and NT is given in table 2. HMN

was extracted into benzene which excluded 6ß-naltrexol while the

combined 6ß-naltrexol-HMN were determined using a modified version of the original method (9).

The following substances were added to 20 ml round-bottom centrifuge tubes: Antifoam A (A.H. Thomas, Philadelphia, PA), 0.2 to 2

ml of plasma and an aqueous internal standard (nalorphine) containing 12.5 ng for the chloroform and 25 ng for the benzene procedure. The pH was adjusted to 7.5 with 1.0 ml tris-maleate buffer
QUANTITATIVE DETERMINATION OF NALTREXONE 41

TABLE 2

and the aqueous phase was shaken with 10 ml of benzene for HMN

or chloroform for naltrexone and combined 6ß-naltrexol-HMN determination. After 10 min shaking and 5 min centrifugation the organic layer was transferred into clean centrifuge tubes containing

5 ml of 0.1 N H2SO4. After back extraction into acid the organic

layer was discarded and 4.3 ml of the H2SO4 phase was transferred

into clean centrifuge tubes and neutralized with 1.0 N NaOH. The

pH was adjusted to 7.5 with tris maleate buffer and the aqueous

phase extracted with 7 ml of benzene for HMN or chloroform for

naltrexone and 6ß-naltrexol containing HMN. After shaking and

centrifugation the organic phase was transferred to a clean 15-ml

test tube and evaporated to dryness on a rotary flash evaporator.

One ml of acetone was added to the benzene phase to facilitate its

evaporation.
42 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

Calibration Curves and Quantitation (Plasma)

Naltrexone and 6ß-naltrexol standard curves were prepared by

adding 2.5, 5.0, 10.0 and 20.0 ng of naltrexone; 5, 10, 20 and 40 ng of

6ß-naltrexol and 12.5 ng of nalorphine to each of 1.0 ml of blank

plasma. For the HMN standards, 5, 10, 20 and 40 ng of HMN with

25 ng nalorphine were added to 1.0 ml of blank plasma. The samples for both standard curves were extracted according to the appropriate method described (benzene or chloroform), derivatized by

PFPA and chromatographed. The peak height or area ratios were

plotted against the respective concentration of standards and the

slope factors determined. The unknown sample peak height or area

ratio was divided by the slope factors of the standards to determine

the concentations of naltrexone, 6ß-naltrexol, and HMN in the unknown samples.

Derivatization Procedure (Plasma)

To the dry residue 150 μl of PFPA was added, the tube was tightly stoppered (00 size hollow Nalgene stoppers; A.H. Thomas) and

placed in a heating block at 110° for 40 min. After the reaction the

samples were stored overnight at -16°C and usually analyzed the

following day. The anhydride was evaporated at room temperature

under a stream of nitrogen. The dried sample was taken up in 50

μl of ethyl acetate and 1 μl aliquots were injected immediately into

the gas chromatograph. At this step one should proceed at the rate

of one sample at a time because the derivatives are not stable in

the ethyl acetate or in any other solvent tested.

Gas-Liquid Chromatographic Conditions (Plasma)

Isothermal conditions were used with a Hewlett-Packard Model

5830A gas chromatograph, equipped with a 63Ni linear electron capture detector. The column was 2 m x 2 mm I.D. packed with 3%

OV-22 on Supelcoport, 80-100 mesh. The carrier gas was a mixture

of 10% methane in argon at a flow-rate of 35 ml/min. The injection

port temperature was 240°C, the detector temperature 300°C and

the column oven temperature 220°C.

Methodological Cautions

A list of suggestions follows; the observations are based on four

years of experience and sometimes frustrations. Implementation of

some or all of these suggestions may determine the success or failure of the adaptation of these methods.
QUANTITATIVE DETERMINATION OF NALTREXONE 43

1. Treat all tubes with siliconizing reagent. Before use,

rinse all test tubes with glass-distilled chloroform.

2. During the extraction procedure, to have the cleanest

possible final sample, avoid the close proximity of the

organic-aqueous interface. Contaminants can easily be

transferred while trying to recover all of a phase.

3. Put chloroform into the refrigerator before final evaporation. A little droplet of water forms on top of the

chloroform; aspirate it off before evaporation.

4. A rotary evaporator is far more efficient than an NEVAP type evaporator in concentrating the little

available bases in the tip of the reaction vessel. With

low concentrations, <10 ng naltrexone/ml, rotary

flash evaporator is necessary.

5. Following derivatization while the bases are in PFPA,

they are stable for at least three weeks when kept in

the freezer.

6. After derivatization put all tubes in freezer and evaporate the samples one by one. After reconstitution, each

sample must be injected into the gas chromatograph

immediately or within minutes. In ethylacetate or any

other solvent, the rate of drug-pentafluoro complex decomposition is rapid.

7. After the injection of 100 to 150 samples into the gas

chromatograph the injection port glass wool should be

changed. Deposits on the glass wool selectively bind

one or more of the bases, causing decrease in sensitivity and errors in quantitation. Use siliconized glass

wool only!

8. After changing the injector port glass wool plug, disconnect the column from the detector and inject approximately 40 μl of Silyl 8. Reconnect 15 minutes

later. This helps to prevent adsorption and/or breakdown of the pentafluoro-drug complexes on the

column.

9. Derivatize a mixture of 100 ng/μl of NT, ß-OL, and

nalorphine with PFPA. Before injection of standards

or unknown, prime the column with these large concentrations of bases (attenuate signal while injecting

primer). This also helps prevent on column binding

and breakdown of samples.

Subjects

To verify the modifications in the original methodology the samples for this study were kindly provided by Professor Volavka, The
44 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

Missouri Institute of Psychiatry, University of Missouri School of

Medicine. The subjects were all male chronic schizophrenic patients who received incremental doses from 50 to 400 mg naltrexone in the first week and 400 mg naltrexone daily in the second

week. The 100 mg doses and above were split and given at 8 am

and 4 pm. Since all the samples were collected just prior to the 8

am dose they represent samples 24 hours past the first and 16

hours past the second half of the daily dosage. Two blood, saliva,

and spot urine samples were collected from each patient on the 7th

day of the 1st and 2nd weeks, thus in both cases the patients were

already receiving 2 x 200 mg (400 mg) naltrexone per day.

RESULTS AND DISCUSSION

Figure 1 shows the structural formula of naltrexone and its

major (ß-OL) and minor metabolite (HMN). The recent availability

of synthetic HMN prompted the development of methods for its

quantitation. Determination of HMN in urine is very similar to the

method reported earlier (6). The three drug-related peaks (NT, ßOL, HMN) and the internal standard (etorphine) were well resolved

by gas liquid chromatography (figure 2). The system using chloroform is suitable for the simultaneous measurement of naltrexone

and its metabolites. However, when 400 mg or larger daily doses of

naltrexone are administered, the excessive amount of 6ß-naltrexol

may interfere with the quantitation of HMN and naltrexone. In

such conditions separate extraction systems should be utilized, one

for ß-OL determination (using chloroform) and another for HMN

and naltrexone determination (using benzene) as outlined in table

1. Since ß-OL is known to be hydrophilic, the utilization of a nonpolar solvent (benzene) and a lower than usual pH (7.5) accomplished the exclusion of ß-OL, while NT and HMN were adequately

recovered. Derivatization using BSTFA requires only one methodological caution: the samples must be very dry; even humidity in

the air must be considered. In our laboratory the tubes are sealed

and flushed with nitrogen for 2 to 3 minutes before the addition of

BSTFA, which is injected through the stopper. If samples for some

reason remain wet, at least 2 derivatives of both NT and ß-OL will

form in different proportions resulting in unreliable results.

Naltrexone, 6ß-naltrexol and HMN are approximately a thousand times less in blood and saliva than in urine. For this reason

halogenated derivatives of the bases were formed using PFPA and

thus greater sensitivity was achieved. The electron capture method

is difficult to adopt because of the many cautions involved in extraction, electrophore derivative formation and the gas chromato-
QUANTITATIVE DETERMINATION OF NALTREXONE 45

FIGURE 1. The chemical structures of naltrexone (I) and its major and minor

metabolites in man: 6ß-naltrexol (II) and 2-hydroxy-3-methoxy-6ß-naltrexol (III)

graphic instrumentation. A list of cautions is given in the methods

section. Using the PFPA derivatives, another problem surfaced.

The retention time of HMN (3.48 min) and that of ß-OL (3.38 min)

are separated only by 0.1 minute (figure 3). When both of these

substances are present in unknown samples only one peak is observed (fig. 3, panel C). This observation explains the years of mystery as to why the retention time of ß-OL was shifting 2 to 4 seconds back and forth while NT and the internal standard retention

time remained constant. It is likely that in all previous studies

when PFPA was used as the derivatizing agent, the values reported

for ß-OL are overestimated because they also include HMN.

Many different stationary liquid phases were tried, but no adequate separation between HMN and ß-OL was achieved. The different solubility characteristics of HMN and ß-OL were utilized for

separation of the two bases during extraction. For the extraction of
46 NALTREXONE SUSTAINEDRELEASE PREPARATIONS

FIGURE 2. The gas liquid chromatographic pattern of the BSTFA derivatives of

naltrexone, its metabolites and the internal standard, etorphine, using flame

ionization detection

HMN and for the exclusion of ß-OL benzene was used and the pH

was lowered to 7.5. Under these conditions HMN recovery was 51

± 1.4% while ß-OL was not recovered. Using chloroform, at pH

9.4, resulted in the recovery of NT, ß-OL and HMN. Thus, for ß-OL

determination the HMN levels were first determined using the

benzene procedure and the HMN values subtracted from the results of the chloroform procedure, which determines the combined

ß-OL and HMN levels. An example of chromatographic tracings,
QUANTITATIVE DETERMINATION OF NALTREXONE 47

FIGURE 3. The gas liquid chromatographic pattern of the PFPA derivatives of

naltrexone (NT), 6ß-naltrexol (ß-OL) and 2-hydroxy-3-methoxy-6ß-naltrexol (HMN)

and nalorphine, the internal standard (IS), using electron capture detection. Panel

A is HMN (3.43 min) standard alone; Panel B is ß-OL (3.38 min), NT (3.94 min), and

the IS (6.16 min) added to blank plasma; Panel C shows a chloroform extract of

plasma of a subject taking naltrexone: both HMN and ß-OL are present, indicated

by the intermediate retention time (3.42 min). Panel D shows a chromatographic

pattern of the plasma extract of the same subject as in Panel C, using benzene:

HMN (3.47 min), NT (3.95 min), and IS (6.16 min) are present but ß-OL is not

recovered under these conditions.

one after benzene and another after chloroform extraction of the

same sample, is shown in figure 3. An ideal method should separate all derivatives; however, at present this has not been accomplished.

For the verification of the modified methods, blood samples

(plasma and RBC), saliva and urine were analyzed for the three
48 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

bases, collected from subjects taking 400 mg NT a day. (See exact

doses and sample collection under “Subjects.”) The data compiled

from the analysis of urine samples is shown in table 3. Since it was

a spot sample collected 24 hr after the dose, only the relative abundance of the bases present in the urine at that time can be evaluated. 6ß-naltrexol accounted for 76.6% of the bases, followed by

HMN (14.4%) and naltrexone (9.0%). In the plasma samples the

order of relative abundance was the same as in urine, with similar

proportion of 6ß-naltrexol (73.5%), significantly more HMN (23.1%)

and slightly less naltrexone (3.4%) present (table 4).

TABLE 3. Urinary Levels of Naltrexone, ß-Naltrexol and 2-Hydroxy-3-

Methoxy-6-ß-Naltrexol (HMN) in a Single Spot Sample 16 and 24 Hrs

After 2X200 mg Naltrexol

Base μg/ml

Subject Naltrexone ß-Naltrexol HMN

1 10.3

1 2.5

2 4.5

2 5.4

3 16.7

3 13.9

4 43.2

4 18.1

Mean ± SD 14.3 ± 13.0

% Of Total Base 8.9

152.0

62.8

59.7

49.4

131.3

77.0

262.3

184.5

122.4 ± 74.8

76.7

20.6

9.1

12.5

9.4

49.7

49.2

18.9

14.6

23.0 ± 16.8

14.4

Naltrexone was approximately equally distributed into plasma

and RBC, while the correlation of naltrexone levels in plasma with

that of saliva is similar but not consistent in this group of subjects,

which may be caused by the method of salivary sample collection.

The relationship of plasma and salivary naltrexone and 6ß-naltrexol levels is presently under investigation.

Distribution of HMN and 6ß-naltrexol into RBC and saliva was

very different. In RBC’s, only the more lypophilic HMN distributed

in the extent of 96.1%; the remaining 3.9% was naltrexone, while

6ß-naltrexol was hardly detectable. Conversely, in saliva, HMN

was absent, the polar 6ß-naltrexol comprised 92.3% of the bases

and naltrexone made up the remaining 7.7%. The two- to threefold

excess of HMN in RBC vs. plasma is interesting but its mechanism

is not at all clear.
TABLE 4. Plasma and RBC Levels of Naltrexone, ß-Naltrexol and 2-Hydroxy-3-Methoxy-6-

ß-Naltrexol (HMN) After 2X200 mg Naltrexone Given 24 & 16 Hrs Prior to Sample Collection

Subject

Naltrexone

Plasma

ß-Naltrexol HMN

Base ng/ml

RBC Saliva

Naltrexone HMN NT ß-OL

1 5.6 143.9 42.7 N.S. N.S. 20.6 160.9

1 4.8 185.0 55.8 4.3 183.1 4.2 106.7

2 8.7 174.8 74.6 7.3 188.3 4.6 43.5

2 8.2 182.7 65.0 7.8 201.4 7.3 74.0

3 105* 572.2* 151.3* 115.0* 342.6* 246.9* 342.1*

3 10.9 251.8 86.3 9.3 211.8 8.4 151.2

4 20.4 234.2 53.2 7.3 102.0 50.3 571.6

4 6.9 231.5 62.8 5.0 112.5 2.0 59.1

Mean ± SD 9.4±5.2 200.6 ± 39.1 62.9 ± 14.4 6.8±1.9 166.5 ± 47.1 13.9 ± 17.1 166.7 ± 184

% of Total 3.4 73.5 23.1 3.9 96.1 7.7 92.3

Base

*Sample was drawn after naltrexone dose; values not included in the calculations,

N.S. = no samples available.
50 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

The first blood sample in subject No. 3 (table 4) was taken shortly after naltrexone administration. Thus the levels of drug and metabolites were substantially higher than in the 24-hour samples.

The high naltrexone, 6ß-naltrexol, and HMN plasma levels indicate excellent absorption and rapid first pass biotransformation of

naltrexone, concurring with data reported in an earlier pharmacodynamic study by Verebey et al. (5).

Based on the data in this report, for toxicological purposes, 6ßnaltrexol in plasma, saliva and urine; and HMN in whole blood,

RBC and urine are better indicators of naltrexone use than the

parent compound itself in any of the body fluids, especially when

the samples are collected long after naltrexone ingestion.

REFERENCES

1. W.R. Martin. Naltrexone, an antagonist for the treatment of heroin dependence.

Arch. Gen. Psychiat., 28, 784-791 (1973).

2. R.B. Resnick, J. Volavka, A.M. Freedman and M. Thomas. Studies of EN-1639A

(naltrexone): A new narcotic antagonist. Am. J. Psychiat., 131, 646-650 (1974).

3. E.J. Cone. Human metabolite of naltrexone (N-cyclopropylmethylnoroxymorphone)

with a novel C-6 isomorphine configuration. Tetrahedron Lett., 23, 2607-2610

(1973).

4. E.J. Cone, C.W. Gorodetzky and S.Y. Yeh. The urinary excretion profile of naltrexone and metabolites in man. Drug Metabol. Disp., 2, 506-512 (1974).

5. K. Verebey, S.J. Mule and D. Jukofsky. A gas liquid chromatographic method for

the determination of naltrexone and beta-naltrexol in human urine. J. Chromatogr., 111, 141-148 (1975).

6. H.E. Dayton and C.E. Inturrisi. The urinary excretion profiles of naltrexone in

man, monkey, rabbit and rat. Drug Metabol. Disp., 4, 474-478 (1976).

7. E.J. Cone and C.W. Gorodetzky. Metabolism of naltrexone and naloxone. Report

to the Committee on Problems of Drug Dependence, Washington, D.C. (1975).

8. K. Verebey, M.A. Chedekel, S.J. Mule and D. Rosenthal. Isolation and identitication of a new metabolite of naltrexone in human blood and urine. Res. Comm.

Chem. Pathol. Pharmacol., 12, 67-84 (1975).

9. K. Verebey, M.J. Kogan, A. DePace and S.J. Mule. Quantitative determination of

naltrexone and beta-naltrexol in human plasma using electron capture detection. J. Chromatogr., 118, 331-335 (1976).

10. K. Verebey, J. Volavka, S.J. Mule and R.B. Resnick. Naltrexone: Disposition, metabolism and effects after acute and chronic dosing. Clin. Pharmacol. Ther., 20,

315-328 (1976).

11. R.A. Sams and L. Malspeis. Determination of naloxone and naltrexone as perfluoroalkyl ester derivatives by electron-capture gas liquid chromatography. J.

Chromatogr., 125, 409-420 (1976).

12. R.A. Sams. Quantitative gas chromatographic determination of naloxone and

naltrexone in plasma and urine by electron capture detection. Doctoral Thesis,

University Microfilms, Ann Arbor, Mi., File No. 75-19 490 (1975).
QUANTITATIVE DETERMINATION OF NALTREXONE 51

13. G.L. Bruce, H.B. Bhat and T. Sokolski. Quantitative determination of naltrexone

prodrugs by electroncapture gas liquid chromatography. J. Chromatogr., 137,

323-332 (1977).

14. E.J. Cone, C.W. Gorodetzky and W.D. Darwin. The identification and measurement of two new metabolites of naltrexone in human urine. Res. Comm. Chem.

Pathol. Pharmacol, 20, 413-433 (1978).

15. G.A. Brine, D.R. Brine, C.D. Welch, V.C. Bondeson, and F.I. Caroll. Carbon-13 nuclear magnetic resonance identification of 2-hydroxy-3-O-methyl-6ß-naltrexol as

a minor naltrexone metabolite. Res. Comm. Chem. Pathol. Pharmacol., 22, 455-

464 (1978).

16. K. Verebey, A. DePace, D. Jukofsky and S.J. Mule. 2-Hydroxy-3-methoxy-6ß-naltrexol determination in human plasma and urine. Federation Proc., 38, 586

(1979).

ACKNOWLEDGMENTS

The author expresses his thanks to Ms. A. DePace, Mr. D. Jukofsky and Dr. M. J. Kogan for their technical assistance. The contribution of biofluids by Dr. J. V. Volavka is acknowledged. Discussion and overview of the manuscript writing and criticism by Dr. S.

J. Mule are deeply appreciated.

AUTHOR

Karl Verebey

New York State Division of Substance Abuse Services

Brooklyn, N.Y. 11217
Naltrexone: Research Monograph 28

R E. Willette and G. Barrett, eds.

National Institute on Drug Abuse, 1980

Analytical Methods for

Quantitative and

Qualitative Analysis of

Naltrexone and

Metabolites in Biological

Fluids

Monroe E. Wall and Dolores R. Brine

Analytical procedures for the determination of naltrexone

and metabolites have been presented. The basic procedure

involves the use of radiolabeled drugs and thin layer chromatography. Naltrexone, 6ß-naltrexol and 2-hydroxy-3-Omethyl-6ß-naltrexol were found by both the TLC procedure

and combined

presence of 3-O-methyl-6ß-naltrexol was indicated by the

as chromatography-mass spectrometry. The

TLC method, but this metabolite could not be found by

mass spectrometry.

INTRODUCTION

In the course of our studies on the metabolism of naltrexone (I)

(1,2) in man, it was necessary to develop quantitative procedures

for I and its metabolites in plasma, urine, and feces. Figure 1 gives

the structure of I and its known metabolites, 6ß-naltrexol (II), 2-hydroxy-3-O-methyl-6ß-naltrexol (III), and a possible but not proven

metabolite, 3-O-methyl-6ß-naltrexol (IV).

Although sensitive gas liquid chromatographic (GLC) procedures

for I, II, and III using flame ionization (3) and electron capture de52
ANALYTICAL CHEMISTRY OF NALTREXONE 53

FIGURE 1. Naltrexone (I), 6ß-naltrexol (II), 2-hydroxy-3-O-methyl-6ß-naltrexol (III),

and 3-O-methyl-6ß-naltrexol (IV).

tectors (4,5) are available, we chose to use thin layer chromatography (TLC) of the radiolabeled drug and metabolites as our basic

procedure. The choice of the procedure was dictated by our need to

study the elimination of total drug and metabolites and to study

both conjugated and nonconjugated metabolites. The TLC-radiolabel procedures had previously been successfully utilized by our

group for the analysis of delta-9-tetrahydrocannabinol and metabolites in biological materials (6) and authenticated by quantitative

combined gas chromatography-mass spectrometry (GLC-MS) (7).

THIN LAYER CHROMATOGRAPHY

Appropriate aliquots of the chloroform extract of the biological

samples (cf. figures 2 and 3) with sufficient I and II added to permit

observation of the standards by ultraviolet quenching were applied

to 20 x 20 cm silica gel-on-glass plates (0.25 mm thickness) prepared by E. Merck, Darmstadt, Germany. The development solvent

was chloroform/ethyl acetate/acetone/triethylamine (40:30:30:2).

As shown in figure 4, excellent separation of I and its actual or potential metabolites can be obtained.
54 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

FIGURE 2. Extraction of naltrexone and its metabolites from human plasma.

After plates were developed in the above system, standards were

visualized under UV light. Zones of silica gel were scraped into

counting vials containing 2.5 ml of distilled water/methanol (1.5:1,

v/v). Samples were then sonicated for 15 min. After addition of 15

ml of scintillation fluor, counting samples were sonicated for an additional 30 min. The radioactivity in each TLC zone was determined by liquid-scintillation counting.

QUALITATIVE IDENTIFICATION OF

NALTREXONE METABOLITES IN URINE

It is desirable to identify metabolites by a procedure of greater

rigidity than TLC. Accordingly, we made a qualitative study of I

and metabolites found in a typical 24-hr human urine sample.

Some of the considerations that are involved in this study are exemplified in figure 5. It will be noted that I and its major metabolite II are closely related structurally, II being formed from I by enzymatic reduction of the 6-ketone to give the 6-hydroxyl. On deriva-
ANALYTICAL CHEMISTRY OF NALTREXONE 55

FIGURE 3. Extraction of naltrexone and its metabolites from human urine.

tization, both I and II give tris-trimethylsilyl (TMS) ethers which

differ by only two mass units. Although as shown in figures 6 and 7

there are characteristic differences in the mass spectra of the trisTMS ethers of I and II, the two compounds are not easily separated

by GLC. Accordingly, a procedure was devised to convert I or possible metabolites with the 6-oxo moiety to the methoxime followed by

conversion of remaining hydroxyl groups to TMS ethers. This

method readily permits separation of methoxime-TMS ethers from

compounds which form only TMS derivatives.

An aliquot (17%) of a 24-hr urine sample was adjusted to pH 9

and extracted three times with 200 ml portions of chloroform. The

chloroform solution was extracted with 2N HCl, which was then

basified and extracted three times with 150 ml portions of chloroform. An aliquot of the acid/base-treated chloroform extract of

urine was dried in vacuo overnight. After addition of 75 μ1 of a 2%

solution of methoxyamine hydrochloride in pyridine, the sample

was tightly capped and shaken at ambient temperature overnight.

347-877 0 - 81 - 5
56 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

FIGURE 4. Thin layer chromatogram of naltrexone and metabolites. Plates (20 x

20 cm silica gel on glass) were developed in chloroform/ethyl acetate/acetone/

triethylamine (40:30:30:2) and were visualized under UV light.

The reagent was evaporated under nitrogen and 75 μl of Tri-Sil

TBT added. Following overnight heating at 115°C excess reagent

was evaporated under nitrogen and 50 μl of hexane was added. The

derivatized urine extracts were analyzed on an LKB model 9000

mass spectrometer using a 1.8 m x 4 mm I.D. column packed with

2% OV-17 on 80/100 mesh Supelcoport (Supelco, Inc., Bellefonte,

PA) at 230°C. Ionizing voltage was set at 70 ev.
ANALYTICAL CHEMISTRY OF NALTREXONE 57

FIGURE 5. Mass spectral parent ions for naltrexone, naltrexol and TMS derivatives.

RESULTS

The data for the qualitative analysis by GLC/MS of urine extract

prepared as described above is shown in figure 8. Shown in the top

portion of this figure are retention times for I as the methoxime

bis-TMS ether, and II as the tris-TMS derivative. It will be noted

that they are easily separated from each other and from the methoxime bis-TMS ether of noroxymorphone. We have found the latter

as a metabolite of rats under in vitro conditions (8). The lower portion of figure 8 shows the total ion current pattern found after subjecting the derivatized urine extract to GLC-MS. Two major and

three minor constituents were noted. The first major peak with Rt

at 8.8 min had a mass spectrum identical to the tris-TMS ether of

6ß-naltrexol (II). The minor constituent with Rt of 10.1 min corresponded to the bis-TMS ether of II, resulting from a slightly incom-
FIGURE 6. Mass spectrum of authentic naltrexone, tris-TMS ether.

FIGURE 7. Mass spectrum of authentic 6ß-naltrexol, tris-TMS ether.
ANALYTICAL CHEMISTRY OF NALTREXONE 59

FIGURE 8. GLC analysis of 24-hr nonconjugated urine fraction from male subject

PM.

plete derivatization. The next major metabolite with Rt of 11.5 min

exhibited a parent ion at 589 mass units corresponding to that of

the tris-TMS derivative of 2-hydroxy-3-O-methyl-6ß-naltrexol (III).

The mass spectrum of III resembled that of metabolites assigned

this structure by Verebey et al. (4) and Cone et al. (9). The data did

not rigidly exclude an alternative structure such as 2-methoxy-6ßnaltrexol. However, 13C-NMR of III isolated from urine and compared with the spectrum of synthetic III readily confirmed the
60 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

structure (10). The minor compound with a peak at Rt of 13.7 min

was identified as the methoxime-bis-TMS derivative of naltrexone

(1). Hence, the parent drug I was a minor constituent of the nonconjugated, 24-hr human urine fraction. The minor constituent

with Rt of 15 min could not be identified. No evidence could be

found in this urine fraction for noroxymorphone, which would have

appeared at 7 min, or for 3-O-methyl-6ß-naltrexol (IV).

QUANTITATIVE VALIDATION OF TLC

PROCEDURE BY GLC/MS

It was desirable to obtain some indication of the accuracy of the

data obtained by the TLC-radiolabeled procedure. Studies were

made of the naltrexone and 6ß-naltrexol fractions comparing the

TLC method with quantitative GLC/MS. The procedure used was

similar to that used in cannabinoid studies (2). Tri-deuterated derivatives of naltrexone and 6ß-naltrexol were prepared and used as

carriers and internal standards.

EXTRACTION OF SAMPLES

The general procedure utilized was a modification of the method

of Cone et al. (3). Figure 2 shows the procedure for the extraction of

nonconjugated I and metabolites from plasma and for the enzymatic hydrolysis and extraction of the conjugated fraction. A slightly

modified procedure was used for red blood samples which were

treated as follows. To each sample (56 ml) was added in order 5 ml

of distilled water, 50 mg of NaF, 0.1N NaOH to adjust the pH to 9,

0.75 ml of 40% (w/v) K2HPO4, and 0.375 g of NaCl. Three extractions with 15 ml of ethyl acetate removed naltrexone and metabolites.

The procedure for the extraction of nonconjugated I and metabolites from urine is shown in figure 3. The extraction of fecal samples was carried out with hot methanol. After removal of methanol

in vacuo, the pH of the aqueous residue (adjusted to 100 ml with

water) was adjusted to 9.0 with sodium hydroxide solution. The procedure then is similar to that shown for urine (figure 3).

DETERMINATION OF RADIOACTIVITY

Aliquots of plasma and urine and extracts of plasma, urine, and

feces were subjected to liquid-scintillation counting in a Triton X100/toluene/Omnifluor (1 liter/2 liter/18 g) scintillation fluor with

use of the external standardization method to correct for quench-
ANALYTICAL CHEMISTRY OF NALTREXONE 61

ing. Samples of red blood cells and fecal residues were combusted

in a Packard sample oxidizer, model 306, and the product collected

in Monophase- for scintillation counting. All samples were counted in a Packard TriCarb model 3375 liquid-scintillation counter.

The procedure for the determination of I in plasma is shown in

figure 9. The procedure for naltrexol is similar, and the procedures

for both I and II in urine are also similar. Derivatization to the

tris-TMS ethers for the quantitative studies was performed by the

forcing procedure described previously. Calibration curves for I in

plasma and II in urine are shown in figures 10 and 11. Linear caliFIGURE 9. Extraction procedure for isolation of naltrexone from plasma.
62 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

bration curves were found in each case over a range of 1-100 ng/

ml.

Table 1 presents a comparison of the TLC and GLC/MS results

for 6ß-naltrexol in urine. In general, satisfactory agreement was

FIGURE 10. GLC/MS calibration curve for naltrexone in plasma.
ANALYTICAL CHEMISTRY OF NALTREXONE 63

found. A similar study with naltrexone in plasma gave less satisfactory agreement because of incomplete separation of naltrexone

and 2-hydroxy-3-O-methyl-6ß-naltrexol by the TLC system in use at

that time.

FIGURE 11. GLC/MS calibration curve for 6ß-naltrexol in urine.
64 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

TABLE 1. Non-conjugated 6ß-Naltrexol Levels in Human Urine After

an Oral Dose of 50 mg of Naltrexone

Subject

Time

(Hours)

μg 6ß-Naltrexol/Urine Sample

By tlc By glc-ms

AG 24 14.000 13.000

40 4.000 3,000

72 1.200 1.400

T A 24 6.400 8,000

48 2,000 2,400

72 420 610

TD 24 3,000 2,700

48 760 670

72 193 300

REFERENCES

1. M.E. Wall, D.R. Brine, and M. Perez-Reyes. Metabolism and physiological disposition of naltrexone in man after oral and intravenous administration. Drug

Metab. Dispos. (In Press).

2. M.E. Wall, D.R. Brine, and M. Perez-Reyes. The metabolism of naltrexone in man.

This volume.

3. E.J. Cone, C.W. Gorodetzky, and S.Y. Yeh. The urinary excretion profile of naltrexone and metabolites in man. Drug Metab. Dispos., 2, 506-512 (1974).

4. K. Verebey, J. Volavka, S.J. Mulé, and R.B. Resnick. Naltrexone: Disposition, metabolism, and effects after acute and chronic dosing. Clin. Pharmacol. Ther., 20,

315-328 (1976).

5. L. Malspeis, MS. Bathala, T.M. Ludden, H.B. Bhat, S.G. Frank, T.D. Sokoloski,

B.E. Morrison, and R.H. Reuning. Metabolic reduction of naltrexone I. Synthesis, separation and characterization of naloxone and naltrexone reduction products and qualitative assay of urine and bile following administration of naltrexone,a-naltrexol, or ß-naltrexol. Res. Commun. Chem. Pathol. Pharmacol., 12, 43-

65 (1975).

6. M.E. Wall, D.R. Brine, and M. Perez-Reyez. Metabolism of cannabinoids in man.

In M.C. Braude and S. Szara (eds.), The Pharmacology of Marihunna, Raven

Press New York, 93-116 (1976).

7. M.E. Wall, T.M. Harvey, J.T. Bursey, D.R. Brine, and D. Rosenthal. Analytical

methods for the determination of cannabinoids in biological media. In R.E. Willette (ed.), Cannabinoid Assays in Humans, National Institute on Drug Abuse

Research Monograph Series 7. DHEW Pub. No. (ADM)76-339. Supt. of Docs.,

U.S. Govt. Print. Off., Washington, D.C., 107-117 (1976).

8. M.E. Wall and D.R. Brine. Annual Report. NIDA Contract Number HSM-42-71-95,

151-160 (1976).

9. E.J. Cone, C.W. Gorodetzky, W.D. Darvin, F.I. Carroll, G.A. Brine, and C.D.

Welch. The identification and measurement of two new metabolites of naltrexone in human urine. Res. Comm. Chem. Path. Pharm., 20, 413-433 (1978).
ANALYTICAL CHEMISTRY OF NALTREXONE 65

10. G.A. Brine, D.R. Brine, C.D. Welch, V.C. Bondeson, and F.I. Carroll. Carbon-13

nuclear magnetic resonance identification of 2-hydroxy-3-O-methyl-6ß-naltrexol

as a minor naltrexone metabolite. Res. Comm. Chem. Path. Pharm., 22, 455-463

(1978).

ACKNOWLEDGMENT

These studies were supported by National Institute on Drug

Abuse (NIDA) Contract HSM-42-71-95. The authors express their

appreciation to Mrs. Carolyn Parker, Dr. Joan Bursey and Mrs.

Valerie Schindler for technical assistance.

AUTHORS

Monroe E. Wall and Dolores R. Brine

Research Triangle Institute

Research Triangle Park, N.C. 27709

Part III

Pharmacology

Naltrerone: Research Monograph 28

R. E Willette and G. Barnett, eds.

National Institute on Drug Abuse, 1980

Background on Animal

Testing in the Drug

Delivery Systems Program

William L. Dewey

In the screening and selection of candidate long-acting narcotic antagonist preparations, careful consideration must be

given to the choice of animal evaluation models. A simple,

rapid method, using analgesia suppression in the mouse

tail-flick test, was chosen for preliminary screening. Suitable candidates were then evaluated by following urinary

excretion rates and blood levels in the monkey. Afterwards,

the most promising preparations were rigorously tested in

their ability to interfere with morphine self-administration

in monkeys. Final evaluation involved toxicological studies

in injection sites and on the whole animal.

INTRODUCTION

The choice of the appropriate animal tests for a particular project must be made on the basis of the purpose of the project. The

purpose of the drug delivery systems program was to formulate a

preparation of a narcotic antagonist that would block the effects of

an administered dose of an opiate over a prolonged period of time.

The goal was to identify two formulations, one that would afford

protection for one month and another that would last for six

months. The duration of the antagonistic activity of the preparation was not an important criterion in the selection of the animal

test. After the appropriate tests have been chosen, they can be used

at various times after the preparation has been administered, to

69
70 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

determine how long the antagonistic action lasts. It is important in

most tests that different animals be used for each time point to

eliminate tolerance, learning, pathologic effects and other factors

which might yield false data.

It was important to choose a battery of animal tests that would

be most predictive of opiate antagonism in man. The specific objective of the preparation in man was not to antagonize the analgesic

activity of an opiate but to block the rewarding, pleasurable or euphorogenic effects of abused opiates. Pleasure and euphoria are

somewhat abstract phenomena which can not be measured in laboratory animals. Other factors, including cost, space, technical difficulty and time requirements, should be considered in the choice of

laboratory tests but always must be given less weight than the

single criterion of predictability. However, these factors play a

more important role in the choice of the preliminary screening

tests which are used to select the few most promising devices for

advanced animal testing.

A number of excellent in vitro techniques such as the opiate

binding, guinea pig ileum and mouse vas deferens assays have been

developed which are good predictors of opiate agonist and antagonist activity. They were not appropriate for this project. Fortunately a number of relatively simple, inexpensive and somewhat predictive screening procedures were available for testing narcotic analgesics in laboratory animals. Each of these screening tests can be

used to predict the narcotic antagonistic activity of a compound or

drug delivery system by administering the test compound or

system at the appropriate test time prior to the injection of a

known antinociceptive dose of morphine or another opiate. The

value of each of these procedures in predicting the antagonistic activity of a drug delivery system is related directly to the value of

the procedure in predicting the antinociceptive activity of an

opiate.

Rodents, usually mice, are used in most of these procedures. Mice

are less expensive, take up less space, and most importantly are as

predictive as any other species for determining the analgesic or antagonistic activity of a number of analgesic compounds. The next

decision to be made was to determine which specific mouse screen

should be used for the drug delivery system experiments. The

major difference in the procedures has to do with the noxious

stimulus. Chemical, heat, cold, electric shock and pressure are the

most frequently used stimuli.
ANIMAL TESTING OF DRUG DELIVERY SYSTEMS 71

TESTING METHOD SELECTION

The tests used most widely today include the tail-flick, the hotplate and the writhing tests. Each of these procedures has limitations and advantages over the others. Any of these tests would be

useful for this project since opiates produce a nice dose response

effect in each of them. The most sensitive of these procedures in

our and other laboratories is the phenylquinone-induced writhing

test (1). The ED-50 for morphine in this procedure is 0.23 mg/kg

while the ED-50 for morphine in the hot plate and tail-flick tests is

about 2 and 4 mg/kg, respectively. Sensitivity is not an important

factor in the selection of the procedure to be used to test the antagonistic activity of the delivery systems, since large doses of an

agonist would be required to simulate the self-administration of

heroin by the postaddict. The writhing test is not very selective for

opiate-like analgesics. Many substances give false positives in this

procedure, and the possibility existed that some other substance

from the delivery system might block the writhing response, falsely

indicating that opiate antagonism was not present. More importantly, the writhing test takes considerable time. A technician can

count the writhes of very few animals at one time. The hot-plate

and tail-flick tests can be much more time efficient.

The hot-plate technique is more specific than the writhing test,

but due to the complexity of lifting and licking of a hind paw or

jumping which are required as end points, a number of the central

nervous system depressants are false positives (2). Still the hotplate method is an excellent screening technique for analgesics.

One of the major advantages of the hot-plate technique, as originally described, is the ability to determine the duration of action of an

antinociceptive agent. This advantage is not useful for testing the

antagonistic activity of the drug delivery systems.

The tail-flick test is more selective than the other test procedures

(3). Sedatives are inactive and weak and moderate analgesics have

limited activity in this procedure. The narcotic analgesics are

active in the tail-flick test and an excellent correlation exists between the potency of a number of opiates in this test and their analgesic potency in man. An even better correlation exists between

the potency of the opiates in this procedure and their dependence

liability in man. This is a particularly useful quality of this procedure for testing the antagonistic properties of the drug delivery

systems. As mentioned above, the specific objective of this project is

to develop a preparation that will release compound to the body

over a prolonged period at a level that will block the effects of a

self-injection of heroin in the postaddict.

347-877 0 - 81 - 6
72 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

The most frequent criticism of the tail-flick test is that the response of the animal is simply a spinal reflex which does not require involvement of the brain. Overwhelming evidence exists to

suggest that the site of the analgesic, and more importantly for

this project, the euphorogenic or rewarding properties of heroin,

morphine and other opiates exists in the brain (4). One might

argue that the correlations of activity in the tail-flick test and man

referred to above are coincidental and that activity in this procedure really has no relation to the action of a drug in the brain.

However, considerable evidence has been presented which strongly

indicates that the inhibitory action of morphine, and presumably

the other opiates, in the tail-flick response is due to their modulating effects on centers higher than the spinal cord. Complete transection of the spinal cord of mice or rats does not block the tailflick response to radiant heat but does significantly decrease or

completely abolish the activity of opiates in this procedure. These

experiments support the hypothesis that this test procedure is

useful in assessing the effects of drugs in the brains of animals and

in predicting their activity in the brains of man.

Although considerable experience is required for a technician to

master the tail-flick procedure, it is the quickest and least demanding of the procedures that were considered as the initial screening

test for this project. Results are reproducible from laboratory to

laboratory and most importantly it, better than the other procedures, may give the best (albeit questionable) indication of opiate

dependence liability. It is my opinion that activity in the tail-flick

test by an unknown compound more strongly suggests opiate-type

dependence liability for the compound than would be predicted by

the other screening tests. Translating this to the narcotic antagonist delivery systems project, one would predict that the ability of

the delivery system to block the activity of an opiate in the tailflick test is a better indication that it would block the effects of an

injection of an opiate in a postaddict than is the blocking of opiate

activity in the other mouse screening tests.

The next aspect of the mouse screening tests to be considered

was the choice of the agonist to be used versus the drug delivery

systems. The opiate most widely used by the addicted population is

heroin. Methadone and other opiates including morphine, meperidine and other prescribed drugs are abused by certain members of

society. The majority of the animal experimentation on mechanism

of analgesic action has been carried out using morphine and it usually serves as the prototype in this class of drugs. Since the characteristics of the antinociceptive actions of the opiates appear to be

similar and the results of the widespread use of morphine among
ANIMAL TESTING OF DRUG DELIVERY SYSTEMS 73

laboratories are in good agreement, it was chosen as the agonist for

this project.

TEST PROCEDURES

The initial test for the screening of the duration of the antagonistic action of a drug delivery system was the mouse tail-flick test

using a subcutaneous injection of morphine as the agonist. The protocol was to implant the delivery system containing the antagonist

in enough mice so that a satisfactory number, usually ranging from

six to ten, could be tested for their control tail-flick reaction time

and then given an injection of morphine, usually an ED-80 dose (in

our bands, 10 mg/kg) and retested 20 or 30 minutes later for tailflick reaction times. An equal number cf mice were implanted with

the delivery system which did not contain the narcotic antagonist.

Mice were used only once, to eliminate the possibility of learning

or pathology of the tail due to repeated long exposures to excessive

heat to give false data. Therefore it was important to implant the

device in enough mice so that a group of mice could be tested at

each desired time point. The control mice, those given only the

drug-free vehicle or delivery system, were to react normally to morphine with a resultant increase in reaction time following the injection of the narcotic. Antagonism was considered to be present

each time the test reaction time of the mice given the antagonist in

the delivery system was shorter than in the mice given the delivery system without the antagonist prior to morphine.

Although the tail-flick test was useful as a primary screening

test, secondary more advanced procedures were necessary to completely evaluate the duration of action of the narcotic antagonist

delivery systems. It has been well established that a direct correlation exists between the blood level and the narcotic antagonist activity of naltrexone in a number of species. Therefore the second

line testing in laboratory animals was to study the pharmacokinetics of the naltrexone released from the delivery system. The release rates of naltrexone could be monitored by quantitating the

excretion rates of radiolabeled naltrexone in urine and feces following the implantation of the narcotic antagonist delivery system in

rats or guinea pigs. It was established early in the project that essentially quantitative recovery of the radiolabel was achieved in

these species (4).

These initial excretion experiments were informative, and along

with the tail-flick data yielded information useful in predicting the

duration of antagonistic action of the delivery systems. A number
74 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

of important problems remained. It was necessary to isolate and

quantitate the parent compound from its structurally related but

pharmacologically relatively inactive metabolites in the body fluids

such as blood and urine and in body tissues before a clear picture

of the pharmacokinetics of naltrexone released from the delivery

systems could be achieved. This necessitated the development of sophisticated analytical methodology for the quantification of naltrexone. The potency of this narcotic antagonist was one of the

strongest reasons for its choice to be used in these studies, since it

afforded the smallest possible size implant. However, it takes less

of a very potent compound to produce the desired pharmacological

effect, which in turn increases the required sensitivity of the analytical procedures used to study its metabolism, distribution, binding to plasma proteins, and other aspects of its pharmacokinetics.

The development of a sensitive gas chromatographic-electron capture assay for naltrexone by Dr. Reuning was a major contribution

in this regard (4).

The second phase on the pharmacokinetics of naltrexone released from the drug delivery systems was carried out in the

monkey. The monkey and possibly the dog were thought to be the

nonrodent species most useful for the testing of these delivery systems. Ideally, the pharmacokinetics should be conducted in a species used for testing the duration of the antagonistic activity of the

delivery system, but it is difficult to obtain sufficient blood and

urine samples from mice. It also was hypothesized that the pharmacokinetics of naltrexone in man would resemble more closely

the pharmacokinetics of the antagonist in the monkey than in the

mouse.

A second, more sophisticated, procedure was necessary to determine the duration of the efficacy of the narcotic antagonist delivery systems to block the effects of an opiate. Even more importantly it was necessary to determine the duration of antagonistic activity versus the rewarding effects of the opiate rather than versus the

antinociceptive activity of the opiate as measured in the tail-flick

test. One of the classical methodologies used to measure the rewarding property of drugs is the self-administration technique

which has been used in both rats and monkeys. The monkey was

the preferred species for this project for a number of reasons, including the fact that the pharmacokinetics of naltrexone in the

monkey more closely resemble its pharmacokinetics in man and

considerable experience exists on the effects of narcotic analgesics

and their antagonists in monkeys.

It has been known for a considerable period of time that monkeys will titrate themselves with narcotic analgesics or for that
ANIMAL TESTING OF DRUG DELIVERY SYSTEMS 75

matter with most of the many other drugs that act on the central

nervous system. A number of other classical conditioning experiments have been developed in an attempt to quantitate the rewarding properties of a drug. None has proven to be as useful or as predictive of the rewarding properties of drugs in man as the self-administration paradigm. The fact that a monkey will work very

hard by pressing a bar many times to receive an injection of these

drugs has led to the conclusion that the injections were rewarding

to the animal. Monkeys will not press a bar for an injection of

saline or other agent known not to act in the central nervous

system and therefore these substances are considered not to be rewarding to the animal.

Monkeys are implanted with an indwelling cannula which is connected to a vessel containing the drug solution or saline. These

monkeys are very valuable animals, due to their inital cost, the

need for sterile surgery during the cannula placement, the recovery time and the training necessary to teach them to press the bar

for the drug injection. Therefore, only the most promising of the

delivery systems could be tested in this procedure. Monkeys used in

the studies with the narcotic antagonist delivery systems were

maintained on a repeated cycle of access to morphine, amphetamine, and saline through the cannula. The monkeys responded

frequently when either amphetamine or morphine was being selfadministered by a lever press, but their response rate was much

less during the saline portion of the cycle. Response rates were determined prior to the implantation of the delivery system and repeatedly until response rates returned to control values after the

implantation. This procedure allowed the investigators to differentiate the selective effect of a narcotic antagonist from the self-administration of only the opiate, without altering the response rate

during the amphetamine portion of the cycle. In contrast, a general

central nervous system depressant would reduce significantly the

response rate in both the morphine and amphetamine portions of

the cycle.

Once again the question arose as to the proper agonist for these

experiments. It is quite possible, and often suggested, that the rewarding effects of heroin are greater than those of morphine in

man. However, it has been demonstrated that naltrexone blocks

equally well the rewarding properties of heroin and morphine as

determined by this self-injection technique in monkeys. Therefore

morphine was chosen as the agonist for these experiments. It was

also felt to be an advantage to use the same agonist as was used in

the mouse tests.
76 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

The duration of the antagonistic activity of naltrexone in the

drug delivery systems in the monkey was correlated to the blood

levels of naltrexone in these animals. Blood samples were taken periodically throughout the time of effective blockade and they were

analyzed for levels of naltrexone by the gas chromatographic-electron capture technique mentioned above. A good correlation was

found between the two parameters and these data overwhelming

support the view that the parent compound is responsible for the

antagonistic activity.

The final aspect of the animal testing paradigm was a safety

evaluation. The toxicity tests for this project can be somewhat

more complicated than usual, since not only the toxicity of naltrexone and the drug delivery matrix themselves need be studied, but

also one must study the toxicity of the combination of the narcotic

antagonist in the drug delivery system. Considerable but certainly

not all of the toxicity work with naltrexone had been completed

prior to the initiation of the project. The majority of the constituents of the drug delivery matrix had been approved for use in

humans for other reasons. Therefore, the one aspect of toxicity

which was of primary concern in this project was the toxicity of

the naltrexone and the drug delivery system combined.

REFERENCES

1. E. Siegmund, R. Cadmus and G. Lu. A method for evaluating both non-narcotic

and narcotic analgesics. Proc. Soc. Exp. Biol., 95, 729-731 (1957).

2. G. Woolfe and A.D. MacDonald. The evaluation of the analgesic action of pethidine hydrochloride (Demerol). J. Pharmacol. Exp. Ther., 80, 300-307 (1944). N.B.

Eddy and D. Leimbach. Synthetic Analgesics. II. Dithienylbutenyl- and dithienylbutzlamines. J. Pharmacol. Exp. Ther., 107, 385-393 (1953).

3. L.S. Harris, W.L. Dewey, J.F. Howes, J.S. Kennedy and H. Pars. Brain acetylcholine levels and inhibition of the tail-flick reflex in mice. J. Pharmacol. Exp.

Ther., 169, 17-22 (1969).

4. R.A. Sams and L. Malspeis. Determination of naloxone and naltrexone as perfluoroalkyl ester derivatives by electron-capture gas-liquid chromatography.

J Chromatogr., 125, 409-420 (1976).

AUTHOR

William L. Dewey

Department of Pharmacology

Medical College of Virginia

Richmond, Va. 23298
Naltrexone: Research Monograph 28

R. E. Willette and G. Barnett, eds.

National Institute on Drug Abuse, 1980

Pharmacological

Evaluation of Narcotic

Antagonist Delivery

Systems in Rhesus

Monkeys

Stephen E. Harrigan and David A. Downs

Rhesus monkeys were chronically restrained, intravenously

catheterized, and allowed to self-administer morphine,

methamphetamine, and saline. Various sustained-release

systems containing naltrexone were then implanted in the

animals and examined for selective morpine blockade.

Similarly, continuous intravenous infusions of naltrexone,

buprenorphine, and methadone were tested against morphine or heroin self-administration.

INTRODUCTION

Our major objective was to determine the degree and duration of

blockade of the reinforcing properties of narcotics in rhesus monkeys after administration of narcotic antagonist delivery systems.

This was accomplished using the intravenous self-administration

preparation described by Harrigan and Downs (1). In brief, minimally restrained rhesus monkeys were prepared surgically with

permanent jugular or femoral venous catheters and trained to

press a response key to receive intravenous injections of morphine,

methamphetamine, or saline. When stable rates of drug and saline

intake were achieved, delivery systems were administered and the

effects were monitored over time until recovery was evident. Ef77
78 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

fects of narcotic antagonist delivery systems were compared

against those of acute or continuous administration of naltrexone,

methadone, and buprenorphine. Morphine and heroin self-administration also were compared for their antagonism by naltrexone.

Some other objectives were to evaluate tissue reactions to various

narcotic antagonist delivery systems at the sites of administration

and to assess the preclinical toxicity of a selected delivery system.

MATERIALS AND METHODS

Self-Administration Procedures

Rhesus monkeys (Macaca mulatta) were housed individually and

were minimally restrained with a jointed metal arm and harness

system (2). About 180 g of Purina Monkey Chow and 1/4 fresh

orange or apple were fed daily, while water was continuously available. Each monkey was prepared surgically with a jugular or femoral intravenous silicone rubber catheter. The distal end of the

catheter exited through the skin of the back and passed through

the hollow restraining arm to a reciprocating syringe driver (Superspenser, Hook and Tucker Ltd., Croyden, England) mounted outside the cage.

A transilluminated plastic key (3.5 x 6.5 cm) served as the operant response device. One key press activated the pump to deliver

0.5 ml of drug solution through the catheter over 5 seconds followed by a 5-second refill. Responses during the infusion cycle had

no consequences. Access to self-administration of drugs was limited

to a 15-minute period every 4 hours. Each access period was initiated by illuminating the key and by one automatic injection. Room

lights were controlled automatically to provide 12-hour diurnal

cycles.

Drug self-administration solutions usually were changed in a repeating cycle of 3 days on morphine sulfate (8 μg/kg/injection), 2

days on methamphetamine hydrochloride (4 μg/kg/injection), and 2

days on 0.9% sterile saline. Thus, within each week the monkeys

had 18 sessions of morphine availability, 12 sessions of methamphetamine availability, and 12 sessions of saline availability. In a few

early studies, morphine, methamphetamine, and saline were each

available for 3 days at a time. All monkeys were exposed to this

schedule for at least 2 months or until drug and saline intakes remained stable prior to the administration of antagonist delivery

systems.

Because self-administration behavior under these conditions was

maintained at low and variable rates at night, only the number of
ANTAGONIST DELIVERY IN MONKEYS 79

injections in each of the three daily “daylight” access periods was

used to compare treatments. Statistical comparisons were made

with analysis of variance and Newman-Keuls multiple range test.

All drug doses were expressed as base. When possible, delivery systems were administered in sufficient quantities to deliver a total

dose of 10 mg/kg of naltrexone.

Administration of Narcotic Antagonist Delivery Systems

Drug delivery systems generally were administered on the first

day of a period of saline self-administration. When necessary, ketamine hydrochloride (10 mg/kg) was used to anesthetize the monkeys prior to administration of a delivery system. Self-administration rates over subsequent cycles of exposure to morphine, methamphetamine, and saline were monitored until morphine intake returned to control levels. Duration of effect was determined as the

last week in which morphine rates were significantly (<0.05) lower

than control values. Implantable delivery systems usually were inserted subcutaneously in the abdominal area via 12-15 gauge trocars. Injectable systems were administered into the rectus femoris

muscle, or in some cases, subcutaneously in the abdominal area.

Tissue Reactions

When possible, implantable devices were removed at the end of

measurable activity; gross visual examination of implant sites was

performed at that time. In some cases, biopsies were made of tissues surrounding implant or injection sites and these were subjected to histopathological analysis by Drs. J. Fitzgerald and S. Kurtz

of the Parke-Davis Department of Toxicology. With some systems,

sites of administration were examined sequentially in nonanesthetized monkeys to determine the time-course of degradation of the

systems. Blood, urine, feces, and recovered systems or system fragments were sent to other NIDA contractors for various analyses.

RESULTS WITH DELIVERY SYSTEMS

The classification of naltrexone delivery systems described below

is summarized in table 1.

Insoluble Salts

The pamoate acid salt of naltrexone in peanut oil (NIDA) blocked

morphine self-administration for less than 5 days, whereas naltrexone zinc tannate and naltrexone aluminum tannate (IITRI) in

peanut oil blocked morphine self-administration for about 24 days
80 NALTREXONE SUSTAINEDRELEASE PREPARATIONS

TABLE 1. Classification of Naltrexone Delivery Systems

lnjections Implants

Insoluble

Salts

Naltrexone Aluminum Tannate

Naltrexone Zinc Tannate

Naltrexone Pamoate

—

Erodrble

Polymer

Matrices

Naltrexone Polylactic Acid

Naltrexone Lactic glycolic Acid

Naltrexone Lactic glycolic Acid

beads

Naltrexone Chronomer Rods

Naltrexone Palmitin Rods

MacroReservoirs

—

Naltrezone Glutamic Acid

Leucine in Glutamic Acid

Leucine tubes.

Naltrexone (Dry or in water or in

sesame oil) in

Capronolactone Lactic Acid tubes

MicroReservoirs

Naltrexone (Base) polylactrc acid

microcapsules

Naltrexone Pamoate polylactrc

acid microcapsules

(figure 1). Biopsy of one injection site 43 days after intramuscular

injection of naltrexone aluminum tannate revealed no marked

change of muscle fiber.

Injectable Erodible Polymer Matrices

Naltrexone polylactide (Yolles) suspended in

carboxymethylcellulose fully antagonized morphine for only a few
ANTAGONIST DELIVERY IN MONKEYS 81

FIGURE 1. Self-administration of morphine (8 μ/kg/injection) (open circles),

methamphetamine (4 μ/kg/injection) (solid squares), and saline (solid triangles)

following intramuscular injection of naltrexone pamoate, naltrexone aluminum

tannate, or naltrexone zinc tannate. Each point represents the mean of four

monkeys. Vertical bars denote ±SEM. Asterisks indicate means significantly

different from corresponding control (C), (P< 0.05). Arrow signifies a single

intravenous injection of naltrexone; drug self-administration for one cycle is not

included in the figure.
82 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

days (figure 2A). Examinations of injection sites revealed considerable tissue encapsulation of injected material.

Naltrexone in micronized polylactic/glycolic acid copolymer

(Dynatech) given subcutaneously in aqueous vehicle suppressed

morphine self-administration only for 3-5 days (figure 2B).

FIGURE 2. Self-administration of morphine (8 μ/kg/injection) (open circles),

methamphetamine (4 μ/kg/injection) (solid squares), and saline (solid triangles)

following subcutaneous injections of naltrexone poly lactic acid (A) or naltrexone

polylactic-glycolic acid (B) polymers. Points in A represent means of two monkeys;

points in B represent means of four monkeys. Vertical bars denote ±SEM.

Asterisks indicate means significantly different from control (C), (P<0.05).
ANTAGONIST DELIVERY IN MONKEYS 83

Implantable Erodible Polymer Matrices

Rods (1.7 x 10 mm) containing 33% naltrexone in a 75/25 dipalmitin/tripalmitin matrix (Battelle Northwest) produced about 20

days of significant morphine blockade (figure 3). The rods caused

very little gross tissue reaction and were easily removed at the end

of the study.

Rods (1 x 14 mm) containing 20% naltrexone, 10% disodium carbonate, and 70% Chronomer (ALZA) suppressed morphine self-administration for about 20 days (figure 3). Recovery after 20 days

was very rapid and corresponded with the near-total absorption of

the rods from the implant sites. Sequential biopsies taken from

normal monkeys showed gradual liquification and eventual total

disappearance of these rods over a 30-day period. Gross tissue reaction was minimal. Seventeen rods were required to deliver 10 mg/

kg naltrexone to each monkey.

Beads (1.5 mm) containing 70% naltrexone in a 90/10 lactic acid

glycolic acid copolymer (Dynatech) caused dose-dependent reductions in morphine self-administration (figure 4). Single beads (about

0.5 mg/kg naltrexone) caused a slight suppression of morphine selfadministration for only one morphine exposure (5 days) after the

implant. Three beads (about 1.5 mg/kg naltrexone) produced a 24%

reduction of morphine self-administration during the first morphine exposure; thereafter morphine intake returned to control

levels within two weeks. Longer and more intense blockade occurred after treatment with six beads (about 3 mg/kg naltrexone);

morphine self-administration was reduced by about 42% for 30

days, although none of the post-implant means were significantly

different from control (P>0.05).

Nine beads (about 5 mg/kg naltrexone) and 18-21 beads (about 10

mg/kg naltrexone) significantly suppressed morphine self-administration for 30 days to rates equivalent to those for saline self-administration (P<0.05). After 33 days, morphine self-injection rates

returned to control levels.

Monkeys receiving control beads (not containing naltrexone)

showed slight increases in morphine injections for the first two

weeks post-implant; thereafter, morphine self-administration returned to near control levels.

Tissue reactions in rata, rabbits, dogs, and monkeys generally

consisted of local induration and chronic inflammatory response

with encapsulation. No suggestion of systemic drug toxicity was

found. Bead material was present at the sites of administration 3

months after implantation.
84 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

FIGURE 3. Self-administration of morphine (8 μ/kg/injection) (open circles),

methamphetamine (4 μ/kg/injection) (solid squares), and saline (solid triangles)

following implanting of naltrexone-containing palmitin or Chronomer rods. Points

in upper figure represent means of three monkeys; points in lower figure are means

of four monkeys, Vertical bars denote ±SEM. Asterisks indicate means significantly

different from control (P<0.05).
ANTAGONIST DELIVERY IN MONKEYS 85

FIGURE 4. Self-administration of morphine (8 μ/kg/injection) following implants of

control (open circles) or 1 (triangles), 3 (solid squares), 6 (diamonds), 9 (solid circles),

or 18 (open squares) naltrexone-loaded beads. Points represent means of four

monkeys. Vertical bars denote ±SEM. Asterisks indicate means significantly

different from control (C) (P<0.05).

Macro Reservoirs

Naltrexone (10%) in 35/65 glutamic acid/leucine copolymer

binder (90%) placed inside sealed tubes (1 cm length, 0.19 cm outer

diameter) composed of copolymer walls 53-63 microns thick (A.D.

Little) blocked morphine self-administration for about 12 days

(figure 5A). The short duration of action and considerable tissue reaction noted when the tubes were removed suggested that the walls

had prematurely failed, rapidly leaking naltrexone and binder. Additional tubes, together with unloaded controls, were implanted in

normal dogs and monkeys to more fully characterize the unexpected tissue reactions. Results indicated that an unusual perivascular

lymphocyte accumulation was associated with the naltrexoneloaded devices and equivocally or not at all with the controls.

Capronolactone/lactic acid copoylmer tubes (2 x 30 mm) (RTI)

containing either naltrexone powder or naltrexone suspended in

water or sesame oil all suppressed morphine self-administration to

about the same degree but for various durations. Figure 5B shows

that tubes 3 cm. in length containing dry naltrexone were effective

for about 30 days; tubes containing naltrexone in sesame oil had

about the same duration of effect. In contrast, identical tubes con-
86 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

FIGURE 5. Self-administration of morphine (8 μ/kg/injection) (open circles),

methamphetamine (4 μ/kg/injection) (solid squares), or saline (solid triangles)

following implants of naltrexone contained in tubes composed of glutamic acid/

leucine (A) or capronolactone/lactic acid (B). Points represent means of three

monkeys. Vertical bars denote ±SEM. Asterisks indicate means significantly

different from control (C) (P<0.05).
ANTAGONIST DELIVERY IN MONKEYS 87

taining naltrexone in water lasted only for about 12 days. These

tubes biodegraded to the extent that large fragments of collapsed

tubes were recovered 50 days after implants. No gross tissue irritations were noted.

Micro Reservoirs

Naltrexone, either as a base or as the pamoate salt contained

within microcapsules (63-106 μ diameter) of polylactic acid (Theis,

Washington University) was tested under a variety of conditions.

The microcapsules were suspended in either peanut oil or an aqueous vehicle and injected either subcutaneously or intramuscularly.

Intramuscular injection of microcapsules in peanut oil produced

very stable morphine blockade for about 50 days (figure 6). By the

same route but suspended in aqueous vehicle, the microcapsules

suppressed morphine self-administration for about as long but with

somewhat less consistency (figure 6). Microcapsules injected subcutaneously in water showed sustained activity, but the naltrexone

release rate appeared to be too low to provide full morphine blockade (figure 6). No biopsies were performed on intramuscular injection sites, but considerable swelling, erythema, and occasional necrosis were observed in subcutaneous sites.

CONTINUOUS INTRAVENOUS INFUSIONS OF

NALTREXONE, BUPRENORPHINE, AND

METHADONE

Continuous intravenous infusions of naltrexone caused dose-related decreases in morphine (8 μg/kg/injection) self-administration.

The suppression of morphine self-administration remained stable

over the 4-week duration of each naltrexone infusion. When naltrexone was discontinued, morphine self-administration returned to

control levels within one or two morphine cycles (1). This study

demonstrated that morphine self-administration rates were sensitive to naltrexone in a dose-dependent manner and that the suppression of morphine self-administration by effective doses of naltrexone was stable over at least month-long intervals. In addition,

it was estimated that steady-state delivery of naltrexone at rates of

5 μg/kg/hour or more were adequate to provide complete blockade

of self-administration of a wide range of morphine doses.

Figure 7 shows that heroin self-administration also was suppressed by continuous intravenous infusions of naltrexone. As with

morphine self-administration, the heroin self-administration dose347-877 0 - 81 - 7
88 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

response curve was shifted to the right and decreased in maximum

as the naltrexone infusion rate increased.

Figure 8 shows that continuous intravenous infusions of methadone also can suppress heroin self-administration. In general, the

suppressant effects of methadone appeared to be similar to those of

naltrexone. However, when the methadone infusion rate was high

enough to block the self-administration of a broad range of heroin
ANTAGONIST DELIVERY IN MONKEYS 89

FIGURE 6. Self-administration of morphine (8 μ/kg/injection) (open circles),

methamphetamine (4 μ/kg/injection) (solid squares), or saline (solid triangles)

following injection of microencapsulated naltrexone pamoate intramuscularly or

subcutaneously. Points represent means of three or four monkeys. Vertical bars

denote ± SEM. Asterisks indicate means significantly different from control

(P<0.05).

doses, the monkeys showed severe debilitation and depression. Two

out of three monkeys died during infusions of methadone above 24

mg/kg/day.

The effect of a continuous intravenous infusion of buprenorphine

at 40 μg/kg/hour was similar to that of naltrexone at 5-20 μg/kg/

hour. A lower rate of buprenorphine infusion (20 μg/kg/hour),

however, had only slight effect on morphine self-administration

(figure 9).

DISCUSSION

Narcotic self-administration by rhesus monkeys was found to be

a reliable, specific and sensitive measure of the effects of the drugs

and drug delivery systems described above. It seems very reasonable to assume that the repeated self-administration of narcotics by

rhesus monkeys and by humans is determined by at least some

common pharmacological factors. This assumption is based on and

supported by the general observation that drugs which act as reinforcers (i.e., maintain self-administration behavior) in rhesus mon-
90 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

FIGURE 7. Heroin self-administration as a function of dose per injection during

continuous infusion of saline (solid circles), or 1 (solid squares), 5 (solid triangles), or

20 μ/kg/hour naltrexone (open circles). Each point represents the mean of four

monkeys. The asterisk at S indicates saline self-administration from a control period

near the start of the study. Standard errors have been omitted for clarity; none was

greater than 3.7 injections per access period.

FIGURE 8. Heroin self-administration as a function of dose per injection during

continuous infusion of saline or 4, 12, or 24 mg/kg/day methadone. Points represent

means of nine access periods in a single monkey.
ANTAGONIST DELIVERY IN MONKEYS 91

FIGURE 9. Number of morphine self-injections as a function of dose during

continuous infusions of 20 (circles) or 40 (triangles) μg/kg/hour buprenorphine.

Points represent means of two monkeys. Dashed line indicates morphine selfadministration during continuous saline infusion; data obtained from other animals.

keys act similarly in humans. A second assumption is that manipulations which alter self-administration of narcotics by rhesus monkeys will act similarly in humans. However, the predictive value of

the procedures and the effects described above remains to be determined in clinical studies with antagonist delivery systems in

humans.

Although most of the delivery systems which were submitted to

us for evaluation contained naltrexone, it is apparent that other

agents are potentially equally effective. Thus, future objectives

could include the development of delivery systems for drugs with

improved efficacy and safety.

Finally, the impressive progress which has been made in the development of narcotic antagonist delivery systems should be of significant value in the development of other drug delivery systems

for a broad range of research and treatment areas.

REFERENCES

1. S.E. Harrigan, and D.A. Downs. Continuous intravenous naltrexone effects on

morphine self-administration in rhesus monkeys. J. Pharmacol. Exp. Ther. 204,

481-486 (1978).
92 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

2. G.A. Deneau, T. Yanagita, and M. Seevers. Self-administration of psychoactive

substances by the monkey: a measure of psychological dependence.

Psychopharmacologica 16, 30-48 (1969).

ACKNOWLEDGMENTS

The authors are indebted to Duncan A. McCarthy for the incep

tion and support of this work. The assistance of Robert J. Labay,

James N. Wiley, Jerry Bowman, and Thomas E. Robinson is gratefully acknowledged. This work was supported by NIDA contracts

271-77-3421 and 42-72-167.

AUTHORS

Stephen E. Harrigan

David A. Downs

Department of Pharmacology

Warner-Lambert/Parke-Davis Pharmaceutical Research Division

2800 Plymouth Road

Ann Arbor, Michigan 48105
Naltrexone: Research Monograph 28

R. E. Willette and G. Barnett, eds.

National Institute on Drug Abuse, 1980

A Comparative Study of

the Oral, Intravenous, and

Subcutaneous

Administration of 3HNaltrexone to Normal Male

Volunteers

Mario Perez-Reyes and Monroe E. Wall

3H-naltrexone was administered orally, intravenously, and

subcutaneously to groups of normal, male, paid volunteers.

The doses given were: 50 mg orally (specific activity 4 μCi/

mg), 1 mg intravenously (specific activity 200 μCi/mg), and

5 mg subcutaneously (specific activity 30 μCi/mg). At these

doses, the subjects did not experience any noticeable effects.

Following intravenous injection, plasma levels of radioactivity were immediately high and declined rapidly during

the first 30 minutes and declined gradually thereafter. Following oral or subcutaneous administration, maximal

plasma levels were observed to occur one hour after dosing,

and reached similar levels to those obtained when the drug

was intravenously injected. This finding indicates the excellent bioavailability of naltrexone following oral or subcutaneous administration. 3H-naltrexone and/or its metabolites were predominately excreted in the urine, and the

renal excretion was similar for all three routes of administration. Fecal excretion is a minor pathway of elimination.

The urinary and fecal excretion of 3H-naltrexone was studied in one subject for 133 hours after drug ingestion, and it

was found that essentially all of the dose administered was

excreted in this period.

93
94 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

INTRODUCTION

Naltrexone is a narcotic antagonist that in clinical pharmacological trials appears to have longer duration of action and to be 2 to 3

times more potent than naloxone (1). Naltrexone has been commonly administered in doses ranging from 50 to 200 mg orally, or 1

to 80 mg subcutaneously, to postnarcotic addicts. The drug appears

to be well tolerated with no side effects if the subjects receiving the

drug have abstained from narcotic drugs for 4 weeks (2).

To obtain information on the bioavailability of naltrexone, pattern of metabolism, and urinary and fecal excretion, the drug was

radiolabeled with tritium and administered orally, intravenously,

and subcutaneously to normal subjects. It is the purpose of this

communication to report the results obtained.

EXPERIMENTAL PROCEDURE

The subjects that participated in the experiments were all male,

healthy, paid volunteers who had never used narcotic drugs and

who were thoroughly informed about the nature and potential

risks of the experiment.

a) Oral administration: Six volunteers received 50 mg of 3Hnaltrexone (specific activity 4 μCi/mg) dissolved in ethanol in two

gelatin capsules 3 hours after a light breakfast. Blood samples

were collected before drug administration, then at 15-minute

intervals for three hours, and at 4 1/2, 6, 12, and 24 hours. Urine

was collected at 3, 6, 12, 24, and 48 hours after drug ingestion, and

feces at 24 and 48 hours.

b) Intravenous administration: Five volunteers received 1.0 mg

of 3H-naltrexone (specific activity 200 μCi/mg), dissolved in 1 ml of

normal saline in a single bolus intravenous injection. Blood samples were collected before drug administration, at 30 seconds, at 1,

3, 5, 10, 15, 30, 60, 90, 120, 150, and 180 minutes, and at 5, 7, 10, and

24 hours. Urine was collected at 3, 6, 12, 24, 48, and 72 hours.

c) Subcutaneous administration: Two volunteers received 5 mg

of 3H-naltrexone (specific activity 30 μCi/mg), dissolved in 1 ml of

normal saline in a single subcutaneous injection. Only two subjects

were tested because of reports about possible carcinogenicity of

naltrexone which temporarily halted our experiments. Blood samples were collected before drug administration, at 5, 10, 20, 30, 45,
ADMINISTRATION OF NALTREXONE 95

60, 75, and 90 minutes, and at 2, 4, 6, 8, 10, 24, 30, and 48 hours.

Urine was collected at 3, 6, 12, 24, 48, and 72 hours.

In the biological samples obtained in the experiments, the total

radioactivity present was measured by liquid scintillation spectrometry in triplicate aliquots. Determination of 3H-naltrexone and

its metabolites was performed by chromatographic procedures and

will be reported elsewhere.

RESULTS

a) Clinical effects: Neither the oral, intravenous, nor subcutaneous administration of naltrexone, at the doses used, produced any

clinical effects other than a slight degree of drowsiness which

could have been produced by a total lack of physical activity for

the first 3 hours of the experiment.

b) Percentage of the dose administered present in the plasma:

Although the same dose of naltrexone was administered to all the

subjects in each of the three routes of administration studied, the

subjects tested within each group had different body weights.

Therefore, the results have been expressed as the percentage of

the total radioactivity administered present in the theoretical

total plasma volume at each of the time intervals sampled. The

total plasma volume of each subject was calculated on the basis

that the average human plasma volume is 45.4 ml/kg of body

weight (3). The plasma volume of each subject was multiplied by

the disintegrations per minute per milliliter of plasma, and from

this value, the percentage of the total radioactivity administered

was calculated.

The plasma levels obtained after oral naltrexone (table 1) show

that the mean gastrointestinal absorption of naltrexone reached

maximal plasma levels at 105 minutes after drug ingestion, remained essentially unchanged for the next three hours, and then

decreased progressively at 6, 12, and 24 hours. However, drug absorption differed significantly among the subjects tested. Thus,

three subjects absorbed the drug rapidly, reaching maximal plasma

levels at 90 minutes; two subjects absorbed more slowly, reaching

maximal plasma levels at 150 minutes; and one subject absorbed so

slowly that he did not reach maximal plasma levels until 4 hours

after drug administration.
96 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

TABLE 1. Percentage of the Total Dose Present in the Total Plasma

Volume Following the Oral Administration of 3H-Naltrexone

TIME R.C. R.B. P.M. E.R. K.B. W.M. MEAN SD.

15' 0 0.01 0.03 0.04 0.03 0.02 0.02 0.01

30' 0.30 0.64 1.51 0.42 0.09 0.15 0.52 0.48

45' 0.52 1.07 1.94 1.31 0.32 0.50 0.94 0.56

60' 0.67 1.25 2.08 1.08 0.47 0.77 1.05 0.53

75' 0.58 1.38 2.23 1.04 0.62 1.17 1.17 0.55

90' 0.55 1.60 1.75 1.31 0.88 0.65 1.12 0.46

105' 0.83 1.51 1.95 1.61 0.96 1.46 1.39 0.38

120' 0.80 1.54 1.91 1.14 0.99 1.67 1.34 0.39

135' 0.99 1.47 1.51 1.41 1.23 1.67 1.36 0.22

150' 1.38 0.97 1.40 1.11 1.22 1.75 1.31 0.25

165' 1.20 1.30 1.39 1.47 1.26 1.73 1.39 0.17

160' 1.16 1.25 1.46 1.32 1.29 1.52 1.33 0.12

41⁄2 hrs 1.34 1.04 0.99 0.19 1.50 1.65 1.29 0.24

6 hrs 1.11 0.69 0.56 0.93 1.10 1.14 0.93 0.22

12 hrs 0.55 0.50 0.54 0.59 0.54 0.73 0.56 0.06

24 hrs 0.18 0.25 0.23 0.27 0.34 0.42 0.28 0.08

TABLE 2. Percentage of the Total Dose Present in the Total Plasma

Volume Following the Intravenous Administration of 3H-Naltrexone

TIME M.H. A.G. R.C. S.B. T.D. MEAN S.D.

30 0 0.12 12.06 1.43 0.64 2.85 5.18

1' 3.03 1.40 5.06 3.21 8.82 4.30 2.84

3' 3.22 1.91 3.09 1.95 2.90 2.61 0.63

5' 2.23 1.76 2.22 1.60 2.12 1.99 0.29

10' 1.68 1.51 1.76 1.50 1.79 1.65 0.14

15' 1.50 1.32 1.67 1.20 1.78 1.49 0.24

30' 1.40 1.17 1.57 1.41 1.45 1.40 0.14

60' 1.11 1.09 1.45 1.36 1.34 1.27 0.16

90' 1.01 1.02 1.41 1.26 1.24 1.19 0.17

120' 0.90 0.93 1.30 1.23 1.12 1.10 0.18

150' 0.93 0.84 1.19 1.07 1.04 1.01 0.13

180' 0.97 0.72 1.06 1.07 1.03 0.97 0.15

5 hrs 0.94 0.80 1.28 1.20 1.13 1.07 0.20

7 hrs 0.86 0.74 1.08 1.05 0.96 0.94 0.14

10 hrs 0.32 0.38 0.79 0.72 0.73 0.59 0.22

24 hrs 0.13 0.13 0.22 0.27 0.22 0.19 0.06

The plasma levels obtained after intravenous naltrexone are illustrated in table 2. Maximal levels of radioactivity were observed

to occur in the plasma one minute after the intravenous injection

of the drug and to decline sharply during the next 15 to 30 minutes. Then the plasma levels of radioactivity remained relatively

steady for the next 7 hours and declined at 12 and 24 hours.
ADMINISTRATION OF NALTREXONE 97

TABLE 3. Percentage of the Total Dose Present in the Total Plasma

Volume Following the Subcutaneous Administration of 3H-Naltrexone

Time L.B. J.W. MEAN S.D.

5' 0.27 0.08 0.18 0.13

10' 0.48 0.26 0.37 0.16

20' 0.91 0.64 0.78 0.19

30' 1.02 0.84 0.93 0.13

45' 1.13 0.90 1.02 0.16

60' 1.28 0.97 1.13 0.22

75' 1.16 0.95 1.06 0.15

90' 1.25 1.04 1.15 0.15

120' 1.24 1.00 1.12 0.17

4 hrs 1.01 1.06 1.04 0.04

6 hrs 0.76 0.67 0.72 0.06

8 hrs 0.51 0.46 0.49 0.04

10 hrs 0.31 0.32 0.32 0.001

24 hrs 0.16 0.16 0.16 0

30 hrs 0.12 0.13 0.13 0.001

48 hrs 0 0 0 0

FIGURE 1

PERCENTAGE OF THE TOTAL DOSE ADMINISTERED PRESENT IN THE TOTAL PLASMA VOLUME

The plasma levels obtained after subcutaneous naltrexone are illustrated in table 3. Maximal levels of radioactivity were observed

to occur in the plasma 60 minutes after the subcutaneous injection

of the drug, remain steady for the next 3 hours, and decline progressively thereafter.

A comparison of the plasma levels of naltrexone and its metabolites obtained after the intravenous, subcutaneous, and oral administration of the drug is illustrated in figure 1.
98 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

FIGURE 2

CUMULATIVE URINARY EXCRETION OF 3H-NALTREXONE

PERCENTAGE OF THE TOTAL DOSE ADMINISTERED

This figure shows that, as expected following the intravenous injection, the plasma levels of the drug are immediately high and

then decline rapidly in a period of 15 to 30 minutes. The low levels

of radioactivity present in the plasma even at peak values suggest

that the drug is poorly bound to the plasma proteins, rapidly leaves

the compartment, and is distributed into the body tissues. Following the oral or subcutaneous administration of naltrexone, the

plasma levels progressively increased to reach a maximum level at

approximately 60 minutes. It would appear that the drug was more

rapidly absorbed after the subcutaneous injection; however, since

only two subjects were studied by this route, this result cannot be

considered significant. The plasma levels of radioactivity observed

one hour after drug administration were similar for all three

routes, which indicates the excellent bioavailability of the drug

after oral or subcutaneous administration. The plasma levels declined progressively during the 24 to 48 hours of observation to undetectable values, probably because of the extensive urinary excretion of naltrexone and/or its metabolites.

c) Percentage of the dose administered excreted in the urine: The

urinary excretion of naltrexone and/or its metabolites was rapid,

extensive, and essentially similar for the three routes of administration investigated (table 4). Thus, more than 12% of the total dose
ADMINISTRATION OF NALTREXONE 99

administered was excreted during the first 3 hours, and more than

45% at the end of 24 hours. Figure 2 again illustrates the similar

average cumulative urinary excretion of the total radioactivity

administered by the three different routes at the time intervals

sampled. This result indicates that the renal clearance of naltrexone

and/or its metabolites is constant and independent of the route of

administration.

TABLE 4. Percentage of the Total Radioactivity Excreted in the Urine

Following the Administration of 3H-Naltrexone

INTRAVENOUS ORAL SUBCUTANEOUS

TIME

MEAN SD. MEAN S.D. MEAN S.D.

3 hrs 15.04 ± 4.43 13.88 ± 3.05 12.34 ± 0.59

6 hrs 10.99 ± 2.60 9.66 ± 2.18 10.85 ± 2.02

12 hrs 9.84 ± 4.61 10.45 ± 5.38 18.14 ± 1.87

24 hrs 10.77 ± 6.02 13.25 ± 2.18 8.63 ± 0.47

48 hrs 8.23 ± 4.17 8.64 ± 2.76 10.82 ± 0.02

72 hrs 3.11 ± 1.68 4.25 ± 1.58

TOTAL 57.99 ± 10.33 55.88 ± 5.85 65.01 ± 1.53

Figures represent the mean of the groups ± the standard deviation.

d) Percentage of the dose administered excreted in the feces: Following oral 3H-naltrexone, the mean fecal excretion of radioactivity

was, on the average, 3.3% of the dose in the 48 hours following drug

ingestion. This result indicates that more than 95% of the dose given

was absorbed from the gastrointestinal tract. Since the fecal excretion following oral naltrexone was minor, no attempt was made to

collect feces after intravenous or subcutaneous dosing.

It became a matter of concern that we could only account for approximately 61% of the dose being excreted (58% in the urine and

3.3% in the feces) following oral 3H-naltrexone. In order to investigate the maximum excretion of 3H-naltrexone, the drug was administered orally to one subject, and plasma, urine, and feces were collected at appropriate intervals for a period of 133 hours. The

plasma levels of radioactivity were similar to those obtained before

and became undetectable after 48 hours. The results of the urinary

and fecal excretion of radioactivity (table 5) indicate that as in our

previous studies, the largest excretion occurred during the first 48

hours and declined progressively thereafter. The total dose excreted after this long interval of time was 79.3% in the urine, and

13.7% in the feces for a total of 93% of the amount ingested. Since
100 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

TABLE 5. Percentage of the Total Radioactivity Administered

Excreted by One Subject Following Oral 3-H Naltrexone

Time Urinary Excretion Fecal Excretion

24 hrs 60.91 0.58

48 hrs 12.88 9.42

72 hrs 3.56 2.80

96 hrs 1.23 0.10

120 hrs 0.55 0.70

133 hrs 0.15 0.10

TOTAL 79.28

Total Excretion = 92.98

13.70

total collection of all urine and especially all the feces is impossible, we consider, for practical purposes, that the entire amount of

naltrexone and/or its metabolites was excreted in this interval of

time.

In conclusion, naltrexone is a narcotic antagonist drug that is

rapidly and completely absorbed from the gastrointestinal tract or

from the subcutaneous tissue and can be intravenously administered in small doses without any untoward effects. The intravenous

administration of naltrexone should be considered in the treatment

of narcotic overdose emergencies, since this drug is 2 to 3 times

more potent than naloxone, a drug that is presently used as the

standard emergency treatment for narcotic overdose.

REFERENCES

1. W.R. Martin, D.R. Jasinski, and P.A. Mansky. Naltrexone, an antagonist for the

treatment of heroin dependence. Arch. Gen. Psychiat., 28, 784-791 (1973).

2. R.B. Resnick, J. Volavka, A.M. Freedman, and M. Thomas. Studies on EN-1639A

(Naltrexone), A new narcotic antagonit. Am. J. Psychiat., 131, 646-650 (1974).

3. L.W. Brady, D.Y. Cooper, M. Colodzin, J.E. McCleanathan, E.R. King, and R. Williams. Blood volume studies in normal humans. Surg. Gynecol. Obstet., 97, 25-32

(1953).
ADMINISTRATION OF NALTREXONE

AUTHORS

Mario Perez-Reyes

Department of Psychiatry

School of Medicine

University of North Carolina

Chapel Hill, NC 27514

Monroe E. Wall

Chemistry and Life Sciences Division

Research Triangle Institute

Research Triangle Park, NC 27709

101

Part IV

Metabolism

347-877 0 - 81 - 8

Naltrexone: Research Monograph 28

R. E. Willette and G. Barnett, eds.

National Institute on Drug Abuse, 1980

The Metabolism of

Naltrexone in Man

Monroe E. Wall, Dolores R. Brine, and Mario PerezReyes

The metabolism and elimination of [15,16-3H2] naltrexone

hydrochloride was studied in man following oral and intravenous administration. The same metabolites, although

in varying proportions, were observed in both cases; conjugated naltrexone and nonconjugated and conjugated 6ßnaltrexol were the major metabolites observed in plasma,

urine, and feces. 2-Hydroxy-3-O-methyl-6ß-naltrexol was

found in minor quantities. Naltrexone was almost completely absorbed following oral administration. About 60%

of the dose was found in the urine and only 5% in the

feces. A similar urinary excretion pattern was observed

after intravenous administration of naltrexone. In early

time periods after oral administration there was a rapid increase in free naltrexone plasma levels up to 1 hr. After

this time, levels remained fairly constant up to 4 hr and

then gradually declined. A similar pattern was observed for

conjugated naltrexone and nonconjugated and conjuated

6ß-naltrexol. These metabolites were found at levels 4-6

times higher than the parent compound at all times sampled. After intravenous administration, nonconjugated naltrexone plasma levels dropped sharply and continuously.

The major metabolites exhibited a pattern closely resembling that found for oral administration. Combined gas

chromatography-mass spectrometry was used to validate the

presence of naltrexone, 6ß-naltrexol and 2-hydroxy-3-Omethyl-6ß-naltrexol in urine. The structure of the latter

was rigorously proven by 13C-NMR. No evidence for the

presence of noroxymorphone or 3-O-methyl-6ß-naltrexol

could be obtained by gas chromatography-mass spectrometry. The metabolism of naltrexone administered subcutaneously was also determined in two subjects. Larger amounts

of 2-hydroxy-3-O-methyl-6ß-naltrexol were found in plasma

than had been present after oral or intravenous administration.

105
106 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

INTRODUCTION

Naltrexone (I) (figure 1) is a potent narcotic antagonist which has

been proposed for the treatment of opiate dependence (1). An excellent review of this subject can be found in the paper by Verebey et

al. (2). Cone isolated and identified 6ß-naltrexol (II) as the major

urinary excretion product of I in man (3). A detailed study of the

urinary excretion of I in man was carried out by Cone et al. (4) and

by Verebey et al. (5), both groups reporting that 6ß-naltrexol was

the major component in urine. Verebey et al. have developed sensitive gas liquid chromatography (GLC) methodology with electroncapture detection for the quantitative determination of naltrexone

and 6ß-naltrexol in human plasma (6). In other studies, Verebey

and coworkers have described the isolation and identification of a

new metabolite of naltrexone in human blood and urine, which

they identified as 2-hydroxy-3-O-methyl-6ß-naltrexol (III) (7). Cone

et al. (8) have recently identified both III and 2-hydroxy-3-Omethyl-naltrexone in urine by GLC techniques. Verebey et al. (2)

studied the oral administration of I and determined plasma levels

of I and its metabolites, II and III, in man. Recent studies in our

laboratory and by Ludden et al. (9) have indicated that other metaFIGURE 1. Naltrexone (I), 6ß-naltrexol (II), 2-hydroxy-3-O-methyl-6ß-naltrexol (III),

3-O-methyl-6ß-naltrexol (IV).
HUMAN METABOLISM OF NALTREXONE 107

bolites such as 6 -naltrexol, 3-O-methyl-6ß-naltrexol (IV), or noroxymorphone can be found in animals and man.

A major objective of our investigation is to obtain information

concerning naltrexone metabolism and pharmacokinetics that may

be subsequently applied to the pharmacokinetic and pharmacologic

evaluation of sustained-release naltrexone delivery systems in man.

The present paper is concerned with the metabolism and excretion

of I administered via oral (po) and intravenous (iv) routes. Some

limited preliminary data obtained by subcutaneous administration

(sc) of I are also included. The iv and sc data are being reported for

the first time in man. The study was designed to take advantage of

the availability of a number of synthetic reference compounds such

as I-IV (figure 1), noroxymorphone, 3-O-methyl-naltrexone, and 2-

hydroxy-3-O-methylnaltrexone, which are available through synthetic studies carried out at the Research Triangle Institute.

MATERIALS AND METHODS

Chemicals and Materials

Naltrexone hydrochloride was obtained from the National Institute on Drug Abuse (NIDA). [15,16-3H2

] naltrexone hydrochloride,

6ß-naltrexol, 2-hydroxy-3-O-methylnaltrexone, 3-O-methyl-naltrexone, 2-hydroxy-3-O-methyl-6ß-naltrexol, and 3-0-methyl-6ß-naltrexol were prepared at the Research Triangle Institute. Ethanol

(95%, USP) was purchased from U. S. Industrial Chemical Co., Louisville, Ky. All other solvents were of reagent grade and were used

without further purification. A solution of ß-glucuronidase (100,000

units/ml) and sulfatase (10,000 μmol of p-nitrocatechol sulfate

cleaved per hr per ml) from Helix pomatia (Type H-2) was purchased from Sigma Chemical Co., St. Louis, MO. Omnifluor was obtained from New England Nuclear, Boston, Mass. Monophasewas purchased from Packard Instrument Co., Downers Grove, Ill.

Thin-layer chromatography plates were 20 x 20 cm silica gel-onglass plates (0.25 mm thickness) prepared by E. Merck, Darmstadt,

Germany. A 2% solution of methoxyamine hydrochloride in pyridine (Mox reagent) and Tri-Sil TBT were obtained from Pierce

Chemical Co., Rockford, Ill. Sephadex LH-20 was purchased from

Pharmacia Fine Chemicals, Piscataway, N.J.

Clinical Procedures

In the study involving oral administration of the hydrochloride

salt of I, six normal males received 50 mg of I hydrochloride con-
108 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

taining 200 μCi of [15,16-3H2]-I hydrochloride. The drug was administered in 1 ml of water/95% ethanol (1:1, v/v) in two gelatin capsules 3 hr after subjects had received a light breakfast. Blood samples were drawn at 15-min intervals for 3 hr and at 4.5, 6, 12, and

24 hr. Plasma and red blood cells were separated by centrifugation.

Urine was collected at 0-3, 3-6, 6-12, 12-24, and 24-48 hr after drug

ingestion. Feces were collected at 0-24 and 24-48 hr.

Five normal male volunteers each received an iv injection of 1

mg of I hydrochloride (containing 198 μCi of [15,16-3Hz]-I hydrochloride) dissolved in 1 ml of normal saline. Blood samples were collected at 0.5, 1, 3, 5, 10, 15, 30, 60, 90, 120, 150, and 180 min and at 5, 7,

10, and 24 hr. Plasma and red blood cells were prepared. Urine was

collected at 0-3, 3-6, 6-9, 9-12, 12-24, 24-48, and 48-72 hr.

Two male volunteers received an sc injection of 4.4 mg of naltrexone hydrochloride (containing 126 μCi of [15,16-3H2

] naltrexone

hydrochloride dissolved in normal saline. Blood samples were collected at 5, 10, 20, 30, 45, 60, 75, and 90 min and at 2, 4, 6, 8, 12, 24,

30, and 48 hr. Urine was collected through 72 hr. After the initial

pilot study was conducted, preliminary reports were received of

possible tumor formation produced when naltrexone was administered to rodents. Although these initial reports have since been

largely discounted, all studies of naltrexone in man were terminated for the time being by our group.

Analytical Methods

These procedures are presented in detail in this monograph in

our paper dealing with analytical methodology (10).

RESULTS

Plasma Concentration of Naltrexone and Metabolites after Oral

Administration

The data for total drug, nonconjugated, and conjugated I plus

metabolites found in the plasma of human volunteers are shown in

figure 2. A rapid increase in total drug concentration occurred between 15 min and 1 hr. Levels of total drug (calculated as naltrexone equivalents) reached maximal values around 240 ng/ml (0.5%

dose/liter of plasma) in 2 hr, remained almost constant through 4.5

hr, and then decreased to 50 ng/ml in 24 hr. The level of the total

conjugated fraction was threefold that of nonconjugated naltrexone

and metabolites. Table 1 presents the analytical data for both nonconjugated and conjugated I and its metabolites, II, III, and IV. In
FIGURE 2. Naltrexone and metabolites found in human plasma after oral administration of

50 mg naltrexone hydrochloride. Values are the average for four subjects.
110 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

TABLE 1. Naltrexone and Metabolites Found in Human Plasma After

Oral Administration of 50 mg Naltrexone Hydrochloride

Values are the average for four subjects.

ng/ml plasma ± SE found nonconjugated as

Time

(min) 2-OH-3-0-

TIc Methyl- 3-0-MethylOrigin 6ß-Naltrexol 6ß-Naltrexol 6ß-Naltrexol Naltrexone

15 0.085 ± 0.044 1.4 ± 1.1 0.67 ± 0.35 0.23 ± 0.11 0.097 ± 0.050

30 0.96 ± 0.40 16 ± 10 0.55 ± 0.22 0.97 ± 0.54 4.4 ± 2.3

45 1.4 ± 0.5 22 ± 10 0.94 ± 0.11 0.85 ± 0.13 6.0 ± 2.1

60 1.6 ± 0.4 24 ± 8 1.3 ± 0.3 0.96 ± 0.08 6.5 ± 2.2

75 1.8 ± 0.5 22 ± 7 1.7 ± 0.5 0.72 ± 0.28 6.5 ± 3.3

90 2.5 ± 0.8 27 ± 7 2.7 ± 0.6 0.78 ± 0.14 5.9 ± 1.6

105 3.0 ± 0.9 29 ± 5 2.6 ± 0.6 1.3 ± 0.6 6.7 ± 1.4

120 2.2 ± 0.8 27 ± 5 2.7 ± 0.7 0.85 ± 0.19 7.5 ± 2.0

135 2.3 ± 0.3 32 ± 4 1.9 ± 0.5 0.72 ± 0.08 7.9 ± 1.9

150 2.5 ± 0.4 28 ± 2 2.4 ± 0.5 0.95 ± 0.24 6.9 ± 2.1

165 2.4 ± 0.3 29 ± 4 2.4 ± 0.7 1.3 ± 0.4 6.8 ± 1.6

180 3.4 ± 1.5 30 ± 4 3.1 ± 1.1 0.86 ± 0.36 6.2 ± 2.0

270 1.8 ± 0.2 32 ± 4 3.2 ± 1.2 1.0 ± 0.26 7.4 ± 2.6

360 1.2 ± 0.2 22 ± 4 2.4 ± 0.6 0.97 ± 0.34 4.4 ± 1.6

720 0.64 ± 0.19 13 ± 2 2.2 ± 1.0 0.62 ± 0.19 3.0 ± 0.9

1440 0.39 ± 0.08 6.3 ± 1.2 1.5 ± 0.6 0.37 ± 0.05 1.1 ± 0.4

Time

(min) TIc

Origin

ng/ml plasma ± SE found conjugated as

2-OH-3-0-

Methyl- 3-0-Methyl6ß-Naltrexol 6ß-Naltrexol 6ß-Naltrexol Naltrexone

15 1.5 ± 0.8

30 17 ± 9

45 18 ± 6

60 18 ± 6

75 16 ± 4

90 19 ± 4

105 22 ± 4

120 25 ± 6

135 2 6 ± 5

150 28 ± 1

165 32 ± 2

180 30 ± 6

270 38 ± 9

360 26 ± 6

720 15 ± 4

1440 4.3 ± 14

0.71 ± 0.23 0.46 ± 0.13 1.2 ± 0.4 1.7 ± 0.7

6.4 ± 2.6 3.6 ± 3.2 9.9 ± 5.6 22 ± 9

19 ± 5 4.4 ± 1.7 11 ± 7 46 ± 12

24 ± 5 5.3 ± 2.0 16 ± 9 50 ± 11

26 ± 8 9.1 ± 3.5 17 ± 8 44 ± 12

29 ± 8 8.8 ± 4.0 22 ± 9 50 ± 8

39 ± 8 9.8 ± 4.6 18 ± 9 52 ± 7

34 ± 6 8.0 ± 1.7 16 ± 5 56 ± 12

32 ± 6 5.8 ± 1.8 15 ± 6 52 ± 15

37 ± 3 8.1 ± 2.5 24 ± 11 67 ± 14

37 ± 3 9.1 ± 0.3 19 ± 4 50 ± 17

29 ± 6 6.7 ± 2.1 16 ± 3 49 ± 11

44 ± 11 7.6 ± 1.1 14 ± 7 34 ± 12

48 ± 16 7.6 ± 2.2 6.4 ± 3.4 24 ± 5

24 ± 4 5.3 ± 1.8 2.8 ± 0.8 15 ± 5

12 ± 2 5.2 ± 2.2 5.8 ± 1.4 4.2 ± 1.5
HUMAN METABOLISM OF NALTREXONE 111

FIGURE 3. Naltrexone and 6ß-naltrexol levels in plasma after oral administration

of 50 mg naltrexone hydrochloride. Values are the average for four subjects.

addition, results are presented for an unidentified polar material

(not noroxymorphone) which did not migrate from the TLC origin.

Figure 3 presents plasma decay curves for naltrexone and 6ß-naltrexol. The data in table 1 and figure 3 show that nonconjugated

naltrexone levels increased rapidly during the first 1530 min following oral administration and attained values of 6-8 ng/ml over

the period from 45 min to 4.5 hr. After this time levels began to

decrease, falling to 1 ng/ml after 24 hr. The plasma half-life of I in

the declining phase (3-24 hr, cf. figure 3) was 8.0 hr. The major metabolite found in the plasma was 6ß-naltrexol, (II), which showed

pharmacokinetics similar to I (plasma half-life 8.9 hr between 3-24

hr). Maximal levels of II were in the neighborhood of 22-30 ng/ml

between 45 min and 4.5 hr. Other metabolites, III and IV, were

minor, their concentrations never exceeding 3 ng/ml and 1 ng/ml,

respectively. In the conjugated fraction, the relationships between I

and II were reversed. In this case I was found to be the major product, with maximal levels between 50-60 ng/ml during 1-3 hr (cf.

table 1). Values of II were about 40% lower. Levels of conjugated I
112 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

did not materially decrease until 12 hr after administration. The

values for III and IV were considerably higher in the conjugated

fraction than in the nonconjugated fraction. The former metabolite

had maximal values of about 9 ng/ml from 1.25 to 2.75 hr; the

latter had values of 15-20 ng/ml from 1 to 4.5 hr.

Urinary Excretion after Oral Administration

Urinary excretion after oral administration of I is shown in table

2 and figure 4. About 30% of the total dose was excreted during the

first 12 hr, 50% in 24 hr, and 60% in 48 hr. The analytical data for

each time period for naltrexone and its metabolites, both nonconjugated and conjugated, are shown in table 2. 6ß-naltrexol was the

major compound found in the urine in the free form. The concentration of nonconjugated I was much lower than that of II (about

0.1) and was even lower than the levels of the minor metabolites,

III and IV. Average renal clearance values for nonconjugated I and

II calculated by the method described by Verebey et al. (2) were 77

and 270 ml/min, respectively.

TABLE 2. Naltrexone and Metabolites Found in Human Urine After

Oral Administration of 50 mg Naltrexone Hydrochloride

(Values are the average for six subjects ± SE.)

μg found nonconjugated as

Time 2-OH-3-0-

(hr) TIc 6ß- Methyl-6ß- 3-0-MethylOrigin Naltrexol Naltrexol 6ß-Naltrexol Naltrexone

3

6

12

24

48

80 ± 7 19OO ± 800 200 ± 98 420 ± 300 130 ± 43

42 ± 20 1200 ± 600 240 ± 110 200 ± 220 85 ± 42

46 ± 18 1400 ± 500 350 ± 140 310 ± 350 84 ± 50

60 ± 33 2000 ± 400 590 ± 388 390 ± 450 140 ± 81

53 ± 14 1400 ± 700 390 ± 270 340 ± 330 150 ± 76

μg found conjugated as

Time

(hr) TIc 2-OH-3-0-

Origin 6ß- Methyl-6ß- 3-O-MethylNaltrexol Naltrexol 6ß-Naltrexol Naltrexone

3 510 ± 130 1600 ± 700 120 ± 50 210 ± 50 2100 ± 680

6 410 ± 100 1300 ± 200 100 ± 40 150 ± 100 980 ± 510

12 410 ± 230 1900 ± 900 160 ± 70 120 ± 90 800 ± 660

24 360 ± 140 2500 ± 400 260 ± 100 100 ± 60 580 ± 260

48 180 ± 70 1600 ± 600 230 ± 110 42 ± 17 180 ± 68
FIGURE 4. Naltrexone and metabolites found in urine after oral administration of

50 mg naltrexone hydrochloride. Values are the average for six subjects
114 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

The conjugated fraction of the urine exhibited markedly different relationships in the relative abundance of I and II. At the initial time period, 0-3 hr, I was found at higher levels than II. The

levels of I then dropped sharply. II was again the major metabolite

at all the other time periods. Only small proportions of III and IV

were present. A considerable amount of highly polar material

which did not move from the origin was also found in the conjugated fraction. Values for the cumulative percent dose excreted as

nonconjugated and conjugated I and II are shown in figure 5. It is

evident from this data that only trace quantities of naltrexone

were excreted in the urine in the nonconjugated form. On the

other hand, significant quantities of the drug were excreted in the

conjugated form. In contrast, the more highly oxygenated minor

metabolites were excreted to a greater extent in the nonconjugated

FIGURE 5. Naltrexone and 6ß-naltrexol excreted in the urine after oral

administration of 50 mg naltrexone hydrochloride. Values are the average for six

subjects.
HUMAN METABOLISM OF NALTREXONE 115

than in the conjugated form. The major excretionary product was

6ß-naltrexol, the concentrations of the nonconjugated and conjugated forms being almost equal.

Fecal Excretion after Oral Administration

The feces were not a major route of elimination for naltrexone

and its metabolites. Analytical data are shown in table 3. In the

first 48 hr after administration of a 50 mg oral dose, only 4.8% of

the total dose was found in the feces, the larger portion of which

was excreted in the 24-48 hr time period. I and metabolites were

determined both in the nonconjugated and conjugated form in the

methanol-soluble feces fraction. 6ß-naltrexol was the major constituent found in both the nonconjugated and conjugated fractions.

There was also a methanol-insoluble fraction somewhat more sizable than the methanol-soluble fraction.

Plasma Concentration of Naltrexone and Metabolites after

Intravenous Administration

The plasma levels of total drug, nonconjugated, and conjugated I

plus metabolites after iv administration are shown in figure 6. The

total and nonconjugated drug fractions showed rather similar

plasma decay curves. Initially a sharp decrease in drug levels occurred in the period between 1-15 min ( phase). This was followed

by a more gradual loss of drug from the plasma over the next 24 hr

(ß phase). In contrast, the total conjugated fraction rose sharply between 1-30 min, increased to a maximum in 1 hr, gradually decreased through 10 hr, and then sharply decreased between 10-24

hr. The complete data for the individual metabolites of naltrexone,

both nonconjugated and conjugated, are shown in table 4.

The plasma disappearance curves for naltrexone and 6ß-naltrexol, nonconjugated and conjugated, are shown in figure 7. Levels

of nonconjugated I decreased rapidly over the 1-30 min time intervals ( phase) with a half-life of 8.5 min; the half-life for the ß

phase calculated from 1.5-10 hr was 1.9 hr. Levels of nonconjugated

II increased rapidly up to 1 hr. The rate of decline for II was much

slower than for I; the half-life from 1-24 hr was 9.3 hr. Conjugated I

and II exhibited plasma decay curves similar to nonconjugated II,

having lengthy retention times. On the other hand, nonconjugated

I showed a much sharper decline in the ß phase. Levels of metabolites III and IV were insignificant.
TABLE 3. Naltrexone and Metabolites Found in Human Feces after Oral Administration of 50 mg Naltrexone

Hydrochloride
FIGURE 6. Naltrexone and metabolites found in human plasma after intravenous

injection of 1 mg naltrexone hydrochloride. Values are the average for five subjects.
118 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

TABLE 4. Naltrexone and Metabolites Found in the Plasma of

Subjects Receiving 1 mg Naltrexone Hydrochloride Intravenously

ng per ml plasma found nonconjugated as

Time 2-OH-3-0

(min) methyl-6ß- 3-0-MethylTLC origin 6ß-Naltrexol naltrexol 6ß-naltrexol Naltrexone

1 2.1 ± 0.8 0.31 ± 0.05 2.2 ± 0.6 2.2 ± 0 7 8.2 ± 0.7

3 0.89 ± 0 17 0.30 ± 0.04 0.61 ± 0.24 0.54 ± 0.22 4.3 ± 1.2

5 0.60 ± 0.11 0.47 ± 0.10 0.49 ± 0.12 0.50 ± 0.30 3.3 ± 0.4

10 0.52 ± 0.11 0.78 ± 0.14 0.16 ± 0.04 0.32 ± 0.22 2.3 ± 0.3

15 0.39 ± 0.07 0.72 ± 0.13 0.17 ± 0.04 0.20 ± 0.05 1.8 ± 0.2

30 0.26 ± 0.03 0.80 ± 0.09 0.11 ± 0.07 0.085 ± 0.019 1.5 ± 0.2

60 0.22 ± 0.04 0.75 ± 0.08 0.082 ± 0.015 0.052 ± 0.019 0.87 ± 0.1

90 0.17 ± 0.03 0.72 ± 0.05 0.11 ± 0.02 0.058 ± 0.004 0.73 ± 0.09

120 0.16 ± 0.04 0.79 ± 0.11 0.15 ± 0.03 0.044 ± 0.008 0.63 ± 0.08

150 0.17 ± 0.02 0.66 ± 0.08 0.11 ± 0.02 0.052 ± 0.005 0.53 ± 0.06

180 0.12 ± 0.03 0.75 ± 0.11 0.14 ± 0.02 0.038 ± 0 002 0.45 ± 0.08

300 0.059 ± 0.008 0.61 ± 0.06 0.11 ± 0.02 0.019 ± 0.002 0.16 ± 0.03

420 0.059 ± 0.013 0.59 ± 0.04 0.12 ± 0.02 0.014 ± 0.005 0.090 ± 0.03

600 0.056 ± 0.011 0.42 ± 0.03 0.094 ± 0.020 0.010 ± 0.008 0.034 ± 0.005

1440 0.016 ± 0.004 0.14 ± 0.02 0.052 ± 0.017 0.003 ± 0.001 0.003 ± 0.001

ng permlplasmafound confugated as

Time 2-OH-3-0

(min) methyl-6ß- 3-0-MethylTLC origin 6ß-Naltrexol naltrexol 6ß-naltrexol Naltrexone

1 0.19 ± 0.01 0.086 ± 0.023 0.072 ± 0.010 0.13 ± 0.03 0.85 ± 0.35

3 0.18 ± 0.01 0.088 ± 0.011 0.084 ± 0.029 0.080 ± 0.012 0.49 ± 0.09

5 0.12 ± 0.01 0.097 ± 0.023 0.058 ± 0.012 0.066 ± 0.021 0.52 ± 0.06

10 0.19 ± 0.04 0.14 ± 0.04 0.098 ± 0.031 0.076 ± 0.021 0.57 ± 0.14

15 0.20 ± 0.07 0.16 ± 0.04 0.089 ± 0.012 0.15 ± 0.03 0.78 ± 0.14

30 0.23 ± 0.04 0.30 ± 0.05 0.15 ± 0.03 0.15 ± 0.05 0.86 ± 0.14

60 0.35 ± 0.11 0.52 ± 0.06 0.15 ± 0.02 0.17 ± 0.03 0.93 ± 0.25

90 0.34 ± 0.08 0.53 ± 0.03 0.12 ± 0.03 0.12 ± 0.05 0 .88 ± 0.23

120 0.39 ± 0.11 0.70 ± 0.08 0.17 ± 0.06 0.11 ± 0.05 0.60 ± 0.12

150 0.34 ± 0.11 0.62 ± 0.08 0.17 ± 0.04 0.095 ± 0.039 0.61 ± 0.21

180 0.42 ± 0.09 0.49 ± 0.02 0.16 ± 0.05 0.097 ± 0.045 0.67 ± 0.14

300 0.39 ± 0.13 0.80 ± 0.04 0.11 ± 0.02 0.11 ± 0.05 0.51 ± 0.16

420 0.36 ± 0.01 0.73 ± 0.12 0.11 ± 0.02 0.039 ± 0.020 0.39 ± 0.09

600 0.23 ± 0.05 0.64 ± 0.12 0.082 ± 0.022 0.038 ± 0.021 0.29 ± 0.09

1440 0.12 ± 0.05 0.31 ± 0.08 0.049 ± 0.018 0.020 ± 0.011 0.11 ± 0.05

Nonconjugated values are the average for five subfects ± SE. confugated values are

the average for four subfects ± SE.
HUMAN METABOLISM OF NALTREXONE 119

FIGURE 7. Naltrexone and 6ß-naltrexol levels in plasma after intravenous injection

of 1 mg naltrexone hydrochloride. Free values are the average for five subjects;

conjugated values are the average for four subjects.

Levels of Naltrexone and Metabolites in Red Blood Cells

Red blood cells from subjects receiving naltrexone hydrochloride

by oral or intravenous administration were analyzed for total drug

and for nonconjugated I and II. In both cases the results were similar to values found in the plasma, which indicates a uniform distribution of naltrexone and its metabolites in plasma and red blood

cells. Minor metabolites were also of the same order.

Urinary Excretion after Intravenous Administration

Urinary levels after iv administration are shown in figure 8 for

total I and metabolites and in figure 9 for naltrexone and 6ß-naltrexol. Table 5 presents the complete analytical information.

During the first 6 hr an average of 26% of the dose was excreted in

347-877 0 - 81 - 9
FIGURE 8. Naltrexone and metabolites found in urine after intravenous injection of

1 mg naltrexone hydrochloride. Values are the average for five subjects.
FIGURE 9. Naltrexone and 6ß-naltrexol excreted in urine after intravenous

injection of 1 mg naltrexone hydrochloride. Free values are the average for five

subjects; conjugated values are the average for four subjects.
122 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

TABLE 5. Naltrexone and Metabolites Found in the Urine of Subjects

Receiving 1 mg Naltrexone Hydrochloride Intravenously

μg ± SE excreted nonconjugated in the urine as

Time 2-OH-3-0-

(hr) TIc 6ß- Methyl-6ß- 3-0-MethylOrigin Naltrexol Naltrexol 6ß-Naltrexol Naltrexone

0-3 3.5 ± 0.6 40 ± 9 5.0 ± 1.4 5.0 ± 1.5 12 ± 3

3-6 1.8 ± 0.2 42 ± 3 4.5 ± 0.3 1.4 ± 0.4 4.2 ± 1.0

6-9 1.1 ± 0.1 28 ± 4 3.5 ± 0.5 0.39 ± 0.12 1.0 ± 0.3

9-12 1.0 ± 0.6 27 ± 17 2.9 ± 0.6 0.50 ± 0.34 0.75 ± 0.25

12-24 1.3 ± 0.3 41 ± 4 6.4 ± 1.6 0.27 ± 0.01 0.71 ± 0.15

24-48 1.9 ± 0.4 55 ± 11 11 ± 2 0.15 ± 0.03 0.24 ± 0.034

48-72 0.51 ± 0.10 12 ± 2 3.5 ± 0.6 0.047 ± 0.016 0.042 ± 0.013

μg ± SE excreted confugated in the urine as

Time 2-OH-3-0-

(hr) TIc 6ß- Methyl-6ß- 3-0-MethylOrigin Naltrexol Naltrexol 6ß-Naltrexol Naltrexone

0-3 6.9 ± 0.9 24 ± 7 4.6 ± 1.3 5.0 ± 0.7 36 ± 7

3-6 5.3 ± 0.8 30 ± 5 3.2 ± 0.4 2.8 ± 1.1 22 ± 6

6-9 3.4 ± 0.8 11 ± 4 1.7 ± 0.4 0.94 ± 0 21 6.5 ± 1.8

9-12 3.4 ± 1.8 24 ± 8 1.6 ± 0.4 0.98 ± 0.18 5.9 ± 2.4

12-24 4.2 ± 1.6 41 ± 1 3.0 ± 0.9 1 .0 ± 0.2 7.5 ± 0.9

2448 4.6 ± 0.7 32 ± 6 3.6 ± 0.8 1.1 ± 0.3 4.6 ± 1.1

48-72 0.92 ± 0.08 9.4 ± 2.4 1.4 ± 0.3 0.15 ± 0.03 0.65 ± 0.07

Nonconjugated values are the average for five subjects ± SE: conjugated values are the

average for four subjects ± SE.

the urine, after 24 hr about 50%, and after 72 hr 60%. Approximately equal quantities of nonconjugated and conjugated urinary

metabolites were observed. In contrast to the oral data, the standard error of the iv data showed much less variation, a not unexpected observation. As shown in figure 9, II, nonconjugated and

conjugated, was the major urinary excretion product found after iv

administration of naltrexone hydrochloride. This metabolite was

excreted continuously in both nonconjugated and conjugated forms

through 48 hr after administration. On the other hand, nonconjugated I was found in low concentration. Excretion of I was rapid,

being virtually complete in 10-12 hr. Conjugated I was found in the

urine in considerably higher concentration than the nonconjugated

drug. As in the oral study, the metabolites III and IV were of

minor magnitude as compared with II. The 2-hydroxy-3-O-methyl
HUMAN METABOLISM OF NALTREXONE 123

metabolite (III) was found during most time periods at significantly

higher levels than the 3-O-methyl metabolite (IV). Renal clearance

values for I and II were 92 and 360 ml/min, respectively.

Subcutaneous Administration

Plasma levels for total, nonconjugated, and conjugated I when

the drug was administered subcutaneously (sc) are shown in figure

10. As was found after iv administration, the conjugated fraction

predominated. After reaching a peak of 28 ng/ml in about 1 hr, the

drug level decreased in a linear fashion over a 50-hour time period.

The data for nonconjugated I and metabolites are shown in figure

11. The results are surprising and definitely require further confirmation with more subjects. Initially the 2-hydroxy-3-O-methyl metabolite (III) predominated but decreased rapidly between 8-50 hr;

6ß-naltrexol (II) showed rather steady levels from 2-50 hr. The

parent drug (I) attained a maximal value of 0.75 ng/ml within 30

min and thereafter decreased rapidly in a biphasic elimination pattern. Urinary excretion patterns are shown in figures 12 and 13.

Rapid excretion of total drug occurred over 3 to 12 hr, about 35-

50% of the total drug being eliminated by 12 hr; thereafter excretion continued slowly, 60-80% being eliminated in 72 hr. The conjugated and nonconjugated fractions were of the same order of magnitude; the former was somewhat larger during the first 12 hr.

Data for the parent drug (I) and metabolites (II) and (III) in the

nonconjugated urine fraction are presented in figure 13. Subjects

LB and JW showed similar values for excretion of 6ß-naltrexol (II);

however, subject JW showed much lower values than LB for naltrexone (II) and metabolite III. In a general way the results found

for urinary excretion via sc administration resembled results obtained by iv administration much more closely than the plasma

values found by the two methods.

DISCUSSION

Administration of naltrexone hydrochloride to human volunteers

by oral or intravenous routes gave similar metabolic patterns but

with some characteristic differences in the plasma levels of naltrexone and its metabolites. As was observed by other workers (2,4),

oral administration of I is followed by rapid metabolism in the

liver. The predominant metabolic reaction involves the reduction of

the 6-oxo moiety in I to the corresponding 6ß-hydroxy group by

liver enzymes. Although the other epimer 6 -naltrexol has been
FIGURE 10. Plasma levels of naltrexone and metabolites following subcutaneous

administration of 4.4 mg naltrexone hydrochloride to Subject LB.
FIGURE 11. Plasma levels of naltrexone and metabolites following subcutaneous

administration of 4.4 mg naltrexone hydrochloride to Subject LB.
FIGURE 12. Urinary excretion of naltrexone and metabolites following

subcutaneous administration of 4.4 mg of naltrexone hydrochloride to human males.
FIGURE 13. Urinary excretion of naltrexone and metabolites following

subcutaneous administration of 4.4 mg of naltrexone hydrochloride.
128 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

found in rodents (11), its occurrence in man is negligible. In our

studies we also isolated a minor metabolite which was established

unequivocally by 13C-NMR (cf. table 6) to be 2-hydroxy-3-O-methyl6ß-naltrexol (III). An analogous metabolite containing the 6-oxo

moiety has recently been shown to occur in the urine in trace

amounts by Cone et al. (8). We also have some evidence for 3-Omethyl-6ß-naltrexol (IV) by TLC techniques but as yet lack positive

GLC/MS confirmation of this compound. Although Malspeis et al.

(11) and our group (12) have shown that noroxymorphone, an Ndealkylated metabolite of naltrexone, is found in the rat and other

animals, a GLC/MS study of urine and feces of human volunteers

failed to detect the presence of this metabolite (10). Our studies and

those of other groups cited previously clearly show that the first

pass of naltrexone through the liver produces several types of

metabolic reactions. One type involves extensive conjugation of naltrexone and the other metabolites. As a consequence, conjugated

metabolites frequently form the major fraction in both plasma and

urine. Reduction of the 6-ketone to give 6ß-naltrexol is the other

major metabolic reaction. Subsequently there are several minor

metabolic transformations produced by microsomal enzymes which

involve methylation of the phenolic 3-hydroxyl and hydroxylation

at the adjacent S-position. Our studies, those of Verebey et al. (7),

and the recent work of Cone et al. (8) indicate (but firm proof is

lacking) that 3-methylation may occur prior to 2-hydroxylation.

Our studies on the metabolism of naltrexone after intravenous administration indicate that the metabolic pattern closely resembles

that found for oral administration.

After oral administration we found that naltrexone was almost

completely absorbed; only 5% of the total dose was eliminated in

the feces. In excellent agreement with previous studies (2,4), we

found that urinary excretion comprised the major route for elimination of naltrexone in man. About 60% of the total dose was

eliminated in 48 hr after administration by the oral route and

about 55% in 48 hr after administration by the iv route. Nonconjugated naltrexone was rapidly eliminated after both oral and iv administration; whereas both nonconjugated and conjugated 6ß-naltrexol and conjugated naltrexone were found in significant quantities over long time periods (cf. figures 6 and 10). Renal clearance

values for naltrexone and 6ß-naltrexol after administration of I by

the oral route were 77 and 270 ml/min, respectively. (Verebey et

al. (2) found 67 and 318 ml/min in their study.) Data obtained after

administration by the iv route were in reasonable agreement with
HUMAN METABOLISM OF NALTREXONE 129

TABLE 6. 13C-NMR Chemical Shifts of Naltrexone Metabolitea

Identification

of Carbon Chemical Shifts

1

2

3

4

5

6

7

a

9

10

11

12

13

14

15

16

NCH2

104.41b

148.74

130.94

145.27

95.83

71.89

25.47

d

61.89

22.88

126.21

123.14

46.00

70.08

46.60

59.06

9.23

3.77, 3.62 3.77, 3.62

60.14 59.80

105.38’

150.15

132.06

146.54

96.46

71.79

27.03

d

61.85

22.64

126.26

122.50

46.00

69.84

d

43.66

58.43

9.23

aChemical shifts are in ppm relative to tetramethylsilane.

bSolvent is chloroform-d.

cSolvent is dimethylsulfoxide-d6

.

d

There are four peaks in the region of 29-31 ppm, two of which are impurity peaks. It

is not possible to assign the 8 and 15 carbons with certainty at this time.

results after oral dosage (92 and 360 ml/min for naltrexone and 6ß

naltrexol, respectively).

A limited pilot study of the sc administration of naltrexone (I) indicates several major differences in the composition of the plasma

metabolites as compared with iv studies. One subject showed surprisingly high initial levels of the normally minor metabolite III.

The parent drug I was found at low levels after sc administration;

whereas after iv injection levels of I were high for several hours. A

comparison of urinary elimination by the sc and iv routes showed

the two methods giving reasonably comparable results. Further

studies with more subjects receiving sc administration of I are evidently required.
130 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

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2. K. Verebey, J. Volavka, S.J. Mule, and R.B. Resnick. Naltrexone: disposition, metabolism, and effects after acute and chronic dosing. Clin. Pharmacol. Therap.,

20, 315-328 (1976).

3. E.J. Cone. Human metabolite of naltrexone (n-cyclopropylmethyl-noroxymorphone) with a novel C-6 isomorphine configuration. Tetrahedron Lett., 28, 2607-

2610 (1973).

4. E.J. Cone, C.W. Gorodetzky, and S.Y. Yeh. The urinary excretion profile of naltrexone and metabolites in man. Drug Metab. Dispos., 2, 506-512 (1974).

5. K. Verebey, S.J. Mule, and D. Jukofsky. A gas liquid chromatographic method for

the determination of naltrexone and beta-naltrexol in human urine. J. Chromatogr., 111, 141-148 (1975).

6. K. Verebey, M.J. Kogan, A. DePace, and S.J. Mule. Quantitative determination of

naltrexone and beta-naltrexol in human plasma using electron capture detection. J. Chromatogr., 118, 331-335 (1976).

7. K. Verebey, M.A. Chedekel, S.J. Mule, and D. Rosenthal. Isolation and identification of a new metabolite of naltrexone in human blood and urine. Res.

Commun. Chem. Pathol. Pharmacol., 12, 67-84 (1975).

8. E.J. Cone, C.W. Gorodetzky, W.D. Darwin, F.I. Carroll, G.A. Brine, and C.D.

Welch. The identification and measurement of two new metabolites of naltrexone in human urine. Res. Commun. Chem. Pathol. Pharmacol., 20, 413 (1978).

9. T.M. Ludden, MS. Bathala, L. Malspeis, M.A. Drum, and R.H. Reuning. Elimination of radioactivity following administration of (15,16-3H) naltrexone to rats

and guinea pigs. Drug Metab. Dispos., 6, 321-328 (1978).

10. M.E. Wall and D.R. Brine. Analytical methods for quantitative and qualitative

analysis of naltrexone and metabolites in biological fluids. This volume.

11. L. Malspeis, MS. Bathala, T.M. Ludden, H.B. Bhat, S.G. Frank, T.D. Sokoloski,

B.E. Morrison, and R.H. Reuning. Metabolic reduction of naltrexone I. Synthesis, separation and characterization of naloxone and naltrexone reduction products and qualitative assay of urine and bile following administration of naltrexone, naltrexone, -naltrexol or ß-naltrexol. Res. Commun. Chem. Pathol. Pharmacol., 12, 43-65 (1975).

12. M.E. Wall and D.R. Brine. Annual Report. NIDA Contract No. HSM-42-71-95,

151-160 (1976).

ACKNOWLEDGMENTS

These studies were supported by National Institute on Drug

Abuse (NIDA) Contract HSM-42-71-95. All synthetic compounds

were prepared at the Research Triangle Institute under NIDA Contract 271-76-3326. The authors express their appreciation to Mrs.

Carolyn Parker, Dr. Joan Bursey and Mr. Horace Parkinson for excellent technical assistance.
HUMAN METABOLISM OF NALTREXONE

AUTHORS

Monroe E. Wall and Dolores R. Brine

Research Triangle Institute

Research Triangle Park, NC 27709

Mario Perez-Reyes

Department of Psychiatry

School of Medicine

University of North Carolina

Chapel Hill, NC 27514

131
Naltrexone: Research Monograph 28

Ft. E. Willette and G. Barnett, eds.

National Institute on Drug Abuse, 1980

Current Status of

Preclinical Research on

Disposition,

Pharmacokinetics, and

Metabolism of Naltrexone

Anand L. Misra

Considerable species-dependent differences exist in the

quantitative pattern of the metabolism of naltrexone. The

metabolic pathways of naltrexone in different species were:

(a) 6-keto group reduction to epimeric 6a- or 6ß-naltrexols,

(b) glucuronide conjugation of naltrexone or naltrexols, (c)

N-dealkylation to nor-compounds, (d) 3-O-methylation and

(e) 2-hydroxylation. Substantially greater quantities of 6ßnaltrexol and/or its conjugates were excreted in urine of

man, monkey, guinea pig and rabbit after naltrexone administration, and very small quantities were excreted in

mouse, rat and dog. Chicken produced predominantly the

6a-naltrexol and the dog excreted mainly the 3-naltrexone

glucuronide, with very small quantities of 6a- or 6ß-naltrexols. Stereoselective metabolism of naltrexone to naltrexols is mediated by two different liver enzymes, and the formation of 2-hydroxy-3-O-methyl naltrexone or the corresponding 6ß-naltrexol is mediated by catechol-O-methyl

transferase. No significant differences were observed in the

extent of plasma-protein binding (20-26%) of naltrexone in

various species, and the low extent of binding indicated a

large apparent volume of distribution for naltrexone in different species and man. Pharmacokinetic studies have

shown a 2-compartmental decay of drug in plasma, and in

man the plasma t1/2 values of naltrexone in acute and

chronic state after 100 mg p.o. doses were 10.3 ±3.3 and 9.7

132
DISPOSITION OF NALTREXONE 133

± 1.1 hr respectively. Steady state plasma concentration of

naltrexone (10.9 ng/ml) was rapidly achieved in man after

a few doses, and there was no accumulation of drug or metabolites in plasma on chronic dosing. Naltrexone did not

induce its own metabolism on chronic dosing. In mice,

chronic administration of naltrexone was shown to depress

mixed function oxidase activity possibly due to a metabolite

of 6ß-naltrexol. A significant portion of the naltrexone dose

remains unaccounted for in different species and man, implying the existence of additional pathways of metabolism.

In general, the comparative antagonist activity of metabolites was much lower than naltrexone, but the much slower

elimination of lipophilic 3-O-methyl metabolites as compared to 6ß-naltrexol or naltrexone may point to their possible role in the overall pharmacologic activity and long

duration of action of naltrexone.

Naltrexone (N-cyclopropylmethylnoroxy morphone, EN-1639A) is

a potent narcotic antagonist (1,2,3) which fulfills the criteria for

clinical usefulness in the treatment of opiate dependence. It is nonaddictive, orally effective, and relatively nontoxic with fairly minor

or well-tolerated side-effects and a large margin of safety. A single

70-120 mg oral dose of naltrexone can provide blockade to 25 mg

intravenous heroin challenges in man for periods from 24 to 72 hr

(4,5,6). In well-motivated opiate-dependent subjects naltrexone provides a promising transitional modality in step-wise treatment

from methadone to a drug-free state (7).

COMPARATIVE POTENCIES OF NARCOTIC

ANTAGONISTS

Previous work (8,3) in mice, rats and in vitro longitudinal muscle

of guinea pig ileum (9) showed that naltrexone essentially had no

agonist activity and did not produce analgesia or respiratory depression in animals. It was approximately 40 times as active as nalorphine and twice as active as naloxone on subcutaneous injection

in rats previously treated with oxymorphone (8). In mice it was

about 8 times more active than naloxone when given orally and in

rabbit twice as active in antagonizing the respiratory depression induced by oxymorphone. The time-course of action of naltrexone

orally in rats was about 3 times longer than that of naloxone (10).

In rhesus monkeys, the precipitation of abstinence syndrome with

naltrexone (s.c.) was achieved at slightly lower doses than with naloxone (11). Martin et al. (3) observed that 30 to 50 mg oral doses of

naltrexone in human subjects produced narcotic blockade equivalent to 2500-3000 mg of naloxone and abrupt discontinuation of
134 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

daily doses resulted in no signs of abstinence, and no subjective responses or physiological changes characteristic of agonist activity

were seen with 80 mg oral doses of naltrexone. In patients dependent on 60 mg morphine daily, naltrexone (s.c.) was 17 times more

potent than nalorphine in precipitating abstinence. The timecourse of action of naltrexone (50 mg p.o.) in man was substantially

longer than naloxone but somewhat shorter than cyclazocine (12).

Thus in experimental animals and man, naltrexone was more

potent and longer-acting than nalorphine and naloxone. The lack

of agonist activity, greater potency and longer duration of action

present distinct advantages of naltrexone over naloxone in the

management of opiate-dependent individuals.

PRECLINICAL TOXICITY OF NALTREXONE IN

EXPERIMENTAL ANIMALS

These studies summarized by Braude and Morrison (13) have

shown that naltrexone was not toxic in any animal species (rat, dog

and monkey) at doses of at least 20 mg/kg which are 20 times

greater than that recommended clinically (1 mg/kg), and data from

rats and dogs have indicated that even a larger margin of safety

was possible. The LD50 (mg/kg) of acutely administered naltrexone

in mice was 180 ± 24 (i.v.), 570 ± 19 (s.c.), 1100 ± 96 (p.o.); in rats

the corresponding values were 1930 ± 338 (s.c.), 1450 ± 265 (p.o.);

in dogs 200 (s.c.) and 130 (p.o.), but oral toxicity in this species was

complicated due to the emesis produced by drug. In rhesus monkeys, the lethal dose of naltrexone was 300 mg/kg (s.c.), while a p.o.

dose of 1 g/kg produced salivation, emesis, and hypoactivity. In all

cases deaths occurred after clonic-tonic convulsions, usually preceded by restlessness, tremor, depression, salivation, or retching and

emesis. Peak effects usually occurred 30-45 min after s.c. injection,

and signs of toxicity usually appeared 2 to 4 hr after dosing, but

there was no delayed lethality.

DISSOCIATION CONSTANT, DISTRIBUTION

COEFFICIENT, PLASMA-PROTEIN AND OPIATE

RECEPTOR BINDING OF NALTREXONE

The pKa’s of naltrexone, naloxone and nalorphine at 37°C were

8.13, 7.82 and 7.59 respectively (14) and octanol/water distribution

coefficients (pH 7.40) were 13.08, 33.55, 28.16 respectively. No significant species differences in extent of binding of [15,16-3H] nal-
DISPOSITION OF NALTREXONE 135

trexone in human, monkey, dog, guinea pig, rat and mouse plasma

were observed, and these values measured by equilibrium dialysis

at 37°C ranged between 21-26% in the concentration range 0.1-500

ng/ml (15). The low extent of plasma-protein binding implies a

large apparent volume of distribution for naltrexone in different

animal species and man and further suggests that potential interactions involving displacement of drug from binding sites will not

be a therapeutic problem with naltrexone. The receptor binding

constants (KRB x 109) using [3H] dihydromorphine and 10,000 g particulate fraction of rat brain homogenate (pH 8, sucrose-tris solution) for naltrexone, naloxone and nalorphine were 0.2, 1.0, 1.5 respectively (16) and there was a good correlation between binding

constant and antagonist potency as determined in the guinea pig

ileum preparations (9). The greater intrinsic activity of naltrexone

as compared to naloxone, however, is not sufficient to explain the

20- to 30-fold dose difference needed for naloxone to produce effects

of similar time-course as naltrexone and underlying differences in

disposition and metabolism may play an important role in differential potencies of these two antagonists.

DISPOSITION, METABOLISM AND

PHARMACOKINETICS OF NALTREXONE IN

EXPERIMENTAL ANIMALS

The isolation and identification of a major metabolite of naltrexone in man was reported by Cone (17). This urinary metabolite, 6ß

naltrexol was formed by the reduction of 6-keto group to an alcohol

and was assigned the ß-isomorphine configuration (with 6-OH in

equatorial position and ring in the chair form) (figure 1). This configuration was subsequently confirmed by its stereospecific synthesis (18) and NMR, IR and mass spectral data (19). A substantial species variation was observed with regard to the formation of 6ß or

6 -naltrexol. Chicken produced predominantly the 6 -naltrexol (6-

OH in axial position and ring in the chair form) (20) and the

guinea pig and rabbit 6ß-epimer along with small amounts of 6

naltrexol (21). Studies by Malspeis et al. (22) showed that substantially greater quantities of 6ß-naltrexol and/or its conjugates were

excreted in the urine of man, monkey, guinea pig and rabbit after

naltrexone administration, and very small quantities of 6ß-metabolite were excreted in mouse, rat and dog. Using a sensitive electron-capture gas chromatographic method for the differentiation of

6 - and 6ß-naltrexol in presence of naltrexone, 6 -epimer was

shown to be present in trace amounts in monkey and guinea pig

347-877 0 - 81 - 10
136 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

urine. Following a 1 mg/kg i.v. injection of [15,16-3H] naltrexone to

guinea pigs, 84 and 14% of radioactivity was excreted in urine and

feces respectively. In guinea pig excreta, 64% of the dose corresponded to naltrexone and conjugates, 19% to 6ß-naltrexol and

conjugates and 2% to 6 -naltrexol and conjugates. In urine, the radioactivity corresponding to 6 -naltrexol and naltrexone was

mainly in conjugated form; whereas 6ß-naltrexol was mainly unconjugated. The radioactivity in feces corresponded principally to

conjugated naltrexone and 6ß-naltrexol. In gall bladder bile of

these animals, conjugated naltrexone and 6ß-naltrexol were found

2 hr after drug administration (23). 6ß-naltrexol (1 mg/kg i.v.) was

excreted in free and conjugated form in bile and urine of guinea

pig. The same dose of 6 -naltrexol gave, in addition to free drug

and conjugated metabolite, another unidentified metabolite with a

gc retention time similar to but not identical to 6ß-naltrexol (22).

Figure 1
DISPOSITION OF NALTREXONE 137

Comparative studies on the 24-hr urinary excretion of naltrexone

and its metabolite in monkey, rabbit and rat (24) have shown that

in monkeys receiving a 12 mg/kg dose p.o. daily for 8 months, 0.3%

of dose was excreted as free 6ß-naltrexol and 3.0% as its conjugate.

Females excreted twice as much total base (38.2%) as males

(16.5%). Rabbits receiving 30 mg/kg dose i.p. daily for 8 days excreted conjugated naltrexone and ß-naltrexol 14.5% and 3.3% of

the dose respectively. Rats receiving 100 mg/kg dose p.o. daily for

12 months excreted less than 1% of the dose as free and conjugated

naltrexone and ß-naltrexol. Studies by Cone and Gorodetzky (25)

also showed very low recoveries in the rat (5.5% of the dose). However recent work by Ludden et al. (23) has shown that rats excreted

42 and 55% of the dose in urine and feces respectively after a 1

mg/kg i.v. injection of [15,16-3H] naltrexone. Other routes of metabolism, therefore, may play a role in the disposition of naltrexone.

Preliminary studies on the pharmacokinetics of naltrexone (1

and 5 mg/kg i.v.) in rabbits using a specific electron capture-gas

chromatographic assay (26) have shown a 2-compartmental decay of

drug in plasma with t1/2ß 70 ± 10 min and 57 ± 6 min respectively with 2 dosages. The concentration of drug in semen was 13 and

11 times that in plasma at 120 min and the t1/2ß values in semen

with 2 dosages were 133 and 94 min respectively. Distribution studies using gas chromatography and [3H] labeled drug showed the following values μg/g) 90 min after a 5 mg/kg i.v. injection: plasma

0.37, brain 2.78, lung 2.39, spleen 7.77, liver 0.08, kidney 3.95,

muscle 1.48, testes 2.33, fat 0.42, stomach 7.08, and small intestine

1.28 respectively. Isolated perfused liver studies with a 10 mg naltrexone dose gave a till of 10 min. Evidence was obtained for several glucuronide metabolites of naltrexone and 6ß-naltrexol in bile.

In dog, the duration of action of naltrexone (27) was surprisingly

short (2 hr) in contrast to man (12 hr) and this species excreted naltrexone predominantly as its 3-glucuronide with little or no free or

conjugated 6ß- or 6 -naltrexol (25). Plasma naltrexone kinetics

after intravenous bolus administration has recently been reported

in the dog and the monkey (28). A 2-compartment open pharmacokinetic model fitted to plasma level-time data for naltrexone in 2

dogs (0.72 mg/kg, i.v. dose) yielded a t1/2ß of 1.0 hr (52-67 min) and

total body clearance of 51-55 ml/min/kg. In 6 rhesus monkeys

given a 1.0 or 10.0 mg/kg iv. dose, a t1/2ß of 7.8 hr and total body

clearance of 64 ml/min/kg (40-100 ml) was observed. The total body

clearance for naltrexone was greater than the hepatic plasma or

blood flow in both dogs and monkeys and this finding together with

extremely low renal excretion of naltrexone (1% of dose) suggested

the existence of other elimination mechanisms besides liver metab-
138 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

olism and renal excretion. Such mechanisms include fecal excretion of drug and metabolites, formation of unknown metabolites

and urinary excretion of conjugates not hydrolyzable by the

enzyme preparation. The serum t1/2ß for naltrexone in the dog has

been reported (29) to be 85.1 ± 9.0 min after a 5 mg/kg i.v. injection.

Recent sensitive methods have shown the presence of small

quantities (< 1%) 6 -naltrexol in the urine of man, rabbit, dog and

rat (25). 2-hydroxy-3-O-methyl 6ß-naltrexol has been identified (30)

as another metabolite of naltrexone in humans receiving 100 mg

naltrexone orally. This lipophilic metabolite had a long bioavailability, much slower excretion rate than naltrexone and 6ß-naltrexol.

Cone et al. (31) have shown that in human subjects receiving a

single 50 mg p.o. dose of naltrexone, excretion of this metabolite

over a period of 5 days was 4.6 ± 2.1% of the dose, with a mean

excretion t1/2 of 20.2 ± 1.8 hr. 2-hydroxy-3-O-methyl naltrexone

was observed as another minor metabolite (0.45 ± 0.08% of the

dose) of naltrexone in human subjects and the rat. These metabolites were predominantly excreted in free form. Unequivocal confirmation of the structure of 2-hydroxy-3-O-methyl 6ß-naltrexol was

provided recently by C-13 nuclear magnetic resonance technique

(32) and this study further ruled out the formation of isomeric 2-Omethyl compound.

Metabolic studies in the rat have shown that a very large portion

of the dose of naltrexone remains unaccounted for (24,251 and

recent studies utilizing gc/mass spectrometry in our laboratory

(Verebey et al., unpublished observations) have shown that naltrexone undergoes 3-O-methylation in this species to give another lipophilic metabolite, 3-O-methyl naltrexone. Disposition of [15,163H]

naltrexone in the central nervous system of the rat was investigated by Misra et al. (33). After a 10 mg/kg s.c. injection, the peak

concentrations in brain and plasma occurred within 0.5 hr. Levels

of naltrexone were sustained in brain between 2 to 24 hr and

barely detectable at 48 hr. Significant amounts of metabolites were

present in brain and plasma at longer time periods. The decay of

naltrexone in brain and plasma was biexponential with t1/2 18

min and t1/2 8.0 hr in brain and 11.4 hr in plasma respectively.

The brain to plasma ratios at 0.5 to 1 hr were higher than those at

later times. 6ß-naltrexol was present in small amounts in brain but

not in plasma. In addition to 7,8-dihydro-14-hydroxy normorphinone and 7,8-dihydro-14-hydroxy normorphine, tentative evidence

was obtained for 3 other lipophilic metabolites in brain which were

cleared from the brain much more slowly than naltrexone or 6ßnaltrexol. These metabolites were also present in plasma in addi-
DISPOSITION OF NALTREXONE 139

tion to free and conjugated naltrexone and its N-dealkylated metabolites. It is conceivable that the 3 lipophilic metabolites in brain

may be 2-hydroxy-3-O-methyl naltrexone, 2-hydroxy-3-O-methyl 6ßnaltrexol and 3-O-methyl naltrexone respectively. The slow decline

of levels of naltrexone in brain would imply that either the binding

of naltrexone to some constituent in brain is particularly strong or

a slow release from extravascular sites into rat plasma may contribute to its prolonged duration in plasma and the long duration

of antagonism.

DISPOSITION AND METABOLISM OF

NALTREXONE IN MAN

The urinary excretion profile of naltrexone as reported by Cone

et al. (34) has been essentially confirmed by subsequent studies of

Verebey et al. (35) and Dayton and Inturrisi (24). Following a single

50 mg oral dose of naltrexone in man, an average of 53.4% of the

dose was recovered in urine of which naltrexone, conjugated naltrexone, 6ß-naltrexol and conjugated 6ß-naltrexol comprised 1.2,

9.7, 26.3 and 16.4% respectively (34). The mean excretion t1/2 of

naltrexone was estimated as 1.1 hr and that of 6ß-naltrexol 16.8 hr.

In 3 human subjects receiving a 180 mg dose of naltrexone daily,

the 24-hr urinary excretion as % of dose was: naltrexone 0.5, conjugated naltrexone 4.5, 6ß-naltrexol 34.6 and conjugated 6ß-naltrexol

14.0%. In another study (24) in maintenance subjects receiving 125

mg naltrexone p.o. 3 times a week, 37% of the dose was excreted in

48 hr, of which naltrexone, conjugated naltrexone, 6ß-naltrexol and

conjugated 6ß-naltrexol comprised 0.8, 7.6, 16.8 and 11.8% respectively.

Detailed studies on the disposition and metabolism of naltrexone

(100 mg p.o.) in 4 acutely and chronically treated human subjects

have been reported by Verebey et al. (36,371. The peak plasma

levels of naltrexone following an acute dose were 43.6 ± 29.9 ng/

ml at 1 hr and of 6ß-naltrexol 87.2 ± 25.0 ng/ml at 2 hr., and these

declined to 2.1 ± 0.5 and 17.6 ± 5.0 ng/ml respectively by 24 hr.

Following chronic administration, the peak levels of naltrexone

and 6ß-naltrexol at 1 hr were 46.4 ± 18.5 and 158.4 ± 89.9 ng/ml

respectively, but by 24 hr these values declined to levels similar to

those in the acute state at 24 hr. Plasma levels of naltrexone and

6ß-naltrexol measured 24 hr after the daily doses of naltrexone indicated that steady-state equilibrium (conc. 10.9 ng/ml) was rapidly

attained and there was no accumulation of these compounds in

plasma on chronic dosing. Biexponential kinetics were observed for
140 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

naltrexone and 6ß-naltrexol in the first 24 hr. The t1/2 of naltrexone and 6ß-naltrexol decreased from the acute to the chronic study

from 10.3 ± 3.3 to 9.7 ± 1.1 hr and from 12.7 ± 2.6 to 11.4 ± 2.0

hr respectively. Plasma levels of naltrexone declined slowly from

24 to 72 hr from 2.4 to 1.7 ng/ml with an apparent t1/2 of 96 hr.

Renal clearance data indicated that naltrexone was partially reabsorbed while 6ß-naltrexol was actively secreted by the kidney.

During acute and chronic treatment, the urinary excretion was 38

and 70% of the dose in 24 hr and the mean fecal excretion 2.1 and

3.6%. Opiate antagonism to 25 mg i.v. heroin challenges was nearly

complete through 48 hr after naltrexone, and at 72 hr the objective

responses reappeared to a greater extent than the subjective ones.

Low plasma levels of drug suggested substantial sequestration in

tissues which released it slowly into the central compartment.

First-pass hepatic metabolism of naltrexone was indicated by rapid

appearance, peaking and substantially higher concentrations of 6ßnaltrexol in plasma. Lack of changes in proportion of naltrexone

and its metabolite from acute to the chronic state indicated that

naltrexone did not induce its own metabolism. The 2-fold recovery

of naltrexone and its metabolites in the chronic study may have resulted from the saturation of tissue binding sites with naltrexone

and its metabolites, forcing larger amounts of daily doses to remain

in the central compartment for more rapid elimination after metabolism. Conjugation mechanism appeared to become more efficient on multiple dosing of naltrexone. During chronic treatment

no tolerance, physical dependence or toxicity was observed in

human subjects,

In a recent study Verebey et al. (38) have developed a sensitive

glc method utilizing derivatization with pentafluoropropionic acid

for the quantitation of 2-hydroxy-3-O-methyl 6ß-naltrexol in

plasma and urine. The relative percentages of this metabolite, naltrexone and 6ß-naltrexol in the plasma of 4 human subjects 18 hr

after 400 mg p.o. dose of naltrexone were 23.2 ± 3.7, 3.4 ± 1.5 and

73.5 ± 3.2 respectively; the corresponding percentages in urine

were 16.0 ± 9.8, 7.4 ± 4.1 and 76.8 ± 10.7 respectively.

COMPARATIVE POTENCIES OF THE

METABOLITES OF NALTREXONE

Although potency ratios between compounds may change substantially depending on the animal species and the experimental

test used to assess the antagonist activity, 6 -naltrexol and 6ß-naltrexol were approximately 1/35 and 1/50 as active as naltrexone in
DISPOSITION OF NALTREXONE 141

morphine-pelleted mice using jumping response as the criterion of

antagonist potency (20). 6ß-naltrexol (s.c.) was about 1/56 as active

as naltrexone in preventing the loss of righting reflex in rats from

morphine sulfate (20 mg/kg s.c.) and about 4 times as long-acting

as naltrexone in tests for duration of action at equi-antagonist ED90,

doses and measurement of time required for 50% decrease in activity. In mice, 6ß-naltrexol was about 1/26 as active as naltrexone in

preventing the Straub-tail reaction with morphine (30 mg/kg s.c.)

and about 9 times as long-acting as naltrexone (39). In chronic

spinal dog following precipitation of abstinence, the potency of 6ßnaltrexol was approximately 1/12 to 1/15 that of naltrexone (341.

The low potencies of the 6 - or 6ß-naltrexol and the rapid onset

of action of naltrexone militate against the formation of these metabolites, contributing substantially to the overall pharmacological

effect of the parent compound. The antagonist potency of 3-Omethyl naltrexone (N-cyclopropylmethyl - 7,8 - dihydro-14-hydroxy

norcodeinone, EN-2994A) in mice was 1/15 and 1/4 that of naltrexone on subcutaneous injection and p.o. administration respectively.

In rat, the antagonist potency was 1/20 and 1/5 that of naltrexone

on s.c. and p.o. administration (Dr. Blumberg, personal communication). The relative affinities of naltrexone, 6 -naltrexol, 6ß-naltrexol and 2-hydroxy-3-O-methyl-6ß-naltrexol for the stereospecific

receptor sites in rat brain homogenate provided IC50 values of 1.3

x 10-9, 1.2 x 10-9 3.2 x 10-9, and 2.7 x 10-6M respectively and in

presence of Na+ the corresponding values were 0.9 x 10-9 2.9 x

10-9, and 2.0 x 10-9, and 1.8 x 10-6M respectively (Dr. Hiller, personal communication). Thus the 2-hydroxy-3-O-methyl-6ß-naltrexol

possessed much less activity as an antagonist compared to naltrexone. The pharmacological activity of 2-hydroxy-3-O-methyl naltrexone and 2-hydroxy-3-O-methyl-6ß-naltrexol in vivo is still unknown.

However, the comparatively slower clearance of these methylated

metabolites than of naltrexone or 6ß-naltrexol may have implications for the long duration of antagonism with naltrexone.

ENZYME SYSTEMS INVOLVED IN THE

METABOLISM OF NALTREXONE

The most common route for the biotransformation of naltrexone

and other opiates having a phenolic group is the glucuronide conjugation in several mammalian species including man. The glucuronides are formed enzymatically by the transfer of glucuronic acid

from uridine diphosphate D-glucosiduronic acid (UDPGA) to the

opiates by glucuronyl transferases found in microsomes of liver and
142 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

other tissues. Sulfate conjugation of phenolic or alcoholic groups of

opiates is a prime pathway in the cat and chicken and occurs in

the soluble fraction of the liver cell, kidney and intestine by several sulfotransferases.

The enzyme system involved in the reduction of 6-keto group of

naltrexone to 6ß-naltrexol requires NADPH and occurs in the soluble fraction of the liver homogenate. Studies by Roerig et al. (40)

have shown that the chicken liver enzyme (which predominantly

produces 6 -naltrexol) was precipitated at 60-70% ammonium sulfate saturation and the rabbit liver enzyme (which predominantly

produces 6ß-naltrexol) at 50-60% saturation. The rabbit enzyme

was more sensitive to inhibition by morphine than was the chicken

enzyme. The reverse order of sensitivity was seen with ketamine.

These differences in properties between the chicken and rabbit

liver enzymes indicate that the stereoselective metabolism of naltrexone to 6 - or 6ß-naltrexol is mediated by two different enzymes.

The formation of metabolites, 2-hydroxy-3-O-methyl naltrexone

and 2-hydroxy-3-0-methyl-6ß-naltrexol is brought about by catechol-O-methyl transferases (COMT) which are found in the soluble

supernatant, of liver, brain and in erythrocytes. This is also an important pathway for biogenic amines and other compounds

(41,42,43). The biochemical characteristics of COMT from brain and

erythrocytes are similar in rat and man (44). The nonspecificity of

COMT towards exogenous substrates usually leads to the formation

of both meta and para-O-methylated derivatives of substrates.

Recent studies (32) have, however, ruled out the formation of 3-hydroxy-2-O-methylated naltrexol as a metabolite of naltrexone. In

view of the recent evidence for the 3-O-methylation of naltrexone

in the rat (Verebey et al., unpublished observations), the sequence

of steps leading to the formation of 2-hydroxy-3-methylated naltrexone metabolites is not clear; i.e., whether 2-hydroxylation of naltrexone occurs first followed by 3-O-methylation or 3-O-methylation

occurs before 2-hydroxylation.

Chronic administration of naltrexone in mice via a long-acting

delivery system depressed the microsomal mixed function oxidases,

MFO (aminopyrine N-demethylase and aniline hydroxylase activity) to 40-50% of the control levels during the 30-35 day period of

naltrexone blockade. A metabolite of 6ß-naltrexol was possibly implicated, and this depression of MFO could inhibit the metabolism

of other drugs (45).
DISPOSITION OF NALTREXONE 143

CONCLUSIONS

It is clear from the above studies that considerable species-dependent differences exist in the quantitative pattern of the metabolism of naltrexone. The metabolic pathways of naltrexone in different species were: (a) 6-keto group reduction to epimeric 6 - or 6ßnaltrexol, (b) glucuronide conjugation of naltrexone and naltrexols,

(c) N-dealkylation to norcompounds, (d) 3-O-methylation, and (e) 2-

hydroxylation. The role of gut wall epithelium in the biotransformation of naltrexone needs to be elucidated in more detail in view

of the considerable first-pass metabolism. As a large portion of the

dose of naltrexone remains unaccounted for in several species including man, there is need for additional studies to completely elucidate its metabolic and dispositional profile. Although the known

metabolites have comparatively much lower antagonist potency

than naltrexone and may not contribute substantially to the overall pharmacologic effect, additional studies are needed to fully

assess the role of 3-O-methyl naltrexone and 2-hydroxy-3-O-methyl6ß-naltrexol metabolites.

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5. J. Volavka, R.B. Resnick, R.S. Kestenbaum, and A.M. Freedman. Short-term effects of naltrexone in 155 heroin ex-addicts. Biol. Psychiat., 12, 679-685 (1976).

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DISPOSITION OF NALTREXONE 145

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29. N.L. Pace. R.G. Parrish, M.M. Lieberman, K.C. Wong and R.A. Blatnick. Pharmacokinetics of naloxone and naltrexone in the dog. J. Pharmacol. Exp. Ther.,

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of naltrexone and 6ß-naltrexol in human plasma using electron-capture detection. J. Chromat., 118, 331-335 (1976).

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(1979).

39. H. Blumberg and C. Ikeda. Comparison of naltrexone and /I-naltrexol for narcotic antagonist action in rata and mice. Fed. Proc., 35, 469 (1976).

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characterisation of enzymes for reduction of naloxone and naltrexone in rabbit

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microsomal hydroxylation of phenols and subsequent enzymatic catechol-Omethylation: substrate specificity. J. Med. Chem., 8, 153-156 (1965).

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O-methylated dihydroxy derivatives. Biochem. Pharmacol, 14, 257-1263

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146 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

45. G.R. Peterson, T. Lehman and P. Pyati. Effects of naltrexone administered via a

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37, 27 (1978).

AUTHOR

Anand L. Misra

New York State Division of Substance Abuse

Services Research Laboratory

Brooklyn, New York 11217
Naltrexone: Research Monograph 28

R. E. Willette and G. Barnett, eds.

National Institute on Drug Abuse, 1980

The Clinical Pharmacology

of Naltrexone:

Pharmacology and

Pharmacodynamics

Karl Verebey

The time-action of opiate antagonist activity of naltrexone

was evaluated in detoxified ex-opiate addicts, using 25 mg

intravenous heroin challenges. A 100 mg naltrexone dose

provided 96% blockade at 24 hr, 86.5% blockade at 48 hr

and 46.6% blockade at 72 hr. Following oral administration, naltrexone was rapidly and completely absorbed. Peak

levels of naltrexone and its major metabolite 6ß-naltrexol

were reached 1 hr after the dose. The high 6ß-naltrexol

plasma concentrations only 1 hr after drug administration

indicate a rapid biotransformation process, convertin a

large fraction of the dose to less active metabolites. Over

70% of the dose was excreted in the 24 hr urine and less

than 0.5% in the feces. No change was observed in the rate

of naltrexone disposition during chronic dosing vs. the

acute study, indicating no metabolic induction. The rapid

achievement of steady state naltrexone plasma levels eliminates the need of stepwise induction at the beginning of

nal trexone treatment.

After intravenous administration of 8 mg naltrexone, the plasma

levels were unexpectedly high for the low dose, ranging between 32

and 3 ng/ml. The intravenous drug administration eliminated

direct exposure to hepatic biotransformation; this is the likely

reason for the higher naltrexone and the significantly lower metabolite (6ß-naltrexol) plasma levels.

A rising dose efficacy study from 100 to 800 mg per day provided

an opportunity for studying naltrexone at much higher than thera147
148 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

peutic doses. No undesirable naltrexone-related side effects were

observed during the study. Two weeks after the 800 mg/day doses

were stopped, the plasma was free of naltrexone and its metabolites, indicating efficient elimination of the drug from the body.

Based on these human studies, a 100 mg dose of naltrexone provided 2 to 3 days protection against 25 mg of intravenous heroin.

Naltrexone seems to be well tolerated even at doses well above

those suggested for opiate antagonist therapy. No toxicity or accumulation of naltrexone and its metabolites was observed in any of

the studies. The lack of dependence liability and absence of pharmacologic or metabolic tolerance during chronic treatment make

naltrexone a safe and efficacious orally effective opiate antagonist.

INTRODUCTION

Naltrexone (N-cyclopropylmethylnoroxymorphone) was synthesized by Blumberg et al. in 1965 (1). In animal (2) and clinical studies (3,4) it demonstrated longer duration of action and greater potency than its N-allyl congener, naloxone. Naltrexone was also

orally efficacious at significantly lower doses than naloxone. This is

important for a drug which is a candidate for the treatment of exopiate addicts. The initial trials of naltrexone in man indicated

good efficacy (opiate antagonism), practicality (long time action and

oral effectiveness) and safety (low doses). In this overview of naltrexone the available data will be combined to examine naltrexone’s opiate receptor blocking activity as it relates to its total biological disposition in human subjects.

The Time Course of Action of Opiate Antagonism

The opiate receptor blocking activity of naltrexone was studied

by challenging it with intravenous heroin injections in four opiate

ex-addicts (5). Control data of various objective and subjective responses to heroin, collected in the absence of naltrexone, in response to a 25 mg heroin injection was considered as 100%. In the

test period, during naltrexone therapy (100 mg/day), 25 mg heroin

challenges were performed 24, 48 and 72 hr after the last naltrexone dose. Individual challenges were at least 10 days apart in the

same patients. The results are shown in figure 1. Averaging both

the objective and subjective results, 96% of heroin-related responses were blocked at 24 hr, 87% at 48 hr and 47% at 72 hr. It is

apparent from the figure that the blockade seems to hold longer

and to a greater extent for subjective responses than for objective

ones. Pooling pupillary miosis and respiratory depression data (ob-
CLINICAL PHARMACOLOGY OF NALTREXONE 149

jective), the blockade was 89% at 24 hr, 73% at 48 hr, and only

20% at 72 hr after naltrexone. The blockade for the subjective responses was 99% at 24 hr, 92% at 48 hr and a respectable 57% at

72 hr after naltrexone. The proposed function of naltrexone is to

block the subjective (or euphorogenic) effects of heroin which, in

fact, were blocked more efficiently than the objective ones. It

should be emphasized that 25 mg of heroin is a substantial dose

and few addicts can obtain such large quantities of heroin routinely. Thus the duration and magnitude of the blockade seem highly

effective for most practical situations.

FIGURE 1. The percent objective and subjective responses are shown after 25 mg

i.v. heroin 24,48 and 72 hr after 100 mg oral naltrexone. The 100% heroin

responses were determined in the absence of naltrexone. For detailed description of

the specific tests, see reference 5.

An interesting observation is that pupillary constriction at 72 hr

is greater than the 100% control heroin effect. It is possible that

some metabolic N-dealkylation occurs, producing the strong agon-
150 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

ist, noroxymorphone, for which Martin et al. (3), Resnick et al. (4),

and Verebey et al. (5) have observed slight agonistic symptoms.

Cone et al. (6) reported the isolation of small quantities of noroxymorphone in the urine of subjects taking naltrexone. The small

amount of agonist may act synergistically with heroin to produce

the higher than control responses.

The Correlation of Narcotic Antagonism With Naltrexone Plasma

Levels

The mean naltrexone plasma levels of 4 subjects declined very

slowly from 2.4 ng/ml at 24 hr to 2.0 ng/ml at 48 hr and to 117 ng/

ml 72 hr after naltrexone (5). The slow exponential decay at this

period translates into a 96-hr terminal half-life and a correlation

coefficient with the total (objective and subjective) heroin-related

responses of r=0.90. The strong correlation indicates that differing

individual biotransformation rates may influence the time action of

naltrexone. Shorter opiate antagonist effects would be expected in

rapid metabolizers than in slow metabolizers of naltrexone. In fact,

the shorter ß-phase half-life values of individuals correlated with

greater responses to heroin at 72 hr after naltrexone (r=0.99). Correlation was also excellent between opiate antagonism and the

combined levels of 6ß-naltrexol and 2-hydroxy-3-methoxy-6ß-naltrexol (HMN) of r=0.82 (5). While opiate antagonist potencies of

these metabolites have been shown to be considerably weaker than

naltrexone in animals (7,8), there is no data in humans. Based on

our observations, it appears that naltrexone itself is responsible for

the major part of opiate antagonism, while the less active metabolites, which are present in large concentrations in the body, also

contribute to the opiate receptor blocking activity.

Absorption and Distribution

After oral administration, naltrexone was rapidly absorbed, as

indicated by the early peaking (1 hr) of naltrexone and 6ß-naltrexol

in the plasma (5). Complete absorption was confirmed by the finding that free naltrexone in the 24-hr feces was only 0.3% of the administered dose during chronic administration of 100 mg oral doses

of naltrexone (5). The low plasma levels of naltrexone indicate that

a large percentage of the dose is rapidly metabolized and the remaining drug is distributed mainly intracellularly, resulting in the

presence of a small fraction of the dose in the systemic circulation

(table 1). Individual variation in the plasma levels of naltrexone

was greatest during the first 8 hours, suggesting that absorption
CLINICAL PHARMACOLOGY OF NALTREXONE

TABLE 1. Plasma Concentration of Naltrexone

151

Hours after naltrexone

Subjects (n) Dose Route 1 2 4 8 12 24

* Postaddicts (4)

* Postaddicts (4)

** Normal volunteers (10)

** Normal volunteers (9)

** Schizophrenic patients (8)

*** Schizophrenic patients (6)

*** Schizophrenic patients (6)

*** Schizophrenic patients (6)

*** Schizophrenic patients (5)

*** Schizophrenic patients (4)

**** Normal volunteer (2)

mg (ng/ml)

100 P.O. (chronic) 44 36 20 8 4 2

100 P.O. (chronic) 46 32 19 10 6 3

50 P.O. (acute) 22 12 5 3

100 P.O. (chronic) 27 17 8 6

2 x 200 P.O. (chronic) 9

100 P.O. (chronic) 2

2 x 100 P.O. (chronic) 3

2 x 200 P.O. (chronic) 5

2 x 300 P.O. (chronic) 6

2 x 400 P.O. (chronic) 9

8 I.V. (acute) 11 8 3

* Data from reference 5

** Courtesy of Dr. Jan Volavka, Dept. of Psychiatry, University of Missouri

*** Courtesy of Drs. N. Klein and G. Simpson, The Rockland Research Institute

**** Courtesy of Dr. Theodore Smith, Dept. of Anesthesiology, University of Pennsylvania

and distribution rates were the most important individual variables (5).

Plasma Levels of Naltrexone and Metabolites

Substantial individual variation of peak naltrexone levels was

observed, ranging from 15 to 64 ng/ml 1 hr after 100 mg oral doses.

After the same dose and time the combined 6ß-naltrexol and HMN

plasma levels ranged from 83 to 288 ng/ml (5). Table 1 shows the

plasma levels of naltrexone in different subjects after various doses

and routes of administration. Irrespective of the size of the dose,

the plasma levels declined to low levels (2 to 9 ng/ml) by 12 to 24

hrs after drug administration, indicating extensive metabolism and

effective distribution of naltrexone. The metabolite plasma levels of

6ß-naltrexol and HMN were 35- and 12-fold higher than naltrexone, respectively (figure 2). With increasing doses from 100 mg to

800 mg naltrexone/day the 24-hr plasma levels of naltrexone, as

well as its metabolites, increased. At the 800 mg/day dose, average

levels reached were 9 ng/ml naltrexone, 123 ng/ml HMN, and 331

ng/ml 6ß-naltrexol. Two weeks after discontinuation of medication

the plasma was free of naltrexone and its metabolites, confirming

the early observations (5) of rapid and efficient elimination of the

drug from the body (figure 2).

347-877 0 - 81 - 11
152 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

FIGURE 2. Naltrexone, 6ß-naltrexol and HMN plasma levels were determined just

prior to each daily dose of naltrexone (24-hr samples). The incremental weekly doses

are shown at the bottom of the figure. The ordinate scale 6ß-naltrexol (ß-OL) and

HMN is from 0 to 400 ng/ml while that of naltrexone is from 0 to 10 ng/ml; (n=6

from 0 to 6th week; n=5 at 7th week and n=4 at 8th and 9th week). The two

patients were dropped from the study for medical reasons, not related to naltrexone

therapy.

Metabolism

The major metabolite of naltrexone in humans is 6ß-naltrexol,

isolated by Cone in 1973 (9), and the structure was confirmed by

Chatterjie et al. in 1974 (10). A minor metabolite, 2-hydroxy-3-methoxy-6ß-naltrexol (HMN), was isolated by Verebey et al. (11) and its

structure was confirmed using synthesis by Cone et al. (12). The
CLINICAL PHARMACOLOGY OF NALTREXONE 153

rapid peaking of 6ß-naltrexol just an hour after an oral dose indicated that immediately after absorption a major portion of naltrexone is biotransformed during its first pass through the liver. HMN

formation from naltrexone can be postulated through various pathways (11). A logical one is the hydroxylation of 6ß-naltrexol at the

2 position, followed by methylation at the 3 position. However,

Cone et al. showed 2-hydroxy-3-methoxynaltrexone in trace

amounts in the urine of subjects taking naltrexone, opening the

possibility for the formation of HMN through another route (12).

In an earlier report, Cone and Gorodetzky showed, by gas chromatography and mass spectroscopy, the existence in the urine of

subjects taking naltrexone of an N-dealkylated metabolite, noroxymorphone, which is a potent agonist (13). This metabolite may be

responsible for the slight agonistic effects observed in some objective and subjective responses following naltrexone administration

(3,4,5).

Quantitatively, 6ß-naltrexol is the most abundant metabolite

formed and is excreted both free and conjugated. Naltrexone was

mostly glucuronidated before excretion and HMN was found only

in the free form. The trace metabolite, 2-hydroxy-3-methoxynaltrexone, was estimated at 0.5% of the administered dose (12). There

is no estimation at present of the quantity of noroxymorphone in

human urine after naltrexone administration.

The opiate antagonistic activity of 6ß-naltrexol varies from

1/50th to 1/12th of that of naltrexone (7,8) depending upon the species and methods used. Preliminary studies of HMN, using the

opiate receptor binding assay from rat brain synaptosome preparation, indicated that HMN binding to opiate receptors was typically

antagonist but its affinity was 1000 times less than that of naltrexone (14). If this potency ratio holds in human subjects, HMN may

not contribute much to the opiate antagonist activity of naltrexone.

In acute vs. chronic dosing, the 6ß-naltrexol over naltrexone concentration in urine, in the same subjects, may have metabolic implications (5). The ratio of 6ß-naltrexol over naltrexone remained

the same (table 2), indicating that naltrexone does not induce its

own metabolism--a desirable feature for a drug given chronically,

because the initial stabilization dose remains effective throughout

the course of the treatment. Based on these studies, the most striking effect of biotransformation on the pharmacological action of

naltrexone is the very high rate of first pass hepatic metabolism of

the drug.
154 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

TABLE 2. Concentration Ratio 6ß-Naltrexol/naltrexone in 24-hour

Urine

Subjects Acute Chronic

1 3.4 2.5

2 3.6 4.0

3 3.6 3.3

4 2.7 3.4

Mean 3.3 3.3

± SD. .4 .6

Naloxone is also biotransformed extensively after oral administration, but into inactive glucuronides. This may be the reason for

its short time action. Fortunately, the active metabolite formation

(6ß-naltrexol and perhaps HMN) prolongs the time-action of naltrexone. This occurs in much the same way as formation of active

metabolites prolongs the time-action of 1- -acetylmethadol, in contrast to methadone, which is converted into inactive metabolites.

Renal and Fecal Excretion of Naltrexone

Urinary excretion of naltrexone and its metabolites is the major

pathway of elimination of the drug from the body. The 24-hr recovery of free and conjugated bases is tabulated in table 3. During

chronic administration of 100 mg oral doses, 23% naltrexone was

excreted, mostly conjugated (93%). 6ß-Naltrexol was the major excretion product, constituting approximately 66% of the total bases

in the 24-hr urine, and was excreted 60% free and 40% conjugated.

HMN was 11% of the bases and was excreted only in the free form

(5). In the acute study an average of 38% of the dose was recovered

in the 24-hr urine, compared with 70% in the chronic study (using

the same subjects). Following an acute dose a certain fraction of

the drug is distributed into tissue stores; however, after chronic

dosing the tissues become saturated and a larger fraction of the

dose is available for renal elimination. This may explain the significantly larger percent recovery of the dose in the chronic study

(table 3 and ref. 5).

In earlier studies, the total recovery of the bases, 6 days after a

50 mg oral dose of naltrexone, was approximately 50% of the dose

(15). In this study HMN must have been included with 6ß-naltrexol, because it was recently found that the HMN and 6ß-naltrexol PFPA derivatives cannot be separated by gas chromatography (16). In another study, after 125 mg oral doses of naltrex-
CLINICAL PHARMACOLOGY OF NALTREXONE 155

one, 34% of the dose was accounted for from 0 to 24 hr, and 3%

from 24 to 48 hr (17). The low recoveries in the early studies may

have resulted from methodological difficulties. In a recent study,

HMN excretion was quantitated for 5 days after a single 50 mg

naltrexone dose (12). The recovery of HMN was 4.6% of the administered dose, which is compatible to the 3.5% reported in another

acute study (table 3). Furthermore, there is evidence for the existence of 2-hydroxy-3-methoxynaltrexone. This minor metabolite was

estimated at approximately 0.5% of the administered dose (12).

Table 4 presents data on the fecal excretion of naltrexone and its

metabolites. In the chronic study a total of 3.6% of the administered dose was excreted in the feces, mostly as 6ß-naltrexol (80%).

Both HMN and naltrexone were present in much lower concentrations, 12 and 8% respectively (table 4 and ref. 5). The data clearly

indicate that fecal excretion is not an important pathway of elimination of naltrexone in human subjects. Collectively, the urinary

and fecal excretion of naltrexone accounted for 73.6% of the administered dose following chronic drug administration. The missing

26.4% may be various metabolic intermediates described in the

pathway to the formation of HMN and other not yet identified metabolites (11). In healthy individuals no problem of naltrexone accumulation is predicted during chronic naltrexone use. Since the

drug is largely dependent on hepatic biotransformation and renal

TABLE 3. Urinary Excretion of Naltrexone and Metabolites,

24-hr Collection

(% of Dose Excreted)

naltrexone 6ß-naltrexol

2-hydroxy-3-methoxy

6ß-naltrexol Total

Free Bound Free Bound Free

Acute 1.0 7.2 19.1 7.1 3.5 37.9

Chronic 1.1 15.0 29.0 17.5 7.6 70.2

TABLE 4. Fecal Excretion of Naltrexone and Metabolites, 24-hr

Collection

(% of Dose Excreted)

naltrexone 6ß-naltrexol

2-hydroxy-3-methoxy

6ß-naltrexol Total

Acute 0.13 1.9 0.08 2.1

Chronic 0.29 2.9 0.44 3.6
156 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

elimination, naltrexone disposition should be studied in subjects

having hepatic and/or renal problems, to evaluate the safety of

naltrexone in such a population.

CONCLUSION

The data collected during intravenous heroin challenges in the

absence and presence of naltrexone showed that the subjective

heroin-related responses were blocked for a longer period of time

and to a greater extent than were the objective ones. This is a definite advantage for the therapeutic purpose of naltrexone; i.e.,

blocking the euphoric effects of heroin is more important than

blocking pupillary constriction. The data also showed that narcotic

antagonism was related to the plasma levels of naltrexone. Individual variation in biotransformation rates may therefore influence

the time course of narcotic blockade. Fortunately, the chronic administration of naltrexone does not induce the microsomal enzyme

system, and therefore the rate of biotransformation appears to

remain unchanged from acute to chronic therapy. This suggests

that the same dose will deliver the same pharmacological effect in

chronic use. The lowest effective naltrexone plasma level was 2.0

ng/ml, providing an average of 86.5% blockade of the effects of 25

mg heroin. Thus, in therapy for effective opiate antagonistic activity, plasma levels of 2.0 ng/ml or greater should be maintained.

Another therapeutic advantage is the rapid attainment of steady

state of naltrexone in plasma, which is a function of the total biological disposition of the drug. Judged from the plasma level data,

steady state is achieved by the second daily dose of naltrexone.

This information is useful for the choice of dose and frequency of

drug administration, especially during the initiation of therapy.

For simplicity of naltrexone use, our data indicates that no induction from lower doses to maintenance dose is necessary. Therapy

may begin with the maintenance dose.

Increasing up to 800 mg naltrexone/day, the plasma levels of

naltrexone and its metabolites also increased to very high levels

without toxicity or any other undesirable side effects. Two weeks

after the discontinuation of 800 mg naltrexone doses the plasma

was free of naltrexone and its metabolites, indicating rapid and efficient drug elimination from the body. This study provides evidence for the high margin of safety of naltrexone even when considerably higher than effective opiate antagonistic doses are used.

In brief, nearly 100% blockade of 25 mg intravenous heroin effects can be maintained for 48 hr after 100 mg oral dose(s) of nal-
CLINICAL PHARMACOLOGY OF NALTREXONE 157

trexone. The absence of pharmacologic or metabolic tolerance

during chronic treatment provides carefree clinical use of naltrexone. Plasma level monitoring can give reliable estimates of the

degree of opiate antagonism. The pharmacodynamic investigations

of naltrexone in man found naltrexone a safe and effective opiate

antagonist for the rehabilitation of well-motivated narcotic addicts.

Used with adequate psychological and social counseling, this chemotherapeutic modality should be most promising.

REFERENCES

1. H. Blumberg, I.J. Pachter, and Z. Matossiank. U.S. Patent 3,332,950 (July 25,

1967).

2. H. Blumberg and H.B. Dayton. Naloxone and related compounds. In H.W. Kosterlitz, H.O.J. Collier and J.E. Villareal (eds.), Agonist and Antagonist Actions of

Narcotic Analgesic Drugs, Proceedings of a British Pharmacological Society

Symposium, Aberdeen, Scotland, July 1971, Macmillan, New York (1973).

3. W.R. Martin, D.R. Jasinski and P.A. Mansky. Naltrexone, an antagonist for the

treatment of heroin dependence. Arch. Gen. Psychiat., 28, 784-791 (1973).

4. R.B. Resnick, J. Volavka, A.M. Freedman and M. Thomas. Studies on EN-1638A

(naltrexone): A new narcotic antagonist. Am. J. Psychiatry, 131, 648650 (1974).

5. K. Verebey, J. Volavka, S.J. Mule and R.B. Resnick. Naltrexone: Disposition, metabolism and effects after acute and chronic dosing. Clin. Pharmacol. Ther., 20,

315-328 (1976).

6. E.J. Cone, C.W. Gorodetzky and W.D. Darwin. The identification and measurement of two new metabolites of naltrexone in human urine. Res. Corn. Chem.

Pathol. Pharmacol., 20, 413-433 (1978).

7. K. Verebey, and S. J. Mule. Naltrexone pharmacology, pharmacokinetics and metabolism: current status. Amer. J. Drug and Alcohol Abuse 2, 357-363 (1975).

8. H. Blumberg and C. Ikeda. Comparison of naltrexone and ß-naltrexol for narcotic

antagonist action in rats and mice. Fed. Proc., 35, 469 (1976).

9. E.J. Cone. Human metabolite of naltrexone (Ncyclopropylmethylnoroxymorphonel with a novel C-6 isomorphine configuration.

Tetrahedron Lett, 23, 2607-2610 (1973).

10. N. Chatterjie, C.E. Inturrisi, H.B. Dayton and H. Blumberg. Stereospecific synthesis of the 6ß-Hydroxy metabolites of naltrexone and naloxone. J. Med.

Chem., 18, 490-492 (1975).

11. K. Verebey, M.A. Chedekel, S.J. Mule, D. Rosenthal. Isolation and identification

of a new metabolite of naltrexone in human blood and urine. Res. Comm. Chem.

Pathol. Pharmacol., 12, 67-84 (1975).

12. E.J. Cone, C.W. Gorodetzky and W.D. Darwin, F.I. Caroll, J.A. Brine, CD. Welch.

The identification and measurement of two new metabolites of naltrexone in

human urine. Res. Comm. Chem. Pathol. Pharmacol., 20, 413-433 (1978).

13. E.J. Cone and C.W. Gorodetzky. Metabolism of naltrexone and naloxone. Report

to the Committee on Problems of Drug Dependence. Washington D.C. (1975).

14. J. Hiller and E. Simon, NYU Medical School, personal communications.

15. E.J. Cone, C.W. Gorodetzky and S.Y. Yeh. The urinary excretion profile of naltrexone and metabolites in man. Drug Metab. Dispos., 2, 506-512 (1974).
158 NALTREXONE SUSTAINEDRELEASE PREPARATIONS

16. K. Verebey, A. DePace, D. Jukofsky, J.V. Volavka and S.J. Mule. Quantitative

determination of 2-hydroxy-3-methoxy-6ß-naltrexol (HMN), naltrexone and 6ßnaltrexol in human plasma, red blood cells, saliva and urine by gas liquid chro

matography. Anal. Toxicol, 4, 33-37 (1980).

17. H.E. Dayton and C.E. Inturrisi. The urinary excretion profiles of naltrexone in

man, monkey, rabbit and rat. Drug Metabol. Disp., 4, 474-478 (1976).

ACKNOWLEDGMENTS

The author wishes to thank the following collaborators for providing biological samples for analysis: Drs. Nathan Klein and

George Simpson of the Rockland Research Institute; Dr. Jan V. Volavka; The Missouri Institute of Psychiatry; and Dr. Theodore

Smith, Department of Anesthesiology, University of Pennsylvania

School of Medicine. The expert technical assistance of Dennis Jukofsky and Ann DePace and the professional assistance of Drs.

M.J. Kogan and S.J. Mule are acknowledged with appreciation.

AUTHOR

Karl Verebey

Director of Clinical Pharmacology and Associate Professor of

Clinical Psychiatry

SUNY Down State School of Medicine

New York State Division of Substance Abuse Services

Testing and Research Laboratory

Brooklyn, NY 11217
Part V

Pharmacokinetics

Naltrexone: Research Monograph 28

R. E. Willette and G. Barnett, eds.

National Institute on Drug Abuse, 1980

Overview of Human

Pharmacokinetics of

Naltrexone

Vojtech Lictko

Experimental data on 3H labelled naltrexone obtained from

the Research Triangle Institute were analyzed. Both

plasma and urine curves were resolved into their exponential components. Mean total clearance of unconjugated naltrexone was 3510 (300) ml/min, and its mean urinary clearance was 76.0 (6.8) ml/min. Fraction of dose excreted

through urine was between 55 and 60%. Orally administered drug is completely absorbed into plasma. Plasma

curve resulting from intravenous administration contained

three exponentials: 1.9 (.5)/min, 0.18 (.07)/min and 0.00106

(.00008)/min. Distribution volumes were 7.6 (.7) liters, 60

(12) liters and 164 (16) liters. After oral administration

there were two exponentials: 0.012 (.002)/min and 0.00122

(.00009)/min. The faster exponential corresponds to gastrointestinal absorption with a half life of about 1 hour.

INTRODUCTION

The purpose of this report is to summarize the pharmacokinetic

analyses of naltrexone data in man collected at the Research Triangle Institute (1).

MATERIALS AND METHODS

Male volunteer subjects were used in the experimental studies.

Five received 198 μCi of 3H naltrexone in the dose of 1 mg intravenously (IV). Out of six subjects who were given 200 μCi of 3H nal161
162 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

trexone in the dose of 50 mg orally (PO) only four were selected for

this analysis since the other two did not have complete sets of data.

The data consist of plasma concentration and amount of drug in

urine samples at various time intervals after administration. There

are seven sets of such plasma and urine data for both routes of administration: total drug (TOTAL), total unconjugated drug (TOTAL

UNCONJ), total conjugated drug (TOTAL CONJ), 6 beta-naltrexol

unconjugated (6ß-OL UNCONJ), naltrexone unconjugated (N-ONE

UNCONJ), 6 beta-naltrexol conjugated (6ß-OL CONJ) and naltrexone conjugated (N-ONE CONJ). In the IV study plasma samples

were obtained at .5, 1, 3, 5, 10, 15, 30, 60, 90, 120, 150 and 180 minutes and at 5, 7, 10 and 24 hours. Urine was collected at 3, 6, 9, 12,

24, 48 and 72 hours. In the PO study plasma samples were taken at

15-minute intervals for 3 hours and at 4.5, 6, 12 and 24 hours.

Urine was collected at 3, 6, 9, 12, 24 and 48 hours. In a preliminary

analysis, averaged data were employed. The failure to recover

about 40% of the dose dictated that the final analysis be carried

out on individual curves.

The sum of exponentials was fitted by a (weighted) nonlinear

least square procedure (weight being proportional to the square of

the reciprocal of the plasma concentration) to the plasma data (formula (1); cf. table 1) and a complement exponential (formula (2)) to

the urine data. Area under the plasma curve, A, was determined

analytically from formula (3) and the total clearance, Ct, was obtained from the dose D and the area A by means of formula (4).

Instantaneous urinary clearance Cu(t) was computed by formula (5)

for the first 12 hours every 3 hours, and the mean value Cu was

taken (formula (6)). The fraction of urinary to total clearance, fc,

was determined by formula (7) and the fraction of the dose excreted

through urine, fd, computed by formula (8). Three distribution volumes, Vi, (i = 1, 2, 3) for different phases of the plasma curve were

obtained by application of formula (9). Standard error, s, of a computed parameter x was estimated from the standard errors, si, of

the parameters xi by formulas (10) and (11). All means are weighted by reciprocal variances, and standard errors are shown throughout in parentheses.

RESULTS

An example of the least square fit of IV data is shown in Fig. 1a

and lb and another one for PO data in Fig. 2a and 2b. The plasma

data are plotted semilogarithmically and the urine data are shown

in linear scales.
OVERVIEW OF HUMAN PHARMACOKINETICS

TABLE 1. Definitions

163

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

p(t) = sum (Ai * exp (-ai*t)); i = 1,2,3

U(t) = Uo * (1 - exp(-k*t))

A = sum (Ai/ai); i = 1,2,3

Ct = D/A

Cu(t) = dU(t)/dt / p(t); t = 3, 6, 9, 12 hours

Cu = sum (Cu(t)/4; t = 3,6,9,12 hours

fc = Cu/Ct

fd = Uo/D

Vi = D/sum (Aj); i = 1, 2, 3; j = i . . . . 3

x = f(x1, x2, . . . . xp)

s = (sum ((df/dxi)*si)**2)**1/2; i = 1,2,...,p

TABLE 2. Weighted Mean Values of Parameters-Total Drug

Parameter IV PO

A/D (min ml) x 10e6 4120 (700)

Ct (ml/min) 167 (25)

Cu (ml/min) 111 (9)

f c ,564 (.085)

f d ,539 (.047)

k (1/min) .00160 (.00007)

al (1/min) 1.9 (.5)

a2 (1 /min) .18 (.07)

a3 (1 /min) .00106 (.00008)

V1 (liters) 7.6 (.7)

V2 (liters) 60 (12)

V3 (liters) 164 (16)

Parameters are defined in Table 1.

Standard errors are shown in parentheses.

4300 (490)

201 (12)

124 (8)

,529 (.021)

563 (.031)

.00128 (.00015)

-

,012 (.002)

.00122 (.00009)

-

-

145 (17)

Tables 2, 3a and 3b summarize the analysis of the curves showing weighted means of the parameters for all subjects. Note that

there are only a few instances when the IV and PO data yield significantly different values. For the total drug there is a significant

difference (p < .03) in the value of the exponential coefficient of

the second exponential, a2. Larger differences are found between

the IV and PO values of unconjugated forms as seen in Tables 3a

and 3b, where a double asterisk (**) indicates significant difference

at level p = .005 and a single asterisk (*) that at level p = .025.
164 NALTREXONE SUSTAINED-RELEASE PREPARATIONS
OVERVIEW OF HUMAN PHARMACOKINETICS 165
166 NALTREXONE SUSTAINED-RELEASE PREPARATIONS
OVERVIEW OF HUMAN PHARMACOKINETICS 167

347-877 0 - 81 - 12
TABLE 3a. Weighted Mean Values of Parameters-All Drug Forms
TABLE 3b. Weighted Mean Values of Parameters-All Drug Forms
170 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

Since the IV and PO values of total and urinary clearances do

not differ at level p = .15, the weighted mean total clearance is

computed as 195 (10) ml/min and the weighted mean urinary clearance as 118 (4) ml/min. Their ratio is 0.605. The area under the

plasma curve for total drug when computed for 1 mg of the drug is

the same for both routes of administration. This implies that drug

administered orally is completely absorbed into plasma.

DISCUSSION

Detailed analysis of individual curves confirmed essentially the

results of the analysis of averaged data. Thus, it can be inferred

that the conclusions drawn here from the analysis of human naltrexone kinetics are independent of the method of data treatment.

Comparison of the fraction of urinary to total clearance with the

computed fraction of dose excreted through urine shows an agreement between both of these mutually independent methods that

only up to 60% of the dose is excreted by urine. The remainder

may be expected to be excreted through feces. Direct experimental

proof of this conjecture does not exist so far. At 48 hours after an

oral administration Wall and coworkers (1) found about 4% of the

dose in feces. Similar data are not available for intravenous administration. However, due to a slow passage through lower intestine,

the peak of the fecal excretion of the drug can be delayed beyond

48 hours.

The values of the exponential coefficient of urinary excretion k

follow very closely those of a3, the exponential coefficient of the

terminal phase of the plasma curve. This observation complies with

the theoretical requirement that at least the terminal portion of

both the plasma and the urine curves must have identical kinetic

character. Then, the ratio of the urinary clearance to the terminal

volume of distribution, Cu/V3, can estimate the fractional removal

rate of the drug through urine. This ratio yields values which are

about 60% to 70% of the value of a3, thus indicating by the third

independent method that no more than two-thirds of the total drug

leaves the body through urine.

While the slowest exponentials (a3) of IV and PO plasma curves

do not differ significantly, there is a substantial discrepancy between the faster components of both curves. After oral administration one cannot find the fast exponentials of the IV curve. In fact,

the faster exponential of the PO curve is still more than one order

of magnitude slower than any of the two of the IV curve. The early

kinetics of naltrexone after oral administration are governed by a
OVERVIEW OF HUMAN PHARMACOKINETICS 171

rate-limiting process which is associated with the route of administration. Therefore, the conclusion can be drawn that the exponential of the PO curve with half life of about 1 hour corresponds to

that of gastrointestinal absorption.

CONCLUSIONS

1. About 60% of naltrexone is excreted by the kidney.

2. All orally administered naltrexone is absorbed into plasma.

3. Half life of gastrointestinal absorption is about 1 hour.

4. Half life of the terminal exponential is about 10 hours.

5. Half life of urinary excretion is about 9 hours.

REFERENCE

1. M.E. Wall, D.R. Brine, and M. Perez-Reyes. Drug Disp. Metab. Submitted.

AUTHOR

Vojtech Licko

BioDyn

San Francisco, Calif. 94122
Naltrexone: Research Monograph 28

R. E. Willette and G. Barnett, eds.

National Institute on Drug Abuse, 1980

Pharmacokinetic

Quantitation of Naltrexone

Release From Several

Sustained-Release Delivery

Systems

R. H. Reuning, S. H. T. Liao, and A. E. Staubus

A method designed to quantitate in vivo naltrexone release

rates from sustained-release systems has been applied to the

evaluation of seven different naltrexone delivery systems in

the monkey. The method consists of two phases: a single intravenous bolus dose quantitation of each monkey’s pharmacokinetic parameters coupled with a delivery system

study in which plasma naltrexone levels are measured

throughout the time period of sustained-release. In vivo release rates and the total amount released are then calculated. It should be noted that these determinations require the

analysis of unchanged naltrexone in plasma as the only experimental measurement. Data from injectable naltrexone

pamoate microcapsule delivery systems indicate that 1)

when these microcapsules are suspended in an aqueous vehicle, a significant part of the dose is released very rapidly,

yielding release rate-time data that parallel a non-sustained-release control; 2) this rapid release for the aqueous

vehicle is followed by a slow release phase lasting to about

24 days for the subcutaneous route and to about 45 days for

the intramuscular route; and 3) when these microcapsules

are suspended in an oily vehicle there is no initial rapid release, substantial release rates are obtained for at least 60

days, and an average of 89% of the dose is calculated to

have been released. Data from implantable naltrexone delivery systems show that 1) the Alza system most closely ap172
QUANTITATION OF NALTREXONE RELEASE 173

proximates a zero-order release rate-time profile; 2) the Battelle system provides a rapid initial release followed by a

slowly declining release mte; 3) the Dynatech system is

characterized by a more rapid initial release rate of 3-8% of

the dose per day over the first 3-5 days followed by a rather

constant 1-3% per day to about day 36; and 4) essentially

corn lete recovery of the dose was obtained for the Battel e

and Dynatech systems,

INTRODUCTION

The rationale for developing sustained-release narcotic antagonist delivery systems for treatment of opiate addiction has recently

been reviewed (1,2) One phase of a scheme for evaluating these

systems consists of a pharmacokinetic quantitation of drug release

rates in vivo (2). The methodology that has been developed for

quantitating naltrexone release in monkeys is characterized by two

phases: 1) calibration of the pharmacokinetics of each individual

monkey from plasma level-time data obtained after an intravenous

bolus dose of naltrexone, and 2) measurement of plasma levels of

unchanged naltrexone over the time period that the sustained-release system yields measurable concentrations. Data from 1) and 2)

above permit calculation of an in vivo release rate-time profile as

well as the total amount of naltrexone released during the study.

The purpose of this report is to summarize the naltrexone release data for those delivery systems that have been evaluated

pharmacokinetically in the monkey. In order to obtain an overview

it was necessary to average the release rate data obtained from the

several monkeys utilized in evaluating each delivery system. Also,

data related to the calibration of each monkey’s pharmacokinetic

parameters has been omitted. Both types of data for individual

monkeys will be included in subsequent manuscripts.

EXPERIMENTAL

Delivery Systems

The following seven delivery systems have been evaluated:

I. Naltrexone in a physical blend with 90% (L+)lactic acid10% glycolic acid copolymer, spherical beads 1.5 mm in diameter, subcutaneous, Dynatech # 24086;

II. Naltrexone pamoate-polyclactic acid) microcapsules suspended in 2% aluminum monostearate-peanut oil and injected intramuscularly, Thies #GL-1-6-76-1;
174 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

III. Naltrexone pamoate-poly(lactic acid) microcapsules suspended in a medium consisting of water, 2% Tween 20,

0.02% anti-foam silicone and 1:10,000 phemerol and injected intramuscularly, Thies #GL-1-6-75-1;

IV. Naltrexone pamoate-poly(lactic acid) microcapsules suspended in an aqueous medium of 0.1% Tween 80 in Macrodex (6% dextran 70 in 5% dextrose/water for injection)

and injected subcutaneously, Thies #GL-3-9-77-3;

V. Micronized naltrexone pamoate (batch # 2M-1869-866-16)

suspended in 2% aluminum monostearate-peanut oil and

injected intramuscularly;

VI. Rods-naltrexone and hydrophobic polymer, Chronomer,

Alza, subcutaneous;

VII. Naltrexone 33% in a dipalmitin (75%) - tripalmitin (25%)

mixture, shaped into rods and administered subcutaneously (Battelle).

These sustained release systems will be referred to by the numerical designation throughout the text. Additional data concerning these delivery systems has been provided by the developers

(2,3). All are intended to be bio- degradable, with systems I, VI and

VII designed for subcutaneous implant and systems II, III and IV

designed for injection. System V was included as a non-sustainedrelease control.

Experiments in Monkeys

Each delivery system was administered to 3 or 4 monkeys at a

dose of approximately 10 mg/kg. With the exception of delivery

system VII, these were self-administrating monkeys and were on a

rotating schedule of morphine, methamphetamine and saline selfinjection. Effects of the naltrexone delivery system on morphine

self-administration were measured as described previously (4) and

will be reported separately. Blood samples were obtained, usually

from a femoral vein, at periodic intervals up to 60 days after administration of the sustained release system.

At least several days were allowed to elapse after the delivery

system either was removed or ceased releasing measurable

amounts of naltrexone. Subsequently, a single intravenous bolus

dose of naltrexone (3-5 mg/kg) was administered and periodic blood

samples were obtained for a sufficient time so that the pertinent

pharmacokinetic parameters of naltrexone could be determined

from the plasma level-time profile.
QUANTITATION OF NALTREXONE RELEASE 175

DISCUSSION

Injectable Systems

The in vivo release rate data for the injectable naltrexone delivery systems are presented in fig. 1. Systems II, III and IV differ

mainly in the vehicle used to suspend the microcapsules and in the

route of injection. System V is a non-sustained-release control formulation included for comparative purposes. Comparison of the

curve for sustained release system II (microcapsules suspended in

the oily vehicle and administered intramuscularly) with the curve

for the control formulation, system V (micronized naltrexone pamoate suspended in the oily vehicle and administered intramuscularly), permits the conclusion that the microcapsule coating is responsible for the pronounced sustained release effect with system

II.

Assay for Naltrexone

A sensitive and specific assay for naltrexone concentrations in

plasma has been described previously (5,6,7). The pharmacokinetic

calculation of in vivo release rates is dependent on an assay that is

specific for unchanged naltrexone and this specificity has been

demonstrated with respect to the known metabolites of naltrexone

(7).

Calculation of Release Rates

Release rates were calculated according to the Loo-Riegelman

method (8). Either a two- or a three-compartment, open pharmacokinetic model was used to fit the plasma level-time data for the intravenous bolus dose of naltrexone in each monkey (9). The threecompartment model was utilized when needed to obtain a good

overall fit to the data. The pharmacokinetic parameters for naltrexone, obtained from the intravenous bolus dose, were then utilized to calculate naltrexone release rates from the plasma naltrexone level-time data obtained in the delivery system study (8). The

total amount released over the entire time period was subsequently

determined according to an equation presented previously (10).

RESULTS

The data for naltrexone concentration in plasma as a function of

time after administration are summarized in table 1 for each of the

seven delivery systems tested. Levels of about 0.25-0.5 ng/ml are

needed to block morphine self-administration in these monkeys

(11). Since this range is also the approximate sensitivity limit of
TABLE 1. Naltrexone Concentration in Plasma During the Time Period in Which the Monkey was Administered a

Naltrexone Sustained Release Delivery System

178 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

the assay, “measurable” levels can be considered to be “effective”

levels in these monkeys. The corresponding average naltrexone release rates for the seven systems are shown in table 2 and in figures 1 and 2. Table 3 contains data summarizing the results of a

comparison of the dose administered with the calculated amount of

naltrexone released and, when available, the amount of naltrexone

recovered from the delivery system after removal from the

monkey.

FIGURE 1. Semilog plot of naltrexone in vivo release rate as a function of time for

injectable sustained release delivery systems II, III, IV and V. Delivery systems are

identified in the text and in table 3. Closed symbols represent an oil vehicle and

open symbols an aqueous vehicle.
QUANTITATION OF NALTREXONE RELEASE 179

The influence of the vehicle used to suspend the microcapsules

on release of naltrexone can also be observed in fig. 1. A comparison of system III (microcapsules suspended in an aqueous vehicle

and administered intramuscularly) with system II suggests that the

influence of the vehicle occurs mainly over the first 15 days after

administration. Subsequently, the release rate declines in approximately parallel exponential fashion for the two systems. During

the first 4-6 days after administration, system III has a release

rate-time profile that parallels the exponential decline of release

rate for the non-sustained-release control. This rapid and extensive

decline in release rate (note the logarithmic y-axis in fig. 1) suggests that a very significant fraction of the naltrexone in delivery

system III was available for rapid release. A similar early rapid release can also be observed for system IV (microcapsules suspended

in an aqueous vehicle and administered subcutaneously). A comparison of aqueous microcapsule suspension systems III and IV (fig.

1) suggests that the intramuscular route (III) yielded much higher

release rates than the subcutaneous (IV) from 10-50 days after administration. Delivery system IV provides the smallest degree of

sustained release of any of these microencapsulated naltrexone pamoate systems. On the other hand, system II provides a significant

release rate for a period of 60 days. Although the release was not

zero order, the rates were within a fairly narrow range (fig. 1). In

addition, it appears that the oil vehicle “protects” the naltrexone

pamoate microcapsules from whatever causes the rapid initial

burst of release with the aqueous vehicle.

The calculated total extent of naltrexone release from each delivery system is compared with the dose administered in table 3. Systems III and IV yielded an incomplete recovery of the administered

dose whereas the average recovery for system II was 89% of the

dose. The microcapsule delivery systems (II, III and IV) were characterized by a high degree of variability in the extent of recovery of

the administered dose (table 3). Part of the reason for the greater

variability observed for the microcapsule systems, compared to the

other delivery systems, may be the difficulty in administering an

accurate dose of the microcapsule suspension (especially for the

aqueous systems). Alternatively, the release of naltrexone may be

more variable with these delivery systems. Unfortunately, the

nature of these microcapsule delivery systems precluded the possibility of removing the microcapsules remaining at the end of the

sustained release study. Therefore, the unabsorbed naltrexone

could not be assayed directly.
TABLE 2. Mean Calculated Release Rates as a Function of Time After Administration for the Various Naltrexone

Delivery Systemsa
QUANTITATION OF NALTREXONE RELEASE 181

TABLE 3. Comparison of the Naltrexone Dose Administered in

Sustained Release Form with the Amount of Naltrexone Accounted for

Experimentally in the Monkey

Delivery

systems

Mean amount

naltrexone

administered

(mg)

Mean

calculated

amount

naltrexone

released (mg)

Mean assayed

amount

remaining

in delivery

system (mg)

Percent of

administered

dose

accounted forb

I 42.1c

38.9c0.0023c92(91-94)C

II 35.3 31.6 - 89(58-116)

Ill 36.3 18.3 - 53(30-74)d

IV 26.2 19.5 - 76(48-91)

V 37.8 32.4 - 86(78-100)e

VI 44.0 30.0 - 66(56-87)

VII 30.7 16.4 16.4f107(92-120)d

aDelivery system identification: I = naltrexone in a physical blend with 90% (L + ) lactic

acid-10% glycolic acid copolymer, spherical beads 1.5 mm in diameter, subcutaneous,

Dynatech #24086; II = naltrexone pamoate-poly(lactic acid) microcapsules suspended in

2% aluminum monostearate-peanut oil and injected intramuscularly, Thies #GL-1-6-76-1:

Ill = naltrexone pamoate-poly(lactic acid) microcapsules suspended in a medium consisting of water, 2% Tween 20, 0.02% anti-foam silicone and 1:10,000 phemerol and

injected intramuscularly (Thies #GL-1-6-75-1); IV = naltrexone pamoate-poly(lactic acid)

microcapsules suspended in an aqueous medium of 0.1% Tween 80 in Macrodex (6%

dextran 70 in 5% dextrose/water for injection) and injected subcutaneously (Thies #GL-3-

9-77-3); V = micronized naltrexone pamoate (batch #2M-1869-866-16) suspended in 2%

aluminum monostearate-peanut oil and injected intramuscularly; VI = rods containing

naltrexone supplied by Alza and administered subcutaneously; VII = naltrexone 33% in

a dipalmitin (75%)-tripalmitin (25%) mixture shaped in rods and administered subcutaneously (Battelle).

bMean of 4 animals, unless stated otherwise. Range in parenthesis.

cMean of 2 values. One animal became ill during the study and another had a sufficient

number of plasma samples with assay interference that a quantitation of total amount

released could not be made.

dMean of 3 animals.

eMean of 3 animals. One animal omitted because of a 163% recovery due to one high

data point and an insufficient number of other data points to obtain an accurate estimate

of the amount released.

The rods removed from each monkey were weighed, dissolved in chloroform, and assayed spectrophotometrically for naltrexone at 282 nm.

Subcutaneous Implants

The release rate-time profiles for the subcutaneous implant delivery systems are shown in fig. 2. System I (Dynatech) is characterized by a more rapid release over the first 6 days followed by a

very constant rate of naltrexone release throughout the remainder

of the study. System VI (Alza) yielded a release rate-time profile
182 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

that was closer to being constant over the entire release period

than any of the other systems. However, this system also was the

shortest of the three in terms of duration. Delivery system VII

(Battelle) is characterized by a rapid initial rate of release followed

by a slowly declining rate from day 2 to about day 38. Overall,

system I provides the longest duration of meaningful release rates

of naltrexone. The “initial burst” of release with this delivery

system is larger than that observed with the other two implantable

systems (fig. 2) but much smaller than that for the aqueous injectable systems (compare fig. 2 with fig. 1, noting the logarithmic ordinate in fig. 1).

The possibility of removal of the implanted delivery systems at

termination of the sustained release study permits a more rigorous

“mass balance” comparison of naltrexone dose with the sum of the

calculated amount released plus the amount remaining in the delivery system. Such a comparison was carried out with systems I

and VII (table 3). Assay of the removed delivery system for naltrexone content yielded negligible amounts in system I. However, about

FIGURE 2. Linear plot of naltrexone in vivo release rate as a function of time for

implantable sustained release delivery systems I, VI and VII. Delivery systems are

identified in the text and in table 3.
QUANTITATION OF NALTREXONE RELEASE 183

half the dose remained in system VII at termination of the study.

Data from both of these delivery systems provided an essentially

complete accounting of the fate of the administered dose, as summarized in table 3. In system VI the device could not be removed at

the end of the study and the lower recovery with this system may

be due to unreleased drug. The variability in recovery between replicate monkeys was less with the three implantable naltrexone delivery systems than with the injectable microcapsule systems (table

3). This may be due to more accurate administration of the intended dose as well as to the ability to remove the system and assay for

unreleased naltrexone.

REFERENCES

1. R.E. Willette. In R.E. Willette (ed.), Narcotic Antagonists: The Search for LongActing Preparations, National Institute on Drug Abuse Research Monograph 4.

DHEW Pub. No. (ADM)76-296. Supt. of Docs., U.S. Govt. Print. Off., Washington, D.C., 1-5 (1976).

2. R.E. Willette. The development of sustained action preparations of narcotic antagonists In R.C. Petersen (ed.), The International Challenge of Drug Abuse, National Institute on Drug Abuse Research Monograph 19. DHEW Pub. No.

(ADM)78-654. Supt. of Docs., U.S. Govt. Print. Off., Washington, D.C., 333-339

(1978).

3. R.E. Willette (ed.), Narcotic Antagonists: The Search for Long-Acting Preparations,

National Institute on Drug Abuse Research Monograph 4. DHEW pub. No.

(ADM)76-296. Supt. of Docs., U.S. Govt. Print. Off., Washington, D.C., 13-42

(1976).

4. SE. Harrigan and D.A. Downs. Continuous intravenous naltrexone effects on

morphine self-administration in rhesus monkeys. J. Pharmacol. Exp. Ther., 204,

481-486 (1978).

5. R.A. Sams and L. Malspeis. Determination of naloxone and naltrexone as perfluoroalkyl ester derivatives by electron-capture gas-liquid chromatography. J.

Chromatogr., 125, 409-420 (1976).

6. R.H. Reuning, V.K. Batra, T.M. Ludden, M.Y. Jao, B.E. Morrison, D.A. McCarthy,

S.E. Harrigan, S.B. Ashcraft, R.A. Sams, M.S. Bathala, A.E. Staubus and L.

Malspeis. Plasma naltrexone kinetics after intravenous bolus administration in

dogs and monkeys. J. Pharm. Sci., 68, 411-416 (1979).

7. R.H. Reuning, S.B. Ashcraft and B.E. Morrison. An electron-capture gas chromatographic assay for naltrexone in biological fluids. This volume.

8. J.C.K. Loo and S. Riegelman. New method for calculating the intrinsic absorption

rate of drugs. J. Pharm. Sci., 57, 918-928 (1968).

9. W.G. Kramer, R.P. Lewis, T.C. Cobb, W.F. Forester Jr., J.A. Visconti, L.A. Wanke,

H.G. Boxenbaum and R.H. Reuning. Pharmacokinetics of digoxin: Comparison

of a two- and a three-compartment model in man. J. Pharmacokin. Biopharm.,

2, 299-312 (1974).

10. M. Gibaldi and D. Perrier. Pharmacokinetics, Marcel Dekker, Inc., New York, p.

137 (1975).

11. S. Harrigan, personal communication.

347-877 0 - 81 - 13
184 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

ACKNOWLEDGMENTS

The authors wish to acknowledge the contributions of Mr. James

N. Wiley and Mr. Stephen E. Harrigan, Warner-Lambert/ParkeDavis, in carrying out all the experimental work with the monkeys.

The developers of the various delivery systems are also acknowledged, among them Dr. C. Thies, (Washington University), Dr. D. L.

Wise (Dynatech R/D Company), Dr. R. Capozza (Alza) and Dr. M.

Sullivan (Battelle). Assays were carried out by S. B. Ashcraft and

B. E. Morrison. Supported in part by Contract HSM-42-73-132 from

The National Institute on Drug Abuse.

AUTHORS

R. H. Reuning, S. H. T. Liao,* and A. E. Staubus

College of Pharmacy

Ohio State University

Columbus, OH 43210

*current address:

Burroughs Wellcome Company

Research Triangle Park, NC 27709
Part VI

Sustained-Release

Systems

Naltrexone: Research Monograph 28

R.E. Willette end G. Barnett, eds.

National Institute on Drug Abuse, 1980

A Review of Parenteral

Sustained-Release

Naltrexone Systems

James L. Olsen and Fred A. Kincl

The ideal naltrexone sustained-release delivery system

should be easy to inject or implant, not cause adverse tissue

reaction, release the drug at a relatively constant rate for at

least 30 days, and biodegrade within a short time afterwards. Mechanisms which can be used for sustaining drug

release include reducing solubility and surface area, coating, encapsulation and microencapsulation, complexation,

binding and hydrophilic gelation. Drug release from such

systems is controlled by diffusion through a barrier/film,

diffusion from a monolithic device, erosion of the surface,

hydrolysis, ion exchange, biodegradation, or a combination

of these. Injectable systems would seem to be ultimately preferred because of the ease of administration and handling,

while the implantable devices may find first use in man

since they are easily removable, should that be necessary.

Maintaining particulate-free products and sterilization

methods are two problems with all parenteral dosage

forms. Production must be particularly well controlled and

validated.

INTRODUCTION

It is almost always advantageous to give fewer doses of a drug

and at the same time to have more constant drug release. In the

case of naltrexone, the relief from a decision process before each

dose is perhaps reason enough for fewer, longer-acting doses. Furthermore, using a sustained-release (SR) parenteral device for

treating narcotic addicts eliminates the need to give the patient

187
188 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

several days’ oral dosage to take home or the need to have the patient return to the treatment center frequently for short-lived

doses.

Another significant reason for using SR preparations for many

drugs including naltrexone is that a lower constant dose instead of

a multiple high dose regimen reduces side effects.

THE IDEAL SYSTEM

A sustained-release parenteral drug delivery system or device of

naltrexone should have the following characteristics:

Be Easy To Inject or Implant

This means a volume of less than 1 cc, using a needle not larger

than 18 gauge for injectables and a small rod, macrocapsule, or

string of beads that are “trocarable” for implant forms should be

considered for convenience of administration.

Be Pharmaceutically Acceptable

The preparation must be sterile, contamination free, pyrogen

free, stable, have the proper strength, content uniformity and reproducible release characteristics, be easily handled at administration (suspendable, wettable particles, easily loaded implant devices)

and be manufacturable on a large scale.

Not Cause Adverse Tissue Reaction

Part of the design of any SR system is to use materials which

will be compatible with the body and not cause allergic reactions,

sterile abcesses and a lump under the skin, pain on injection, or

even discomfort.

Give Relatively Constant Drug Release

Few SR systems can give zero order naltrexone release, although

some can. Fortunately, with naltrexone, there is a wide range between the effective dose and the toxic dose, so that SR systems

giving a higher initial release rate can be used without much penalty. The 30-day release period is rather a compromise between

achievable technology and desirability for an even longer-acting

system. When our ability to produce acceptable drug delivery systems improves, it will then be advantageous to extend the duration

of action.
PARENTERAL SUSTAINED-RELEASE 189

Biodegrade

Constructing a SR system for naltrexone means adding at least

one other component, be it polymer, polyglyceride, or whatever.

This or these other components including solvents used in production must not only be compatible with the drug and the body but

must also degrade into fractions which in turn can be eliminated

without causing any side reaction.

Degradability must be present so that there are no “ghosts of

doses past” present in the patient. Further, the last remnants of

the device should be gone within a reasonable time after the drug

itself is eliminated. We have used a rule of thumb that the entire

system should be gone within a period of time equal to the release

period, that is, if a system is effective for 30 days, then every component should disappear within another 30-day period. Even that

way a patient would have two devices within his body, one active

and one degrading.

MECHANISMS FOR ACHIEVING SUSTAINED

RELEASE

Theoretical

If we look at one of the common formulas for describing rate of

drug release from a solid, e.g.,

dm

dt = k A(S-C)

Where m = mass dissolved

C = concentration at time t

A = surface area of the solid

S = saturation solubility

k = dissolution rate constant,

k = D = diffusion coefficient

h = thickness of the diffusion layer

we see that there are three basic ways to reduce rate of dissolution.

a) Reduce surface area of the device. Use a sphere or rod; for

example, one 1.5 mm bead has A = 7 mm2. The same bead

milled to 100μ particles = A = 106 mm2.
190 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

b) Reduce (saturation) solubility. Make an insoluble complex,

e.g., zinc tannate, or an insoluble mixture, dispersion or

solid solution, or form an ester or ether.

c) Control the rate of diffusion or desorption from an inert

matrix. These can be used in combination as has been

done in several instances by combining hydrophobic sub

stances with naltrexone in a rod or bead form. For example, specific mechanisms have been used for many years

with oral dosage forms and may be adapted to parenterals.

Coating

A coating on a drug particle or device acts as a film or barrier

through which the drug must diffuse, degrade, or rupture, releasing all of the contents. Coated products can further be classified

into macrocapsules and microcapsules. There do exist nanocapsules; however these are considered too small to remain in situ and

will not be considered further.

Macrocapsules are single units which contain all of the drug in

one capsule. They are therefore implantable devices. The release of

drug is governed by transport through the intact film barrier. The

outstanding characteristics of such devices are that the effective

surface area of the capsule is constant throughout the life of the

film (at least until degradation starts), and also that “excess” drug

inside the capsule maintains a constant saturated solution inside

the capsule. The serious disadvantage of a macrocapsule (besides

implanting) is that the film could rupture and release all of the

drug at once.

Microcapsules are those having a diameter of from a few microns

to perhaps 500 microns. Here the drug is present in many individual capsules which collectively resemble a powder for a given dose.

The dose ostensibly could be given, for example, as a suspension of

the microcapsules in isotonic saline solution.

The mechanism of drug release from microcapsules could be as

in macrocapsules, that is, diffusion through a film. However, given

the nature of the microencapsulation process, the biodegradability

of the coating and the thickness of the films, drug release is most

probably accompanied by film rupture. A SR dose would consist of

many capsules, the most perfectly coated not releasing drug for

some time, and the imperfect coatings releasing the drug sooner. In

actuality both diffusion and rupture probably occur in any drug release profile.
PARENTERAL SUSTAINED-RELEASE 191

Insoluble Salts, Complexation and Binding

These systems reduce the solubility of naltrexone, thus prolonging release of the drug. Examples would include zinc tannate, ionexchange, solid solutions, and dispersions of the drug in insoluble

carriers. Physical forms of these systems can be injectable suspensions of particles or implantable rods and beads. They are generally monolithic particles in any case, that is, having the same composition throughout the particle from surface to the center.

Hydrophilic or Hydrophobic Vehicles (matrices)

Hydrophilic materials are water loving to such an extent that

they form a hydrated gel around the particle, thus increasing the

diffusion layer and “protecting” the drug from rapid dissolution.

Materials such as sodium alginate have been used although there

is a question whether or not the FDA would regard them as safe

for human parenteral use. Hydrophobic polymers such as Alza’s

Chronomer (1) series effectively waterproof the particle until they

hydrolyze and are eroded from the surface, thus releasing more dispersed drug. An advantage of the hydrophobic mechanism is that

release rate is zero order and not dependent upon the amount of

drug remaining in the device.

Prodrugs

A prodrug is a mechanism for sustaining release of a drug by

chemically binding to a polymer backbone. The drug would be essentially insoluble until the bonds are hydrolyzed or enzymatically

cleaved. The release rate is governed by the degradation rate of the

drug-polymer bond.

The prodrug must still be formulated into an appropriate dosage

form, such as a suspension or an implant. The particular form is

dependent upon the hydrolysis rate of drug from the polymer.

Combinations of the above mechanisms can be and have been

made. Microcapsules were made by coating an insoluble salt of naltrexone, for example.

PRODUCTION PROBLEMS

The process in which SR preparations are made is important for

a number of reasons, most having to do with the fact that the systems are parenteral solids.
192 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

Scale-up

Roth directions of scale-up are important; that is, some production methods cannot be dependably scaled up and some cannot be

well scaled down. There was difficulty in scaling down the microencapsulation process of coacervation for development purposes, for

example, because it is basically a large-scale operation. Agitation,

surface area and cooling rates all vary with batch size. Some operations like molding and compression are easily done with small

quantities but are almost impossible in large-scale production. High

energy production is also easily done with small batches (e.g.,

blenders and ultrasonification) but difficult in even medium size

batches.

Raw Material Supplies

Material supplies are a scale-up problem to some extent in that

larger supplies of all components will be required if any of the devices proves successful. Can a large batch of polymer be made with

the same characteristics as the small developmental ones? Some

components are just plain expensive (tripalmitin, for example).

How does this cost translate to a large batch? Availability, regardless of cost, may be a problem. Production capacity may not exist

for what was once only an interesting, little-used chemical.

Batch Production

Rear in mind that parenterals must be sterile, pyrogen free and

foreign particle free. In addition the products must be made according to current good manufacturing practices (GMP’s) and also according to part of the current Large Volume Parenteral GMP’s in

lieu of published Small Volume Parenteral GMP’s. Copious records

are required, and in fact few facilities are available that can undertake such production.

CONCLUSION

Regardless of the many difficulties in development and production, a good deal of progress has been made in the last several

years. We are now on the verge of entering phase I human trials

with the Dynatech PLGA bead preparation (2). Supplies are on

hand with which to begin such trials and the toxicity studies are

almost completed.

It is an exciting time for all who have been involved with the

project, from Dr. Seymour Yolles, of the University of Delaware,
PARENTERAL SUSTAINED-RELEASE 193

who first demonstrated sustained release of a narcotic antagonist

with his PLA particles, to Dr. Robert Willette, who has been masterminding the SA narcotic antagonist program. There has been

significant input from the National Institute for Child Health and

Human Development, from the consultants in various fields, and

from the contractors.

All who have contributed have not only furthered the NIDA

effort but have also expanded the scientific and practical knowledge surrounding sustained action parenteral systems.

REFERENCES

1. R.C. Capozza, E.E. Schmitt, L.R. Sendelbeck. Development of chronomers for narcotic antagonists. In R.E. Willette (ed.), Narcotic Antagonists: The Search for

Long-Acting Preparations. National Institute on Drug Abuse Research Monograph 4. DHEW Pub. No. (ADM)76-296. Supt. of Docs., U.S. Govt. Print. Off.,

Washington, D.C., 39-42 (1976).

2. A.D. Schwope, D.L. Wise, J.F. Howes. Development of polylactic/glycolic acid delivery systems for use in treatment of narcotic addiction. In R.E. Willette (ed.),

Narcotic Antagonists: The Search for Long-Acting Prepamtions. National Institute on Drug Abuse Research Monograph 4. DHEW Pub. No. (ADM)76-296.

Supt. of Docs., U.S. Govt. Print. Off., Washington, D.C., 13-18 (1976).

AUTHORS

James L. Olsen

Department of Pharmaceutics

School of Pharmacy

University of North Carolina

Chapel Hill, N.C. 27514

Fred A. Kincl

Department of Biology

College of Staten Island

City University of New York

Staten Island, N.Y. 10301
Naltrexone: Research Monograph 28

R.E. Willette and G. Barnett, eds.

National Institute on Drug Abuse, 1980

Development of Drug

Delivery Systems for Use

in Treatment of Narcotic

Addiction

A. Carl Sharon and Donald L. Wise

The long term goal of the NIDA narcotic antagonist program of developing an implantable, biodegradable, naltrexone/PLGA matrix system which will sustain the delivery of

naltrexone to biological systems for one month has been

achieved. The dosage forms which provide the desired delivery characteristics are 1.5 mm diameter beads composed

of 70% by weight naltrexone base in 40,000 molecular

weight, 90L/10G poly (L(+)lactic-co-glycolic acid). Other

dosage forms, including 1.5 mm diameter rods which provide 6 months’ naltrexone release, finely divided injectable

powders which provide up to 30 days’ naltrexone release,

and 1.5 mm diameter rods which provide 2 weeks’ sustained delivery of morphine, have also been investigated. In

vitro and in vivo release rates have been determined by

measuring chemical concentrations in pH 7 buffer solution

and urine, respectively. In vivo efficacy of naltrexone sustained delivery devices has been measured by direct challenge with morphine (Dewey-Harris mouse tail-flick test)

and inhibition of morphine self-administration in monkeys.

Good Manufacturing Practices documentation has been developed and used to produce a large batch of the 1.5 mm

diameter naltrexone bead dosage forms at an FDA-registered pharmaceutical manufacturer. These beads, produced

at the University of North Carolina School of Pharmacy,

are awaiting use in human clinical trials.

194
DEVELOPMENT OF DELIVERY SYSTEMS 195

INTRODUCTION

A problem universal to chemotherapeutic programs is the administration of drugs to patients at safe and effective levels for extended periods of time. Much technical activity has been devoted to

devising novel systems, and improving currently existing ones, for

regulating drug delivery.

One important category of the dosage forms under investigation

is an implantable, tissue absorbable preparation which releases

chemical (e.g., pharmaceuticals, fertilizers, and pesticides) at a constant or controlled rate to biological systems. There are two fundamentally different approaches to achieve this end. One relies on

chemical alteration of the agent so that absorption by the system is

delayed. The second leaves the active agent chemically unchanged,

but physically restricts its bodily availability. This is achieved by

injecting the agent in a carrier solution (e.g., oil and saline) and by

encapsulating the chemical in an absorbable, release-regulating

material.

Polymers of lactide and glycolide have been evaluated as releaseregulating materials at Dynatech R/D Co. Polylactic/glycolic acids

are tissue compatible and hydrolyze to lactic and glycolic acids,

which are then absorbed into the body. The rate of hydrolysis is

controllable, and dependent on the lactic/glycolic moiety ratio and

copolymer molecular weight. Solid solutions/mixtures of the narcotic antagonist naltrexone and copolymers of lactic and glycolic

acids were prepared and evaluated in vitro and in vivo for utility

as long term, controlled delivery dosage systems.

Two delivery systems have been developed by Dynatech R/D Co.

for NIDA under Contract Number HSM-42-73-267. One, a 70% w/w

naltrexone base in 90L/10G PLGA, 1.5 mm diameter “bead,” provides controlled delivery of the drug for approximately 30 days

when subcutaneously implanted. Good Manufacturing Practices

documentation has been developed for the production of this dosage

form. A large batch of these beads has been produced under GMP

documentation, suitable for use in human clinical trials, at the

UNC Chapel Hill School of Pharmacy, under the supervision of

Dynatech engineers. The other system, composed of 33% w/w naltrexone pamoate in high molecular weight 90L/10G PLGA, 1.5 mm

diameter rods, coated with the same polymer, provides over six

months’ controlled delivery of naltrexone. This paper summarizes

the development, testing, and production of these drug delivery systems.
196 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

PREPARATION AND ANALYSIS OF MATERIALS

Polymer Synthesis

L-lactide and glycolide were obtained commercially or synthesized from their respective acids, and purified by multiple recrystallization. Polylactic acid, and poly-(L(+)-lactic-co-glycolic acid)

(PLGA) were synthesized in the ratio 75% w/w L-lactic acid (L)/

25% glycolic acid (G) and 90% w/w L/25% w/w G by oven bulk

polymerization. Para-toluene sulfonic acid was used as catalyst.

Molecular weights of these polymers were determined by gel permeation chromatography (GPC), membrane osmometry, and light

scattering to range from 40,000 to 200,000. Molecular weights of

poly-dl-lactic-acid polymers were obtained by absolute methods. A

comparison of these results with the intrinsic viscosity of poly-dllactic acid solutions in tetrahydrofuran (THF) at 37°C, yielded an

approximate correlation between molecular weight and intrinsic

viscosity. All polymer molecular weights reported here are weightaverage molecular weights (Mw) obtained by gel permeation chromatography, unless otherwise noted.

Preparation of Delivery Vehicles

The free base and pamoate forms of the narcotic antagonist, naltrexone (supplied by NIDA), were formulated with polymer into delivery vehicles for this study. Naltrexone pamoate was chosen for

use with longer term delivery systems because of its low solubility

in pH 7 buffer solution (approximately 0.5 mg/ml) relative to that

of naltrexone base (solubility 4 mg/ml). Radio-labelled naltrexone base, tritiated at the 15 and 16 positions, was used to follow

naltrexone excretion in the urine and feces of some animals.

Naltrexone and PLGA were codissolved and intimately mixed in

THF or methylene chloride (CH2Cl2) by overnight rolling on a ball

mill. The solutions were cast in thin films (<0.25 mm) on clean

glass plates, and the solvent allowed to flash off. The resulting

films were of uniform consistency. Naltrexone pamoate/PLGA

films appeared to contain small naltrexone pamoate crystals encased in polymer. Naltrexone base/PLGA films appeared clear at

low naltrexone loadings, and cloudy at high drug loadings. This indicates that a naltrexone base forms a solid solution with PLGA, at

least at low drug loadings.

After the solvent flashed off, the naltrexone/PLGA films were

removed from the glass plates, and the remaining solvent removed

by evacuation (< 2 Tori) at 65°C for two to five days. Residual solvent contents of these films were determined to be less than 2000
DEVELOPMENT OF DELIVERY SYSTEMS 197

ppm. The films were then formed into implantable shapes: “beads”

of 1.5 mm (nominal) diameter were prepared by transfer molding,

and cylindrical rods of 1.5 mm diameter were prepared by extrusion. A thin polymer coating was added to selected samples by dipping the beads or rods into a 10% w/v polymer solution in CH2Cl2

.

Dip-coated samples were dried under vacuum at 50°C for 48 hours.

The resultant coating was estimated to be 25 to 75 mm thick.

In Vitro Analysis

The in vitro release of drug from sustained-delivery vehicles was

monitored by suspending beads or rods in Whatman extraction

thimbles in test tubes containing 30 ml of Fisher pH 7 phosphate

buffer solution. The test tubes were stoppered and maintained at

37°C in an American Optical Co. shaker bath. Naltrexone concentrations of the buffer solutions were determined

spectrophotometrically for samples containing unlabelled drugs,

and by means of a Beckman LS100 liquid scintillation counter for

3H-labelled drugs. After each concentration measurement, the extraction thimble was drained, and buffer solution replaced in order

to keep the naltrexone concentration in the buffer below 20% of

saturation. Above this level, the bulk buffer solution no longer acts

as a perfect sink for naltrexone, and drug concentration in the

buffer begins to affect drug release from the sustained release

device.

In Vivo Analysis

In vivo testing of naltrexone sustained-delivery devices was performed in mice and monkeys. Mouse testing was performed at

SISA, Incorporated, Cambridge, MA, and monkey testing at

Warner-Lambert/Parke-Davis, Ann Arbor, MI. After implantation

with the sustained delivery device, all mice were tested for narcotic

antagonism effect by morphine challenge. Urine and feces, or urine

alone, of those mice which were implanted with 3H-labelled naltrexone were also collected. Excretion of drug and metabolite was

thus monitored. Monkeys trained to self-administer morphine were

implanted with sustained-release devices, and their demand for

morphine was monitored regularly. Urine and feces, or urine alone,

of those monkeys implanted with 3H-labelled naltrexone were collected for drug excretion analysis. Blood samples were also drawn

periodically from selected monkeys for naltrexone blood level analysis by Reuning, Ashcraft, and Morrison, at Ohio State University

(this volume).
198 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

In vivo test methods are briefly described below. Complete test

method descriptions, along with results, are fully described elsewhere in this monograph.

In Vivo Analysis in Mice

Male albino Charles River mice (18-22 g) were used throughout

these studies. The mice were anesthetized with Penthrane (Abbott)

and a small slit was made in the scapular region. A single rod or

three beads were inserted below the slit and the wound was closed

by a silk suture. The animals were allowed to recover. Groups of

ten animals were then placed in cages and allowed to develop normally until the test date. Ten individual mice were placed in separate metabolism cages for collection of urine.

The urine was collected daily for the duration of the experiment.

Cages were rinsed with distilled water and the washings were

added to the daily urine collections, which were made up to ten ml

with distilled water. A 1.0 ml sample was taken and added to 10.0

ml of Aquasol liquid scintillation cocktail (New England Nuclear)

in a vial. The radioactivity was measured using a Beckman LS-230

liquid scintillation counter. A quench curve was constructed using

a series of 3H quench standards and this curve was used to correct

all counts.

The mouse tail-flick procedure was used throughout this study. A

ten second cut-off time was employed, and a control reaction time

of two to four seconds was used. A rheostat, incorporated into the

instrument, was used to adjust the intensity of the light falling on

the tail of the mouse such that each reaction time fell within the

stated range. Animals with a control reaction time outside the

stated range were rejected. The rheostat adjustment was only made

if a significant proportion (more than two out of every ten mice) of

the reaction times was outside the range of two to four sec. Groups

of ten mice were medicated with an ED80 dose (14.0 mg/kg ip) of

morphine sulphate, intraperitoneally and re-exposed to the noxious

stimulus twenty minutes later. The analgesic response was calculated by Harris and Pierson as the percentage of the maximum

possible response time (1). The following formula was used for

these calculations: (test-control/10-control) x 100 = % maximum

possible morphine analgesia effect.

Each group of animals was tested by the narcotic antagonist procedure only once. Groups were planned so that testing was performed every 7 or 10 days.

At the termination of each experiment, the animals were sacrificed and the implant site was examined. The condition of the implanted material and signs of encapsulation, injection, or irritation
DEVELOPMENT OF DELIVERY SYSTEMS 199

were noted. All testing was performed under the supervision of Dr.

John S. Howes at SISA, Inc., Cambridge, MA.

In Vivo Analysis in Monkeys

In vivo analysis of sustained-release naltrexone delivery systems

in rhesus monkeys was carried out by Harrigan and Downs, at

Warner-Lambert/Parke-Davis. The reader is referred to their description elsewhere in this volume.

RESULTS AND DISCUSSION

The objectives of this study were to develop implantable, biodegradable naltrexone-polylactic/glycolic acid matrix systems which

will sustain the delivery of chemical to a biological system for both

one and six months. Primary to achieving these objectives are the

development of reproducible polymerization procedures using catalysts acceptable for use in parenterals, the fabrication of matrices

from polymers which hydrolyze within specified times, and the selection of a practical sterilization process.

Experiments were performed to ascertain the effects on chemical

release rate and release duration by the

degree of chemical loading,

solubility of the chemical in both the surrounding environment

and the polymer,

surface area/volume ratio and porosity of the matrix,

hydrolysis rate of the polymer (i.e., composition of polymer), and

polymer molecular weight.

Quantitative evaluations of most of these parameters have been

completed.

The degree of chemical loading and the solubilities have been

identified as most influential on the performance of the implant.

The duration of chemical delivery is inversely proportional to the

drug loading level and the solubility of the chemical in the surrounding environment. Thus, matrices designed to meet the sixmonth goal are prepared with naltrexone pamoate, which is less

soluble in aqueous solutions than naltrexone base. Release duration is directly proportional to the solubility of the drug in the

polymer. The effects of chemical solubility in the polymer have

been especially important in this study--the base form of naltrex347-877 0 -81 - 14
200 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

one forms a solid solution with polylactic/glycolic acids. This behavior permits the use of the moderately water soluble naltrexone

base for long duration implants.

In other experiments it has been determined that dip-coating the

implants with a thin layer of polylactic/glycolic acid reduced the

initial surge of chemical from the surface of the implant and extended the duration of delivery. The releases of chemical from rods,

spheres, and micron-size particle matrices have been measured.

Based on the above findings, matrices have been fabricated specifically to meet the aforementioned goals. These systems have

been tested in vitro and in vivo. One matrix system, dip-coated rods

of 33% by weight naltrexone pamoate in 90 L(+)/10G polylactic/

glycolic acid, has proven antagonistic to morphine challenge for

220 days in mice (dosage 8 mg naltrexone pamoate/mouse).

Another system, composed of 70% by weight naltrexone base in

90L/10G PLGA has reproducibly proven antagonistic to morphine

challenge for about 30 days in mice and monkeys. Related work

has also shown that an injectable form of this matrix system shows

promise of providing up to 30 days antagonism to morphine challenge.

Polymer Synthesis and Selection

Polymers have been synthesized from dl-(racemic) and L(+)-lactic

and glycolic acids (G), in ratios ranging from 25% w/w L(+) or dl

and 75% w/w G, to 100% L(+) or dl-lactic acid. Molecular weights

ranging from 40,000 to 300,000 (measured by intrinsic viscosity)

have been obtained. The catalysts triethyl aluminum (TEAL), triphenyltin (T T), and para-toluene sulfonic acid (PTSA) were used

to initiate the reactions, and both oven-bulk and solution polymerization techniques were used. Polymers made from dl-lactic acid

became sticky and amorphous when blended with naltrexone base.

L(+)-lactic acid polymers remained crystalline to 48°C (the glass

transition temperature of PLGA’s) and so were selected for use in

implants. In vitro and in vivo testing was conducted with naltrexone base and pamoate in PLGA composites. Dosages were in the

form of 1.5 mm diameter rods and beads. Drug loading ranged from

17% to 90%. Experiments with various dosage forms and loadings

indicated that 90% L/10% G polymers displayed the most desirable

release characteristics. It was also determined that polymers synthesized with PTSA catalyst, in the molecular weight range of

30,000-50,000 were suitable for a 1-month delivery system, and polymers synthesized with TEAL catalyst Mw 200,000, were suitable

for a 6-month duration delivery system.
DEVELOPMENT OF DELIVERY SYSTEMS

Batch to Batch Polymer Reproducibility

201

A critical task in the development of any drug/polymer sustaineddelivery system is demonstration of polymer reproducibility,

enabling the production of a number of batches of dosage forms.

When a promising 1-month sustained delivery system had been developed by this program, efforts turned to reproducibly manufacturing the polymer component of the sustained-delivery system. To

that end, the synthesis of five ten-gram batches of 90L/10G polymer was undertaken. One percent PTSA was used as a catalyst.

Polymers with a molecular weight of approximately 40,000 were

sought.

Table 1 contains the characteristics of the five polymer batches

produced in this study. The table clearly shows the reproducibility

of the polymer synthesis operation; the five batches possess Mw's

within ± 2000 of the mean, 43,000. Other polymer characteristics

also demonstrate batch-to-batch similarity, and are approximately

identical with those desired.

TABLE 1. Summary of Polymer Characteristics Demonstrating

Batch-to-Batch Reproducibility

% Lactide

Batch Mw from Specific from Sp.

Number Mw Mn Dispersity Viscosity Viscosity Rotation Rotation

31547 41,000 23,000 1.81 0.4 60,000 2.95 91.8

31553-A 42,000 24,000 1.72 0.37 53,000 2.85 89.0

31553-B 41,000 24,000 1.72 0.4 60,000 2.90 90.6

31563-A 45,000 25,000 1.80 0.35 50,000 2.95 92.2

31563-B 41,000 22,000 1.88 0.39 59,000 2.95 92.2

Development of Sustained-Delivery Systems for Naltrexone

One-Month System

The primary goal of this work was the development of an implantable sustained-delivery system that would provide antagonistic levels of naltrexone to the patient for about 30 days, and would

be removable in case of adverse reaction to the implant. Other desirable features of the system would include high drug loading, biodegradability, and ease of implantation. These characteristics were

all satisfied by the 70% w/w naltrexone, 30% w/w, 40,000 Mw,

90L/10G PLGA, 1.5 mm diameter bead dosage form. This form was

selected after extensive testing with polymers of Mw's of 40,000 to

200,000, lactide/glycolide ratios of 25L/75G to 100 L, and formulations with drug loadings from 17% to 90% by weight.
202 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

Experimental release rate and narcotic antagonism results from

in vitro and in vivo (mice and monkeys) testing are presented in

figure 1. These data reveal approximately 30 days’ controlled delivery of sufficient naltrexone to animals to effect morphine antagonism. Urinary, and in some cases fecal, recovery of tritium-labelled

naltrexone yielded in vivo release data.

FIGURE 1. Comparison of in vitro and in vivo release rates and in vivo narcotic

antagonism of two batches of 70% by weight naltrexone, 90/10 PLGA, 1.5 mm

diameter beads (batch numbers 22843 and 24086) (references 2,3,4,5,6,7)
DEVELOPMENT OF DELIVERY SYSTEMS 203

Another important result reflected in the graph is that bead release rate and antagonism are consistent from batch to batch.

Figure 1 shows narcotic antagonism in mice from two batches of

beads: #22843, produced in January 1976, and #24086, produced

during December 1976-March 1977. Figure 2 compares narcotic antagonism in monkeys for the same two batches. This figure also

shows the narcotic antagonism effects of continuous naltrexone infusion (10 mg/kg/hr) into monkeys (open circles). Consistent, complete narcotic antagonism is shown through thirty days, for both

FIGURE 2. Superimposed graphs of morphine self-administration following:

naltrexone infusion (10 μg/kg/hr for 37 days; open circles), and implantation of 70%

naltrexone beads (#22843, open triangles; # 24086, open diamonds). Saline selfadministration corresponding to infusion or bead implantation is represented by

solid figures. Batch numbers for each solid figure correspond to morphine selfadministration open figures. Solid squares indicate methamphetamine selfadministration during continuous naltrexone infusion (references 2,3,4).
204 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

batches of beads tested (open triangles and diamonds). Rapid recovery of monkeys to control levels of morphine self-administration is

observed after 35 days. It is notable that monkeys dosed with beads

display this rapid recovery, very much as monkeys in whom naltrexone infusion was abruptly discontinued.

Six-Month System

A secondary goal of this work was the development of a sixmonth sustained-delivery system. As with the one-month system, it

was desired that this system be implantable and removable. These

characteristics were satisfied with 1.5 mm diameter rods containing

33% by weight naltrexone pamoate. The rods were composed primarily of 90L/10G PLGA, and dip-coated in a 10% w/v solution of

the same polymer. This system has proven antagonistic to morphine challenge for 220 days in mice. Dosage of naltrexone pamoate implanted was 8 mg/mouse.

Figure 3 summarizes the results of experiments with this sustained-delivery system. Significant antagonism to morphine challenge was observed beginning at 140 days. Erratic performance

prior to this time was attributed to a lower than effective release

rate caused by a too-thick coating. After 220 days, antagonism remained at 100%. At day 240, all mice but one showed 100% antagonism. No polymeric residue could be recovered from this mouse. At

day 26, three mice exhibited 100% antagonism, two mice 0%. At

day 280 all mice exhibited no antagonism to morphine challenge,

and no polymeric residue could be isolated from any of the mice.

Complete degradation of a PLGA sustained-release device has thus

been observed.

Effect of Surface Area/Volume Ratio on Release Rate

The apparent independence of release rate on surface area/

volume ratio of sustained-release naltrexone devices was observed

in experiments conduced at Dynatech. A casting containing 50% by

weight naltrexone in a 90/10 PLGA copolymer of 100,000 molecular

weight was prepared. Rods of 0.26, 0.80, 1.60, and 3.18 mm diameter

were extruded from this casting. Samples weighing 0.030 g were selected from each rod size. Since the surface area/volume ratio increases as 1/R (neglecting ends) for constant weight cylindrical

samples, one would expect an equivalent increase in release rate by

most diffusion-type models. The radii of the rods vary by a factor of

twelve; thus one would expect the release rate to vary by the same

factor.

The results of the release study shown in figures 4 and 5 show

nearly identical release for all sizes of rods. There appears to be
FIGURE 3. Performance and polymer degradation of 6-month naltrexone pamoate

sustaineddelivery system (reference 8)
206 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

FIGURE 4. Comparison of in vitro release from extruded rods of various diameters

of 100,000 Mw 90/10 PLGA with 50% w/w naltrexone loading (reference 9)

FIGURE 5. Same as figure 4, but cumulative release is compared to t

1⁄2

. Solid line

corresponds to that in figure 4 (reference 9)
DEVELOPMENT OF DELIVERY SYSTEMS 207

little dependence of release rate on surface area/volume ratio. The

cumulative release profiles of these systems also appear to be

linear with the square root of time, as shown in figure 5. This release profile is not one which would be expected from cylindrical

sustained-release devices, when estimated by most diffusion-type

models. Rather they are similar to that which would be expected

from a sustained-release device with flat plate geometry. This

result carries the outstanding implication that finely divided, injectable dosage forms may provide the same sustained-release characteristics as molded bead or extruded rod implantable dosage forms.

Injectable System

A long term goal of the NIDA narcotic antagonism program has

been the development of an injectable sustained-delivery device for

naltrexone. Ideally, this device would display the same release

characteristics as the 1-month system described above. The results

of the study of the effect of surface area/volume ratio on release

rate conducted at Dynatech indicate that this may be physically

possible. Injectable naltrexone base/PLGA sustained delivery systems have been produced by the cryogenic grinding of beads and

rods. The use of cryogenic temperatures makes the polymer/drug

mixture brittle. This in turn makes it possible to grind the preparations into very small particles. The resulting powder is then sieved

into narrow ranges of particle sizes, usually 45-90 μ or 90-180 μ.

Dosage is accomplished by suspending the sieved powder in an

agent such as Macrodex or Methocel, and injecting the slurry

through a suitably sized needle.

Several promising injectable sustained-release preparations have

been formulated at Dynatech. Figures 6 and 7 summarize the in

vitro release and in vivo narcotic antagonism performance of the

devices. Figure 6 shows the performance of a 90-180μ size, 50% by

weight naltrexone base in 39,000 Mw, 90/10 PLGA powder. Figure 7

shows the performance of 90-180μ, 70% by weight naltrexone base

in 39,000 Mw, 90/10 PLGA powder. This figure also shows the in

vitro release of naltrexone from naltrexone/PLGA rods from which

the powders were ground. Ten milligrams of each powder were

poured into a surgical incision in the scapular region of mice. The

wounds were then sewn closed. Narcotic antagonism was tested

using the mouse tail-flick test.

Figure 6 reveals high levels of antagonism, and near-constant in

vitro release for more than 30 days. Figure 7 shows erratic levels of

antagonism, but similar in vitro release for both powders and rods

(1.5 mm diameter).
208 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

FIGURE 6. In vitro release and in vivo antagonism data for sample #24085, 39,000

Mw 90/10 PLGA, 50% w/w naltrexone loaded powder (reference 9)

FIGURE 7. In vitro release and in vivo antagonism data for sample # 24085, 39,000

Mw 90/10 PLGA, 70% w/w naltrexone loaded preparations (reference 9)

An injectable system with the same composition as that shown in

figure 7, but sieved to 45-90μ particle size, was recently prepared.

The naltrexone in this system was 3H-labelled at the 15 and 16 positions. Monkeys trained to self-administer morphine were subcu-
DEVELOPMENT OF DELIVERY SYSTEMS 209

taneously injected with this preparation. Inhibition of morphine

self-administration was observed. Urine was collected for about 30

days.

This preparation inhibited morphine self-administration for more

than 5 but fewer than 12 days in these monkeys. Tritium was

measured in monkey urine for 12 to 24 days after injection. While

this preparation did not provide 30 days’ narcotic antagonism, it

does show promise for the further development of an injectable

system. Mouse antagonism data with the 90-180μ powders shows

much promise for development of injectable sustained-release

system which will provide complete narcotic antagonism for 30

days.

Development of a Sustained-Delivery System for Morphine

In addition to extensive development work on naltrexone delivery systems, Dynatech has developed an implantable dosage form

which provides about 2 weeks’ sustained delivery of morphine. The

system is composed of 50% by weight morphine in 30,000-50,000

Mw, 50/50 PLGA. The physical form of the sustained-delivery

device is a 1.5 mm diameter, 1 cm long rod. Several thousand of

these dosage forms have been produced at Dynatech for use in

animal studies by other investigators.

Preparation of Beads for Human Clinical Trials

The successful development of an implantable 30-day naltrexone

delivery system by Dynatech resulted in a decision by NIDA to proceed to human clinical trials with the Dynatech beads. These beads

are composed of 70% by weight naltrexone base, in 40,000 Mw, 90/

10 PLGA. It was decided that a 2000-bead batch would be required

to begin human clinical trials. It was also recommended by consultants that the production of the beads be undertaken at an FDAregistered drug manufacturing facility. The University of North

Carolina, School of Pharmacy, Chapel Hill, was chosen as the site

for this production.

Good Manufacturing Practices Documentation

Good Manufacturing Practices documentation was developed by

Dynatech for the bead production operation. GMP documentation

is designed to be “a statement of the methods, facilities and controls used for the manufacturing, processing and packing of the

new drug to establish and maintain appropriate standards of identity, strength, quality and purity as needed for safety and to give

significance to clinical investigations made with the drug.” (FD
210 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

Form 1571 [11/75], required attachment 5). GMP documentation is

based on the techniques of bead manufacture developed by Dynatech R/D Co. Analytical test methods were also developed by Dynatech in response to the requirements of the GMP documentation

and the suggestions of consultants. The GMP documentation developed includes the preparation of all necessary documentation to

support an application for a Claimed Investigational Exemption for

a New Drug (IND) from the U.S. Food and Drug Administration

(FDA) by NIDA. Mr. Ed Adams of Endo/Dupont Laboratories, and

Dr. James L. Olsen, Director of the UNC, Chapel Hill, School of

Pharmacy Drug Product Program served as consultants for the

preparation of the GMP documentation (10,11). A flowchart of the

bead manufacturing process, including the tests required by GMP

regulations, is depicted in figure 8.
DEVELOPMENT OF DELIVERY SYSTEMS 211

Continuation of Figure 8

FIGURE 8. Flowsheet of bead manufacturing procedure with Good Manufacturing

Practices documentation (reference 12). 70% w/w naltrexone, 30% w/w 90 L(+)/

10G PLGA bead manufacturing and quality control flowsheet
212 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

Production of Batch #L780901X

The UNC School of Pharmacy was supplied with all equipment

necessary for bead manufacture. The equipment loaned was the

same as that used to produce most previous batches of beads at

Dynatech. Included among these is batch number 24086, which underwent exhaustive testing for sterility, toxicity, and in vivo release rate and narcotic antagonist effect. These beads were produced using the analytical and manufacturing procedures detailed

in the GMP documentation developed for this process.

All equipment was assembled at UNC during November 1978.

Starting and intermediate material analyses, equipment assembly,

and initial bead moldings were all supervised by Dynatech staff

(12). Packaging, labelling, package seal testing, and visual inspection of the product beads were performed during January 1979.

Sterilization by exposure to 2.5 Mrads of Cobalt-60 -radiation, and

testing to assure sterility, were conducted during February 1979.

Two thousand eighty-five acceptable beads, and a number of rejected beads, were packaged 15 to a vial. Rejected beads are available for analytical testing, and for display purposes. The acceptable

beads are now ready for use in clinical trials, and are in storage at

the UNC, Chapel Hill, School of Pharmacy (13).

SUMMARY

The long term goal of the NIDA narcotic antagonist program of

developing an implantable, biodegradable, naltrexone/PLGA

matrix system which will sustain the delivery of naltrexone to biological systems for 1 month has been achieved. Dosage forms have

been reproducibly prepared and tested in vitro and in vivo for consistent release and efficacy. Analytical and manufacturing procedures have been developed and proven effective. Good Manufacturing Practices documentation for the production of these dosage

forms has been developed. The documentation was also used for

producing a large batch of dosage forms at an FDA registered pharmaceutical manufacturing facility. This batch is suitable for use in

human clinical trials.

Several other sustained-release dosage forms have also been developed by Dynatech. These include an implantable sustained-delivery system capable of 6 months’ release of naltrexone, and a finelydivided injectable form of the 1-month duration system, which

shows promise of successful application with further development.

An implantable dosage form which provides 2 weeks’ sustained-delivery of morphine has also been developed and produced in quantity for use in animal experiments. Thus the development of sus-
DEVELOPMENT OF DELIVERY SYSTEMS 213

tained-release devices at Dynatech has resulted in the production

of several systems with potential for practical application.

REFERENCES

1. L.S. Harris and A.K. Pierson. Some narcotic antagonists in the bensomorphan

series. J. Pharmacol. Exp. Ther., 143, 141-148 (1964).

2. S. Harrigan and D.A. McCarthy. Preclinical Pharmacologic and Toxicologic Evaluation of Compounds Used in the Treatment of Narcotic Addiction. NIDA Contract #HSM-42-72-167. Report #78, May 1976.

3. S.E. Harrigan and D.A. Downs. Continuous intravenous naltrexone effects on

morphine self-administration in rhesus monkeys. J. Pharmacol. Exp. Ther., 204,

2 (1978).

4. S.E. Harrigan and D.A. Downs. Preclinical Pharmacologic and Toxicologic Evaluation of Compounds Used in the Treatment of Narcotic Addiction. NIDA Contract #271-77-3421. Report #111, June 1978.

5. T.M. Lehman, P. Pyati, and G.R. Peterson. Inhibition of drug metabolism by

chronically administered naltrexone. Article Submitted to Life Sciences.

6. A.D. Schwope and D.L. Wise. Development of Drug Delivery Systems for Use in

Treatment of Narcotic Addiction. 11th Quarterly Report on NIDA Contract

#HSM42-73-267. Dynatech R/D Co., Cambridge, Massachusetts, April 1976.

7. J.E. Sanderson and D.L. Wise. Development of Drug Delivery Systems for Use in

Treatment of Narcotic Addiction. 15th Quarterly Report on NIDA Contract

# HSM-42-73-267. Dynatech R/D Co., Cambridge, Massachusetts, July 1977.

8. A.C. Schwope and D.L. Wise. Development of Drug Delivery Systems for Use in

Treatment of Narcotic Addiction. 12th Quarterly Report on NIDA Contract

#HSM-42-73-267. Dynatech R/D Co., Cambridge, Massachusetts, July 1976.

9. A.C. Sharon and D.L. Wise. Development of Drug Delivery Systems for Use in

Treatment of Narcotic Addiction. 16th Quarterly Report on NIDA Contract

#HSM-42-73-267. Dynatech R/D Co., Cambridge, Massachusetts, October 1977.

10. A.C. Sharon and D.L. Wise. Development of Drug Delivery Systems for Use in

Treatment of Narcotic Addiction. 17th Quarterly Report on NIDA Contract

#HSM42-73-267. Dyantech R/D Co., Cambridge, Massachusetts, January 1978.

11. Ibid. 21st Quarterly Report. October 1978.

12. Ibid. 22nd Quarterly Report. January 1979.

13. Ibid. 23rd Quarterly Report. April 1979.

AUTHORS

A. Carl Sharon and Donald L. Wise

Dynatech R/D Company

Cambridge, Massachusetts 02139
Naltrexone: Research Monograph 28

R. E. Willette and G. Barnett, eds.

National Institute on Drug Abuse, 1980

Use of Synthetic

Polypeptides in the

Preparation of

Biodegradable Delivery

Systems for Narcotic

Antagonists

Kenneth R. Sidman, Arthur D. Schwope, William D.

Steber, and Stephen E. Rudolph

The goal of this program was the development of biocompatible sustained-release systems that would release naltrexone at a rate of 20 to 25 μg/hr for 30 days, and that would

biodegrade within 90 days. The focus was on the use of macrocapsules prepared from synthetic polypeptides, specifically copolymers of glutamic acid and ethyl glutamate (i.e.,

Glu/EGlu copolymers). Tubular capsules prepared from 18/

82 Glu/EGlu were the most promising systems developed.

Capsules 1 cm in length, 0.19 cm in outside diameter, and

0.005 cm in wall thickness released naltrexone in mice at

rates in the range of 20 to 40 μg/hr for 18 days. The rates

then decreased during the next 12 days as the capsules

became exhausted of drug. These capsules were biocompatible and they appeared to biodegrade within 90 days. In general, the Glu/EGlu copolymers exhibit permeation and degradation rates that increase as the glutamic acid content is

increased. Radiotracing studies revealed &hat the ultimate

degradation product was carbon dioxide, which appeared

in the expired air. This result is consistent with a polypeptide degradation- process that involves hydrolysis of the

214
SYNTHETIC POLYPEPTIDE SYSTEMS 215

ethyl esters followed by hydrolysis of the peptide bonds to

produce glutamic acid, which enters the metabolic pool.

INTRODUCTION

We have been working on the development of injectable, biodegradable drug delivery systems that would provide a sustained release of naltrexone. The goal was a delivery system (i.e., depot) that

would release drug at a relatively constant rate of 20 to 25 micrograms per hour for 30 days, and then biodegrade within 90 days.

Synthetic polypeptides were used to form the depots. Specifically,

copolymers of glutamic acid with ethyl glutamate (i.e., Glu/EGlu

copolymers) were studied. In this paper we describe the synthesis

procedures and the studies that were carried out in order to assess

the permeability and biodegradability of the copolymers.

MATERIALS AND METHODS

Copolymer Synthesis

The preparation of Glu/EGlu copolymers was carried out in

three stages: 1) synthesis of monomers; 2) polymerization of benzylblocked intermediates; and 3) debenzylation of the intermediates.

The monomers, N-carboxyanhydrides (i.e., NCA’s) of -benzyl-L-glutamate and -ethyl-L-glutamate, were prepared from the respective

amino acid esters using the procedures of Fuller et al. (1). Polymerization of the monomers to form copolymers of benzyl and ethyl

glutamate was carried out in solvent at 25°C, following the procedures of Anderson (2). Triethylamine (1.3 M in benzene) was used

as the initiator at a level of 2 mole percent based on the monomers.

The reaction mixture was stirred under nitrogen for 3 days. The

product was then isolated by precipitation into methanol.

After vacuum drying, the benzyl-blocked intermediate was dissolved in toluene and debenzylated using anhydrous HBr, according to the methods of Fasman et al. (3). This procedure typically

resulted in the loss of all of the benzyl groups and some ethyl

groups so that the final Glu/EGlu copolymer exhibited a higher

glutamic acid content than would be expected if the procedure

were totally selective for benzyl ester acidolysis.

The copolymers exhibited inherent viscosities in dichloroacetic

acid (0.2% solution at 25°C) ranging from 0.9 to 1.5 dl/g. Composition was determined by proton NMR in trifluoroacetic acid solutions.

347-877 0 - 81 - 15
216 NALTREXONE SUSTAINEDRELEASE PREPARATIONS

Biodieposition Studies

Radiolabelled Copolymer Synthesis

One millicurie of L-glutamic acid [

14C(U)] was converted to the

ethyl ester via procedures described by Greenstein and Winitz (4).

The ester was then converted to the NCA, copolymerized with

benzyl glutamate, and the intermediate was debenzylated according to the procedures described above. The product exhibited a

Glu/EGlu mole ratio of 45/55, an inherent viscosity in dichloroacetic acid of 1.05 dl/g, and a specific activity of 660,000 dpm/mg. A

schematic representation of the polymer is shown.

Film Implantation

Films, approximately 0.006 cm thick, were cast from a solution of

the copolymer in N,Ndimethyl formamide. After drying at 60°C for

3 days, the films were cut into 1 cm square pieces and implanted

subcutaneously (via a midline incision) in the sacral area of the

back; one piece was positioned to lie on one flank, a second piece to

lie approximately over the kidney.

Recovery and Analysis of CO2

One or two mice at a time were housed in a glass metabolic cage.

Food and water were available ad libitum. Cage air was continuously evacuated from the top and passed through 60 ml 4 M KOH

via a fritted tube. The rate of air flow was 300-400 ml/min. Room

air was allowed to enter the cage at the bottom to replace the evacuated air. The KOH was replaced about every 24 hours. Aliquots

were counted in Modified Bray’s scintillation cocktail.

Whole-Body Autoradiography

At various times following implantation of the Glu/EGlu film,

mice were killed by immersing them rapidly in hexane/dry ice.

Animals were stored frozen at -25°C.
SYNTHETIC POLYPEPTIDE SYSTEMS 217

For microtomy, an animal was imbedded in a mold filled with

methylcellulose. The mold was kept in a -25°C freezer until the

methylcellulose was frozen. Whole-body sections, 60 microns in

thickness, were cut with a Jung (Heidelberg) microtome housed in

a Harris cryostat (Harris Manufacturing Co., Cambridge, Mass.).

The sections were picked up on Scotch #810 transparent tape,

transferred over dry ice to a low-humidity cold room, and stored at

-25°C until excess tissue moisture was removed. The whole-body

sections were taped to Kodak No-Screen Medical X-ray film, in a

photographic darkroom at room temperature.

After suitable exposure time (which ranged from one week to

several months, depending upon the intensity of radioactivity), the

sections were removed and the X-ray films processed in Kodak Xray developer and fixer.

Film Permeability Studies

The experiments were carried out using a two-chambered, glass

cell which exposed one side of a test film (5.1 cm2 area) to either an

aqueous or a sesame oil solution saturated with 3H-naltrexone and

containing undissolved drug. The solution was agitiated by means

of a magnetic stirrer. The other chamber-the downstream sidecontained 50 cm3

 of Earle’s Balanced Salt solution. A 5% CO, gas

was bubbled into the Earle’s solution in order to maintain its pH

and bicarbonate concentrations. Cell temperature was 37°C. Permeant concentration in the Earle’s solution was determined by

scintillation counting; 1 cm3 samples were taken daily. The influence of downstream permeant concentration on permeation rate

was minimized by replacing the Earle’s solution before the naltrexone concentration approached 20% of the saturation value.

Capsule Preparation and Evaluation

Capsules were assembled from tubes and caps, which were prepared by dip coating glass mandrels into a solution of the copolymer in N,N-dimethyl formamide. The capsules were filled with a

solid rod of naltrexone (tritium-labelled in the 15,16 positions--specific activity equal to 1060 dpm per microgram of drug). The rods

were prepared by extrusion of a stiff paste consisting of drug and

tetrahydrofuran (in some cases, sesame oil was used in place of tetrahydrofuran), through a stainless steel die (see figure 1). The rods

were dried at 70°C for 3 to 5 days to remove the solvent. After the

naltrexone rods were inserted into the tubes the caps were cemented in place using the copolymer solution. The size of a typical capsule is shown in figure 2.
218 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

FIGURE 1. Compression Mold Used to Extrude Solid Rod

FIGURE 2. Capsule Filled With Naltrexone (Each scale division is 1 mm.)

The capsules were implanted subcutaneously in mice, and the

urine was monitored for radioactivity using the liquid scintillation

counting procedures. The release rates were calculated by assuming that 74% of the naltrexone released from the capsules appeared in the urine (as parent compound and metabolites). This

fractional recovery was established in separate experiments wherein mice were administered subcutaneous injections of radioactive

naltrexone and then studied to determine urinary and fecal distribution of radioactivity. In addition, the areas under the release

rate vs. time curves were routinely measured to determine if the

total drug recovered was equivalent to the initial drug contained in

the capsules.

In previous studies (5), we determined that a release rate of approximately 1 μg/hr was sufficient to block an AD80 dose of morphine in mice. In the following studies, the release rates greatly exceeded this minimum effective level, so the tail-flick bioassay procedure was used infrequently, only to confirm that naltrexone was

being released in an active form.
SYNTHETIC POLYPEPTIDE SYSTEMS 219

RESULTS AND DISCUSSION

Biodegradation and Biodisposition of Glu/EGlu Copolymers

Glu/EGlu copolymers degrade in vivo in a continuous process

that was postulated to involve hydration of the film, hydrolysis of

the ethyl esters, dissolution of the soluble molecules (i.e., copolymers with high Glu/EGlu ratios), enzymatic hydrolysis of peptide

bonds, and finally passage of glutamic acid into the “metabolic

pool.” The glutamic acid should be disposed of by two principal

routes: 1) decarboxylation in the tricarboxylic acid cycle; and 2) incorporation into protein.

Two studies of the biodegradation process were carried out in a

program supported by the National Institute of Child Health and

Human Development under Contract No. N01-HD-4-2802 (6). In one

study, films exhibiting a range of copolymer compositions and

thicknesses were evaluated to determine their biodegradation

rates. In the second study, the ultimate biodisposition of a 14C-labelled Glu/EGlu copolymer was determined.

Biodegradation

Films with Glu/EGlu mole ratios of 13/87, 18/82, 23/77, 30/70,

40/60, and 50/50 Glu/EGlu were implanted subcutaneously in

mice. Periodically, animals were sacrificed and the implant sites

were examined. The results are summarized in table 1.

The time for biodegradation of the films decreased as the glutamic acid content was increased. For example, films containing 13

mole percent glutamic acid remained intact for more than 79 days,

while films containing 40 mole percent disintegrated within 7 days.

As might be expected, the degradation time increased with increasing film thickness.

Biodisposition

The ultimate disposition of the copolymer was determined in

studies involving the implantation of radiolabeled films of 45/55

Glu/EGlu in mice. Table 2 shows typical results. Approximately

73% of the implanted radioactivity was recovered within 8 days,

and 96% of this was 14CO2.

Autoradiograms of the animals, shown in figures 3 and 4, revealed that the residual radioactivity concentrated at the implant

site, suggesting that the slow step in the elimination of the copolymer was its dissolution in body fluids. Once the copolymer dissolved, it appeared to be transported rapidly to sites where it was

metabolized. After only 7.5 hours, there were significant levels of
220 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

TABLE 1. Interim Biodegradation Results

Film Appearance

(Numbers in parenthesis denote percent

weight loss)

Copolymer Film

Ratio Thickness,

Days Post-Implantation

Glu/EGlu c m 7-10 28-35 78-79

13/87

18/82

23/77

30/70

40/60

0.0025 - Intact (15%)

0.0051 - Intact (0%)

0.0025 Intact (30%‘) Fragmented (33%)

0.0051 Intact (0%) Intact (17%)

0.0051 Intact (15%) Fragmented (75%)

0.0064 Intact (19%) Fragmented (33%)

0.0038 Disintegrated

o r

Dissolved

Intact (15%)

Intact (13%)

Disintegrated

Fragmented

Disintegrated

Disintegrated

50150 0.0038 Disintegrated

or

Dissolved

*Film showed a 30% weight gain, not a loss.

TABLE 2. Ultimate Biodisposition of Glu/EGlu Film

Percentage of the Administered Dose of Radioactivity Recovered in Expired Air and

Excreta from a Mouse Implanted with 12 mg of Glu/EGlu Radiolabelled Copolymer Film

Time (hrs) % Recovery of

Radioactivity

Trapped 14CO2 0 - 6.5

6.5 - 23.5

23.5 - 48.5

48.5 - 72

72 - 105

105 - 121

121 - 145

145 - 168

2.3%

15.5%

20.7%

10.2%

6.4%

2.7%

5.3%

3.9%

168 - 192 3.0%

70%

Excreta 0 - 51 1.8%

51 - 147 1.0%

2.8%

Total 72.8%
SYNTHETIC POLYPEPTIDE SYSTEMS 221

radioactivity in kidney, liver, bone marrow, para-orbital glands,

and the intestinal epithelium. In general, the radioactivity was associated with the more metabolically active tissues. By day 11,

there was a significant reduction in the total level of radioactivity.

This is not fully apparent in the figure because the X-ray film exposure time was quadrupled in this case in order to obtain good

contrast.

FIGURE 3. Autoradiogram Taken 7.5 Hours Post-Implantation

FIGURE 4. Autoradiogram Taken 11 Days Post-Implantation

The results of these studies were consistent with the hypothesized metabolic disposition of Glu/EGlu copolymers; that is, dissolution, hydrolysis of the ester in ethyl glutamate, hydrolysis of the

polyglutamate to glutamic acid, and finally, passage of glutamic

acid into the metabolic pool.

Biocompatibility

To be useful for drug delivery systems, the Glu/EGlu copolymers

must be well tolerated by the body. This means they should not

elicit any adverse tissue response in an acute exposure, and they
222 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

should not induce hypersensitivity reactions (i.e., an antigenic response) on chronic or repeated exposure.

Work to date has been focused on copolymers that contained glutamic acid concentrations in the range of 10 to 35 mole percent, because these materials exhibit degradation rates especially appropriate for capsules. Films and depots (in the form of rods and cap

sules) were implanted in mice and the implant sites were examined

at frequent intervals. Gross observation consistently revealed little

or no tissue inflammation associated with the copolymers.

When an insoluble Glu/EGlu copolymer is implanted, we believe

it begins to hydrolyze at the ester group, thereby generating a variety of watersoluble, partially esterified polymers that diffuse from

the implant site and are ultimately biodegraded to glutamic acid.

During this process it is assumed that the body will be exposed to

copolymers exhibiting various ratios of Glu/EGlu. In a previous

study (6), we evaluated the antigenicity of a 60/40 Glu/EGlu copolymer which was used as a model for this diverse mixture. Guinea

pigs, sensitized with three injections of the copolymer over a 20-day

interval, were skin-tested on day 30.

The results indicated that there were essentially no differences

between the initial (control) and final reactions. No evidence of hypersensitivity reactions was observed. Since the 60/40 ratio is considered a typical intermediate in the degradation process of solid

implants to glutamic acid, we infer that slowly degrading drug delivery vehicles would be nonantigenic in guinea pigs.

Development of Naltrexone Depots

A series of Glu/EGlu copolymers with glutamic acid contents

ranging from 13 mole % to 30 mole % were evaluated for their potential usefulness as capsules. First, the moisture sorption capacity

of each copolymer was determined because this property has been

found to be an expeditious means of obtaining insight to probable

permeability of the copolymer-high moisture sorption is predictive

of high naltrexone permeability. Film permeation studies were

then carried out with selected copolymers and, finally, capsules

were prepared and evaluated.

Film Permeability Studies

Experiments were carried out using films prepared from copolymers exhibiting the following Glu/EGlu mole ratios: 13/87, 18/82,

20/80, and 30/70 Glu/EGlu. Two suspending agents-water and

sesame oil-were investigated. The latter agent was studied because

it is a biocompatible fluid that may be conveniently used in cap
SYNTHETIC POLYPEPTIDE SYSTEMS 223

sules to ensure good contact of the drug with the interior surfaces

of the capsule. (Sesame oil exhibits low volatility and is retained

during capsule assembly and drying operations.)

The results are shown in table 3. The measured film permeabilities are given in the last column. When these numbers are multiplied by film surface area and then divided by film thickness, the

permeation rate is obtained in terms of μg/hr. The permeability

generally correlated with the measured moisture pickup for the copolymers. However, when sesame oil was used as the suspending

agent, an unusually high permeability resulted. In separate experiments, the ability of the 20/80 copolymer to become plasticized

with sesame oil was determined by immersing films into both

sesame oil and sesame oil saturated with naltrexone. After 24

hours at 37°C, the films picked up only 4% to 5% by weight, and

this did not increase during a total of 7 days immersion. This small

weight gain does not appear to be sufficient to-account for the high

permeability, but at the present time no better explanation has

been found.

TABLE 3. Permeation Results

Naltrexone Permeability Study

Batch Moisture Film

Copolymer Identifi- Pick-up Thickness Upstream Film Permeability

Glu/EGlu cation (%) (cm) Medium [μg-cm(thick)/hr-cm2

(area)] x 102

13/87 19599-142 6 0.003 Water 4.3

13/87 19599-142 6 0.011 Water 2.0

18/82 19599-151 80 0.005 Water 75

20/80 19599-131 175 0.004 Water 103

20/80 19599-131 175 0.012 Sesame Oil 240

30/70 19599-140 100 0.006 Water 128

The permeabilities in the table were used to make predictions of

capsule performance. Typically, the capsules were prepared with a

surface area of 0.8 to 1.0 cm2 and a wall thickness of 0.005 cm.

When these values were multiplied by the permeabilities, the calculations indicated that release rates ranging from a low of 3 μg/

hr to a high of 340 μg/hr could be obtained.

Capsule Studies

Capsules were prepared from each of the copolymers. In certain

cases, sesame oil was used as an extrusion aid in the preparation of

the naltrexone cores. Some capsules were filled with additional
224 NALTREXONE SUSTAINEDRELEASE PREPARATIONS

sesame oil in order to investigate the value of maintaining an oilfilled reservoir. The results are described below, beginning with the

30/70 Glu/EGlu depots, which released antagonist at the fastest

rates, and progressing to the 18/82 and 13/87 depots, which sustained release for 30 days and longer.

30/70Glu/EGlu

Figures 5a and 5b show the release rates for capsules 1.5 cm in

length, 0.18 cm in inside diameter, and 0.004 to 0.005 cm in wall

thickness. These were filled with 40 mg cores composed of 22 mg

naltrexone and 18 mg sesame oil (all the oil was absorbed in the

core.) The results in figure 5a were for capsules that received addi-
SYNTHETIC POLYPEPTIDE SYSTEMS 225

tional sesame oil to ensure that liquid contacted the interior surface of the capsules. The results in figure 5b were for capsules that

received no additional oil. In both cases, the rates were extremely

rapid, peaking at approximately 300 μg/hr and then dropping off

within 8 days. No significant difference in behavior of depots with

and without additional sesame oil was observed.

These release results are in close agreement with predictions

that would be made on the basis of the film permeability shown in

Table 3, 1.28 μg x cm/hr x cm2

.

 For example, the predicted rate

for capsules that exhibit a surface area of 1 cm2 and an average

wall thickness of 0.0045 cm is given below.

Predicted Release Rate Capsule Area/Wall Thickness x Permeability

1 cm2/0.0045 cm x 1.28 μg · cm/hr · cm2

= 284 μg/hr

20/80 Glu/EGlu

Capsules were prepared 1 cm in length, 0.18 cm in inside diameter, 0.005 cm in wall thickness, and they were filled with pure naltrexone rods weighing 22 mg. Sesame oil was added to some of the

tubes, while others were left free of oil to provide a basis for comparison. The release rates are shown in figure 6a. Capsules with oil

exhibited slightly higher rates, peaking at 260 μg/hr and then rapidly falling off. Capsules without oil peaked at 200 μg/day, but

they, too, were exhausted of drug within 12 days.

A second set of capsules was prepared in order to study the influence of wall thickness and sesame oil. These capsules were longer

than those above, 1.5 cm, and they were either 0.009 cm or 0.011

cm in wall thickness. They were filled with 40 mg rods composed of

22 mg naltrexone and 18 mg sesame oil. Additional oil was added

to fill the tubes. As is shown in figure 6b, there was little difference that could be ascribed to wall thickness, and the magnitude of

release was similar to that shown in figure 5a. This suggests that

the larger surface of the 1.5 cm capsules compensated for their

thicker walls.

18/82 Glu/EGlu

The release rates from capsules 1 cm in length, 0.005 cm in wall,

and filled with 24 mg rods (composed of 16 mg naltrexone and 8 mg

of sesame oil) are shown in figure 7. The rate gradually rose over

the first 3 days to a range of 20 to 40 μg/hr, which was maintained

for approximately 18 days. During this interval, approximately 13
226 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

mg was administered out of the initial 16 mg loading of naltrexone.

The remaining 3 mg was released over the next 12 days.

The release behavior of these capsules was quite unlike that of

the 20/80 capsules discussed above, despite the small difference in

copolymer composition. However, the moisture absorption results

(80% for the 18/82 copolymer and 175% for the 20/80) appeared to

provide an indication of the real differences between the copolymers and, therefore, moisture absorption will be utilized in the

future to characterize capsule materials.

The 18/82 capsules represent the best depots developed in the

program. They exhibited essentially the target release rate, and

they would have maintained this rate for at least 30 days if the

cores had contained the maximum naltrexone load (i.e., 30 mg,

based on full utilization of the interior volume). As has already
FIGURE 7 NALTREXONE RELEASE FROM 18/82 GLU/EGLU CAPSULES
228 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

been described, the Glu/EGlu copolymers are biocompatible in

mice, rats, and guinea pigs, and they exhibit the requisite biodegradability. Only residues of the 18/82 capsules were found 90 days

after implantation. The typical pattern of degradation is shown in

figure 8.

It should be noted that the predicted release rate based on film

permeability was higher than the actual capsule results:

Predicted Release = (Area of 1 cm long capsule)/

(Thickness)

= 0.7 cm2/0/005 cm

x 0.75 μg · cm/hr · cm2

= 105 μg/hr

One possible explanation for the discrepancy is that the capsules

did not contain the additional sesame oil necessary to ensure good

contact with the interior walls. Conceivably, if the capsules were

FIGURE 8. Biodegradation sequence of naltrexone capsules. Figure 8a shows depots

prior to implantation. Figures 8b, 8c, and 8d show capsules excised after 30, 60, and

90 days, respectively. Note that the depot in Figure 8b is collapsed, indicating

depletion of drug. At 90 days the copolymer was difficult to distinguish from living

tissue.
SYNTHETIC POLYPEPTIDE SYSTEMS 229

filled with oil or higher drug loadings, then actual release would be

closer to the predicted value, 105 μg/hr. Since this rate would be 4

times the target level, 25 μg/hr, it would be necessary to quadruple

the wall thickness in order to reduce the rate to the desired range.

Clearly, additional development effort on the 18/82 capsules is required in order to meet all the program objectives and, based on

the positive results to date, such effort appears to be warranted.

13/87 Glu/EGlu

Capsules 1 cm in length and 0.008 cm in wall thickness were fabricated from the 13/87 copolymer. These were filled with 22 mg

rods that contained 12 mg of naltrexone and 10 mg of sesame oil.

Figure 9 shows that the release rate was quite slow, peaking at approximately 8 μg/hr by day 56, and it was prolonged for almost 100

days. Residues of this slowly degrading copolymer were still evident at the implant sites 180 days after implantation.

SUMMARY

Tubular capsules made from glutamic acid-ethyl glutamate copolymers offer wide-ranging potential for controlled narcotic antagonist release. The physical dimensions and copolymer composition

are readily varied to meet specific delivery rate and duration objectives, while satisfying equally important biocompatibility and degradation requirements.

REFERENCES

1. W.D. Fuller, M.S. Verlander, and M. Goodman. A Procedure for the facile synthesis of amino-acid N-carboxyanhydrides. Biopolymers, 15, 1869-1871 (1976).

2. J. Anderson, Case Western Reserve University. Personal Communication (1977).

3. G.D. Fasman, M. Idelson, and E.R. Blout. The synthesis and conformation of high

molecular weight poly-E-carbobenzyloxy-Llysine and polylysine, HCl. J. Am.

Chem. Soc., 83, 709-712 (1961).

4. J.P. Greenstein and M. Winitz. Chemistry of the Amino Acids, Vol.2, John Wiley

& Sons, Inc., N.Y., 297 (1961).

5. K.R. Sidman, D.L. Arnold, W.D. Steber, K.K. Stevenson, and L.C. Wu. Develop

ment and evaluation of a biodegradable drug delivery system for the controlled

release of narcotic antagonists. Seventh Quarterly Report, NIDA Contract No.

ADM-45-74-218 (1976).

6. K.R. Sidman, W.D. Steber, A.D. Schwope, and S.E. Rudolph, Development and

evaluation of a biodegradable drug delivery system. Sixteenth Quarterly Report,

NICHD Contract No. NO1-HD-4-2802 (1976).
FIGURE 9 NALTREXONE RELEASE FROM 13/87 GLU/EGLU CAPSULES
SYNTHETIC POLYPEPTIDE SYSTEMS 231

ACKNOWLEDGMENT

The work reported in this paper was supported by Contract No.

ADM-45-74-218 from the National Institute on Drug Abuse and

Contract No. NO1-HD-4-2802 from the National Institute of Child

Health and Human Development.

AUTHORS

Kenneth R. Sidman, Arthur D. Schwope, William D. Steber, and

Stephen E. Rudolph

Arthur D. Little, Inc.

Acorn Park

Cambridge, MA 02140

347-877 0 - 81 - 16
Naltrexone: Research Monograph 28

R. E. Willette and G. Barnett, eds.

National Institute on Drug Abuse, 1980

Biodegradable Drug

Delivery Systems Based

on Aliphatic Polyesters:

Application to

Contraceptives and

Narcotic Antagonists

Colin G. Pitt, Thomas A. Marks, and Anton Schindler

The classes of polymer which form the basis of different

types of drug delivery systems are discussed, and the relationships between the chemical structure of the polymer

and its permeability, morphology, biodegradability, and mechanical properties are considered, using polyesters as specific examples. Studies of the permeability and biodegradability of poly -caprolactone), poly(DL-lactic acid), and various copolymers are described and used to illustrate how

these properties may be varied by the choice of polymer

structure. An induction period prior to bioerosion of these

polymers, coupled with high permeability, permits their use

as reservoir devices (capsules) which exhibit constant, diffusion-controlled drug release rates and which erode after the

drug is exhausted. The applications of this approach to the

long term delivery (1 year) of levonorgestrel, a contraceptive

agent, and the short term delivery (1-2 months) of naltrexone, a narcotic antagonist, are described.

232
ALIPHATIC POLYESTERS DELIVERY SYSTEMS 233

INTRODUCTION

There are three fundamentally different approaches to the

design of polymeric drug delivery systems - control by osmotic pressure, by diffusion, and by erosion (1). Only the last, by definition,

necessarily involves polymer biodegradation. Regardless of the ap

proach selected, it is necessary to carefully control the properties of

the polymer, the most important of which are its permeability, biodegradability (or stability), biocompatibility, and mechanical prop

erties. Since these properties are often interdependent, their optimization can present a challenging exercise in polymer design.

Polymers used for drug delivery may be separated into two distinct classes, those that are in the glassy state (Tg>37°C)2

 and

those that are in the rubbery state (Tg<37°C) at body temperature.

If the polymer is in the glassy state it will generally be tough and

inflexible, with a low permeability, and drug release by leaching

and polymer erosion will be favored relative to diffusion. The only

possibility of achieving a constant rate of drug release from this

class of polymer is by constant bioerosion of a device with a constant surface area, i.e., a slab, or by diffusion-controlled release

from a microcapsular reservoir device where the very thin walls

and increased surface-to-volume ratio compensate for the reduced

permeability. Poly(lactic acid), Tg 57°C, formulated as microcapsules, is an example of the latter system (2-4).

Polymers in the rubbery state will have a high permeability but

will often lack sufficient mechanical strength and form stability

unless reinforced by the presence of cross-links, crystalline domains, or strong interactions between chains, e.g., hydrogen bonding. Silicone rubber is an example of a non-degradable rubbery

polymer (Tg -120°C) which must be strengthened by both filler and

light cross-linking. If the polymer is to be biodegradable, the means

of reinforcement must also be biodegradable. This can present

some difficulties, for if diffusion-controlled drug release is operative, degradation of filler or cross-links may change the polymer

morphology and hence its permeability. Changes in device dimensions and crystallinity produced by biodegradation will also affect

the rate of drug diffusion. These problems can be minimized if

bioerosion occurs after the useful life of the device, i.e., after drug

depletion. This is the approach which we have preferred in our laboratory.
234 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

Polymer Design

Excluding polymers which are bioerodable by virtue of their

water solubility, e.g., polyvinyl alcohol, the classes of polymer

which have been shown to be degraded at an appreciable rate in

vivo are:

The selection of any one of these classes for study is somewhat

subjective, for within each class one can find a spectrum of properties. The permeability, biodegradability and mechanical properties

may all be changed substantially by logical but rather small

changes in the chemical structure of the substituent or chain. The

polyesters (figure 1) provide a good illustration. Thus, on the basis

of electronic effects and the properties of monomeric esters, one

can anticipate that the rate of non-enzymatic hydrolysis of polymers of -hydroxy acids, e.g., poly(glycolic acid), will be greater

than polymers of ß, and -hydroxy analogs; aromatic esters

will be least readily hydrolyzed. Alkyl or aryl substituents adjacent

to the ester group can retard hydrolysis by steric hindrance.

The polymer morphology and permeability are superimposed

upon these chemical effects, determining the accessibility of the

bulk of the polymer to aqueous hydrolysis and the loss of cleaved
ALIPHATIC POLYESTERS DELIVERY SYSTEMS 235

FIGURE 1. The Chemical Structures and Relevant Physical Properties of Some

Typical Polyesters.

chain fragments, as well as the rate of drug diffusion and mechanical properties. Here the Tg

 and the melting point (Tm) provide a

good indication of structure-morphology relationships (figure 1).

Poly(glycolic acid), the simplest aliphatic polyester, is a practically

insoluble, high melting, crystalline polymer with a Tg of 36°C.
236 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

These properties reflect the polarity and structural order of this

polymer. Stereospecific introduction of a single methyl group, as in

poly(D or L-lactic acid) has little effect on the Tg and Tm. Non-stereospecific introduction of a methyl group, as in racemic poly(DLlactic acid) does not change Tg

 greatly but completely eliminates

the crystallinity because of the random arrangement of the asymmetric centers. Similarly, nonspecific introduction of two different

alkyl groups will both eliminate the crystallinity and reduce the

Tg, and a polymer such as poly(DL-methylethylglycolic acid) is rubbery at 37°C. However, if both the alkyl groups are identical, as in

poly(dimethylglycolic acid), the symmetry and crystallinity are restored.

It is possible to reduce the Tg substantially without changing the

crystallinity by increasing the length of the methylene repeat unit,

e.g. poly(alkylene adipates), poly( -caprolactone), and their homologs. These polymers are permeable (low Tg

) and mechanically

strong (crystallinity). The crystallinity can be reduced or eliminated by introduction of an asymmetric center, as in poly(DL- -decalactone). This modification increases the permeability but reduces

the mechanical strength to the extent that the latter polymer is a

viscous gum.

Introduction of aromatic groups into the polymer backbone increases the Tg

 and the crystallinity, and a polymer such as

poly(ethylene terephthalate) is a tough, high melting, inflexible,

impermeable material.

Plasticization, blending, and copolymerization represent other

ways in which the polymer properties may be tailored, and specific

examples are given below.

Polymer Permeability

There is a good correlation between the Tg and the permeability

of those polyesters which have been tested. Poly( -caprolactone)

and its copolymers have low Tg's and are as permeable as silicone

rubber; poly(DL-lactic acid) and its copolymers with poly(glycolic

acid) are in the glassy state and are relatively impermeable to

drugs such as steroid hormones. Using progesterone as an example,

the maximum steady state flux (Jlim), solubility, partition and diffusion coefficients of this steriod in poly( -caprolactone) and its copolymers with DL-lactic acid are compared with silicone rubber in

table 1. The permeability of poly( -caprolactone), as reflected by

the value of Jlim, is almost equal to that of silicone rubber, largely

because of the greater drug solubility in poly( caprolactone). (The

maximum steady state flux, Jlim, is the product of the diffusion coefficient and the solubility of the drug in the polymer and is equal
ALIPHATIC POLYESTERS DELIVERY SYSTEMS 237

to the maximum drug diffusion rate through a membrane of unit

area and thickness.) While poly(DL-lactic acid) is 104 times less permeable than poly( -caprolactone), copolymers of DL-lactic acid and

-caprolactone have the same permeability as poly( -caprolactone).

This is understandable, for copolymerization affords a polymer

with a Tg between the Tg's of the two homopolymers, as illustrated

in figure 2, and with less crystallinity than poly( -caprolactone).

TABLE 1. The Maximum Steady State Flux (Jlim), Partition Coefficient

(K), Solubility (C,) and Diffusion Coefficient (D) of Progesterone in

Poly( -caprolactone), Poly(DL-Lactic Acid), Poly( -caprolactone-co-DLlactic acid), and Silicone Rubber

Polymer

Jlim x 1011 Partition Solubility D x 109

(g.cm 1.sec 1) Coefficient (mg/g) (cm2

.sec 1)

Poly( -caprolactone) 6.1 1200

1:1 copolymera5.6 1554

Poly(DL-lactic acid) 0.33 x 10346

Silicone Rubberb26 50

a

1:1 (wt%) Copolymer of -caprolactone and DL-lactic acid

bData for polymer containing filler (Reference 10).

16.9 3.6

21.9 2.7

0.65 5.1 x 103

0.57 450

Progesterone is released very rapidly from thin films (100-300

μm) of poly( -caprolactone) and its copolymers with lactic acid and

glycolic acid, release being essentially complete within 24 hrs (6).

The fraction of drug load released is proportional to (time)1/2, although the kinetics are moderated by an aqueous boundary layer

which produces some curvature in a plot of the fraction released vs

t

1/2 (figure 3).

Release of progesterone from poly(DL-lactic acid) films is very

much slower, even from thinner films (10 mm), and leaching may

be partly responsible for the rate which was observed (figure 4).

Similar results were obtained with copolymers of glycolic acid and

DL-lactic acid except that in some instances there was a marked

increase in rate after several weeks (figure 5). This coincided with

the mechanical deterioration of the film and is an example of erosion-enhanced release (6).

The rate of diffusion could also be enhanced by plasticization of

poly(DL-lactic acid) with tributyl citrate (5). This served to reduce

the glass transition below 37° and transformed the polymer to the

rubbery state. However, the effect was short lived (figure 6), as the

plasticizer was rapidly leached into the external aqueous medium.

The permeability could also be enhanced (200-fold) by casting
238 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

FIGURE 2. Variation of Glass Transition Temperature (Tg) of Copolymers of

Caprolactone and DLLactide as a Function of DL-Lactide Content. Solid Line is

Calculated Relationship, Based on Fox Equation (from Reference 8).

poly(DL-lactic acid) from a solution containing glycerine, then soaking the film in water (5). Here the permeability increase was the

result of a highly porous structure easily detected by scanning elec-
ALIPHATIC POLYESTERS DELIVERY SYSTEMS 239

FIGURE 3. Fraction of Progesterone Released from Poly( -caprolactone) Films (10%

Drug Load) as a Function of Time1/2, and Film Thickness. Solid Lines are

Calculated from Theoretical Relationship (Reference 11) Which Incorporates Effect

of Aqueous Boundary Layer.

tron microscopy (figure 7). This structure may mimic that of a film

partly depleted of drug.

Polyester Biodegradation

Most reports of polymer biodegradation are qualitative and confined to evaluation of a single property; in many cases the specifications of the polymer are not reported. This is unfortunate because residence times in the body may vary by an order of magnitude depending on the molecular weight, morphology, physical

form, etc. As a consequence, the factors which contribute to polymer degradation are still poorly understood.

In these laboratories, poly( -caprolactone) has been studied most

extensively, and changes in viscosity, weight, molecular weight distribution, and tensile properties have been determined in vivo
RELEASE OF PROGESTERONE FROM POLY(DL-LACTIC ACID)

FILM IMPLANTED IN RABBIT

FIGURE 4. Daily Rate of Release of Progesterone from Poly(DL-Lactic Acid) Film

Implanted in Rabbit, Measured as Radioactivity Excreted in Urine (from Reference 7).
ALIPHATIC POLYESTERS DELIVERY SYSTEMS 241

FIGURE 5. In Vitro Rates of Release of Progesterone From Two Poly(DL-Lactic

Acid-co-Glycolic Acid) Films, Thickness 100 mm, Drug Load 10%, Plotted as the

Fraction of Drug Released Versus the Square Root of Time. Break in Line is

Indicative of Fragmentation of One of the Films.

(rabbit, rat) and under accelerated in vitro conditions (7,8) In

common with other polyesters studied, biodegradation of poly( -caprolactone) is characterized by an induction period prior to weight

loss. There is a continual decrease in the viscosity and molecular

weight (GPC) resulting from hydrolytic cleavage of the ester linkages, and semi-log plots of MW or the intrinsic viscosity versus

time are linear until the MW has decreased to about 30,000. Several different polymers are compared in figure 8, which shows that

poly(DL-lactic acid) is degraded more rapidly than poly( -caprolac-
242 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

DIFFUSION OF PROGESTERONE THROUGH A MEMBRANE

OF POLY(DL-LACTIC ACID) PLASTICIZED WITH TRIBUTYL CITRATE

FIGURE 6. Rate of Diffusion of Progesterone Through a Membrane of Poly(DLLactic Acid) Plasticized with Tributyl Citrate. Break in Plot is Indicative of Loss of

Tributyl Citrate (from Reference 5).

tone). This can be attributed to chemical effects, for the same relative rates are observed with the monomeric esters. The greater rate

of degradation of the copolymers must be attributed to morphological changes. Others have observed that copolymers of lactic acid

and glycolic acid are degraded more rapidly than the homopolymers (9), presumably for the same reason.

The semi-logarithmic relationship between the molecular weight

and time in vivo is consistent with bulk hydrolytic cleavage of the

ester linkages autocatalyzed by the generated carboxylic acid end

groups. The same rate is observed in vitro for (H20, 40°) and for

films and cylinders, despite a large surface-to-volume difference.

These observations rule out any enzymatic contribution to the hydrolysis rate.

For poly( -caprolactone), the decrease in molecular weight is accompanied by a slow increase in crystallinity, elastic modulus, and

a decrease in the % elongation at break and ultimate tensile

strength. Weight loss commences when the MW is less than about
Magnification: 330 Magnification: 1020

FIGURE 7. Scanning Electron Micrographs of Films Prepared From Poly(DL-Lactic Acid) and 10% Glycerine (from Reference 5).
244 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

RELATIVE RATES OF BIODEGRADATION OF DIFFERENT

ALIPHATIC POLYESTERS

FIGURE 8. Fractional Change in the Intrinsic Viscosity of Different Polyesters as a

Function of Time Implanted in Rabbit, Illustrating Dependence of Degradation Rate

on Chemical Structure and Morphology.

30,000. Changes in both the weight and viscosity of various copolymers of -caprolactone and DL-lactic acid are shown in figure 9.

Application to Contraception and Narcotic Antagonist Therapy

The substantial induction periods prior to weight loss, coupled

with the high permeability of poly( -caprolactone) and its copolymers, have permitted the development of biodegradable reservoir

devices which function by sustained, diffusion-controlled drug delivery and which erode after the drug reservoir is depleted. These devices retain the advantages of subdermal poly(dimethylsiloxane)

systems, i.e., constant and sustained drug delivery, but the need for

surgical removal after drug exhaustion is eliminated.

The primary objective of our studies has been the development of

a device capable of subdermal delivery of contraceptive levels i.e.,

-50 μg/day, of levonorgestrel for a period of at least one year.
ALIPHATIC POLYESTERS DELIVERY SYSTEMS 245

RATES OF BIODEGRADATION OF DlFFERENT

COPOLYMERS OF -CAPROLACTONE/DL-LACTlC ACID

FIGURE 9. Semi-logarithmic Plots of Intrinsic Viscosities of Poly( -caprolactone-co

DL-Lactic Acid) Capsules Versus Time In Vivo (Rabbit). Numbers at Individual Data

Points Show Averaged Magnitude of Weight Loss.

Poly( -caprolactone) is the most suitable polymer for this purpose,

for it is permeable to levonorgestrel, and an induction period of 1

year prior to the onset of bioerosion is easily achieved. Examples of

long term levonorgestrel release rates in vitro and in vivo (rat) are

shown in figures 10 and 11. Sterilization of the device is achieved

b y -irradiation, and toxicology studies have shown no adverse

local or systemic effects. Initial pharmacokinetic studies in women

are now planned.

The requirements of a narcotic antagonist maintenance program

are very different from those of birth control programs. Naltrexone, the most potent narcotic antagonist, is only effective at a dose

of at least 3 mg/day and present treatments are restricted to 1-2

months. The properties of poly( -caprolactone-co-DL-lactic acid) are
FIGURE 10. Daily Rate of Release of Norgestrel from Poly( -Caprolactone)

Capsules, In Vitro, Per Centimeter Length of Capsule, Using Two Different Capsule

Wall Thicknesses.
RELEASE OF LEVONORGESTREL FROM POLY( -CAPROLACTONE)

CAPSULES IMPLANTED IN RAT AFTER 33 DAYS IN VITRO

FIGURE 11. Daily Rate of Release of Norgestrel from a Poly( Caprolactone)

Capsule Implanted in Rat After 32 Day In Vitro. Release Rate Determined by

Measurement of Radioactivity in Urine and Feces. Difference in the In Vitro and In

Vivo Rates is the Result of Metabolic Loss of Radiolabel by Routes Other Than

Urine and Feces.
248 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

suitable for a sustained-delivery device with these requirements.

Thus, by proper choice of molecular weight and chemical composition, it is possible to obtain a polymer which is biostable for a

period of anywhere between 4 and 12 weeks (drug delivery phase),

and which undergoes rapid bioabsorption subsequently (figure 9).

This copolymer is also in the rubbery state and very permeable.

A copolymer with an intrinsic viscosity of 1.5 dl/g and 20 mole%

lactic acid was selected for evaluation. The maximum steady state

flux (Jlim) and the diffusion coefficient (D) of naltrexone in this copolymer were determined from the kinetics of transport through a

thin film in a standard diffusion cell.

Jlim = 2.7 (± 0.4) x 10-10g.sec-1.sec-1

D = 5.9 (± 2.4) x 10-9cm2.sec-1

Based on the value of Jlim and the kinetic expression for the diffusion controlled release of a cylindrical reservoir (Eq 1), one can

estimate

Release rate = 2 DK C/ln (od/id) = 2 Jlim/ln (od/id) . . . (1)

FIGURE 12. In Vitro Rate of Release of Naltrexor-3H from a Poly( Caprolactoneco-DL-Lactic Acid) Capsule Containing Naltrexone as 50% Suspension in Water.
ALIPHATIC POLYESTERS DELIVERY SYSTEMS 249

that two 2.5 cm capsules, 2.7 mm outer diameter and 2.1 mm

inner diameter, filled with a 50% aqueous slurry of naltrexone,

will release 3 mg of drug per day for 30 days. This is within the

range believed desirable for an antagonist maintenance program.

Rates of release were first measured in vitro and found to be relatively constant over a 36-day period. Using capsules with a 2.05

mm inner diameter and 2.35 mm outer diameter, the release rate

was between 300 and 400 μg/day per cm capsule length for 24 days,

declining as the drug was exhausted over the next 20 days (figure

12a). A lower but more sustained release rate was achieved when

capsules with a slightly thicker wall were used (figure 12b). These

rates are lower than the values estimated from Jlim, but this is not

surprising in view of the errors of measurement of the thin walls of

the polymer capsule, the sensitivity of the rate to the od/id ratio in

the range used, and the fact that the method of tube preparation

produces some irregularity in the dimensions and possibly some

differences in morphology.

Since poly( -caprolactone-co-DL-lactic acid) is permeable to

water, the rate of drug released from a capsule containing only naltrexone was measured in vitro. There was a slow increase in rate,

t o 300 μg/day/cm length (figure 13). Presumably the increase was

related to water diffusion into the capsule, the water being necessary to maximize dissolution of the drug in the capsule wall. The

maximum release rate is dependent on maintaining the drug concentration gradient ( C) across the polymer wall. The rate slowly

declined after day 30, until the rate was 100 μg/day at day 95.

When such capsules, 3 cm length, were subdermally implanted in

three monkeys, one per animal, morphine self-administration was

immediately suppressed and remained so for 33 days.

The naltrexone used in this study was tritium labeled, and blood

radioactivity levels were measured to assess the constancy of the

drug release rate. The rapid increase in tritium levels after about

20 days in two cases (figure 14) strongly suggests that capsule degradation commenced at that time. At the end of the study (day 46)

the capsules were recovered as partially degraded fragments. Evidently, the time prior to polymer bio-erosion was somewhat less

than that estimated from figure 9, and the morphine blockade

could have been maintained for a greater time period by use of a

copolymer with a slightly different composition.

These studies with naltrexone only amounted to exploratory experiments, to test the feasibility of using a polyester in the form of

a biodegradable reservoir device. It would appear that the approach is feasible and that it is only necessary to optimize the various components to achieve a practical device.
FIGURE 13. In Vitro Rate of Release of Naltrexone-3H from a Poly( -Caprolactone-coDL-Lactic Acid) Capsule Containing Dry Naltrexone. Capsule Dimensions: 2.4 mm od,

2.05 mm id, 1 cm length.
FIGURE 14. Plasma concentrations of Naltrexone and its Metabolites Resulting From

Implantation of Poly( Caprolactone-co-DL-Lactic Acid) Capsules Containing Dry

Naltrexone in Three Monkeys.

NALTREXONE AND METABOLITE LEVELS IN MONKEY PLASMA

AFTER IMPLANTING A 3 cm CAPSULE OF PCL-LA
252 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

REFERENCES

1. R.W. Baker and H.K. Lonsdale. An emerging use for membranes. Chem. Tech., 5,

668-674 (1975).

2. D.L. Gardner, A.J. Patanus, and D.J. Fink, Steroid release from microcapsules. In

H.L. Gabelnick (ed.), Proc. Drug Delivery Systems, DHEW Pub. No. (NIH) 77-

1238, pp 265-278 (August 1976).

3. E.S. Nuwayser, D.L. Williams, P.M. Meier, T.C. Wu, S.Z. Merchant, and J.K. Kerrigan. A microcapsule system for drug delivery. In H.L. Gabelnick (ed.), Proc.

Drug Delivery Systems, DHEW Pub. No. (NIH) 77-1238, pp 193-251 (August

1976).

4. C. Thies. Characterization of microcapsules containing naltrexone or naltrexone

pamoate. In D.R. Paul and F.W. Harris (eds.), Controlled Release Polymeric Formulations, ACS Sumposium Series 33, American Chemical Society, Washington,

D.C., 190-194 (1976).

5. C.G. Pitt, A.R. Jeffcoat, R.A. Zweidinger, and A. Schindler. Sustained drug delivery systems I. The permeability of poly(E-caprolactone), poly(DL-Lactic acid),

and their copolymers. J. Biomed. Mater. Res., 13, 497-507 (1979).

6. C.G. Pitt, M.M. Gratzl, A.R. Jeffcoat, R. Zweidinger, and A. Schindler. Sustained

drug delivery systems II: Factors affecting rates of release from poly(E-caprolactone) and related biodegradable polyesters. J. Pharm. Sci., submitted for publication.

7. C.G. Pitt, D. Christensen, R. Jeffcoat, G.L. Kimmei, A. Schindler, M.E. Wall and

R.A. Zweidinger. Biodegradable polymers and sustained delivery of contracep

tive drugs. In H.L. Gabelnick (ed.), Proc. Drug Delivery Systems, DHEW Pub.

No. (NIH) 77-1238, pp 141-192 (August 1976).

8. A. Schindler, R. Jeffcoat, G.L. Kimmel, C.G. Pitt, M.E. Wall, and R. Zweidinger.

Biodegradable polymers for sustained drug delivery. In E.M. Pearce and J.R.

Schaefgen (eds.), Contempomry Topics in Polymer Science, Vol. 2, Plenum Pub.

Corp., New York, 251-289 (1977).

9. R.A. Miller, J.M. Brody, and D.E. Cutright. Degradation rates of oral resorbable

implants (polylactates and polyglycolates): Rate modification with changes in

PLA/PGA copolymer ratios. J. Biomed. Mater. Res., 11, 711-719 (1977).

10. T.J. Roseman. Release of steroids from a silicone polymer, J. Pharm. Sci., 61, 46-

50 (1972).

11. T.J. Roseman and S.H. Yalkowsky. Influence of solute properties on release of p

aminobenzoic acid esters from silicone rubber: Theoretical considerations. J.

Pharm. Sci., 63, 1639-1641 (1974).

ACKNOWLEDGMENTS

This work was supported by the Contraceptive Development

Branch, Center for Population Research, National Institute of

Child Health and Human Development, NIH, under Contract No.

NO1-HD-3-2741.
ALIPHATIC POLYESTERS DELIVERY SYSTEMS 253

AUTHORS

Colin G. Pitt, Thomas A. Marks, and Anton Schindler

Research Triangle Institute

P.O. Box 12194

Research Triangle Park, NC 27709
Naltrexone: Research Monograph 28

R. E. Willette and G. Barnett, eds.

National Institute on Drug Abuse, 1980

An Improved Long-Acting

Delivery System for

Narcotic Antagonists

Anand L. Misra and Ronald B. Pontani

The feasibility of using cholesterol-glyceryltristearate

matrix for prolonged release of naltrexone was evaluated in

mts. Implantable cylindrical pellets (cholesterol 105 mg,

glyceryl tristearate 15 m and naltrexone 30 mg), diameter

4.5 mm, length 9 mm, locked the antinociceptive action

(hot plate 55°C) of 10 mg/kg s.c. challenge dose of morphine

in rats for 3 months. The release rate of naltrexone from 10

or 50 mg pellets approximated first-order kinetics with t1/

2

 of 20-26 days and t1/2ß 40-60 days. The factors affecting

the release of drug rom the delivery system were he ratio t

of cholesterol to naltrexone, drug loading level and surface

area to unit volume of dosage form. The minimum release

rate of naltrexone to block the effect of 10 mg/kg s.c. dose

of morphine in rats was about 4 to 5 μg/kg/hr. The cumulative urinary excretion of radioactivity rom 10 mg [

3H]

naltrexone pellets implanted s.c. in rats after 30, 60, and 90

days was 17.7, 23.7, and 25.7% of dose respectively and the

percent dose released from pellets at these times was 55.8,

68.8, and 78.2 respectively. The devices possess the desirable

characteristics of simplicity, nontoxicity, nonirritability,

ease of sterilization with ethylene oxide, small size for easy

insertion and removal, absence of encapsulation by surrounding tissue, and an extended period of drug release unaffected by body metabolism. Neither deterioration of implant nor gross anatomic or histological changes at the site

of implant occurred 6 months to 1 year after implantation

and aside from some enhanced sexual activity (e.g., spontaneous penile erections) no side effects were observed in rats,

which fed well and gained weight during the entire treatment. The concentrations o free morphine in brains of 30

mg naltrexone pellet-implanted rats were significantly

254
AN IMPROVED DELIVERY SYSTEM 255

lower (2.4 and 15%) as compared to the placebo controls 0.5

and 1.0 hr after a 10 mg/kg s.c. dose of [6-3H] morphine. We

are currently evaluating these long-acting devices for the

duration of effective antagonism to morphine in rhesus

monkeys.

Long-acting narcotic antagonists, e.g., naltrexone, have potential

clinical usefulness in combatting opiate dependence and prevention

of relapse to opiate-seeking behavior (1,2,3,4,5). The urgent need for

long-acting delivery systems for naltrexone and the status of research on such systems have recently been indicated in a monograph (6). It is felt that such systems will eliminate the need for

clinic visits in opiate-dependent individuals without reducing protection against self-administered opiates and will in addition

reduce the emphasis on medication alone and refocus it on rehabilitation aspects. The systems design and some specifications for a

clinically acceptable and useful delivery system for narcotic antagonists with a long biological t1/2 have been outlined by Archer

(7) and Willette (8). These are: (a) adequate and smooth drug-release rate, preferably obeying zero-order kinetics; (b) relative ease

of insertion and removal in the event of a need for analgesic medication; (c) biocompatibility or lack of adverse tissue reaction; (d)

biodegradability; (e) ease and reasonable expense of manufacture;

(f) stability to sterilization and storage conditions; (g) patient and

physician acceptability.

Previous work has utilized biodegradable polylactic and lacticglycolic acid composites for cyclazocine and naltrexone (9,10,11),

nonbiodegradable polymer for naloxone (12), synthetic glutamic

acid-leucine copolymers, polyglycerides, insoluble salts and metal

complexes of antagonists (13) as prolonged-release preparations (6).

These vehicles have in general produced blockade of antinociceptive action of morphine for periods ranging from 15 to 30 days or

longer. Woodland et al. (9) obtained t1/2 values of 11 to 13 days on

release of antagonist, a poor correlation between in vitro and in

vivo data and an inflammatory process at the site of film implant;

Yolles et al. (10) obtained effective blocking action to morphine in

rats for 24 days (t1/2 22 days); Fishman et al. (12) observed the duration of antagonism for 22 days in rats and slight encapsulation of

implant by surrounding tissue. The release rates of narcotic antagonist from lactic/glycolic acid polymers approximated zero-order

kinetics for most of the release period and antagonist response to a

challenge dose of morphine was maintained from 1 to 6 months depending on the formulation tested (11). The procedures involved in
256 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

the preparation of above copolymers are complex, and much work

remains to be done on the reproductibility of polymerization conditions, use of pharmacologically suitable catalysts, and overall tissue

compatibility and toxicity of such copolymers. Previous reports on

the sustained-release hormonal preparations (14,15,16) prompted us

to evaluate compressed pellets of cholesterol and glyceryltristearate as long-acting delivery systems for narcotic antagonists. We

report here a promising delivery system for naltrexone which may

have clinical usefulness in treatment of narcotic dependence.

MATERIALS AND METHODS

Naltrexone hydrochloride and [15,16-3H] naltrexone (sp. act. 47.7

mCi/mg) were provided by Research Triangle Institute, N.C.,

through the courtesy of National Institute on Drug Abuse, Rock

ville, Md. [6-3H] Morphine (New England Nuclear Corporation,

Boston, Mass.), cholesterol m.p. 146-149°C (J.T. Baker Chemical

Company, Philipsburg, N. J.) and glyceryltristerate m.p. 65-67°C

(K.K. Laboratory, Inc., Plainview, N. Y.) were commercial samples.

Preparation of Naltrexone Pellets

Cholesterol (360 mg), glyceryltristerate (40 mg) and naltrexone

base (100 mg) were dissolved in 10 ml chloroform and the solution

evaporated to dryness in vacuo in a Rota Vapor and the residue

further dried in vacuo overnight. The thoroughly mixed powder (50

mg) was compressed to a cylindrical pellet (54,500 lb/sq in), diameter 3 mm, length 6.35 ± 0.13 (s.e.) mm, weight 48.6 ± 0.8 (s.e.) mg,

surface area 56.5 mm2, volume 42.3 mm3 in a Carver Laboratory

Press (Fred Carver, Inc., Summit, N.J.). Radioactive naltrexone pellets containing 10 mg naltrexone were prepared using [15,16-3H]

naltrexone by the above procedure. Cylindrical pellets (diameter

4.5 mm, length 9 mm) of naltrexone (30 mg) comprising cholesterol

(105 mg) and glyceryltristerate (15 mg) and 50 mg naltrexone pellets of same dimensions comprising cholesterol (90 mg) and glyceryltristearate (10 mg) were also prepared by the above procedure.

ANIMAL EXPERIMENTS

Cylindrical pellets of naltrexone and placebo pellets were implanted subcutaneously in the dorsal area behind the right hind
AN IMPROVED DELIVERY SYSTEM 257

limb of male Wistar rats, pushed away from incision and the incision sutured. The response iatencies (sec) of animals in 2 groups

were measured on a hot plate (55°C) with a cut-off time of 30 sec

before morphine injection and 0.5, 1, 1.5, 2 and 3 hr after 10 mg/kg

s.c. morphine injection (ED80, 6.5 mg/kg). The criterion of reaction

of the rat was licking of one paw or jumping on hind legs. The duration of effective antagonism to challenge dose of morphine was

tested at 15 days, 1, 1.5, 2 and 3 months after implantation. After

being in place for 2 or 3 months, the naltrexone pellets were removed in the experimental group and their responses to a challenge dose of morphine compared with the placebo group.

Radioactive naltrexone pellets (10 mg) were also implanted s.c. in

a group of 7 rats, and the excretion of radioactivity in urine collected in metabolism cages every 24 hr for 30 days was measured by

counting the radioactivity in an aliquot of urine and correcting it

for quenching, using (

3H) toluene as an internal standard. These

pellets were removed 30, 60 and 90 days after implantation, dissolved in chloroform, and radioactivity counted in an aliquot.

Disposition Of [6-3H] Morphine In Rats Implanted With LongActing Naltrexone Pellets

The rats were implanted s.c. with 30 mg naltrexone or placebo

pellets, and, after 2 weeks, a 10 mg/kg dose of [6-3H] morphine was

injected subcutaneously in these animals. The concentration of [6-

3H] morphine in brain, liver, plasma obtained at different times

after injection was determined by a method previously described

(17).

RESULTS AND DISCUSSION

The response latencies in 30-day 10 mg naltrexone pellet-implanted animals at 0.5 to 2 hr after the morphine dose were not

significantly different from the reaction times before morphine injection by correlated t-test (18). At 1.5 and 2 months, the degree of

analgesic blockade to this morphine dose was approximately 90 and

35% respectively. Three days following the removal of naltrexone

pellets, the reaction times observed 0.5 to 3 hr after the morphine

dose were not significantly different from those in placebo-pelleted

rats, indicating that the implanted naltrexone pellets also blocked

the development of tolerance to morphine. The results (18) showed

that the delivery system (10 mg naltrexone) produced a complete
258 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

and effective blockade of analgesia due to a 10 mg/kg (s.c.) dose of

morphine in test animals for 1 month.1 Similarly, naltrexone pellets (30 mg) provided effective antagonism to this challenge dose of

morphine for at least 3 months (19).

The daily urinary excretion of radioactivity from 10 mg radioactive naltrexone pellets is shown in figure 1. From an initial value

of 2.5 ± 0.3%, there was a steep fall to 0.8 ± 0.15% in 3 days, the

values then declined slowly to 0.3 ± 0.05% on day 30. The cumulative urinary excretions of radioactivity (mean s.e.) (figure 2) in 30

and 60 days were 17.7 ± 0.4 and 23.7 ± 0.9% respectively, and in

90 days this excretion amounted to 25.7%. The pellets were removed 30, 60 and 90 days after implantation and the radioactivity

remaining in the pellets was estimated. The percentages of radioactivity (mean ± s.e.) released from the implanted pellet in 30, 60

and 90 days were 55.8 ± 1.3, 68.8 ± 0.7 and 78.2% respectively

(figure 3). The plot of percentage dose remaining at the site of implant vs. time was bioexponential with t1/2 values of 26 and 60 days

respectively. The excretion of radioactivity via fecal or other routes

by subtraction therefore amounted to 38.1, 45.1 and 52.6% of implanted dose in 30, 60 and 90 days respectively. Thus the 10 mg

naltrexone pellet gave a satisfactory release of drug over an extended period of time.

The release rate in vivo from 50 mg radioactive naltrexone pellets (figure 4) gave t1/2 values of 20 and 40 days respectively, The

FIGURE 1. Daily amounts of radioactivity excreted in urine in rata in 30 days from

10 mg (3H) naltrexone pellets implanted subcutaneously. Data represent the mean

±S.E.M. percent of implanted dose from 7 animals.
AN IMPROVED DELIVERY SYSTEM 259

FIGURE 2. Cumulative percent of implanted dose excreted as total radioactivity in

urine over a period of 60 days from 10 mg (3H) naltrexone pellets implanted

subcutaneously in rats. Data represent mean ±S.E.M. from 7 animals up to 30 days

and 4 animals thereafter.

ratio of cholesterol to naltrexone in 50 mg pellets was 1.8 and that

in 30 and 10 mg naltrexone pellets 3.5 and 3.6 respectively.

Although plasma levels of naltrexone at various times after 10

mg pellet-implantation in the same animal were not determined in

this study, the values at 30-day period of narcotic blockade amounted to 4 to 5 μg/kg/hr. Harrigan and Downs (20) have reported that

continuous naltrexone infusion of 1-20 μg/kg/hr produced effective

blockade of morphine self-administration in the rhesus monkey and

proposed a minimum value of 5 μg/kg/hr as an effective release

rate of naltrexone for a drug-delivery system.

In our experiments neither deterioration of implant nor gross

anatomic or histological changes at the site of implant occurred 6

months or longer after implantation. Aside from some enhanced

sexual activity (e.g., spontaneous penile erections), no obvious side

effects were observed in rats, which fed well and gained weight

during treatment. The method thus has the merits of simplicity,

nontoxicity, nonirritability, small size for ease of insertion and removal, ease of sterilization with ethylene oxide, biocompatibility,

absence of encapsulation by surrounding tissue, and an extended

period of drug release unaffected by body metabolism. The results

clearly establish the feasibility of using cholesterol-glyceryltristearate as a matrix for the controlled release of naltrexone. Cholesterol

provided a simple barrier effect on the diffusion of drug and it is

conceivable that increasing the size of pellets while holding the
FIGURE 3. In vivo release of radioactivity

from 10 mg (

3H) naltrexone pellets

implanted subcutaneously in rats.

FIGURE 4. In vivo release of radioactivity

from 50 mg (

3H) naltrexone pellets

implanted subcutaneously in rats.
AN IMPROVED DELIVERY SYSTEM 261

ratio of cholesterol to naltrexone constant may reduce the rate of

drug release, thus extending its duration. Currently we are conducting experiments in rhesus monkeys using 50 mg and 666 mg

naltrexone pellets (diameter 13 mm, length 16 mm), having the

same cholesterol to naltrexone ratio (1:8). In rhesus monkey implanted s.c. with 50 mg naltrexone pellets the threshold plasma

concentration of naltrexone during blockade of pupillary dilatation

due to hydromorphone challenges was 2.9 ng/ml.

Data on the comparative distribution of free morphine in placebo

and naltrexone pellet-implanted rats after a 10 mg/kg s.c. injection

of [6-3H] morphine appear in figure 5. The concentration of free

morphine in brains of naltrexone pellet-implanted rats was significantly lower (24 and 15%) as compared to placebo controls 0.5 and

1.0 hr after morphine injection. The values of morphine in plasma

of naltrexone pellet-implanted animals were 45 to 26% lower and

those in liver 39 to 27% lower as compared to those of placebo-pelleted rats 0.5 and 1.0 hr after morphine injection. The brain to

plasma ratios at 0.5 and 1.0 hr were comparatively higher in naltrexone pellet-implanted animals (0.21, 0.381 as compared to the

placebo controls (0.16, 0.33). The values of total radioactivity comprising free morphine plus metabolites (μg-equivalent per ml of

fluid or μg equivalent per g of tissue) at 0.5 and 1.0 hr after morphine injection in the two groups were as follows. In plasma: placebo pellet-implanted rats, 5.73 ± 0.34 and 5.90 ± 0.49; naltrexone

pellet-implanted rats, 5.23 ± 0.79 and 4.77 ± 0.29 respectively. The

corresponding values for liver were: placebo, 23.24 ± 2.24 and 24.04

± 2.42 respectively; naltrexone pellet-implanted rats, 20.29 ± 1.30

and 17.08 ± 0.25 respectively. Of these values, only those in liver

at 1 hr were significantly different (p<0.05) in the two groups.

Due to the chronic blockade of opiate receptors in naltrexone

pellet-implanted rats, the dose of morphine did not produce any antinociceptive effect, in contrast to the placebo pellet-implanted rats

which clearly showed the opiate effects. Naltrexone has much

greater receptor affinity than morphine (21). The lowered distribution of morphine to the brain in naltrexone pellet-implanted animals as compared to the placebo controls could possibly involve

competitive interactions and changes in drug binding between naltrexone and morphine at the opiate receptor sites. However, physiological factors involving an altered blood flow or improved blood

circulation in antagonist-implanted rats, as compared to placebopelleted rats which were fully sensitive to morphine effects, may

also play an important role in the differential disposition of morphine in the two groups. Recent work by Manara et al. (22) has

shown a similar lowering of etorphine levels in brains of naloxone-
262 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

FIGURE 5. Comparative distribution of morphine in placebo and 30 mg naltrexone

pellet-implanted rate after a single 10 mg/kg subcutaneous injection of [6-3H]

morphine. Data represent ±S.E.M. (ng/g tissue or ml of fluid) from 3 animals in

each group. *, **, denote significant differences from placebo at p<0.05 and p<0.01

respectively.

pretreated animals and in these animals the clearance from

plasma of Bromsulphalein, a dye reflecting hepatic blood flow, was

four-fold greater than that of controls. Chronic administration of

narcotic antagonist naloxone in the rat has recently been shown

(23,24) to cause supersensitivity to the analgesic actions of morphine, which was correlated with a 40% increase in the number of

opiate receptor binding sites with no changes in the dissociation

constants for naloxone and saline groups. The supersensitivity did

not involve changes in morphine distribution or metabolism resulting from chronic naloxone infusion.
AN IMPROVED DELIVERY SYSTEM

CONCLUSIONS

263

This study has demonstrated the feasibility of cholesterol-glyceryltristearate matrix as a means of providing sustained delivery of

naltrexone for periods up to 3 months or more in rats on subcutaneous implantation. Although the release of drug from the matrix

obeyed first-order kinetics, these devices meet several other criteria

of clinically acceptable delivery systems. Our current efforts are directed towards improving these devices to achieve longer periods of

blockade to the challenge doses of morphine in rhesus monkeys.

FOOTNOTE

The preceding data in the Results and Discussion section are reprinted with permission of the Pharmaceutical Society of Great

Britain, London, copyright 1978, from the Journal of Pharmacy and

Pharmacology. See reference 18.

REFERENCES

1. M. Fink. Narcotic antagonists in opiate dependence. Science, 169, 1005-1006 (1970).

2. H. Blumberg and H.B. Dayton. Naloxone, naltrexone and related oxymorphones.

In MC. Braude, L.S. Harris, E.L. May, J.P. Smith and J.E. Villarreal (eds.),

Adv. Biochem. Psychopharmacol, Raven Press, New York, 8, 33-43 (1974).

3. W.R. Martin, D.R. Jasinski, and P.A. Mansky. Naltrexone, an antagonist for

treatment of heroin dependence. Arch. Gen. Psychiat., 28, 784-791 (1973).

4. R.B. Resnick, J. Volavka, A.M. Freedman, and M. Thomas. Studies of EN-1639 A

(naltrexone, a new narcotic antagonist. Amer. J. Psychiat., 131, 646-650 (1974).

5. R.B. Resnick. Prospects, problems, side-effects and safety of narcotic antagonists.

Int. J. Addictions, 12, 863-867 (1977).

6. R.E. Willette (ed.). Narcotic Antagonists: The Search for Long-Acting Preparations,

National Institute on Drug Abuse Research Monograph 4. DHEW Pub. No.

(ADM)76-296. Supt. of Docs., U.S. Govt. Print. Off., Washington, D.C. (1976).

7. S. Archer. Design considerations for long-acting antagonists. In M.C. Braude, L.S.

Harris, E.L. May, J.P. Smith and J.E. Villarreal (eds.). Narcotic Antagonists,

Adv. Biochem. Psychopharmacol., Raven Press, New York, 8, 549-554 (1974).

8. R.E. Willette. The development of sustained action preparations of narcotic antagonists. Narcotic Antagonists: Naltrexone Progress Report, National Institute

on Drug Abuse Research Monograph 9. DHEW Pub. No. (ADM)76-387, Supt. of

Docs., U.S. Govt. Print. Off., Washington, D.C., 31-34 (1976).

9. J.H.R. Woodland, S. Yolles, D.A. Blake, M. Helrich, and F.J. Meyer. Long-acting

delivery systems for narcotic antagonists. J. Med. Chem., 16, 897-901 (1973).

10. S. Yolles, T.D. Leafe, J.H.R. Woodland, and F.J. Meyer. Long-acting delivery systems for narcotic antagonists II, Release rates of naltrexone from poly (lactic

acid) composites. J. Pharm. Sci., 64, 348-349 (1975).

11. A.D. Schwope, D.L. Wise, and J.F. Howes. Lactic/glycolic acid polymers as narcotic antagonist delivery systems. Life Sci., 17 1877-1886 (1976).

347-877 0 -81 - 18
264 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

12. J. Fishman, E.F. Hahn, B.I. Norton, A. Ronai, and F.F. Foldes. Preparation and

evaluation of a sustained naloxone delivery system in rata. Pharmacology, 13,

513-519 (1975).

13. A.P. Gray and D.S. Robinson. Naltrexone-zinc tannate complex: a prolonged

action narcotic antagonist complex. J. Pharm. Sci., 63, 159-161 (1974).

14. F.A. Kincl and H.W. Rudel. Sustained release hormonal preparation. Acta Endocrinol. Suppl, 151, 5-30 (1971).

15. G.N. Gupta. Sustained absorption of 3H-norgestrel from subcutaneous fused pellets in rats: A potential totally bioabsorbable implant for human contraception.

Fed. Proc., 36, 997 (1977).

16. L.R. Beck and S.R. Lowry. In vivo evaluation of cholesterol as a matrix for the

release of progesterone. Contraception, 18, 491-505 (1978).

17. A.L. Misra, C.L. Mitchell, and L.A. Woods. Persistence of morphine in the central nervous system of rata after a single injection and its bearing on tolerance.

Nature, 232, 48-50 (1971).

18. A.L. Misra and R.B. Pontani. An improved long-acting delivery system for narcotic antagonists. J. Pharm. Pharmacol., 30, 325-326 (1978).

19. A.L. Misra, R.B. Pontani, and N.L. Vadlamani. Plasma corticosteroid levels in

rata maintained on a long-acting naltrexone delivery system. Res. Comm. Chem.

Pathol. Pharmacol., 20, 43-50 (1978).

20. SE. Harrigan and D.A. Downs. Continuous intravenous naltrexone effects on

morphine self-administration in rhesus monkeys. J. Pharmacol. Exp. Ther. 204,

481-486 (1978).

21. C.B. Pert and S.H. Snyder. Properties of opiate receptor binding in rat brain.

Proc. Nat. Acad. Sci. (U.S.A.), 70, 2243-2247 (1973).

22. L. Manara, C. Aldinio, C. Cerletti, P. Coccia, A. Luini, and G. Serra. In vivo

tissue levels and subcellular distribution of opiates with reference to pharmacological action. In M.L. Adler, L. Manara, and R. Samanin (eds.), Factors Affecting the Action of Narcotics, Raven Press, New York, 271-296 (1978).

23. A.H. Tang and R.J. Collins. Enhanced analgesic effects of morphine after chronic

administration of naloxone in the rat. Europ. J. Pharmacol., 47, 473-474 (1978).

24. R.A. Lahti and R.T. Collins. Chronic naloxone results in prolonged increases in

opiate binding sites in brain. Europ. J Pharmacol., 51, 185-186 (1978).

AUTHORS

Anand L. Misra and Ronald P. Pontani

New York State Office of Substance Abuse Services

Testing and Research Laboratory

80 Hanson Place

Brooklyn, New York 11217
Naltrexone: Research Monograph 28

R. E. Willette and G. Barnett, eds.

National Institute on Drug Abuse, 1980

Solid Solutions as LongActing Naltrexone Delivery

Systems

James L. Olsen

Solid solutions and solid dispersions have been used both

to increase and to decrease solubility. Solid solutions may

extend the release of naltrexone depending upon composition and production methods. Several combinations of naltrexone and cholesterol esters were prepared and evaluated.

These combinations did prolong release of the drug in some

cases, but none acceptably long enough. Preparation of

these solutions and dispersions involved compression and

heat as well as solvent mixing. Drug release from particles

is dependent upon surface area, composition, and method of

preparation. Suspendable, injectable particles can be produced which will give acceptable release rates. The preparation and evaluation are complex and will require much

more research effort.

INTRODUCTION

Solid solutions can be viewed as homogeneous, molecular scale

mixtures of one solid in another. The molecular particles in such a

mixture are considered to retain their individual characteristics.

The mixing viewpoint can be extended to define solid dispersions as

molecular aggregates of one solid in another. A physical mixture is

taken to be simply a mixture of large particles of both solvent and

solute associated in the same solid form.

In the course of their work involving naltrexone mixtures with

polylactic/glycolic acids (PLGA), Schwope, Wise and Howes (1) observed that certain preparations of naltrexone appeared to form

265
266 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

solid solutions with the polymer. This observation led to consideration of solid solutions as mechanisms for reducing the dissolution

rate of naltrexone.

Recently Misra et al. (2) and Misra and Pontani (3) formed compressed mixtures of cholesterol and naltrexone which gave sustained release following subcutaneous implantation in rats. Gupta

(4) also used fused pellets of cholesterol and a progestin in the

same manner to obtain sustained release.

Solid solutions have been used for many years to increase the

dissolution rate of poorly soluble drugs. Goldberg et al. (5) considered the pharmaceutical use of solid solutions to improve absorption and demonstrated this in subsequent articles (6,7). A comprehensive review of solid dispersions was done by Chiou and Riegelman (8) on many aspects of such mixtures. A recent article by

Maulding (9) indicates that solid solutions are still being examined

for altering the dissolution rate of drugs.

A great volume of work has been done utilizing solid solutions of

poorly soluble drugs in hydrophilic solids to increase the drugs’ dissolution rates and thus improve absorption. In view of the demonstrated need for sustained release injectable or implantable devices

of naltrexone (10), we investigated solid solutions of naltrexone in

poorly water soluble solid solvents (cholesterol and its esters) in an

attempt to reduce dissolution rates. Kim and Jarowski (11) used

similar solvents in an investigation of hydrocortisone dissolution

rates, as did Joseph et al. (12) with progesterone.

MATERIALS AND METHODS

Naltrexone and naltrexone HCl were obtained from the National

Institute on Drug Abuse (NIDA) and were used without any further purification. All other chemicals were reagent grade commercial chemicals of at least 99% purity.

Melting points were determined by the capillary tube method

after dissolution of the components in chloroform and drying in

vacuo.

In vitro dissolution studies were carried out at 37° in phosphate

buffer pH 7.4 with slow horizontal shaking at a rate of 77 cycles

per minute. About 15 mg equivalent of naltrexone was placed in 10

ml of dissolution medium. Individual samples were run for each

time period and were assayed spectrophotometrically at 283 manometers.

In vivo studies were performed at NIDA on some of these preparations using the tail flick response.
SOLID SOLUTIONS AS DELIVERY SYSTEMS 267

The naltrexone-lipid combinations were prepared either by melting and subsequent cooling of the resultant solution, or by mixing

and granulating with chloroform followed by air drying at 60°C for

10 hours. The dry granules were compressed at overload pressure

(25,000 lb/in2) on a Stokes single punch tablet press. The slugs were

broken down into particles which were screened into various particle sizes.

RESULTS

Naltrexone hydrochloride was combined with various esters in

all proportions from 90:10 to 10:90 and the melting points determined. Those tested were:

Stigmasterol Cholesterol

Cholesterol nonanoate Cholesterol acetate

Cholesterol palmitate Cholesterol trimethyl acetate

Cholesterol propionate Sitosterol

Sitosterol palmitate Sitosterol trimethyl acetate

Naltrexone base was similarly tested for melting point depression in combinations with:

Cholesterol

Cholesterol acetate

Sitosterol

Sitosterol trimethyl acetate

Cholesterol nonanoate

Cholesterol palmitate

Cholesterol propionate

Sitosterol palmitate

Stigmasterol

In all cases the naltrexone began to discolor prior to melting. We

selected those cholesterol esters and naltrexone combinations

which melted at the lowest temperature so that degradation would

be minimized. A typical melting point-composition curve is shown

in figure 1.

None of the melting point-composition data supported the eutectic composition by demonstrating a marked melting-point reduction. Those esters and compositions selected were chosen because of

a slight reduction in melting point. These combinations were:

60/40 Naltrexone/cholesterol

60/40 Naltrexone HCl/cholesterol

50/50 Naltrexone HCl/cholesterol

30/70 Naltrexone/cholesterol

80/20 Naltrexone/cholesterol
268 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

FIGURE 1: Melting Point Depression of

Naltrexone/Cholesterol Mixtures

In vitro dissolution rate studies were performed on these preparations. A typical run is shown in figure 2. Notice that about 40%

of the naltrexone is released within the first 5 minutes. This was a

consistent result for all dissolution studies and indicates that much

of the drug is available from the surface of the particles.

In vivo analgesia studies confirmed that none of the devices gave

effective release for more than 3-5 days, correlating well with the

rapid dissolution rates.

Particles made by fusion were found to be brittle and none of

these were used in the release evaluations.
FIGURE 2: Dissolution Profile of Naltrexone/Cholesterol

Mixture 60:40 (150-250 micron)
270 NALTREXONE SUSTAINED-RELEASE PREPARATIONS

DISCUSSION

Particles in the 100 micron diameter range have such high surface area (200 mm2

/mg naltrexone) that the dissolution rates were

also high. Compared to the Misra (2) cylinder of 20% naltrexone in

cholesterol (5.6 mm2/mg naltrexone), one would expect rapid drug

release. There was some sustaining of release after the initial

burst, and this degree of sustained release warrants the continued

investigation of solid solution systems.

CONCLUSIONS

The particles tested could be classified as solid dispersions of naltrexone in cholesterol esters. Particles in the 100 micron diameter

range have a high surface area and show rapid drug release.

True solid solutions of these chemicals are possible in nearly all

proportions and should be investigated for sustained release.

REFERENCES

1. A.D. Schwope, D.L. Wise, and J.F. Howes. Development of polylactic/glycolic acid

delivery systems for use in treatment of narcotic addiction. In R.E. Willette

(ed.), Narcotic Antagonists: The Search for Long-Acting Preparations, National

Institute on Drug Abuse Research Monograph 4. DHEW Pub. No. (ADM)76-296.

Supt. of Docs., U.S. Govt. Print. Off., Washington, D.C., 13-18 (1976).

2. A.L. Misra, R.B. Pontani and N.L. Vadlamani. Plasma corticosteroid levels in rats

maintained on a long-acting naltrexone delivery system. Res. Commun. Chem.

Pathol. Pharmacol., 20, 43-50 (1978).

3. A.L. Misra and R.B. Pontani. An improved long-acting delivery system for narcotic antagonists. Communications, J. Pharm. Pharmac., 30, 325-326 (1978).

4. G. Gupta. Sustained absorption of 3H-norgestrel from S.C. fused pellets in rats: a

potentially bioabsorbable implant for human contraception. Fed. Proc., Fed. Am,

Soc. Exp. Biol., 36. 997 (1977).

5. A.H. Goldberg, M. Gibaldi and J.L. Kanig. Increasing dissolution rates and gastrointestinal absorption of drugs via solid solutions and eutectic mixtures I. J.

Phorm. Sci., 54, 1145-1148 (1965).

6. A.H. Goldberg, M. Gibaldi and J.L. Kanig. Increasing dissolution rates and gastrointestinal absorption of drugs via solid solutions and eutectic mixtures II. J.

Pharm. Sci., 55, 482-487 (1966).

7. A.H. Goldberg, M. Gibaldi and J.L. Kanig. Increasing dissolution rates and gastrointestinal absorption of drugs via solid solutions and eutectic mixtures III. J.

Pharm. Sci., 55, 487-492 (1966).

8. W.L. Chiou and S. Riegelman. Pharmaceutical applications of solid dispersion systems. J. Pharm. Sci.. 60, 1281-1302 (1971).
SOLID SOLUTIONS AS DELIVERY SYSTEMS 271

9. E.V. Maulding. Solid-state dispersions employing urethan. J, Pharm. Sci., 67, 391-

394 (1978).

10. R.E. Willette. Introduction. Narcoric Antagonists: The Search for Long-Acting

Preparations. National Institute on Drug Abuse Research Monograph 4. DHEW

Pub. NO. (ADM)76-296. Supt. of Docs., US. Govt. Print. Off., Washington, D.C.,

13-18 (1976).

11. K.H. Kim and C.I. Jarowski. Surface tension lowering and dissolution rate of hydrocortisone from solid solutions of selected n-acyl esters of cholesterol. J.

Pharm. Sci., 66, 1536-1540 (1977).

12. A.A. Joseph, J.L. Hill, J. Patel, S. Patel and F.A. Kincl. Sustained release hormonal preparations XV: Release of progesterone from cholesterol pellets in

vivo. J. Pharm. Sci., 66, 490-493 (1977).

AUTHOR

James L. Olsen

Department of Pharmaceutics

School of Pharmacy

University of North Carolina

Chapel Hill, North Carolina 27514
PARTICIPANTS

TECHNICAL REVIEWS ON NALTREXONE

TECHNICAL REVIEW

MODERATORS

Robert E. Willette, Ph.D.

Division of Research

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, Maryland 20857

Gene Barnett, Ph.D.

Division of Research

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, Maryland 20857

CHAIRMEN

Fred Leonard, Ph.D.

George Washington Medical School

2300 Eye Street, N.W.

Washington, D. C. 20037

Salomon Stavchansky, Ph.D.

College of Pharmacy

University of Texas

Austin, Texas 78712

PARTICIPANTS

Sydney Archer, Ph.D.

Department of Chemistry

Rensselaer Polytechnic Institute

Troy, New York 12181

William L. Dewey, Ph.D.

Department of Pharmacology

School of Basic Science

Medical College of Virginia

Richmond, Virginia 23298

David A. Downs, Ph.D.

Department of Pharmacology

Warner-Lambert/Parke-Davis

2800 Plymouth Road

Ann Arbor, Michigan 48106

Stephen E. Harrigan

Department of Pharmacology

Warner-Lambert/Parke-Davis

2800 Plymouth Road

Ann Arbor, Michigan 48106

Fred A. Kincl, Ph.D., D.Sc.

Department of Biology

City University of New York

50 Bay Street

Staten Island, New York 10301

Vojtech Licko, Ph.D.

Cardiovascular Research Institute

Univ. of Calif. School of Medicine

San Francisco, California 94143

Anand L. Misra, Ph.D.

N. Y. State Substance Abuse Serv.

Testing and Research Laboratory

80 Hanson Place

Brooklyn, New York 1121’7

James L. Olsen, Ph.D.

College of Pharmacy

University of North Carolina

Chapel Hill, North Carolina 27514

272
273

GUESTS

Henry L. Gabelnick, Ph.D.

Center for Population Research

National Institute of Child Health and

Human Development

Bethesda, Maryland 20014

Gail L. Levine

Biometric Research Institute

1010 Wisconsin Avenue, #505

Washington, D. C. 20007

William K. Schmidt, Ph.D.

Stine Laboratory/Endo Laboratories

E. I. DuPont Company

Newark, Delaware 19711

NIDA STAFF

Jack D. Blaine, M.D.

Richard L. Hawks, Ph.D.

Pierre F. Renault, M.D.

Division of Research

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, Maryland 20857

TECHNICAL REVIEW

COORDINATION

Marcia G. Goldstein

Paulette B. Skalka

Macro Systems, Inc.

8630 Fenton Street, #300

Silver Spring, Maryland 20910

Mario Perez-Reyes, M.D.

Department of Psychiatry

School of Medicine

University of North Carolina

P. 0. Box 1020

Chapel Hill, North Carolina 27514

Colin G. Pitt, Ph.D.

Research Triangle Institute

P. 0. Box 12194

Research Triangle Park, NC 27709

Richard H. Reuning, Ph.D.

College of Pharmacy

Ohio State University

500 West 12th Avenue

Columbus, Ohio 43210

A. Carl Sharon

Dynatech R/D Company

99 Erie Street

Cambridge, Massachusetts 02139

Kenneth R. Sidman

Arthur D. Little, Inc.

Acorn Park

Cambridge, Massachusetts 02140

Karl Verebey, Ph.D.

N. Y. State Substance Abuse Services

80 Hanson Place

Brooklyn, New York 11217

Monroe E. Wall, Ph.D.

Research Triangle Institute

P. 0. Box 12194

Research Triangle Park, NC 27709

Donald L. Wise, Ph.D.

Dynatech R/D Company

99 Erie Street

Cambridge, Massachusetts 02139
1 FINDINGS OF DRUG ABUSE RESEARCH. Not available from NCDAI.

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276

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29 DRUG ABUSE DEATHS IN NINE CITIES A SURVEY REPORT. Louis A.

Gottschalk, M.D., et al. Epidemiologic study providing data on drug-involved deaths

and procedures for their investigations. Not available from NCDAI

GPO Stock #017-024-00982-2 $4.25 NTIS PB #80-178882 $12

30 THEORIES ON DRUG ABUSE: SELECTED CONTEMPORARY PERSPECTIVES. Dan J. Lettieri, Ph.D.; Mollie Sayers; and Helen Wallenstein, eds. Volume

presents summaries of the major contempomry theories of drug abuse by each of 43

leading theorists.

GPO Stock #017-024-00997-1 $8.50 Not available from NTIS

31 MARIJUANA RESEARCH FINDINGS: 1980. Robert C. Petersen, Ph.D., ed. The

text of the 8th Marijuana and Health report to Congress and the background scientific papers on which this summary report was based.

GPO Stock #017-024-01010-3 $5.00 NTIS PB #80-21571 $18.50

32 GC/MS ASSAYS FOR ABUSED DRUGS IN BODY FLUIDS. Rodger L. Foltz,

Ph.D.; Allison F. Fentiman, Jr., Ph.D.; and Ruth B. Foltz. A collection of methods for

the quantitative analysis of several important drugs of abuse by the technique of gas

chromatography-mass spectrometry.

GPO Stock # 017-024-01015-4 $5 NTIS PB #81-133746 $17

TO ORDER

While limited supply lasts, single copies of the publications listed may be obtained free

of charge from the National Clearinghouse for Drug Abuse Information (NCDAI).

Additional copies may be purchased from the U.S. Government Printing Ofice (GPO)

and/or from the National Technical Information Service (NTIS). NTIS prices are for

paper copy. Microfiche copies, at $3.50, are also available from NTIS. Prices from

either source are subject to change. When ordering from GPO or NTIS, be sure to list

the appropriate stock number. Addresses are:

NCDAI

National Clearinghouse for Drug Abuse Information

Department “RM”

P.O. Box 1080

Washington, D.C. 20013

GPO NTIS

Superintendent of Documents National Technical Information

U.S. Government Printing Office Service

Washington, D.C. 20402 US. Department of Commerce

Springfield, Virginia 22161

U.S. GOVERNMENT PRINTING OFFICE : 1981 0 - 347-877
DHHS Publication No. (ADM) 81-102

Printed 1981