Opioid Peptides:

Molecular

Pharmacology,

Biosynthesis,

and Analysis

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES • Public Health Service • Alcohol, Drug Abuse, and Mental Health Administration
Opioid Peptides:

Molecular Pharmacology,

Biosynthesis, and Analysis

Editors:

Rao S. Rapaka, Ph.D.

Richard L. Hawks, Ph.D.

Division of Preclinical Research

National Institute on Drug Abuse

NIDA Research Monograph 70

1986

DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, Maryland 20857
NIDA Research Monographs are prepared by the research divisions of the

National Institute on Drug Abuse and published by its Office of Science. The

primary objective of the series is to provide critical reviews of research problem

areas and techniques, the content of state-of-the-art conferences, and

integrative research reviews. Its dual publication emphasis is rapid and targeted

dissemination to the scientific and professional community

Editorial Advisors

MARTIN W. ADLER, Ph.D.

Temple University School of Medicine

Philadelphia, Pennsylvania

SYDNEY ARCHER, Ph.D.

Rensselaer Polytechnic Institute

Troy, New York

RICHARD E. BELLEVILLE, Ph.D.

NB Associates Health Sciences

Rockville, Maryland

KARST J. BESTEMAN

Alcohol and Drug Problems Association

of North America

Washington, D.C.

SIDNEY COHEN, M.D.

Los Angeles, California

MARY L. JACOBSON

National Federation of Parents for

Drug Free Youth

Omaha, Nebraska

REESE T. JONES, M.D.

Langley Porter Neuropsychiatric Institute

San Francisco, California

DENISE KANDEL, Ph.D.

College of Physicians and Surgeons of

Columbia University

New York, New York

HERBERT KLEBER, M.D.

Yale University School of Medicine

New Haven, Connecticut

RICHARD RUSSO

New Jersey State Department of Health

Trenton, New Jersey

GILBERT J. BOTVIN, Ph.D.

Cornell University Medical College

New York, New York

JOSEPH V. BRADY, Ph.D.

The Johns Hopkins University School of

Medicine

Baltimore, Maryland

THEODORE J. CICERO, Ph.D.

Washington University School of

Medicine

St. Louis, Missouri

NIDA Research Monograph Series

CHARLES R. SCHUSTER, Ph.D.

Director, NIDA

JEAN PAUL SMITH, Ph.D.

Acting Associate Director for Science, NIDA

Acting Editor

Parklawn Building, 5600 Fishers Lane, Rockville, Maryland 20857
Opioid Peptides:

Molecular Pharmacology,

Biosynthesis, and Analysis
ACKNOWLEDGMENT

This monograph is based upon papers and discussion from the

technical review on the medicinal chemistry and molecular

pharmacology of opioid peptides and the opiates which took place on

September 4 - 6, 1984, at Bethesda, Maryland. The meeting was

sponsored by the Office of Science and the Division of Preclinical

Research, National Institute on Drug Abuse. The papers on molecular

pharmacology, biosynthesis, and analysis are presented in this

volume. Those on the medicinal chemistry of opioid peptides appear

in NIDA Research Monograph 69.

COPYRIGHT STATUS

The diagram on the cover of this volume is reprinted by permission

from Camerman et al. Crystal structure of leucine-enkephalin.

Nature 306:447-450. Copyright 1983, Macmillan Journals Limited.

The National Institute on Drug Abuse has also obtained permission

from the copyright holders to reproduce certain previously published

material as noted in the text. Further reproduction of this

copyrighted material is permitted only as part of a reprintlng of

the entire publication or chapter. For any other use, the copyright

holder's permission is required. All other material In this volume

except quoted passages from copyrighted sources is in the public

domain and may be used or reproduced without permission from the

Institute or the authors. Citation of the source is appreciated.

Opinions expressed in this volume are those of the authors and do

not necessarily reflect the opinions or official policy of the

National Institute on Drug Abuse or any other part of the U.S.

Department of Health and Human Services.

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

commercial product or company. Trade, proprietary, or company names

appearing in this publication are used only because they are

considered essentlal in the context of the studies reported herein.

DHHS Publication No. (ADM)87-1455

Alcohol, Drug Abuse, and Mental Health Administration

Printed 1986 Reprinted 1987

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

selectively included in the coverage of American Statistics Index,

Biosciences Information Service, Chemical Abstracts, Current

Contents, Psychological Abstracts, and Psychopharmacology Abstracts.

iv
Foreword

The discovery of opioid peptides in the mid-seventies came at an

opportune time, as all the technologies required for identification

and synthesis of opioid peptides were readily available. The

identification of opioid genes followed shortly, facilitated by

recent advances in recombinant technology. This in turn led to the

rapid identification of many more opioid peptides. Concurrently,

recognition of opioid receptor heterogeneity brought to the fore

questions about the role of the multiple receptors in analgesia and

abuse liability.

Another area of research which quickly developed following these

discoveries was the analysis of opioid peptides in Diofluids, a

difficult area which presents unique problems due to the low levels

present. Most analytical development has been based on

radioimmunoassays, which are quite sensitive but which suffer from

variable specificities. Development of chemical methods that are

both specific and sensitive has been urgently needed, particularly

in anticipation of the development of peptides for clinical trials

and the need to have adequate pharmacokinetic analyses of such

drugs.

This volume is primarily concerned with the molecular pharmacology,

biosynthesis, and analysis of the opioid peptides. It is the

companion volume to NIDA Research Monograph 69, Opioid Peptides:

Medicinal Chemistry.

We stand at an exciting point in this rapidly expanding field. It

is hoped that this volume and the preceding one will serve as useful

reference sources for researchers and will provide new incentives

for drug abuse research in the opioid peptide field.

Marvin Snyder, Ph.D., Director

Division of Preclinical Research

National Institute on Drug Abuse

v

Contents

Foreword

Marvin Snyder. . . . . . . . . . . . . . . . . v

Introduction

Rao S. Rapaka. . . . . . . . . . . . . . . . . 1

Folding and Enzymatic Processing of Precursors of

Biologically Active Peptides and Proteins

Irwin M. Chaiken; Tatsuhiko Kanmera; and Reginald P.

Sequeira . . . . . . . . . . . . . . . . . . 3

Biosynthesis of Opioid Peptides

Olivier Civelli; Jim Douglass; Haim Rosen; Gerard Martens;

and Edward Herbert . . . . . . . . . . . . . . . 21

Proenkephalin Biosynthesis in the Rat

Richard D. Howells . . . . . . . . . . . . . . . 43

Enzymes in the Metabolism of Opioid Peptides: Isolation, Assay,

and Specificity

Neville Marks; Myron Benuck; and Martin J. Berg . . . . . 66

Isolation and Identification of Opioid Peptides

Hisayuki Matsuo . . . . . . . . . . . . . . . . 92

B-Endorphin: Naturally Occurring or Synthetic Agonists and

Antagonists

Choh Hao Li . . . . . . . . . . . . . . . . .109

Enkephalin Degrading Enzyme Inhibitors: A Physiological Way

to New Analgesics and Psychoactive Agents

Bernard P. Roques and Marie-Claude Fournie-Zaluski . . . . 128

Progress in the Characterization of the Opioid Receptor

Subtypes: Peptides as Probes. Future Directions

Eric J. Simon. . . . . . . . . . . . . . . . . 155

Regulation of Agonist Binding to Opioid Receptor Types by

Sodium and GTP: Relevance to Receptor Function

Brian M. Cox; Linda L. Werling; and Gary Zarr . . . . . . 175

Opioid Receptors for the Dynorphin Peptides

Iaian F. James . . . . . . . . . . . , . . . . 192

vii
Computer Analysis of Radioligand Data: Advantages, Problems,

and Pitfalls

David Rodbard; Rudolph A. Lutz; Ricardo A. Cruciani;

Vincenzo Guardabasso; Guido 0. Pesce; and Peter J. Munson . . 209

Recent Developments in Bioassay Using Selective Ligands and

Selective In Vitro Preparations

Hans W. Kosterlitz; Alistair D. Corbett; Maureen G. C.

Gillan; Alexander T. McKnight; Stewart J. Paterson; and

Linda E. Robson . . . . . . . . . . . . . . . . 223

Endorphins and Memory Regulation

Ivan Izquierdo; Carlos A. Netto; and Renato D. Dias . . . . 237

Current Status of RIA Methods for the Analysis of Enkephalins

and Endorphins

R. Wayne Hendren. . . . . . . . . . . . . . . . 255

The Analysis of Endogenous Opioid Peptides With HPLC,

Radioreceptor Assay, Radioimunoassay, and Mass Spectrometry

Dominic M. Desiderio; Hisayoshi Takeshita; Hiroshi Onishi;

Genevieve Fridland; Francis S. Tanzer; Claire Wakelyn;

and Chhabil Dass. . . . . . . . . . . . . . . . 304

Reverse Phase HPLC of Peptides: Application to the Opioid

Peptides

Tatsuhiko Kanmera and Reginald P. Sequeira . . . . . . . 319

Peptides as Drugs in the Treatment of Opiate Addiction

Hemendra N. Bhargava . . . . . . . . . . . . . . 337

Progress in the Potential Use of Enkephalin Analogs

Robert C. A. Frederickson. . . . . . . . . . . . . 367

Opioid Peptides as Drug Products: FDA Regulatory Requirements

Charles P. Hoiberg and Rao S. Rapaka . . . . . . . . . 385

A Few Thoughts on the Development and Regulation of

Neuropeptides

John L. Gueriguian and Yuan-Yuan H. Chiu. . . . . . . . 405

viii
Introduction

Rao S. Rapaka, Ph.D.

In order to bring into focus the rapidly expanding areas of research

associated with the opioid peptides, the National Institute on Drug

Abuse sponsored a technical review in September 1984 on the

medicinal chemistry and molecular pharmacology of opioid peptides

and the opiates. As stated in the introduction to NIDA Research

Monograph 69, a companion volume to NIDA Research Monograph 70, this

is an area of major interest for both the short-term and long-term

goals of the Institute because of its potential usefulness in

further research and in treatment applications.

This monograph presents contributions both from the symposium

speakers and from other invited authors in the various aspects of

the molecular pharmacology, biosynthesis, and analysis of opioid

peptides. Highlights of these reviews are presented here.

Medicinal chemistry aspects are presented in NIDA Research Monograph

69.

Biosynthesis of neuroendocrine and opioid peptides and the

processing of precursors is not only dependent on their primary

sequence, but on their three-dimensional conformation. This subject

is reviewed by Chaiken et al. Civelli and colleagues discuss the

biosynthesis of opioid peptides with emphasis on opioid peptide

genes, transcriptional and posttranscriptional regulation of opioid

peptide gene expression, and translational and posttranslational

regulation of opioid peptide production. Dr. Howells discusses the

general biosynthetic aspects of opioid peptides and proenkephalin

biosynthesis in rats. All these metabolic processes involve a

number of specific enzymes. An account of their isolation, assay,

and specificity is presented by Marks et al. An account on the

synthesis of specific enzyme inhibitors of enkephalinase as new

analgesic drugs is given by Drs. Roques and Fournie-Zaluski, an area

yet to be more fully explored.

To follow the release of the processed precursors and to establish

their structures involves a number of chemical and biochemical

techniques. A discussion on isolation and identification of the

opioid peptides, along with a table of the known peptides and a

1
demonstration of these techniques with adrenorphin and neuromedins,

is presented by Dr. Matsuo. Similar isolation studies and synthesis

of ß-endorphin analogs on naturally occurring ß-endorphin peptides

have resulted in the hypothesis by Dr. Li that segments of the

hormone may act as inhibitors to the hormonal action.

An understanding of the types and structures of receptors is

critical in understanding mechanisms of action and also in aiding in

the design of new analogs. A review of this subject is presented by

Dr. Simon and on opioid receptors for dynorphin by Dr. James, while

a discussion of regulating factors of agonist binding is presented

by Dr. Cox and associates. As analysis of binding data is critical,

an account on computer analyses of ligand data is given by Dr.

Rodbard and colleagues. In the binding studies, receptor-specific

ligands have played a critical role; recent developments in bioassay

are described by Dr. Kosterlitz and colleagues. The role of

endorphin in memory regulation is discussed by Dr. Izquierdo and

colleagues.

Great progress in research on opioid peptides has been made possible

by simultaneous advances in the techniques of synthesis,

purification, and analysis of peptides. RP-HPLC purification and

analysis techniques are discussed by Drs. Kanmera and Sequeira, and

analysis of endogenous peptides using advanced techniques by Dr.

Desiderio and colleagues. Current status of RIA methods for the

analysis of enkephalins and endorphins is reviewed by Dr. Hendren.

The ultimate goal of the medicinal chemist and biologist is to

develop therapeutic drugs. Progress in this area with clinical data

on FK 330824 (Sandoz) and Ly 127623 (Metkephamid, Lilly) is

discussed by Dr. Frederickson. Other potential uses of the peptide

drugs, such as in the treatment of opiate addiction, are described

by Dr. Bhargava.

As more and more peptides are likely to be clinically evaluated in

the near future, it is appropriate to update information on

regulatory requirements for new drugs from the FDA perspective.

Hence, these requirements are presented in an introductory chapter

by Drs. Hoiberg and Rapaka, and in a chapter by Drs. Gueriguian and

Chiu which specifically addresses the regulation of neuropeptides.

Based on the presentations and discussions of scientists from

various disciplines and nations who participated in the conference

and others who submitted papers, an effort has been made in this

monograph and its companion volume to bring together a substantial

body of information and to summarize its potential applications in

future research and treatment.

2
Folding and Enzymatic Processing of

Precursors of Biologically Active Peptides

and Proteins

Irwin M. Chaiken, Ph.D.; Tatsuhiko Kanmera, Ph.D.; and

Reginald P. Sequeira, Ph.D.

INTRODUCTION: NEUROENDOCRINE PRECURSORS AS LINEAR AND THREE

DIMENSIONAL MACROMOLECULES

The molecular events which lead from protein precursors to active

peptides are governed both by a cascade of specific converting

enzymes for posttranslational processing and by precursor structure which encodes the action of these enzymes. A rapidly expanding list of biologically active polypeptides which are derived

from precursors has been identified (Docherty and Steiner 1982;

Udenfriend and Kilpatrick 1983; Douglass et al. 1984). And,

though the biosynthesized proteins themselves often are difficult

to obtain in large amounts due to their transient existence in

vivo, amino acid sequences have been defined through genomic or

complementary DNA structure determination. Such sequence information has been helpful to identify what peptides may be derived

from a particular precursor as well as to define and characterize

the types of posttranslational enzymatic conversions, including

limited proteolysis and such modifications as acetylation, phosphorylation, sulfation, and glycosylation, which must occur to

produce active peptides. Yet, because of nonavailability of precursors per se, the molecular understanding of these proteins

remains rudimentary.

The sequences of several neuroendocrine precursors are shown in

figure 1, including those for the opioid peptides as well as the

neurohypophysial hormones. What marks our current view of all such

precursors is that we typically draw them as linearly connected

blocks of sequence; each block or sequence domain represents either an ultimately active polypeptide, an activity domain, or a

region of no or uncertain function bordering or between activity

domains. These precursor structures also reveal the repeated

occurrence of cleavage signals, such as dibasic pairs between sequence domains. In spite of this conceptual linearization, proteins do fold and this should be true of precursor proteins as

well. While local sequence provides chemically defined sites for

3
FIGURE 1

Schematic representation of the primary structures of proAVP/NPII (propressophysin), pro-ACTH/ED (proopiomelanocortin),

pro-ENK (proenkephalin), pro-DYN (prodynorphin), and pro-OT/NPI

(prooxyphysin) deduced by c-DNA sequencing. The positions are

shown of paired and single basic amino acid residues which

serve as processing sites for trypsinlike and carboxypeptidase

B-like enzymes. Enzymatic amidation sites are indicated by

"G"; the residue amino to G is amidated. Domains of sequence

which yield active peptides (activity domains) or other peptides accumulated upon precursor processing are labeled as

follows: AVP, arginine vasopressin; NPII, AVP-associated

neurophysin; GP, glycopeptide; ACTH, adrenocorticotropic hormone;

ß-EDO, beta endorphin; ß-MSH, beta melanocyte stimulating hormone; ENK, enkephalin; ME, methionine enkephalin; ME', methionine enkephalin-Arg6

-Gly7-Leu8; ME", methionine enkephalin-Arg6-

Phe7; DYN, dynorphin; ßNEDO, beta neoendorphin; LE, leucine

enkephalin; RIM, rimorphin; OT, oxytocin; and NPI, OT-associated

neurophysin. Amino acid residue abbreviations are: G, glycine; K, lysine; R, arginine; and H, histidine. The scale at

the bottom denotes the length of the sequence, in residues from

the amino terminus.

4
processing, precursor folding is expected to provide overall guidance and control. Understanding the biosynthetic origin of opioid

and other neuroendocrine peptides thus is both a three-dimensional

and a linear problem.

The interplay of sequence and conformation is reflected elegantly

in classical protein chemistry by such well-studied cases as protease zymogens. Perhaps the best example is the chymotrypsinogenchymotrypsin system, for which the activation pathway is known and

the crystal structures of both precursor and processed forms have

been solved (Blow 1971; Kraut 1971). Chymotrypsinogen is activated by limited proteolysis, with the critical step being tryptic

cleavage at the Arg 15-Ile 16 bond to liberate the Ile 16 a-amino

group which forms an essential component of active site organization (Blow 1971). But neither trypsin nor the chymotrypsin generated during processing act significantly on chymotrypsinogen at a

large number of other peptide bonds which are possible as cleavage

sites baaed on sequence alone. Thus, while protease specificity

dictates cleavage based on linear sequence, precursor conformation

limits the availability of proteolysis sites. In addition, while

chymotrypsinogen can refold and form correct disulfide bonds from

an unfolded, disulfide disordered state, chymotrypsin cannot. This

suggests that the three-dimensional organization of the processed

form needed for activity depends on the prior attainment of correct conformation by precursor folding.

It is evident from the chymotrypsinogen example that folded precursor structure can play at least two major roles in the biosynthesis of active polypeptides: (1) control of posttranslational

enzymatic reactions by steric access to scissile bonds and residue

side chains; and (2), at least for diaulfide-containing polypeptides, preformation of nativelike conformation of ultimate endproducts. Based on the above, two major and interrelated goals

may be addressed in considering neuroendocrine precursor structure. One is to define the primary sequence and to use this as a

guide to describe the linear pathway of processing reactions which

yield active neuroendocrine peptides. The second is to define the

higher order secondary, tertiary, and quaternary structure of

precursors and to understand hou such conformational features

regulate processing reactions and the nature of polypeptides produced.

THE PROCESSING PATHWAY OF NEUROHYPOPHYSIAL HORMONE/NEUROPHYSIN

PRECURSOR

In our own work, we have tried to correlate neuroendocrine precursor processing and precursor structure in the neurohypophysial

hormone/neurophysin system. Figure 1 schematically shows the

linear sequences of composite precursors identified for orytocin

and vasopressin. These sequences were defined directly by cDNA and

genomic DNA cloning (Ivell et al. 1983). The DNA sequencing was a

culmination of prior studies by in vivo pulse labeling (Brown5
stein et al. 1980) and in vitro translation (Chaiken et al. 1982;

Ivell et al. 1983). Each neurohypophysial hormone precursor se-.

quence contains at least two activity domains, one for hormone and

a second for the associated binding protein, neurophysin. A tripeptide spacer links the hormone and neurophysin domains, while

the C-terminus is either a single His residue in the oxytocin case

or an arginyl linker followed by a glycopeptide of unknown functional significance (but known to be an accumulated product of

processing) in the vasopressin case. Both pro-forms are translated with a leader (signal peptide) sequence, which is removed in

vivo by the time translation is complete.

The sequences of neuroendocrine precursor proteins, such as those

for neurophysins and hormones, infer the presence of a small corps

of enzymes which must act to produce the final set of active peptides (see review by Marks, this volume). The peptide cleavage

conversions in the hormone/neurophysln case can be inferred, as

shown in figure 2, to include three types of enzymatic reactions:

an endoprotease step in the tripeptide linker region and, in the

AVP case, an additional endoprotease step at the linker between

neurophysin and glycopeptide; carboxypeptidase B (CPase B) trimming of basic residues from the linker vestiges on the C-termini

of the hormone and neurophysin domains; and amidation to generate

the active, C-terminal amidated form of hormone.

FIGURE 2

Schematic diagram depicting the steps in enzymatic processing

of neurophysin (NP)/hormone (H) biosynthetic precursor proteins

to produce, in each case, a mature neurophysin and either

oxytocin or vasopressin. The precursors are visualized to be

compact, folded macronmolecules in which processing sites are

accessible in external surface regions. The enzymatic steps

of endoproteolysis are expected to occur in the linkage region

between hormone and neurophysin domains and, in the case of

the vasopressin precursor, between the neurophysin and carboxyl

terminal glycopeptide domains. Exproteolytic trimming by

CPase B to produce mature neurophysin and hormone-Gly, and

amidation to convert the latter to mature amidated hormone, are

viewed as occurring sequentially after endoproteolysis.

6
Based on these inferences on processing, one tactic that ve are

using to study the enzymatic reactions is to chemically synthesize

local segments of the precursor sequence suspected to contain

processing sites or to be processing intermediates in conversion

of intact precursor, and then to use these segments as substrates

to identify, isolate, and characterize processing enzymes. A

family of such synthetic segments related to the oxytocin precursor haz been made (Kanmera et al. 1983; Rapaka et al.. in press),

including orytocinyl-Gly-Lys-Arg (OT-GKR), OxytocinylGly-Lys (OT-GK), and oxytocinyl-Gly (OT-G). The first of these waS

obtained by solid phase peptide synthesis, the second by immobilized trypsin cleavage of OT-GKR, and the third by pancreatic CPase

B digestion of OT-GKR. All peptides were purified by reverse

phase high performance liquid chromatography (HPLC).

Both OT-GKR and OT-GX have been used to detect and characterize

the CPase B of posterior pituitary neurosecretory granules, the

enzyme which is expected to act on hormone/neurophysin precursor

and intermediates in vivo. When OT-GKR vaz incubated vith whole

granule lysate, the sequential release of Arg and Lys was detected

(figure 3). The release of Lys Prom OT-GK occurred with the same

pH-dependence as that of Arg Prom OT-GKR. The rates of release

and nature of products detected suggest that no significant amount

Of “dipeptidase” cleavage occurred to produce OT-G and Lys-Arg in

a single step. The sequential CPase B activity had a pH optimum

of about 5.5 to 6, a value similar to the internal pH of posterior

pituitary neurosecretory granules (Gainer 1981). Of note, the

properties found for the CPase B activity at the crude (granule

lysate) level of isolation are similar in our own work (Kanmera et

al. 1983; Kanmera and Chaiken, in press and in that of Hook and

L0h (1984).

Partial purification of the crude CPase B, which is active against

the oxytocinyl peptides, was achieved by gel filtration on Sephacry1 S-300 (Kanmera et al. 1983). What has made this step particularly useful was that it allowed separation of two carboxypeptidase activities, the later-eluting of which is the CPase B

vith clear preference for basic residues and relatively little

tendency, for example, to cleave Gly Prom OT-G to give oxytocinoic

acid (OT acid). The earlier-eluting CPase has little preference

for exoproteolytic removal of Arg Prom OT-GKR verzus Gly Prom

OT-G. The later-eluting specific CPase B has several enzymatic

properties similar to those reported for a CPase B that can act on

enkephalinyl peptide (Supattapone et al. 1984).

Based on prior conversion studies vith model peptides by pituitary

amidating enzyme (Bradbury et al. 1982; Eipper et al. 1983), oxytocin in Its active C-terminal amidated form is expected to be

derived from conversion of the CPase B product OT-G. This enzymatic conversion was detected using 125I-OT-G (labeled at Tyr 2

using the lactoperoxidase-glucose oxidase method). By reverse

phase HPLC, 125I-OT could be detected as a product of reaction

7
with lysates of granules obtained by differential centrifugation

(figure 4). However, with the latter as the source of enzyme, a

competing and presumably nonspecific (possibly lysozymal) proteolytic degradation led to loss of product as well as substrate and

to the appearance of early-eluting iodinated species, presumably

degradation products. The amidating enzyme was found to be substantially enriched over the nonspecific proteolytic activity in

granule subfractions obtained by Percoll density gradient ultracentrifugation of posterior pituitary granules. Thus, subfractions migrating as the most dense in the Percoll gradient were

relatively more free of degrading activity and led to a more obvious accumulation of product (125I-oT) in RP-HPLC and little of the

early-eluting degradation peaks evident in figure 4 (Kanmera and

Chaiken, in press). The specific granule fractions obtained

FIGURE 3

Reverse phase HPLC analysis of conversion of the oxytocinyl

precursor fragment GT-GKR by neurosecretory granule lysate.

OT-GKR (80 nmles, prepared by solid phase peptide synthesis)

MS incubated with 10 μl of granule lysate (granules prepared

by differential centrifugation) in 200 μl of 0.1 M sodium phosphate buffer, pH 5.5. Aliquots of reaction mixture taken at

0, 0.7, 5, and 20 hours at 37°C were applied to a cyanopropyl

silyl RP-HPLC column (Zorbax CN, 0.46 x 25 cm, Dupont) using a

Varian LC 5000 system and eluted with a linear gradient, at 0.8

ml/min., from 93% triethylammonium phosphate (TEAP, 67 mM, pH 3)/7%

acetonitrile at 0 time to 70% TEAP/30% acetonitrile at 20 min.

Peaks, identified by amino acid analysis, are: (a) OT-GKR; (b)

OT-GK; and (c) 0T-G.

8
FIGURE 4

Reverse phase HPLC analysis of conversion of [125I]OT-G

(substrate peak, S) to C-terminal amidated [125I]OT (product

peak, P) by neurosecretory granule lysate (see figure 3

legend). A: Reverse phase HPLC profile of aliquot of reaction

at 135 minutes after addition of granule lysate. The reaction

mixture consisted of 200 μl of lysate in 10 mM HEPES, pH 7.0,

and 100 μl of a solution of 0.03 mM CuSO4 1 mM sodium ascorbate, 0.3 mg catalase/ml, and 3 x 105

 cpm [125I]OT-G (<.1 μg)

in 50 mM TES, pH 7.0. Aliquot of 50 μl was mixed with 100 μl

of 50 mM ammonium acetate, pH 5.0, and injected onto an octadecyl silyl column (Zorbax ODS, Dupont, 0.46 x 25 cm) and

eluted with a linear gradient of 85% 50 mM ammonium acetate,

pH 5.0/15% acetonitrile at 0 time to 60% 50 mM ammonium acetate,

pH 5.0/40% acetonitrile at 30 min. S and P were identified by

comparison with elution of starting substrate and iodinated

authentic orytocin. Peaks centered at 4 min. (breakthrough

volume) and 15 min. increase with time of reaction and are

assumed to arise from nonspecific proteolytic degradation of

S, P, or both. B: Time course of decrease of Sand transient

increase followed by decrease of P. Conversion reactions carried out with lysates of selected neurosecretory granules prepared by Percoll density gradient centrifugation (Kanmera and

Chaiken. in press; Rapaka et al., in press) show

a more obvious and prolonged increase in P with time and a reduced degradation to early-eluting forms.

9
by density gradient centrifugation provide a partially

purified amidation enzyme preparation suitable for further isolation and study.

In terms of observable reactions, the enzymatic conversions expected from the oxytocinyl precursor sequence--CPase B trimming

followed by amidation — can occur with the precursor fragments

OT-GKR , OT-GK, and OT-G. Yet, several data argue that processing

reactions of the hormone/neurophysin precursors must involve overall precursor structure, and the neurophyzin domain in particular.

First, the initial endoproteolytic conversion of precursor to

yield precursor intermediates of the type OT-GKR would be expected

to occur with the full-sequence and, therefore, fully- folded precursor as substrate. Data reviewed below show that such a precursor

has a well-defined folded conformation. Second, the putative

precursor intermediate OT-GKR was found, by analytical affinity

chromatography on Sepharoze-immobilized neurophyzin II, to bind

noncovalently to neurophyzin (Kanmera et al. 1983; Kanmera and

Chaiken, in press). Based on the degree of retardation

verzuz that for OT and Met-Tyr-Phe amide, OT-GKR binding to neurophysin is concluded to have a Ka value close to that of OT (a

greater value cannot be excluded by the data obtained so far). At

the high concentrations of neurophyzin and hormone expected to

exist in neurozecretory granules, OT-GKR likely remains bound to

neurophysin noncovalently after endoproteolytic cleavage. Thus,

the actual substrates for CPase B and amidating enzymes are likely

to be, not free peptides, but rather those folded into relatively

fixed conformations as parts of noncovalent peptide/neurophysin

complexes. The conformations of the peptide/protein complexes are

likely to mimic those of the precursors themselves. This conclusion makes it important to define the conformation of precursors

and of the intermediate complexes that arise from them.

THE FOLDED NATURE OF NEUROENDOCRINE PRECURSORS

Describing the degree of ordered structure in neuroendocrine precursor proteins depends largely on obtaining sufficient amounts of

precursor for conformational characterization, including crystallographic analysis where possible. This need contrasts with the

realization that such precursors are only transiently persistent

species in situ and obtainable in only very small (subfemtomole)

amounts by such procedures as in vitro translation or in vivo

pulse-labeling. The gap between avability and need is likely

to be reduced, but only partially, by using micromethods for characterization.

In order to obtain workable amounts of hormone/neurophysin precursor, we have used semisynthesis, an approach in which synthetic

and natural polypeptide components are reconstituted to rebuild a

larger protein (Chaiken 1981). As a first target, we have chosen

the oxytocin/neurophysin I precursor, which consists essentially

of a dodecapeptide hormone-linker domain (OT-GKR) attached to a

10
neurophyzin I domain (see figure 1). The reaction scheme used for

the formation of this precursor (Kanmera and Chaiken 1985) employed coupling of an α-amino protected synthetic OT-GKR

([Nα1,ε11

−diBoc]OT-GKR) as an active ester to a selectively ε−

amino-blocked native protein ([dl-acetimidyl] NPI). In our studies so far, the partially protected species [des His 106, dl-Acet

30,71]pro-OT/NPI has been prepared, with the product purified by

reverse phase HPLC. The acetimidyl groups on the two ε− amino

functions of NPI can be removed, but have been retained in studies

so far since they are useful for incorporation of radiolabel and

do not affect the functional and folding properties examined (see

below). The lack of C-terminal His in the semisynthetic precursor

is assumed, as a first order approximation, not to lead to a significant aberration in the major conformational properties of

precursor protein. The semisynthesis scheme we have developed is

general and is being used at present to build vasopressin-containing, precursorlike molecules and, ultimately, a set of mutant

precursors.

[des His 106, di-14C- acetimidyl 30,71]Pro-OT/NPI has been used to

Investigate the question of whether neurohypophyzial hormone precursors fold to form well-defined conformations that act as “blueprints” for the ultimate conformation-dependent interaction properties of the mature hormone/neurophyzin noncovalent complexes.

One aspect of this study has been to examine both the binding

properties of the semisynthetic precursor for hormone binding site

ligands and the self-association potential by analytical affinity

chromatography (Chaiken 1979; Angal and Chaiken 1982). [des His

106, di-Acet]Pro-OT/NPI was found (Kanmera and Chaiken 1985)

not to bind significantly to Met-Tyr-The-Affigel 102. The latter

Immobilized tripeptide acts as a mimic of hormone and binds to the

hormone binding site of neurophyzin (Angal and Chaiken 1982). The

tripeptide affinity matrix does bind to [di-Acet 18,59]NPI, with

an affinity essentially equal to that of native NPI (Kanmera and

Chaiken 1985). The blocking of the hormone binding site of the

neurophyzin domain of the precursor is concluded to be due to

intramolecular hormone domain-neurophysin domain interaction.

Interestingly, intramolecular domain-domain interaction such as

that considered likely in the precursor is increasingly considered

as a common feature of folded protein conformation in general

(Wetlaufer 1981; Fontana et al. 1983).

The obserevation of intramolecular domain-domain interaction In

[des His 106, di-Acet]pro-OT/NPI suggests that neurohypophysial

hormone precursors also can self-associate with relatively high

affinity. This prediction is based on the known self-association

of native neurophysin to dimers and the potentiatlon of the selfassociation by ligand binding to the neurophyzin subunits (Cohen

et al. 1979; Angal and Chaiken 1982). Indeed, [des His 106, diAcet]pro-OT/NPI Is retarded on [NPII]Sepharose, with a net elution

volume (observed elutlon volume, V, minus unretarded elution volume Vo) about an order of magnitude greater than that of

11
[diAcet]NPI (figure 5). The results show that precursor, with an

intramolecularly liganded neurophysin domain, associates with

unliganded immobilized neurophysin and that the association has a

hi her affinity (1.7 x 106M

-1) than that of [di-Acet]NPI (1.3 x 6 

105M

-1). Moreover, precursor retardation is increased by addition

of close to saturating amounts of hormone (Lys 8-vasopressin) to

the elution buffer. This shows that liganding of the immobilized

neurophysin potentiates precursor-protein association further. He

take the analytical affinity chromatographic data together to

argue that hormone/neurophysin precursor self-associates upon

storage in secretory granules after biosynthesis and that the

self-association is stabilized by intramolecular hormone domain

interactions which persist after proteolytic processing.

During the events leading from precursors to neurohypophysial

hormones, the well-defined folded structures of precursors may

FIGURE 5

Analytical affinity chromatography assay of protein-association

property of semisynthetic oxytocin/neurophysin I precursor and,

for comparison, neurophysin I. Zones containing 1500-3000 cpm

(<10.5 μg) of [di-14C-Acet 18,59]NPI and [des His 106, di-14C-Acet

30,71]pro-oT/NPI were eluted on bovine NPII-Sepharose (70 nmole

BNPII/ml of bed volume, 198 μl bed volume) with 0.4 M ammonium

acetate/0.5% bovine serum albumin, pH 5.7. Fraction size was 4

drops (157 μl) for NPI and 10 drops (391 μl/for semisynthetic

precursor. Flow rate was 5 ml/min.; chromatography was at ambient temperature.

12
well help control the subsequent enzymatic processing reactions

leading to active neuroendocrine peptides. This hypothesis is

shown schematically in figure 6. One prediction of this scheme is

that hormone/neurophysin precursors should be able to fold spontaneously from a disordered state, in a manner expected for intact

biosynthetic precursors but not observed for native neurophysin

itself (Chaiken et al. 1975). This feature now has been examined

with the semisynthetic precursor by testing whether the precursor

is stable to disulfide shuffling: nonbiosynthetically intact

proteins such as neurophysin are not stable, but biosynthetically

intact proteins as a rule are (Givol et al. 1965). We have observed that, in the presence of dithioerythritol, [des His 106,

di-Acet]pro-OT/NPI exhibits such disulfide stability but [diAcet]NPI does not. Neurohypophysial hormone precursors (proforms) of the sequence type shown in figure 1 thus are vieued as

having sufficient sequence information to code for stabilization

of the correct disulfide pairing. It is concluded that the precursors fold spontaneously to a defined native conformation upon

completion of translation and before packaging and enzymatic processing.

FIGURE 6

Schematic model depicting relationship of biosynthetic precursor

structure to molecular events occurring in neurohypophysial

hormone/neurophysin biosynthesis. The filled and open lines

denote hormone and neurophysin sequence domains, respectively.

The cross-hatched line represents the C-terminal glycopeptide

occurring in pro-Arg 8 vasopressin/neurophysin. Folding of

the precursor leads to establishment of self-association through

the NP domains of the precursors. The NP-NP and H-NP interaction

surfaces are retained after enzymatic processing, which leads to

formation of noncovalent complex between Hand NP and its dimer

in secretory granules until released exocytotically.

13
FIGURE 7

Analysis of bovine ovary extract by reverse phase HPLC for

immmoreactive (ir) OT and bovine NPI. Pooled acetic acid

extracts of bovine ovaries were fractionated on Sep-PakR;

the eluate was dissolved in 0.2 ml triethylammonium phosphate

buffer (67 mM TEAP; pH 3.0) and injected onto an octylsilyl

column (Zorbax C8, Dupont, 0.46 x 25 cm). One minute fractions

were collected, dried in a speed vac, reconstituted in 0.99 ml

of radioimmunoassay buffer (50 mM phosphate-150 mM NaCl, pH 7.61

containing 0.5% bovine serum albumin and 0.1% sodium aside, and

the pH adjusted to 7.6 with 10 μl of 5 M NaOH. Duplicate aliquots of 0.1 ml of the fractions were analyzed by radioimmunoassay for ir-OT and -bNPI. Note the ir-peaks at or close to

those of neurohypophysial OT and bNPI. An additional ir-OT

peak having a longer retention time is also observed. Upon

re-examination using gradients allowing more refined separation

in the neurophysin elution regions, it was found that most of

the ir-bNPI species (about 80%) have a retention time significantly shorter than that of neurohypophysial bNPI.

While the scheme of figure 6 denotes a single molecular pathway

for the biosynthesis of the neurohypophysial hormone/neurophysin

system, it is oversimplified in at least one important way. It is

becoming more evident that there are sites of occurrence of the

hormone/neurophysin system besides the classically defined hypothamo-neurohypophysial tract. It is likely that some of these

other sites represent independent anatomical pathways of de novo

biosynthesis. We have been examining one such site, namely the

ovary. Data obtained by reverse phase HPLC mapping of bovine

ovary extracts show the presence of both ir-neurophysin and irhormones (Sequeira and Chaiken 1984; Chaiken et al. 1984a). The

data for oxytocin and NPI are shown in figure 7. The co-occur14
rence of HP with oxytocln and a growing body of other data argue

that the ovary is likely to be a site of Independent hormone synthesla (Rodgers et al. 1983; Suann et al. 1984). Indeed, there is

a significant peak of ovarian OT which essentially co-eluted with

pituitary OT (figure 7). However, the ovarian NPI identified in

the ovary, which elutes close to pituitary NPI in figure 7, has

been found to be a different molecular form than the pituitary

form (R.P. Sequeira and T. Kanmera, unpublished data) when examined both by RP-HPLC using a flattened gradient more suitable for

separation of neurophysin isoforms (Chaiken et al. 1984b) and by

HPLC peptide mapping (Chaiken and Hough 1980). This analysis

suggests that products of processing of the pro-OT/NPI precursor

synthesized in the ovary apparently are different than those produced in the hypothalamo-neurohypophysial tract. This view is

supported by the observation of a second, prominent ovarian ir-OT

FIGURE 8

Schematic diagram of neuroendocrine peptide/protein pathways.

Depicted are the biosynthesis, folding, and granule packaging

of precursors containing multiple activity domains; enzymatic

processing of precursors to produce mature, active polypeptides;

axonal transport and storage of matured granules; exocytotic

release of neuroendocrine polypeptides; and ultimate action of

active peptides at postsynaptic or peripheral target receptors.

15
form with a much greater retention time than that of pituitary OT.

this later-eluting form has immunoreactivity and neurophysin-binding properties consistent with it containing the oxytocln sequence, but it is not biologically active in the rat uterus (R.P.

Sequelra, R. Medway,and W.H. Sawyer, unpublished data). Such a

species was not observed in pituitary extracts. That this form

may be an alternatively processed, stable oxytocinyl precursor

intermediate, containing OT with a C-terminal extension of indeterminate length, is an intriguing possibility currently being

examined. Taken together, the data indicate that some species of

ovarian ir-OT and ir-NPI are not fully identical with the molecular species stored in pituitary. This suggests that, if there is

a local synthesis in the ovary by a precursor akin to that identifled for pituitary OT, it may well occur by a different processing pathuay than that of the hypothalami-neurohypophysial tract.

These results suggest that, if we wish to define how precursors

fold and are processed, as in figure 7, the mechanisms we examine

may be different at different sites of synthesis.

OPIOID PEPTIDES, NEUROHYPOPHYSIAL HORMONES, AND COMMONALITY IN

NEUROENWCRINE BIOSYNTHETIC PATHWAYS

Both molecular similarities and co-occurrence mark the emerging

view of the relationship of neuroendocrine pathways for classical

neurohypophysial hormones and oplold peptides. A common view has

evolved of the origin and fate of neuroendocrine peptides by pathways in which precursors are posttranslationally processed enzymatically to produce a aet (most often more than one) of biologically active peptides that can function as neurotransmitters,

neuromodulators, or endocrine hormones (figure 8). Met- and Leuenkephalins, dynorphin, ß-endorphln, oxytocln, vasopressin, and

neurophysins among other neuroendocrlne peptides all are produced

by such a pathway. Several of the features described in this

chapter for precursor structure and processing in the hormone/neurophysin system also are repeated themes for oplold peptides. These include multidomain precursors (figure l), types of

endo-and exo-proteases and nonproteolytic-converting enzymes

(e.g., amidatlng enzymes) to process precursors (Loh et al. 1984),

and tissue-specific processing (Watson and Akil 1982; Weber et al.

1982). More than similarity, opioid and hormone/neurophysin pathways apparently are co-localized in some anatomical sites. This

has been observed in magnocellular neurons of the hypothalamus for

Met-enkephalin and oxytocin (Rossier 1982; Vanderhaeghen et al.

1983) and in neurohypophysial nerve terminals for dynorphin and

vasopressin Whitnall et al. 1983) and for [Met]-enkephalin and

oxytocin (Martin and Volgt 1981).

While apparent similarities exist, our understanding of the detailed mechanistic relatedness between oplold and hormone/neurophysin pathways is not well developed. Future studies need to

address at least two sets of questions. First, to what extent is

conformation a controlling feature? are all precursors conforma16
tionally defined structures (as with the hormone/neurophyzin

cases)? is the conversion to intermediates and products marked by

transition to less or more conformational order? do most mature

neuroendocrine peptides assume more disordered structures (at

least when dissociated from other interactive components in storage granules) which make them amenable to productive recognition

(in a sense, capture) of a particular conformation by receptors?

Second, do pathways for different neuroendocrine peptides have

common enzymatic processing machinery? for example, are carboxypeptidases B, dibasic endoproteases, or amidating enzymes similar

or even the same for producing different peptides from different

precursors or are there instead sequence-specific enzymes for each

type of precursor system? Describing the molecular mechanisms

controlling the biosynthetic origin for any single neuroendocrine

peptide pathway is certainly a far-from-simple task. How closely

similar these molecular mechanisms are for a family of neuroendocrine pathways, including those for hormoneineurophyzin and opioid

peptides, remains an even more provocative challenge for future

investigation.

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Chaiken, I.M.; Kanmera, T.; Sequeira, R.P.; and Swaisgood, H.E.

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Proenkephalin, [Met]enkephalin, and oxytocin immunoreactivities

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19
AUTHORS

Irvin M. Chaiken, Ph.D.

Tatsuhiko Kanmera, Ph.D.

Reginald P. Sequeira, Ph.D.

Molecular, Cellular, and Nutritional Endocrinology Branch

National Institute of Arthritis, Diabetes, and Digestive and

Kidney Diseases,

National Institutes of Health

Bethesda, Maryland 20205 USA

20
Biosynthesis of Opioid Peptides

Olivier Civelli, Ph.D; Jim Douglass, Ph.D.;

Haim Rosen, Ph.D.; Gerard Martens, Ph.D.; and

Edward Herbert, Ph.D.

INTRODUCTION

In the last decade, a number of peptides have been described

which have opioid activity. Although these peptides induce

diverse reactions in animals, they share two important physical

features: they have an enkephalin sequence at their aminoterminus which confers upon then their biolgical activity and

they have a small size ranging from 5 to 40 amino acids (figure

1). While many researchers have concentrated on the implication

of the opioid peptides in behavior, others have concerned

themelves with the mode of biosynthesis of the opioid peptides,

with the goal of understanding regulation of synthesis in the

animal.

In this review, we summarize the results obtained on the

biosynthesis of the opioid peptides. By applying recombinant

DNA technology, it has been possible to show that all of the

opioid peptides are derived from three precursors:

proopiomelanocortin (POMC), proenkephalin, and prodynorphin.

The complete sequence of these precursors has been determined in

different species, as well as the sequences of their

corresponding genes. These studies have provided researchers

with the DNA probes necessary to analyze the regulation of

opioid gene expression under different physiological conditions.

Although only a few physiological changes have been analyzed

thus far, they reveal a great diversity in regulatory mechanisms

and in the opioid gene sequences. Finally, we review the

experiments which deal with the generation of the bioactive

opioid peptides from the polypeptide precursors. Probably the

most intriguing results are those which show that important

regulatory steps occur after the translation of the precursors.

21
FIGURE 1

Structure of some opioid peptides

The enkephalin domains are in boxes.
While these discoveries open the way for looking at specific

processing factors, they add another level to the diversity in

the production of opioid peptides.

THE OPIOID POLYPEPTIDE PRECURSORS

Because of the small size of the opioid peptides (5 to 40 amino

acids) (figure 1), it was thought initially that their synthesis

might not involve the ribosome-dependent protein synthesizing

machinery of the cell. However, numerous neuropeptides and

peptide hormones share this size characteristic. The studies

carried out on the biosynthesis of adrenocorticotropin (ACTH)

finally revealed the key of the enigma It was shown that ACTH

is synthesized in the form of a large polypeptide precursor and

that this precursor also contains the sequence of an opioid

peptide ß-endorphin. This discovery ignited numerous studies on

the biosynthesis of other small bioactive peptides, which

confirmed that all of these peptides are synthesized in the form

of large polypeptides precursors and that in the majority of the

cases, these precursors code for more than one bioactive

peptide. This gave rise to the concept of polyproteins for

polyfunctional precursors (Douglass et al. 1984).

The first opioid precursor protein to be characterized by

recombinant DNA methods was POMC, which gives rise to the opioid

ß-endorphin and a variety of other peptides, including ACTH and

α, ß, and γ−melanocyte stimulating hormones (MSH) (Nakanishi et

al. 1979). The second precursor protein to be characterized was

proenkephal in, which contains six copies of Met-enkephalin and

one copy of Leu-enkephalin (Comb et al. 1982; Gubler et al.

1982; Noda et al. 1982a). In the adrenal medulla, this

precursor produces a number of different opioid peptides that

include Met- and Leu-enkephalins: peptide E, which contains one

copy of Met-enkephalin and one copy of Leu-enkephalin; and

peptide F, which contains two copies of Met-enkephalin and Metenk-Arg-Gly-Leu. The third precursor to be sequenced was

prodynorphin, which contains three copies of Leu-enkephalin and

gives rise to the opioids dynorphin, ß-neo-endorphin, and

rimophin (dynorphin B) (Kakidani et al. 1982) (figure 2). Some

remarkable similarities in the structure of the opioid peptide

precursors are revealed in figure 2. First, the precursors are

almost all the same length and the sequences of the biologically

active peptides are confined almost exclusively to the Cterminal half of the precursors. Then, the N-terminal region of

each precursor is rich in cysteine residues and the distribution

of these residues is similar in each case, indicating that

formation of disulfide bridges may be essential for stabilizing

the protein in conformations required for correct processing.

Finally, almost all of the biologically active domains in each

precursor are flanked on both sides by pairs of basic amino acid

residues, a feature not specific to precursors of opioid

peptides but common to all the polyproteins, which implies that

trypsinlike cleavages are involved in the maturation of the

active peptides.

23
FIGURE 2

Schematic representation of the three opioid polypeptide precursors

The black box at the N-terminus represents the signal sequence.

Cys indicates the presence of a cystein residue In the precursor.

Some of the bioactive peptides are indicated. The black boxes In

the precursors indicate the presence of an enkephalin sequence.

Pairs of basic amino acid residues are indicated by a bar.
The similarities in structure between these different precursors

suggest that they arose by similar evolutionary mechanisms.

This suggestion is supported by similarities in the structures

of the opioid peptide genes.

THE OPIOID PEPTIDE GENES

The three peptide genes share the characteristics common to the

majority of the eukaryotic genes; their coding regions (exons)

are separated by noncoding intervening sequences (introns). In

the opioid genes, however, the introns do not separate

functional regions of the polypeptide precursors as is the case

for some other polyprotein (Douglass et al. 1984).

Opioid Peptide Gene Structure

The proopiamelanccortin gene structure has been determined for

human (Cochet et al. 1982), bovine (Nakanishi et al. 1981), rat

(Drouin and Goodman 1980), and mouse (Notake et al. 1983; Uhler

et al. 1983). The overall structure of the POMC gene is highly

conserve among these different species. The human, rat, and

amphibian proenkephalin (Noda et al. l982b; Comb et al. 1983;

Rosen et al. 1984; Martens and Herbert 1984) and human

prodynorphin genes (Horikawa et al. 1983) have also been

isolated. The general organization of the proenkephalin and

prodynorphin genes is remarkably similar to that of the human

POMC gene and is schematically diagramed in figure 3. All three

genes have large 3'exons which contain the nucleotides coding

for all of the biologically active peptides and the majority of

the N-terminal portions of the precursors. Note that the 3'

untranslated region of the prodynorphin gene is much larger than

that of the other two genes. Some 3 kb upstream (5') is a

smaller exon which contains sequences coding for the remainder

of the amino-terminal portion of the precursor molecule, the

initiator methionine, and a few bases of the 5' untranslated

region of the mRNA. The remaining sequences coding for the 5'

untranslated region of the mRNA are found further upstream on

either one (POMC) or two (proenkephalin and prodynorphin) exons.

The prodynorphin gene differs from the other two genes in that

its 5' untranslated region is large.

The structural similarities between these two genes in the human

suggest that they may have arisen via a common evolutionary

mechanism. The human POMC and proenkephalin genes are not,

however, closely linked in the genome. POMC has been localized

to chromosome 2, while the proenkephalin gene is located on

chromosome 12. The location of the prodynorphin gene is not

known.

The 3' exons of all the opioid genes contain regions of intrasequence homology. In POMC, three repetitive nucleotide regions

are present (approximately 50 nucleotides in length) which code

for α−, ß-, γ−MHS. In human proenkephalin, there are seven

regions of internal nucleotide sequence homology (approximately

25
FIGURE 3

Structural comparison of the human POMC, proenkephalin, and prodynorphin genes

Lines indicate introns; boxes indicate exons. ATG Indicates the

initiator methionine; TERM Indicates the mRNA 3'-end. CAP shows

the start of transcription. Dashed boxes indicate the signal

peptide; hatched boxes the extent of the coding region. The

different enkephalin sequences are indicated.
25 base pairs in length) which code for the biologically active

enkephalin moieties. This observation has led to the suggestion

that the POMC and proenkephalin genes have evolved via a series

of duplication and rearrangement events of ancestral MSH-like

sequences and enkephalinlike sequences, respectively (Noda et

al. 1982b).

Southern blotting experiments and the isolation of genanic

clones fran genomic libraries have implied that only one

proenkephalin and POMC gene is present in the human genome. The

same holds true for the number of POMC genes in the bovine and

rat gene. In contrast, two groups (Notake et al. 1983; Uhler

et al. 1983) have reported the presence of two POMC genes in the

mouse gene, one of which is a pseudogene. The pseudogene

exhibits 92% homology with 533 base pairs of the functional POMC

gene, including the coding regions for ACTH and ß-LPH. However,

the presence of a premature translation termination codon and a

mutation in a codoon for a dibasic amino acid cleavage site

within the protein predicts that ß-endorphin would not be

present in the translation product, and ACTH would not be

cleaved from the precursor by a trypsinlike cleavage enzyme.

Thus, the POMC pseudogene in mouse cannot encode a functional

precursor protein similar to the POMC precursor. Finally, the

mouse POMC pseudogene sequence is flanked on both sides by

direct repeats 10 basepairs in length. This observation raises

the interesting possibility that the pseudogene may have arisen

via the formation of an aberrant transcript of the functional

gene, followed by the insertion of its cDNA copy into the mouse

genome uptake et al. 1983).

Comparative Aspects of Proenkephalin Genes in the Human,

and Amphibian

Analysis of protein sequences in different species in order to

determine conserved regions constitutes an approach to

understanding which sequences may be functionally significant

The proenkephalin amino acid sequences are known in the human,

bovine, and rat as well as the toad Xenopus laevis and,

therefore, can serve such a comparison. The toad proenkephalin

contains five copies of Met-enkephalin and one copy of Metenkephalin-Arg-Gly-Tyr and one Met-enkephalin-Arg-Phe (Martens

and Herbert 1984). Met-enkephalin-Arg-Gly-Leu and Leuenkephalin, two enkephalin sequences present in human, bovine,

and rat proenkephalin, are not found in the Xenopus sequence.

Thus, amphibian proenkephalin contains no Leu-enkephalin

sequences, suggesting that a switch from a Met-enkephalin to a

Leu-enkephalin sequence in the mammalian proenkephalins occurred

less than 350 million years ago (time of divergence between the

Xenopus line and the main vertebrate line).

The distribution of enkephalin sequences appeared to be very

similar among human, bovine, rat, and Xenopus proenkephalin.

Some of the spacer regions between the enkephalin sequences have

a high degree of amino acid homology, while others have diverged

27
to a considerable extent It is interesting to note that the

highly conserved regions between enkephalin units 2 and 3, 5 and

6, and 6 and 7 (figure 4) correspond to enkephalin-containing

peptides isolated from bovine adrenal medulla (peptides F, E,

and B, respectively). The high degree of conservation of these

peptides (especially of the highly potent opioid peptide E)

might point to an important physiological role for then, both in

mammals and lower vertebrates. The enkephalin sequenoes in both

mammalian and amphibian precursors are flanked by pairs of basic

amino acids, suggesting that similar processing mechanisms are

used in both mammals and amphibians In mammals, the cysteine

residues in three opioid precursor proteins are located in

almost identical positions in the N-terminal region It has

been suggested that this region is important for proper folding

of the precursor molecule in order to ensure correct processing.

The conservation of the cysteine residues in the proenkephalin

sequences of the four species reinforces this concept. Thus,

the high conservation of proenkephalin sequences in mammals and

amphibians suggests that the enkephalins and enkephalincontaining peptides have an important physiological function(s)

in a wide range of vertebrates (Martens and Herbert 1984).

TRANSCRIPTIONAL AND POSTTRANSCRIPTIONAL REGULATION OF OPIOID

PEPTIDE GENE EXPRESSION

The tissue levels of opioid peptides can be altered by a variety

of synthetic as well as naturally occurring substances. In this

section, we will dccument sane examples in which various

regulators are altering opioid peptide levels as the result of

changes in the level of mRNA that codes for these peptides. The

following questions will be addressed: (1) Which tissues are

actively transcribing polyprotein genes? (2) What are some of

the characteristics of these genes that make them

transcriptionally active? (3) How are transcriptionally active

opioid genes regulated in vivo?

Detection of mRNA Coding for Various Polyproteins

An opioid cDNA clone or genomic clone can be used as a

hybridization probe for detecting and quantifying the

corresponding mRNA. Study of the distribution of POMC mRNA in

various rat brain tissues shows that the hypothalamus, amygdala,

and cerebral cortex contain POMC transcripts (mRNA) while the

cerebellum and midbrain do not (Civelli et al. 1982). In

addition, POMC transcripts in the amygdala and cortex appear to

be slightly smaller in size than POMC transcripts isolated from

the hypothalamus. The distribution of proenkephalin mRNA in

these same tissues has been measured (Tang et al. 1983) and

proenkephalin transcripts were detected (in order of abundance)

in the striatum, hypothalamus, cerebellum, midbrain,

hippocampus, and cortex.

These data provide two important pieces of information. First,

the distribution of POMC mRNA in the rat brain is different from

28
FIGURE 4

Nucleotide sequences comparisons between the rat (A), human (B),

bovine (C), and Xenopus laevis (D) proenkephal in genes.

The exons are shorn by blocks, the introns by lines. The "TATA”

sequence, capping site, translational initiator ATG and

terminator TGA, and poly (A) addition sites are indicated. The

enkephalin sequences are represented In the rat gene by black

boxes. Three degrees of homology are used to characterize the

nucleotide sequence comparison: open box, shaded box and sol Id

box representing homology of less than 65%, between 65-86%, and

more than 85%, respectively.
that of proenkephalin mRNA. For example, the rat cerebellum

contains relatively high levels of proenkephalin transcripts but

no detectable POMC mRNA. Since opioid peptides are derived from

both precursor molecules, proenkephalin-derived peptides may

play an important role in this region of the brain while ßendorphin (from POMC) does not. Second, the levels of mRNA can

be correlated with the levels of the bioactive peptides derived

from POMC or proenkephalin. In most cases, a direct correlation

is observed, confirming that the opioid peptides previously

detected in these tissues by radioimmunoassay are present as a

result of direct synthesis in those tissues, and not transport

to those tissues from a secondary site of synthesis. Rec`ently,

this has been demonstrated to be also true for POMC mRNA and

POMC-derived peptides in the testis (Pintar et al. 1984).

Several groups (Hudson et al. 1981; Gee and Roberts 1982) have

used POMC cDNA as a hybridization probe to study the

differential expression of the POMC gene in the various lobes of

the rat pituitary. Using these probes, approximately 3% to 5%

of anterior lobe cells appear to contain POMC transcripts, while

greater than 903 of the intermediate lobe cells show cytoplasmic

localization of POMC mRNA. By combining immunohistochemical

methods with in situ hybridization histochemistry, it has also

been shown that the same cells contain both POMC peptides and

POMC mRNA (Gee and Roberts 1982). These data suggest that the

presence of the POMC peptides in these cells is due to their

local synthesis and is not the result of uptake from the plasma.

Characteristics of Transcriptionally Active Opioid Genes

Gene expression in eukaryotic cells is influenced by a wide

variety of factors. Of particular importance are the DNA

sequences situated upstream to the transcription start, since

they can potentially change the level of transcription. Gene

transfer experiments have been applied in order to localize

these DNA sequences.

A cloned human POMC gene has been ligated to SV40 DNA,

introduced into CDS monkey cells, and transcribed from its own

promoter (Mishina et al. 1982). Deletion mutants in the 5'

region of the gene were generated to determine the effects of

these sequences on transcription of the gene in vivo, If

nucleotide +l is the first be of the primary transcript,

deletion of sequences from -20 to -40 (containing the TATA box

start of transcription) completely abolished accurate

transcription from the POMC promoter. This result suggests that

the TATA box region is a distinct promoter element of the human

POMC gene.

A unique feature of the human POMC promoter region is that the

deletion of sequences from -53 to -59 results in a threefold

enhancement of the transcriptional efficiency. This region

overlaps with a continuous stretch of G-C pairs present -48 to

-56 bp upstream from the cap site. The G-C stretch itself, or

30
its specific pattern of methylation, may depress transcription

of the gene; and its partial removal may therefore increase the

transcriptional efficiency of the human POMC gene.

Regulation of Expression of Cpioid Peptide Genes

Many compounds alter the levels of bioactive peptides. Some of

these substances change the rate of processing of the precursor

molecules. Others regulate the rate of transport or secretion

of the bioactive peptides following their release from the

precursor molecule.

Regulation of POMC Gene Expression The secretion of POMCderived peptides is regulated differently in the anterior lobe

(AL) and neurointermediate lobr (NIL) of the rat pituitary. In

the AL, the secretion of POMC-derived peptides is positively

regulated by corticotropin-releasing factor (CRF and negatively

regulated by endogenous glucocorticoids released from the

adrenal cortex. Glucocorticoids also inhibit POMC production in

AL corticotrophs but have no effect on NIL cells that produce

POMC.

In the NIL, dopaminergic neurons from the hypothalamus impinge

on POMC-containing cells and inhibit the release of POMC-derived

peptides. As expected, the administration of dopamine

antagonists, such as haloperidol, stimulates the release of POMC

peptides from the NIL and increases the levels of POMC peptides

within the NIL. These agents have no effect on the release of

POMC peptides from AL corticotrophs (Douglass et al. 1984).

POMC cDNA clones have been used as hybridization probes to

accurately determine the effects of adrenalectomy and subsequent

dexamethasone (DM) administration on POMC mRNA levels in the

rat AL (Birnberg et al. 1983). Eight hours after adrenalectomy

the levels of POMC mRNA levels increased markedly, reaching

fifteenfold to twentyfold the control level at 18 days

postoperation When DEX was administered to rats 8 days after

adrenalectomy, the above events were reversed (Bimberg et al.

1983), and after 5 daily injections of DEX, POMC mRNA had

returned to control levels (figure 5). These results confirmed

previous experiments which used POMC mRNA translational activity

as a measure of POMC mRNA content.

The large changes in POMC mRNA levels in anterior pituitary

after adrenalectomy raised the question of whether

glucocorticoids regulate POMC gene expression at the level of

transcription. To test this possibility, POMC transcription

rates were assayed by the nuclear transcription method which

measures the number of RNA polymerase molecules transcribing

POMC genes in a given time period (1 and 4 hours after

adrenalectomy). The results show that the rate of transcription

increases by twentyfold in the anterior pituitary within 1 hour

of adrenalectomy. The observed increase in transcription rate

was completely suppressed by administration of DEX to animals

immediately after adrenalectomy.

31
FIGURE 5

Time-specific processing pathways of the POMC precursor

PRE: Presequence; N-TERM: N-terminal peptide; J: joining

peptide; CLIP: corticotiopinlike intermediate lobe peptide;

ß-END: ß-endorphin.

32
The above effects are specific to POMC gene expression in the

AL; POMC mRNA levels and the rate of transcription of the POMC

gene in the NIL are not altered by adrenalectomy or DEX

administration (Birnberg et al. 1983).

In situ hybridization has been used to determine that the

increase in POMC mRNA levels in the AL following adrenalectomy

is due to a number of factors, including enlarged cell volume

and an increase in the number of POMC-prducing cells (Gee and

Roberts 1982). This technique clearly allows for a more refined

analysis of the factors involved when a heterogeneous tissue is

being studied.

Cell-free translation studies and RNA dot blotting (Chen et al.

1983) have been used to study the effects of dopamine agonists

and antagonists on POMC mRNA levels in the rat NIL.

Administration of the dopamine antagonist haloperidol results in

a fourfold to sixfold (time-and-dose-dependent) increase in the

level of NIL POMC mRNA. This stimulatory effect is observed as

early as six hours after administration In contrast,

ergocryptine, a dopamine agonist, decreases twofold to threefold

the level of POMC mRNA in the rat NIL (Chen et al. 1983). The

time-dependent changes in POMC mRNA levels and the magnitude of

these changes suggest that dopaminergic compounds modulate POMC

mRNA levels in the NIL in the same fashion as they regulate POMC

peptide secretion. The mechanisms underlying dopminergic

modulation of POMC mRNA levels in the NIL remain to be

elucidated.

It is also worthwhile to note that dopminergic compounds have

no effect on POMC mRNA levels in the AL (Chen et al. 1983).

Proenkephalin Gene Regulation. Daily injections in rats for 2

to 3 weeks with haloperidol, a dopamine receptor antagonist,

increases twofold the level of Met-enkephalin in the striatum.

From these data, it was suggested that the prolonged blockage of

dopamine receptors by haloperidol accelerates the synthesis of

the enkephalin precursor molecule.

To investigate the possibility that haloperidol was modulating

the level of proenkephalin mRNA in the striatum, cell-free

translation and immunoprecipitation (Sabol et al. 1983) and

Northern blotting techniques (Tang et al. 1983) were employed.

Following chronic haloperidol treatment for 3 weeks, the levels

of proenkephalin mRNA in the striatum were increased twofold

(Sabol et al. 1983) to fourfold (Tang et al. 1983), consistent

with the concomitant elevation of striatal Met-enkephalin

content Thus, haloperidol elevates the Met-enkephalin content

in the striatum primarily by increasing the proenkephalin in

mRNA content in that tissue. This effect was specific to the

striatum since haloperidol had no effect on maintaining

proenkephalin mRNA levels in the rat hypothalamus, cortex, or

hippocampus (Tang et al. 1983).

33
TRANSLATIONAL AND POSTTRANSLATIONAL REGULATION OF OPIOID PEPTIDE

PRODUCTION

Opioid peptides become active only after they are cleaved out of

their precursor molecules. In some cases, other modifications

such as glycosylation, phosphorylation, amidation, or

acetylation must also occur in order to activate these peptides.

Amino acid sequences specify the enzymatic processes that lead

to activation of the opioid peptides. These processes usually

occur in a well-defined order as the proteins and peptides move

through compartments in the secretion pathway.

Most of the domains of the opioid peptides in the precursors are

flanked by pairs of basic amino acid residues (either Lys-Arg,

Lys-Lys, or Arg-Arg), suggesting that trypsinlike enzymes are

involved in the cleavage reaction. A carboxypeptidaselike

enzyme is thought to remove the C-terminal basic amino acid to

produce the bioactive peptide. However, other types of cleavage

recognition sites are also used, including a single Arg site in

proenkephalin and prodynorphin.

Carboxyterminal amidation has been observed in the formation of

α−MSH from POMC and a Met-enkephalin octapeptide from

proenkephalin. The C-terminal amino acid of peptides that

undergo amidation is followed by a glycine residue in the

precursors which is involved in transfer of the amino group and

is cleaved from the peptide during the amidation reaction.

Glycosylation of a protein at asparagine (Asp) residues requires

the sequence asparagine-x-threonine or -x-serine. Glycosylation

can also occur at serine or threonine (Thr) residues, POMC is

known to be glycosylated. Human and bovine proenkephalin also

have Asp-x-Thr sequences. However, as not all such sequences

are glycosylated in a protein, it is not possible to predict

which precursors will contain these oligosaccharides. In order

to demonstrate the existence of oligosaccharides, one must

isolate the protein and analyze its carbohydrate content or

carry out pulse label studies with radioactive sugars.

Other posttranslational modifications have been detected in the

processing of POMC, including phosphorylation, acetylation,

sulfation, and methylation (Herbert et al. 1984).

Tissue-Specific Processing of POMC, Proenkephalin, and

Prodynorphin

Processing of POMC in Anterior and Neurointermediate Lobes of

the Rodent Pituitary. Cultures of AL and NIL of rat pituitary

provide viable and convenient systems for studying expression of

POMC-derived peptides. In addition to the tissue-specific

differences in regulation of hormone release described earlier,

there are marked differences in the types of peptides derived

34
from the ACTH-ß-LPH portion of the precursor in the two lobes of

the pituitary. The AL contains predominantly the steroidogenic

hormone, ACTH1-39, while the intermediate lobe of the pituitary

contains high levels of α-MSH (ACTH1-13 with an acetylated Nterminus and an amidated C-terminus) and corticotropinlike

intermediate lobe peptide (CLIP) (ACTHl8-39) as shown in figure

5 (Scott et al. 1974; Roberts et al. 1978; Mains and Eipper

1979; Eipper and Mains 1980). Thus, ACTH]-39 is further

pressed in the neurointermediate lobe to yield α-MSH and CLIP

(Scott et al. 1973). The lobes of the rodent pituitary also

differ in the amounts of ß-LPH, ß-endorphin, and acetylated

derivatives of ß-endorphin that they contain. While the AL

contains mainly ß-LPH, the neurointermediate lobe has

predominantly ß-endorphin and derivatives of ß-endorphin

(Herbert et al. 1984) (see figure 5). Despite the differences

in the ACTH/endorphin peptides in the two lobes of the

pituitary, the forms of POMC that they contain are very similar

(Roberts et al. 1978).

Pulse-label and pulse-chase studies with rodent pituitary cells

show that the initial cleavages and glycosylation steps in the

processing of POMC are the same in the two lobes of the

pituitary (Roberts et al. 1978; Hinman and Herbert 1980). As

shown in figure 5, glycosylation of the N-terminal portion of

POMC occurs first in the γ-MSH region of the molecule. About

half of the POMC molecules are also glycosylated at Asn residue

29 in the ACTH portion of the molecule. After core

glycosylation is complete, cleavage occurs between ACTH and ßLPH, resulting in the formation of glycosylated ACTH

intermediates and B-LPH as in AtT-20-D16v cells. Another

cleavage then occurs to release glycosylated and unglycosylated

forms of ACTH (Roberts et al. 1978; Mains and Eipper 1979;

Eipper and Mains 1980; Hinman and Herbert 1980) and an Nterminal fragment. In the rodent anterior pituitary processing

essentially ceases at this point; but in the NIL ACTH is

processed to α−MSH and CLIP by cleavage in the middle of the

molecule. The N-terminus ACTH is then trimmed back to 13

residues (ACTHl-13) presumably by carboxypeptidases, amidated at

the C-terminus and acetylated at the N-terminus (Scott et al.

1973). The ß-LPH portion of POMC is cleaved in the NIL to form

γ−LPH and ß-endorphin. ß-Endorphin is then acetylated at its Nterminus and shortened by removal of four C-terminal amino

acids. Acetylation of ß-endorphin at its N-terminus destroys

its analgesic activity. Hence, this modification might be a way

of regulating the amount of active endorphin available in the

NIL.

Recent evidence suggests that the N-terminal portion of POMC is

also cleaved extensively in the pituitary. In the rodent NIL,

proteolytic cleavages occur at both pairs of basic amino acids

in the N-terminal portion of POMC. These cleavages give rise to

γ−MSH, which has been shown to be present in its glycosylated

form, and to an acidic peptide called a joining peptide (J)

(Seidah et al. 1981). Formation of these peptides is

35
essentially confined to the NIL since γ−MSH peptides are not

detected in the anterior pituitary. Hence, the N-terminal of

POMC is more correctly processed in the NIL of the pituitary

than in the anterior pituitary (Herbert et al. 1984; Douglass et

al. l984), as already demonstrated for the ACTH and ß-LPH

domains.

These results show that cleavage occurs at all of the pairs of

basic amino acid residues contained in POMC (figure 5).

Almost all of the POMC-derived products shown in figure 5 arise

by proteolytic cleavages at Lys-Arg sites. It appears that in

mammals this sequence is preferred as a cleavage site over ArgArg or Lys-Lys (as in the case of conversion of proinsulin to

insulin).

Processing of Proenkephalin and Prodynorpin. Detection of

imnunoreactive enkephalin peptides in the bovine adrenal medulla

provided impetus for using this tissue to study biosynthesis of

enkephalins. Since these subjects have been reviewed in depth

recently (Udenfriend and Kilpatrick 1983; Lewis and Stem 1983),

we will present only a brief summary emphasizing tissue-specific

differences in processing of proenkephalin.

A variety of enkephalin-containing peptides (ECPs), in addition

to the pentapeptides Met- and Leu-enkephalin, are present in

adrenal medulla and brain. Figure 6 shows the biosynthetic

relationship of these peptides in the adrenal medulla and brain.

The arrangement of the six Met- and one Leu-enkehalin units in

preproenkephalin is presented first. Met-enkephalin-Arg-Phe is

present at the C-terminal part of the precursor and the

octapeptide product, Met-enkephalin-Arg-Gly-Leu, comprises the

amino acid sequence 186 to 193 in the precursor. Larger ECPs

containing more than one enkephalin sequence are also observed,

including peptide E which has a Met-enkephalin at its N-terminus

and a Leu-enkephalin at its C-terminus,and peptide F which has

a Met-enkephalin sequence at each end. These smaller ECPs are

derived from larger peptides. For example, peptide E arises

from peptide I by cleavage at a Lys-Arg site and peptide F comes

from still larger fragments which have also been characterized

(figure 6). It is still not clear which of these peptides are

and products of processing and which are intermediates.

Bioassays indicate that peptide E, Met-enkephalin-Arg-Phe and

Met-enkephalin-Arg-Gly-Leu are very potent opioids. However,

peptide F and larger ECPs are not very active in assays of

opioid activity. The bioactivity assays suggest that peptide E

and the smaller ECPs are the true end products of biosynthesis,

whereas the larger fragments are intermediates in processing.

The proenkephalin peptide sequences flanked by Lys-Arg or LysLys sites are cleaved out of the precursor, whereas, the Metenkephalin sequence in peptide E that has an Arg-Arg on its Cterminus is not released from the precursor.

36
FIGURE 6

Tissue-specific processing pathways of the proenkephalin and

prodynorphln precursors

The products of proenkephalin processing are compared between the

adrenal medulla and brain. The anterior and the neurointermediate

lobe of the pituitary.
An amidated octapeptide, ECP, has recently been isolated from

bovine brain and human pheochromocytoma (a tumor of the adrenal

medulla). This peptide, which results from cleavage at a single

Arg residue in peptide E, is the first amidated opioid peptide

that has been isolated Cleavage at a single Arg residue also

occurs in the processing of several other precursors, including

proAVP (producing a glycopeptide), prodynorphin,

procholecystokinin, and progrowth-hormone-releasing-factor

(Douglass et al. 1984).

As in the case of POMC, the processing of proenkephalin is

tissue-specific. The major products of processing in the

adrenal medulla are the higher molecular weight ECPs, whereas in

the brain, one finds mainly the enkephalins and other small ECPs

(Udenfriend and Kilpatrick 1983; Weber et al. 1983; Liston et

al. 1983). Indeed, it has been shown recently that three of the

proenkephalin-derived peptides, synenkephalin, Met-enkephalinArg-Phe, and the octapeptide Met-enkephalin-Arg-Gly-Leu, are

present in different processing intermediates in the brain as

compared to the intermediates in the adrenal medulla (Liston et

Although the processing of prodynorphin has been

less studied, its proteolytic cleavages appear also to be

tissue-specific in the two lobes of the rat pituitary (Seizinger

et al. 1984). Dynorphin(l-17) and rimorphin, two of the opioid

peptides present in this precursor, exist in these forms in the

NIL but are only detected as part of 6000 MW polypeptide in the

AL Also, dynorphin(l-8) which is present in the NIL, is

undetectable in the AL, The peptides containing neoendorphin

sequences also differ in the two lobes of pituitary. While both

lobes contain α− and ß-neoendorphin, an 8000 MW polypeptide

immunoreactivity against neo-endorphin antibodies is detected in

the AL and not in the NIL. These data are summarized in figure

6 and show that, as for the two other opioid polypeptide

precursors, the pressing pathways of prodynorphin are tissuespecific.

Several mechanisms can be proposed to explain tissue-specific

processing of neuroendocrine peptide precursora One is the

existence of a different set of processing enzymes in different

tissues. The environment at the sites of processing could also

differ in different tissues. Another possibility is a

difference in the structures of the precursor molecules in

different tissues. In the latter case, a sequence difference in

the precursor molecule would dictate how the precursor is

processed in each tissue. Different translational modifications

of the precursor might also account for tissue differences in

processing. Different precursors could arise by selective

expression of one or more of a family of genes in each tissue or

from a single gene by alternate modes of splicing. Most of the

evidence to date suggests that differential processing of POMC

occurs at the level of processing enzymes in the pituitary.

First, there is only one POMC gene in the mouse (Uhler et al.

1983) and rat (J. Drouin, personal communication, 1984); second,

an extensive search for different forms of mRNA that might code

38
for different POMC forms in the two lobes of the pituitary has

been negative (Herbert et al. 1984); and third,

posttranslational modifications of POMC appear to be the saame in

the two lobes of the pituitary.

Thus, different sets of processing enzymes appear to be

responsible for tissue-specific expression of neuroendocrine

peptides. Posttranslational processing comprises the last steps

in the numerous events leading to expression of the opioid genes

and appears to be a major mechanism for generating diverse sets

of peptides in the opioid peptide system

In conclusion, we have tried at the generation of the opioid

peptides as the products of a complex cascade of events. All

the steps involved in the expression of a gene are used to

generate the greatest diversity of bioactive peptides. The

synthesis of the opioid peptides from only three precursors

provides a means of coordinating the synthesis of functionally

related peptides which could act together to mediate distinct

behavioral responses. Future experiments will provide

information about the role of opioid peptides in behavior. On

the other hand, the transfer of opioid genes from one cell to

another will reveal the basic mechanisms directing the genetic

express ion of the opioid peptides. We will then be in the

position of being able to control the production of the opioid

peptides and, therefore, to test specifically their actions in

the organism

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AUTHORS

Olivier Civelli, Ph.D.

Jim Douglass, Ph.D.

Edward Herbert, Ph.D.

Institute for Advanced Biomedical Research

The Oregon Health Sciences University

Portland, Oregon 97201

Haim Rosen, Ph.D.

The Hebrew University Medical School

Hadassa, Jerusalem, Israel 91010

Gerard Martens, Ph.D.

Katholieke Universiteit

The Netherlands

42
Proenkephalin Biosynthesis in the Rat

Richard D. Howells, Ph.D.

The discovery of opiate receptors in mammalian brain (Pert and

Snyder 1973; Simon et al. 1973; Terenius 1973) naturally stimulated speculation on the purpose of these sites. Surely they did

not survive eons of evolution for the chance consumption of

opiate alkaloids. Since none of the many substances which had

been proposed as neurotransmitters or neuromodulators had an

appreciable affinity for opiate receptors, the search began for

an endogenous ligand. Shortly thereafter, two substances were

isolated from porcine brain which could interact with opiate

receptors and mimic the pharmacological activity of morphine in

the guinea pig ileum bioassay (Hughes et al. 1975). These

substances were pentapeptides with the structures Tyr-Gly-GlyPhe-Met and Tyr-Gly-Gly-Phe-Leu and were named methionine

enkephalin and leucine enkephalin, respectively. It was fortuitously noticed that the sequence of [Met]enkephalin was

contained within ß-lipotropin (ß-LPH), a protein that was first

isolated from porcine pituitary glands (Li 1965). At about the

same time, several groups isolated and determined the structure

of other peptides with opiate activity that were derived from

ß-LPH which had C-terminal extensions of the [Met]enkephalin

sequence. These peptides became known as α−endorphin, γ−endorphin

(Ling et al. 1976), C'-fragment (Bradbury et al. 1976a), and

ß-endorphin (Cox et al. 1976; Li and Chung 1976; Bradbury et al.

1976b; Graf et al. 1976). ß-LPH and adrenocorticotrophic hormone

(ACTH) were shown to be derived from a common 31 kilodalton

precursor, proopiomelanocortin (Mains et al. 1977; Roberts and

Herbert 1977; Rubinstein et al. 1978; Kimura et al. 1979). The

entire primary structure of bovine preproopiomelanocortin

(prePOMC) was determined using molecular cloning and cDNA

sequencing (Nakanishi et al. 1979). PrePOMC contains 265 amino

acids with a molecular weight of 29,259. The precursor contains

the sequences of ACTH, ß-LPH, and another melanocyte stimulating

hormone-related peptide, γ-MSH. These peptides are bounded by

two basic amino acids (Lys or Arg), which presumably serve

as signals for a processing enzyme (Steiner et al. 1980)

to selectively release the active peptide. POMC is processed

differently in the anterior and intermediate lobes of the

pituitary (Mains and Eipper 1980). Although ß-endorphin was

originally thought to be the precursor of [Met]enkephalin, it

became apparent rather quickly that the opioid pentapeptides were

43
derived from a different precursor (Rossier et al. 1977;

Watson et al. 1978; Bloom et al. 1978; Lewis et al. 1978;

Nakanishi et al. 1979). It was clear from these studies that the

enkephalins and ß-endorphin were contained within discrete,

separate neuronal systems in the brain. Also, the sequence of

[Leu]enkephalin did not occur in prePOMC. Work then began anew

for the precursor to the enkephalins.

Bovine striatum was used originally as the starting point for

the extraction and purification of enkephalin precursors;

however, it became apparent that bovine adrenal medulla would be

more suitable for these studies. In 1978, Hökfelt and colleagues (Shultzberg et al. 1978) reported the presence of

enkephalin immunofluorescence in adrenal medulla. It was noted

that rat adrenal glands contained significantly less enkephalin

immunofluorescence compared with guinea pig adrenals. The

difference in enkephalin content was found to be even greater

between the adrenals of rats and that of dogs and cattle

(Viveros et al. 1979; Hexum et al. 1980). The bovine adrenal

gland was found to have several advantages over brain tissue as a

starting point for the isolation of enkephalin precursors (Lewis

et al. 1979). The total amount of enkephalin was greater and,

more importantly, the bulk of the enkephalin activity (90%) was

found to be contained within higher molecular weight (1-18 kD)

peptides which were presumably intermediates in the biosynthetic

pathway of the enkephalins. These larger peptides did not

crossreact with enkephalin antibodies or bind to opiate receptors

unless they were treated with trypsin; subsequent treatment with

carboxypeptidase B (CPB) increased the activity even more.

Digestion with these enzymes would liberate enkephalin sequences

if they were bounded by dibasic amino acids. Another advantage of

the bovine adrenal medulla was that the enkephalin-containing

(EC) peptides could be greatly enriched by purifying the secretory granules which contained almost all of the opioid

activity in the chromaffin cells. Udenfriend's group began the

systematic isolation and sequencing of these peptides. A totally

unexpected finding which arose from this work was that some of

the purified EC peptides contained more than one copy of the

enkephalin sequence. The first such peptide that was discovered

was named peptide F (Kimura et al. 1980), which has the Try-GlyGly-Phe-Met sequence at its N- and C-termini. Peptide I (Kimura

et al. 1980) was the first EC peptide shown to contain the

[Leu]enkephalin sequence; it also contained [Met]enkephalin. A

total of 16 different EC peptides (figure 1) were sequenced (see

Udenfriend and Kilpatrick 1983); the largest of these, an 18.2 kD

polypeptide, was shown to contain four copies of [Met]enkephalin

(Kilpatrick et al. 1982a). Although the putative enkephalin

precursor, proenkephalin (Lewis et al. 1980), had not been

isolated, there was considerable evidence to support a biosynthetic pathway beginning with proenkephalin which was processed subsequently by specific trypsin- and CPB-like enzymes to

yield smaller active opioid peptides. Ultimately, the entire

coding regions of bovine and human preproenkephalin were obtained

via recombinant DNA technology (Gubler al al. 1982; Noda et al.

44
FIGURE 1

Structure of Endogenous EC Peptides Isolated

Medulla.

From Bovine Adrenal

Adrenal medulla tissue was used to prepare partially purified

chromaffin granules. The granules were extracted in acid and

chromatographed on Sephadex G-75. The chromatogram was divided

arbitrarily into five peaks; some of the EC peptides contained in

these peaks were purified using HPLC. Regions for which the amino

acid sequence was not determined are in parentheses. The

sequences of [Met]enkephalin, [Leu]enkephalin, [Met]enkephalin-Arg-Gly-Leu and [Met]enkephalin-Arg-Phe are underlined.

45
1982; Comb et al. 1982). Preproenkephalin was shown to have

within its primary structure four copies of [Met]enkephalin and

one each of [Leu]enkephalin, [Met]enkephalin-Arg-Gly-Leu, and

[Met]enkephalin-Arg-Phe. All of these sequences are bound by two

basic amino acids, except for the heptapeptide, which is followed

by a chain termination codon. It had been predicted earlier that

the heptapeptide was at the C-terminus of proenkephalin (Lewis et

al. 1980). Both the octapeptide and heptapeptide have been shown

to bind to opiate receptors (Kilpatrick et al. 1981a; Stern et

al. 1979). In addition, [Met]enkephalin-Arg-Phe was found to be

eight times more effective than [Met]enkephalin on a molar basis

for eliciting antinociception in mice (Inturrisi et al. 1980).

Elucidation of the primary structures of prePOMC and preproenkephalin did not account for two other peptides which contained

[Leu]enkephalin, α−neo-endorphin (Kangawa et al. 1981) and

dynorphin (Goldstein et al. 1981). Evidently there was still

another precursor which contained these peptides. Investigations

into their origin led to the discovery of a third [Leu]EC peptide

(Kilpatrick et al. 1982b), rimorphin (figure 2), and a 32 amino

acid peptide which contained dynorphin at its N-terminus and

rimorphin at its C-terminus, the two being linked by a Lys-Arg

sequence (Fischli et al. 1982). It was shown by Kakidani et al.

(1982) that all of these [Leu]EC peptides were derived from a

single precursor named proenkephalin B (also known as prodynorphin and pronorphin). The cloned cDNA from pig hypothalamus

contained one copy each of α−neo-endorphin, dynorphin, and

rimorphin. Like the other opioid peptide precursor proteins, the

active peptide sequences have two basic amino acids at the NH2-

and COOH-termini.

All of the opioid peptides that have been isolated are derived

from one of the three precursors: POMC, proenkephalin, or

proenkephalin B ([Leujenkephalin can arise from proenkephalin or

proenkephalin B). Several structural similarities exist between

the three gene products besides the presence of enkephalin

sequences flanked by paired basic residues (figure 3). They are

all approximately the same size. All three contain a cysteinerich amino terminal region that is devoid of typical processing

recognition sites and is preceded by a signal peptide sequence.

The presence of an even number of cysteine residues in each

precursor suggests that disulfide linkages are likely present in

the mature proteins. These similarities suggest an evolutionary

relationship among the three different precursors.

Having elucidated the biosynthetic origin and details of the

pathway whereby [Met]- and [Leu]enkephalin are produced, we

began experiments to understand the regulation of proenkephalin

biosynthesis. The previously mentioned paper by Schultzberg

et al. (1978) was important not only for establishing the

presence of opioid peptides in adrenal medulla, but also for

discovering the influence of the splanchnic nerve on the enkephalin content of the rat adrenal medulla. The authors observed

that the rat adrenal ordinarily contained very little enkepha46
FIGURE 2

Structures of [Leu]EC Peptides Isolated From Posterior Pituitary

Glands.

47
FIGURE 3

Schematic Representation of the Three EC Peptide Gene Products.

The positions of cysteine residues and paired and single basic

amino acids serving as processing sites are indicated. Characterized products of processing are also shown.

48
lin like immunoreactivity; however, when the splanchnic nerve

which inners the gland was severed, there was a marked

increase in enkephalin immunoreactivity. Our group followed up

this interesting phenomenon on a quantitative basis (Lewis et al.

1981; Fleminger et al. 1984a). After denervation, there was a lag

period of 12 hours before EC peptides levels began to rise

()figure 4). The levels peaked 72 to 96 hours following surgery,

then declined slowly over several weeks. The observed changes in

EC peptiodes following denervation were in marked constrast to

those seen with catecholamines. While the EC peptide content of

the denervated glands was increasing, the catecholamine content

of the same glands was decreasing, reaching levels that were 30%

to 40% of the innervated glands after 24 hours. Thus, despite

evidence that enkephalins and catecholamines are stored and

concomitantly secreted from the same secreoty vesicles (Viveros

et al. 1979), they appear to be under seperate control.

We found that the increase in EC peptides at all time points was

due almost entirely to a polypeptide which was 20-30 kD (figure

5). This material is believed to be intact proenkephalin, based

on the following criteria (Fleminger et al. 1984b): it migrates

with an apparent molecular weight of 25,000, as estimated by gel

filtration chromatography; on high performance liquid chromatography, it eluted at much higher propanol concentrations than

the largest previously characterized EC peptide which is 18.2 kD;

treatment of the polypeptide with Lys-C endoproteinase yielded

[Met]enkephalin, [Leu]enkephalin, [Met]enkephalin-Arg6-Phe7, and

[Met]enkephalin-Arg-Gly-Leu (figure 6) in the same ratios that

are found in rat proenkephalin as deduced from sequencing rat

preproenkephalin cDNA (see below.)

Some processing of the "proenkephalin" was observed with time in

the denervated adrenals. As shown in figure 7, a peak of 3-6 kD

slowly increased with time. This peak reacted with [Met]enkephalin antisera, but not [Leu]enkephalin antisera, following

digestion with trypsin and CPB; and it crossreacted with

[Met]enkephalin-Arg-Phe antibodies without digestion. Based on

these results, we assume that this peptide is probably peptide B

(Stern et al. 1981).

Having characterized the increase in EC peptides following

denervation of the rat adrenal gland, we wanted to learn more

about the mechanisms underlying the effect. Were the increases

due, for instance, to increased transcription of the proenkephalin gene, increased translation of a fixed amount of

proenkephalin mRNA, decreased release, or decreased degradation

of the EC peptides? We found that after splanchnicectomy, there

was a several-fold increase in the steady-state levels of

preproenkephalin mRNA (figure 8) which became maximal (>tenfold)

after 24 to 48 hours (Kilpatrick et al. 1984). These results

indicated that the increased EC peptide levels following denervation were due to pretranslational mechanisms. It is interesting to note that the increase in preproenkephalin mRNA was

accompanied by a twofold to fourfold decrease in total poly(A)

49
FIGURE 4

Changes in [Met]EC Peptides And Catecholamines in Rat Adrenal

Glands After Denervation.

Aliquots of the tissue extracts of innervated (0) and denervated

glands (10-200 μl) were lyophilized, redissolved in 100 μ1

of 0.2 M N-ethylmorpholine acetate buffer (pH 8.0), and treated

with trypsin and carboxypeptidase B. [Met]enkephalin released

was assayed by a specific radioimmunoassay. Aliquots of tissue

extracts in 5% trichloroacetic acid/100 mM HCl (20 μ1) of the

innervated (0) and denervated gland were assayed for

catecholamines.

50
FIGURE 5

Size Distribution of [Met]EC Peptides in Tissue Extracts at

Various Time Intervals After Denervation on a Sephadex G-75

Column.

Aliquots (20-200 μ1) from each fraction were lyophilized, treated

with trypsin and carboxypeptidase B, and assayed for [Met]enkephalin, as described in figure 4. All panels, except for the

first two, show [Met]EC peptides in denervated rat adrenal

glands. The column was calibrated with markers showing the

elution position of hemocyanin (HC), RNase, and tyrosine (Tyr),

respectively.

51
FIGURE 6

Mapping of EC Peptides Derived From Proenkephalin.

An aliquot (1 ml, equivalent to 8 pmol of proenkephalin) of the

pooled fractions from RP-8 chromatography was evaporated to

dryness and redissolved in 200 μl of 0.2 M N-ethylmorpholine

acetate buffer, pH 7.4. Lys-C endoproteinase (40 μ1, 1 mg/ml)

was added and the solution was incubated at 37 °C for 16 hrs. The

solution was then heated to 100 °C and, after cooling, purified

carboxypeptidase B (40 μl, 5 μg/ml) was added. The solution was

again incubated at 37 °C for 4 hrs. 2-Mercaptoethanol was then

added (0.5% final concentration) and the solution was heated to

100 °C for 30 mins. After cooling, 125I-[Met]enkephalin was

added and the solution was applied to an RP-18 column (0.46 x 10

cm) and developed with a gradient of I-propanol. Each fraction

(1 ml) was dried down and redissolved in 200 μl of 0.2 M N-ethylmorpholine buffer, pH 8.0. [Met]enkephalin and [Leu]-

enkephalin immunoreactvities were assayed after treatment

with trypsin and carboxypeptidase B. Octapeptide and heptapeptide immunoreactivities were assayed directly without any

further enzymatic treatment. "Peptide E Derivative" refers to the

peptide Argo -Des(Arg6

[Leu]enkephalin) peptide E (rat proenkephain (187-206).

52
FIGURE 7

Processing of Proenkephalin in Denervated Rat Adrenal Glands.

(Lower) Aliquots (200 μl) of the fractions of a Sephadex G-75

column (see figure 5) were lyophilized and assayed with specific

antibodies to the heptapeptide [Met]enkephalin-Arg6-Phe7. (Upper)

Enlargement of the corresponding panels in figure 5. Numbers

represent the amounts of [Met]enkephalin that are higher than 1

pmol/mg of protein.

53
FIGURE 8

Time Course For The Effect of Unilateral Denervation on Rat

Adrenal Preproenkephalin mRNA.

Poly(A) mRNA (12 μg)from innervated (I) and denervated (D) rat

adrenal glands, rat liver (RL) and rat brain (RB) were denatured

by heating to 60 °C for 15 mins in the presence of 7.4% formaldehyde/0.9 M NaCl/0.09 M Na citrate (denaturing buffer).

Threefold serial dilutions of each sample were made up in

denaturing buffer and applied in a volume of 0.5 ml to nitrocellulose filters (8.0, 2.7, and 0.9 μg of adrenal samples and

8.0 to 0.1 μg of liver and brain samples, from left to right).

Wells were rinsed with 0.5 ml denaturing buffer, and the filter

was then baked, prehybridized, and hybridized with nicktranslated human proenkephalin cDNA (107

 cpm) at 42 °C, as

outlined in the legend to figure 9. Numbers refer to the hours

following denervation after which adrenal glands were removed.

54
mRNA. This indicated that neuronal activity exerts an influence

on proenkephalin gene expression and RNA metabolism in rat

adrenals. A drawback of the studies involving assay of preproenkephalin mRNA was that neither synthetic oligodeoxyribonucleotides nor human and bovine preproenkephalin cDNAs were

sensitive enough to detect preproenkephalin mRNA in control or

innervated adrenal glands (figure 9). We hoped that rat

preproenkephalin cDNA would provide a more sensitive probe for

these types of studies involving very low levels of the message,

so the molecular cloning of rat cDNA was undertaken.

Preproenkephalin mRNA was enriched by sucrose gradient centrifugation of poly(A)-containing mRNA from rat brain and was used

as a template for double-stranded cDNA synthesis. The cDNA was

inserted into the Eco RV site of the plasmid pBR322 using the

GC-tailing method and the resulting recombinant plasmids were

used to transform E. coli RR1 cells. A 30-base-long synthetic

oligodeoxyribonucleotide with a sequence that had been shown to

be identical in bovine and human preproenkephalin cDNA (Gubler et

al. 1982; Noda et al. 1982; Comb et al. 1982) was prepared to

screen the clone bank. The sequence of this probe is shown in

figure 10. Approximately 28,000 bacteria were screened from

which 10 positive clones were obtained. The plasmid (referred to

as pRPE-1) with the longest cDNA insert (about 1,200 bases) was

selected for sequencing, which was accomplished using the Maxam

and Gilbert (1980) technique. It was found that pRRE-1 cDNA

contained the entire protein coding region of preproenkephalin

(Howells et al. 1984; figure 11). Like the bovine and human gene

products, rat preproenkephalin has four [Met]enkephalin sequences and a single copy each of [Leu]enkephalin, [Met]enkephalin-Arg-Gly-Leu, and [Met]enkephalin-Arg-Phe. Each of these

sequences is bracketed by two basic amino acids, except the

heptapeptide which is followed by the termination codon UGA. It

should be noted that the sequence of the highly active peptide E

(Kilpatrick et al. 1981b) is entirely conserved in all three

species, even though there are a significant number of single

base changes.

The initiation site for translation has been tentatively assigned

to the methionine codon at positions 1 to 3, based on the amino

acid sequences of bovine and human preproenkephalin (figure 12).

Rat preproenkephalin would consist then of 269 amino acids with a

molecular weight of 30,936. This compares to 267 residues for

human and 263 residues for bovine preproenkephalin. The amino

terminus of rat preproenkephalin is most likely the Asp (coded

for by GAC) at position 24 of the amino acid sequence by analogy

to the bovine and human proteins. In those species, the Nterminal amino acid is Glu, which is coded for by GAA. The

signal peptide would then contain 24 largely hydrophobic amino

acids and terminate in Ala. This is typical of signal peptides

(Steiner et al. 1980). The rat signal peptide contains two

cysteine residues at positions 8 and 17, whereas the human and

bovine forms contain only a single Cys at position 8. All three

species contain six additional Cys residues (excluding those in

55
FIGURE 9

Effect of Splanchnicectomy on Rat Adrenal Preproenkephalin mRNA.

Innervated and denervated adrenal glands (0.3-0.4 gm) were

obtained from rats that had been unilaterally denervated for 24

hrs. Poly(A) mRNA was prepared and the entire sample from each

group of glands (30 μg from innervated adrenals and 6 μg from

denervated adrenals) was subjected to electrophoresis on 1%

agarose gels containing 10 mM methymercury hydroxide. Sixteen μg

of rat brain poly(A) mRNA were included as a standard. After

transfer onto nitrocellulose, the filters were baked and then

prehybridized for 4-12 hrs at 30 °C in 0.7 M NaCl/0.07 M sodium

citrate/0.05 M sodium phosphate (pH 7)/0.02% BSA/0.02%

Ficoll/0.02% polyvinylpyrrolidone/0.1 mg/ml salmon sperm DNA and

50% formamide. Hybridization with 32P-labeled synthetic oligodeoxyribonucleotide (2 x 107

 cpm) was carried out at 30 °C for

12-18 hrs in prehybridization buffer containing 10% dextran

sulfate. The filter was washed four times in 0.3 M NaCl/0.03 M

sodium citrate/0.1% SDS at room temperature for 10 mins each,

followed by four washes at 50 °C in 15 mM NaCl/l.5 mM sodium

citrate/0.1% SDS for 30 mins. An autoradiogram was exposed after

an overnight incubation at -70 °C.

56
FIGURE 10

Sequence of The Synthetic Oligodeoxyribonucleotide Used For

Probing Cloned cDNA For Preproenkephalin.

The partial amino acid sequence of peptide E and the corresponding codons that were shown to be present in both bovine and

human preproenkephalin mRNA are shown on top. Below is the

complementary sequence of the 30-base-long oligodeoxyribonucleotide probe that was synthesized for use as a probe. Numbers

above the amino acids refer to their positions within the

sequence of peptide E, which contains 25 amino acids.
FIGURE 11

Partial Nucleotide Sequence of Rat Preproenkephalin Messenger

RNA.

Nucleotides are numbered in the 5' to 3' direction beginning with

the first residue of the initiator codon AUG; nucleotides

preceding the AUG codon are given negative numbers. The sequence

is incomplete at both the 5'- and 3'-termini of the mRNA. The

predicted amino acid sequence. is displayed below the mRNA. Amino

acids are numbered beginning with the methionine residue coded

for by nucleotides 1-3. The sequences of [Met]enkephalin,

[Leu]enkephalin,[Met] enkephalin-Arg-Gly-Leu and [Met]enkephalin-Arg-Phe are boxed.

58
FIGURE 12

Comparative Amino Acid Sequences of Rat, Human, and Bovine

Preproenkephalin.

Rat preproenkephalin is displayed on top in each row, human

preproenkephalin in the middle, and bovine preproenkephalin on

the bottom. The human and bovine proteins are compared with rat

preproenkephalin; regions of homology with the rat sequence are

boxed. Deletions in the human and bovine sequences are denoted

with a line under the corresponding rat amino acid. Numbering is

as in figure 11. The sequences of [Met]enkephalin, [Leu]enkephalin, [Met]enkephalin-Arg-Gly-Leu and [Met]enkephalln-Arg-Phe are

shaded.

59
FIGURE 13

Comparative Hybridization of Different Preproenkephalin cDNAs

to Rat Brain Poly(A) mRNA.

Rat, human and bovine cDNAs (250 ng each) were labeled by nick

translation to a specific activity of approximately 108 cpm/μg.

Rat brain poly(A) mRNA was blotted onto nitrocellulose in

threefold serial dilutions (left, 6 μg; middle, 2 μg; right, 0.67

μg) in triplicate and each replicate was hybridized with one of

the preproenkephalin cDNAs (3 x 106 cpm). Autoradiograms of the

washed filters were developed after overnight exposure at -70 °C

and relative band intensities were measured by scanning laser

densitometry.

60
the signal peptide) which are located in the amino-terminal

region of proenkephalin; all occur at exactly the same positions

(amino acid residues 26, 30, 33, 48, 52, and 65). The amino acid

sequence of rat preproenkephalin is 82% homologous to both bovine

and human preproenkephalin, allowing for the additional amino

acids at positions 170 and 182 in the rat molecule relative to

the human and bovine molecules and at positions 85-87 and 183

relative to the bovine form. Rat preproenkephalin cDNA is 80% and

83% homologous to the bovine and human cDNAs, respectively, at

the nucleotide level.

As mentioned earlier, it was hoped that rat proenkephalin cDNA

would be a more sensitive probe than bovine or human cDNA. A 435

base pair (bp) PvuII fragment of pRPE-1 cDNA, extending from

nucleotides 165 to 600, was chosen as the probe, which was

compared with a 1000 bp bovine cDNA and a 918 bp human cDNA

fragment to hybridize to preproenkephalin mRNA in extracts of rat

brain (figure 13). When the band intensities were quantified

using scanning laser densitometry, the rat cDNA was shown to be

four times more sensitive as a probe for the detection of rat

preproenkephalin mRNA than the heterologous cDNAs. Moreover,

only rat cDNA was able to detect consistently the message in

normal rat adrenal glands. This probe is currently being used to

monitor changes in preproenkephalin mRNA following denervation of

the rat adrenal gland and to study proenkephalin metabolism in

other tissues.

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both a [Met]- and [Leu]enkephalin sequence. Proc Natl Acad Sci

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Kilpatrick, D.L.; Wahlstrom, A.; Lahm, H.W.; Blacher, R.; and

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Kilpatrick, D.L.; Howells, R.D.; Fleminger, G.; and Udenfriend,

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1984.

Kimura, S.; Lewis, R.V.; Gerber, L.D.; Brink, L.; Rubinstein, M.;

Stein, S.; and Udenfriend, S. Purification to homogeneity of

camel pituitary pro-opiocortin, the common precursor of opioid

peptides and corticotropin. Proc Natl Acad Sci USA

76:1756-1759, 1979.

Kimura, S.; Lewis, R.V.; Stern, A.S.; Rossier, J.; Stein, S.; and

Udenfriend, S. Probable precursors of [Leu]enkephalin and

[Met]enkephalin in adrenal medulla: Peptides of 3-5 kilodaltons. Proc Natl Acad Sci USA 77:1681-1685, 1980.

Lewis, R.V.; Stein, S.; Gerber, L.D.; Rubinstein, M.; and

Udenfriend, S. High molecular weight opioid-containing

proteins in striatum. Proc Natl Acad Sci USA 75:4021-4023,

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Lewis, R.V.; Stern, A.S.; Rossier, J.; Stein, S.; and Udenfriend,

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Science 208:1459-1461, 1980.

Lewis, R.V.; Stern, A.S.; Kilpatrick, D.; Gerber, L.D.; Rossier,

J.; Stein, S.; and Udenfriend, S. Marked increases in large

enkephalin-containing polypeptides in the rat adrenal gland

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Li, C.H. Lipotropin, a new active peptide from pituitary

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Li, C.H., and Chung, D. Isolation and structure of an untriakontapeptide with opiate activity from camel pituitary gland.

Proc Natl Acad Sci USA 75:1145-1148, 1976.

Ling, N.; Burgus, R.; and Guillemin, R. Isolation, primary

structure and synthesis of α-endorphin and γ-endorphin, two

peptides of hypothalamic-hypophysial origin with morphinomimetic activities. Proc Natl Acad Sci USA 73:3942-3946, 1976.

Mains, R.E., and Eipper, B.A. Biosynthetic studies on ACTH,

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Mains, R.E.; Eipper, B.A.; and Ling, N. Common precursor to

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64
Watson, S.J.; Akil, H.; Richard, C.W.; and Barchas, J. Evidence

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ACKNOWLEDGMENTS

Dr. G. Fleminger performed the peptide and catecholamine assays

and Dr. D. Kilpatrick quantified the changes in preproenkephalin

mRNA following denervation. The research reported herein was

carried out in the laboratory of Dr. S. Udenfriend.

AUTHOR

Richard D. Howells, Ph. D.

Roche Research Center

Roche Institute of Molecular Biology

Nutley, New Jersey 07110

65
Enzymes in the Metabolism of Opioid

Peptides: Isolation, Assay, and Specificity

Neville Marks; Myron Benuck; and Martin J. Berg

This chapter provides an overview of opioid peptide metabolism

with emphasis placed on inactivation (as illustrated by some work

from this laboratory) rather than conversion. This limitation is

necessary since processing covers a wide field, including the

genome itself, translation and translocation of the growing peptide chain, sorting and posttranslational modifications within the

rough endoplasmic reticulum or Golgi apparatus, and transport and

packaging to the extracellular compartments (see figure 1). Since

inactivation cannot always be differentiated from activation

(biodegradation may result in generation of a new active species

as, for example, conversion of a kappa to a delta agonist), I have

included a brief section on recent aspects of conversion (or

transformation) and its implications to regulation of opioid

peptide activity.

Despite rapid progress in the cloning and sequence of preprohormones (see Civelli and Howells, this volume), very little is known

about the enzymes associated with processing. Since it is

difficult to reconstitute in vitro a full processing system (other

than through the use of whole cell preparations), the strategy for

many investigators has been to use prohormonal intermediates in

order to identify processing or inactivating enzymes (see Chaiken,

this volume). Some common enkephalins are listed in figure 2,

along with enzymes identified as cleaving at one or more sites.

In sane cases, more than one category of enzyme can cleave the

same site; for convenience, enzymes acting at the same site are

compared in this chapter in tabular form. Much of the older

literature has been reviewed (see Marks 1977; Marks et al. 1982a)

amd only recent citations are given. Enzymes acting at sites 1,

2, and 3 result in complete loss of opioid properties for all

forms shown (figure 2). Action at the other sites can result in

inactivation or conversion to smaller active opioid peptides.

The existence of multiple pathways for the degradation of a neuropeptide poses questions on the significance of each enzyme.

Several strategies have been used to evaluate the importance of

66
FIGURE 1

Co- and Frost-translational events associated with maturation of

gene product (PO). Convertases can act at sites adjacent to pairs

of basic residues as depicted, or at other linkages (renin acts at

Leu-Leu). Those acting at pairs of basic residues yielding Cterminal extensions, such as Arg-Lys, require further trimming by

carboxypeptidases.

individual. enzymes. These include localization with respect to

opioid receptors and substrate, and inferences that can be drawn

from the biological effects of biostable analogs or specific

inhibitors of the enzyme. Among factors that have to be considered in extrapolating from in vitro to in vivo are: environmental influences (pH, ion, cofactors, inhibitors) and the physiological status of the tissue (addictive states, physical dependency, stress, etc). There is also a question on whether there

are specific hormonal degrading peptidases or ones with broad

specificity which recognize peptide domains having the correct

67
FIGURE 2

Abbreviations: ME, Met-enkephalin; MEA, Met-enkephalin-Arg; MEAP,

Met-enkephalin-Arg-Phe; MEAGL, Met-enkephalin-Arg-Gly-Leu.

Dynorphin l-8 and 1-13 are derived from dynorphin A.

Exopeptidases: , aminopeptidase(ayrlamidase, LAP);

diaminopeptidase I or III (cathepsin C, DAP-III); angiotensin

converting enzyme (PD-A, ACE); lysosomal carboxypeptidase A

(cathepsin A); carboxypeptidase B; neutral

carboxypeptidase.

Endopeptidases: , metalloendopeptidase (E.C.3.4.24.11);

soluble (phosphoramidon insensitive) metalloendopeptidase;

synaptic endopeptidase; cysteine endopeptidase (cathepsin B).

For citations, see text.

conformational and structural requirements. The discovery of

enkephalins and other neuropeptides has spurred the enzymologist

to reinvestigate a number of enzymes in the contact of their

physiological roles. In some cases, enzymes have been rediscovered and, in others, there is evidence for presence of new

enzymes hitherto unsuspected. The characterization of such

enzymes provides a basis for the rational design of potent analogs

or specific inhibitors having clinical potential.

METHODS

This chapter on brain neuropeptidases is illustrated with work

from this laboratory. Purification schemes have appeared for

aminopeptidase (Marks et al. 1968; Berg and Marks 1984), metalloendopeptidase (Benuck et al. 1981), cysteine proteinase (Suhar and

Marks 1979; Suhar et al. 1981), and lysosomal carboxypeptidases

(cathepsin A) (Grynbaum and Marks 1976; Marks et al. 1976, 1981).

68
Measurements with chromogenic or fluorogenic substrates were

undertaken as previously described in the publications. For

studies with opioid peptides, the products were separated by

hydrophobic chromatography;or by high pressure liquid chromatography (HPLC) using isocratic or gradient procedures (Eenuck et

al. 1982a, 1982b, 1984). In some instances, peaks separated by

HPLC were identified further by amino acid or end-group analysis

using dansyl or other procedures as described. The C-termina1

residues were examined by use of carboxypeptidase Y followed by

prederivatization and HPLC separations. The potency of various

enkephalin analogs was evaluated by the hot-plate or tail-flick

assays (Jacquet and Marks 1976).

SITE ONE CLEAVAGES

Soluble Aminopeptidases

Several groups have isolated soluble site 1 cleaving enzymes as

summarized in tabular form in table 1. The term “soluble” is

operational since since groups used hypotonic buffer or even water

(see Schnebli et al. 1979; Hersh and McKelvy 1981) and thus may

have included loosely bound (membrane) or vesicle forms.

Different methods of extraction and, in some cases, assay probably

account for the wide divergence in Mr and other properties,

although species differences also may be a factor. Enzyme has

been purified 50-to 2,000-fold from man, monkey, rat, mouse, and

bovine brain, and consist of two groups: puromycin sensitive

(column 1, the maiority of enzymes purified) and puromycin insensitive (mouse brain leucine aninopeptidase is the only example

available).

Aminopeptidases in table 1 are nonspecific and degrade a variety

of polypeptides with N-terminal free groups, such as enkephalins,

angiotensins, melanostatin, and adrenocorticotropic hormone

(ACTH). Within the group of puromycin-sensitive enzymes, separate

categories exist based on preferential specificity toward basic

(-B) or neutral (-N or -M) naphthylamide substrates. There was

some evidence in earlier studies for presence in brain of enzymes

hydrolyzing Arg- preferentially to Leu- or Ala-2NA (Marks et al.

1968). These enzymes, termed “arylamidases,” were shown active

toward a large number of peptide substrates (Marks 1977).

Hayashi (1978) showed that monkey brain arylamidase purified with

the aid of Arg-2NA sequentially degraded enkephalin to release all

substituents, and this was confirmed for enzyme purified from rat

(Schnebli et al. 1979) and bovine brain (Hersh and McKelvy 1981)

with the aid of Tyr-2NA. Traficante et al. (1980) observed the

presence of an aminopeptidase in soluble extracts of human striatum having a restricted specificity and purified with the aid of

labeled enkephalin. Arylamidases purified from different species

are inhbited by metal chelating reagents and can be reactivated

with Zn2+, Co2+, or Mn2+.

69
TABLE 1

Aminopeptidases Degrading Enkephalin at Sites 1 and 2

(Tyr-Gly or Gly-Gly)

Aminopeptidase Leucineamino- Diaminopeptidase peptidase III

(E.C.3.4.11) (E.C.3.4.11.l) (E.C.3.4.14)

pH optima 6.5-7.5 8.5 8.5-9.0

Mr(Kd)

62-192 > 350 80-110

Specificity Arylamides Leu.NH2 Arg-Arg-2NA

di, tripeptides di, tripeptides Tetrapeptides

Enkephalins Enkephalins Enkephalins

Angiotensins MIF Angiotensin-II

MIF Proteins

Kinins

ß-endorphin

Dynorphins

Endorphins

Inhibitors Chelators Chelators

-SH agents -

Puromycin

Bestatin Bestatin

Asbtin

Chelators

-SH agents

DFP

Comments Known also as Known also as Properties of a

arylamidases. cytosolic amino- serine protease.

Consists of peptidase-I, but Largely

more than one particulate forms cytosolic.

group in observed in brain.

cytosol and

membranes.

purified 70-2,000 x Purified 2,000 x purified 50-1,500 x

Data taken from the following sources with species in parentheses. Puromycin sensitive aminopeptidases (column 1): Marks et

al. 1968 (rat); Suszkiv and Brecher 1970 (bovine); Hayashi and

Oshino 1977 (monkey); Traficante et al. 1980 (human striatun);

Schnebli et al. 1979 (rat); Wagner et al. 1981 (rat); Hersh and

McKelvy 1981 (bovine); Shimmura et al. 1984 (monkey membranes);

Hersh 1981 (rat membranes); Hui et al. 1983 (rat membranes). The

second column lists puromycin sensitive leucine aminopeptidase

purified from mouse brain by Neidle (1981). The third column

lists DAP-III purified from brain or pituitary (McDonald and

Schwabe 1977; Lee and Snyder 1982; Marks and Sachs 1983) and

unpublished findings.

Hydrolysis of peptides at the N-terminal is related to peptide

size and conformation as shown for a series of dynorphin-related

peptides (table 2); values for peptide are 50 to 100 times

70
those with 13 and 17 residues. Rapid inactivation of pentapeptides is in keeping with the notion that they act as putative

neurotransmitters. Given the high concentration of aminopeptidases and the low concentrations of peptide in the range of ng per

g wet weight, along with a Km of about 20 μM plus a high turnover

rate, it is understandable that enkephalins have a relatively

short half-life in vivo (Jaquet and Marks 1976). Analogs with

D-Ala or other residues blocking the action of aminopeptidases

have longer duration of action in vitro and sometimes in vivo (see

Marks et al. 1982a).

TABLE 2

Degradation of Dynorphins by Purified Aminopeptidase

Peptide

YG

YGG

YGGF

YGGFL

TGGFL-NH2

YGGFLR

YGGFLRRI

YGGFLRRIRPKLK

YGGFLRRIRPKLKWDBQ

Relative activity

1.0

0.8

0.8

14.3

15.2

6.1

4.4

0.3

0.14

Aminopeptidase activity (cleavage of Tyr1-Gly2 bcmd) was assayed

by ultra violet detection of tyrosine by HPLC on a Spherisorb C18

column with particle size of 5 u. Activities expressed relative

to Tyr-Gly degradation (4.4 nmol mg-1 min-1). Values are the

means of three or four determinations agreeing within 5%. The

single letter code used for amino acids was: Y-Tyr, G-Gly , F-Phe,

L-Leu, R-Arg, I-Ile, P-Pro, K-Lys, W-Trp, D-Asn, B-Asp, Q-Gln,

M-Net, Reprinted from Berg and Marks (1984) by permission of Alan

R. Liss, NY. Copyright 1984, Alan R. Liss, Inc.

Another strategy to enhance action of injected enkephalins is to

use aminopeptidase inhibitors. Surprisingly, there have been few

studies on puromycin with respect to its action on degradative

enzymes, albeit it has been subject to studies concerning its role

in elongation of the growing polypeptide chain. In an early

study, we found that substituting the amino acid moiety (methoxyPhe) for other residues (Tyr, Leu, beta -alanine, methoxy Tyr-Gly ,

Tyr methoxy Gly) led to loss of potency (Marks and Lajtha 1970)

with even lower activity for the aminonucleotide (see also Hersh

1982). Aminopeptidases are inhibited by bestatin and amastatin

(Barclay and Phillipps 1980) : analogs of bestatin (replacing Leu

with Lys) were observed by Wagner and Dixon (1981) to be more

potent toward rat brain "soluble" enzyme (compare to membranebound forms below). Aminopeptidases are inhibited also by various

metal chelating reagents or hydroxamates (Blumberg et al. 1981).

71
Formation of des-Tyr Enkephalins

Sane studies have been directed to the formation of des-Tyr

derivatives and their metabolism. Many of these derivatives

appear to have nonopioid properties when injected centrally

(Burbach et al. 1980; Walker et al. 1982). Action by soluble or

membrane-bound aminopeptidases can provide a pathway for their

formation. Some aminopeptidase preparations sequentially release

all five amino acids of enkephalin while others preferentially

release only Tyr. The Gly-Gly is believed to retard the action of

aminopeptidases and this may account for the accumulation of GlyGly subunits in digests of enkephalin made with brain homogenates

(Marks 1977; Stem and Marks 1979).

Des-Tyr enkephalin serves as a substrate for angiotensin converting enzyme, yielding two dipeptides (see Marks et al. 1982a),

and for a peptide dipeptidase recently purified fron brain to

homogeneity by Neidle and Kelly (1984). The latter acts preferentially on tetrapeptides with an aromatic residue in the third

position (e.g., des-Tyr enkephalin, ACTH 7-10).

Morphiceptin

The synthetic mu agonist Tyr-Pro-Phe-Pro.NH2 illustrates the

potential for characterizing novel enzymes using biologically

active materials. Morphiceptin was not a substrate for purified

site 1 enzymes described in the preceding paragraphs, yet was

degraded by brain homogenates (Marks et al. 1984). Further

studies revealed the presence of an aminopeptidase P-like enzyme

active toward X-Pro peptides, such as morphiceptin itself, TyrMIF, substance P, and kinin-9 (Neidle et al. 1984). This enzyme

demonstrates that proline (Pro) in the second position can affect

significantly the specificity of an aminopeptidase. Pro in the

first position is recognized by arylamidases, e.g., MIF (Pro-LeuGly.NH2) (Marks 1977). Another class of enzyme with restricted

specificity is aminopeptidase A. Recently, Kelly et al. (1983)

described an enzyme purified with Asp-Phe-MeE(aspartame) that

cleaved aspartyl peptides or ACTH 5-10 (Glu- ) but was inactive

toward enkephalin.

Menbrane-Bound Aminopeptidases

Early studies with brain membranes pointed to the presence of a

site 1 enzyme in brain membranes having a significantly lower

affinity than other enkephalin degrading enzymes (see Schwartz et

al. 1981). In our studies, illustrated in figures 3 and 4, Tyr

was found to be released in higher amounts than Tyr-Gly-Gly at all

substrate concentrations, although at 50 uM the ratio was smaller

than at 450 uM. Membranes may contain more than one site 1

cleaving enzyme responsible for the changes seen with alteration

in substrate concentration. As in the case of soluble enzyme,

there was a relationship between peptide size and rates; rates for

the pentapeptide were 27-to 40-fold higher than for dynorphin l-10

or 1-13 (table 4).

72
Enzyme cleaving site 1 has been purified from membranes of rat and

monkey brain (Hersh 1981; Hui et al. 1983; Shimamura et al.

1984). For the most part, these resemble arylamidases of the

“soluble” fractions. Hersh (1981) separated two kinetically different forms, with one more active toward Arg than Ala 2NA; the

type that was active against the neutral substrate had the highest

affinity for enkephalin (Km 20 uM) and was inhibited by low concentrations of puromycin (Ki 10-6M). The membrane-bound enzyme of

Hui et al. (1983) was inhibited by bestatin and anastatin. In a

comparison of bestatin analogs, Shimamura et al. (1984) observed

highest inhibition by those with Leu or Met and some activity for

analogs with dipeptide Leu-Arg, Leu-Asp. The use of bestatin analogs with differently charged amino acids provides potential

FIGURE 3

Comparison of Leu-enkephalin (E5) and Met-enkephalin-Arg6-Phe7

(E7) breakdown by time of incubation with rat striatal membranes.

Results were plotted as nmol of product recovered from 100 ul of

incubation mixture containing 45 nmol E5 14 ug protein as compared

to 14.5 nmol E7

were Tyr and Gly-Gly-Phe-Leu and for E7 were Arg-Phe and

and 0.7 ug protein. Note major products for E5

Met-enkephalin. Benuck et al. 1982b. Copyright 1982, Pergamon

Press.

73
FIGURE 4

Effect of E5 concentration on rate of Tyr vs Tyr-Gly-Gly release

incubated in presence of whole brain SPM. Benuck et al. 1982b.

Copyright 1982, Pergamon Press.

probes to separate aminopeptidase N and B. Amastatin, a microbial

peptide with the structure amino-2-hydroxyl-5-methyl hexanoylVal-Val-Asp, is an inhibitor of aminopeptidase A and LAP. Analogs

prepared with C-terminal peptides of varying length wer shown by

Tobe et al. (1982) to be specific inhibitors of aminopeptidase A.

In our studies, amastatin was equipotent to bestatin and puromycin

for the inhibition of purified brain aminopeptidase from cytosol

(Berg and Marks 1984).

SITE 2 CLEAVAGE (GLY-GLY)

Since the yield of Tyr-Gly from opioids is low, less attention has

been paid to the role of diaminopeptidases (IMP-enzymes). Such

enzymes are widely distributed in tissues and can be grouped according to their chromogenic substrate, pH, localization, and -SH

or Cl- requirements (Marks 1968; McDonald and Schwabe 1977).

Those implicated in enkephalin metabolism include DAP-1 (cathepsin

C, a lysosomal enzyme active at pH 5.5 in presence of -SH and Cl-)

and DAP-III (largely cytosolic, pH 9.0 optimum for hydrolysis of

Arg-Arg-2NA or cogeners) (Lee and Snyder 1982; Marks and Sachs

1983).

74
Incubation of purified brain P2 or SPM membranes at pH 5.5 with

enkephalin led to release of Tyr-Gly by a pathway stimulated by

-SH and Cl-, leading to our conclusion that DAP-I is a potential

enkephalin degrading enzyme (Marks and Sachs 1983). This enzyme

has been purified from pituitary and other tissues and has been

extensively studied; it is regarded as nonspecific and can be used

to sequentially degrade peptides such as glucagon. Hazato et al.

(1982) isolated a DAP enzyme from monkey particulates and observed

release of Tyr-Gly at pH 7.6; they indicated that their DAP enzyme

required metals for activity. Lee and Snyder (1982) purified a Mr

86,000 protein from rat brain cytosol and observed cleavage of

enkephalin and angiotensin-II or its related peptides; 15% of the

activity was observed at pH 6.5 and 60% at pH 7.4, indicating

actions within the physiological range. Enzyme was high in pituitary when soluble extracts were compared but lower in pituitary

particulates compared to other brain areas. Enzyme was unaffected

by 10-4M puromycin and could be differentiated from other aminopeptidases. Specific inhibitors are not available, but W-III

was shown to be inhibited by tyrosyl dipeptides among others. In

our studies on purified preparations of DAP-II and DAP-III

obtained from brain, we showed that the former acted only on tripeptides (as a carboxytripeptidase) and the latter degraded larger

opioids except dynorphin 1-l3; the action of cytosolic and membrane-bound forms decreased with peptide size (table 3).

TABLE 3

Substrate Specificity of Rat Brain DP-III

Substrate Cytosolic Membrane bound

Leu-enkephalin

Met-enkephalin

Met-enkephalin-Arg-Phe

Dynorphin 1-13.

H-beta endorphin

100* 100

184 77

43 11

0 0

12 11

100 ul contained 4-12 ug purified enzyme protein incubated with 40

nmol peptide for 75 min and analyzed for Tyr-Gly by HPLC.

*Represented release of 7-8 nmol of Tyr-Gly. From Marks and Sachs

(1983) and unpublished findings.

SITES 6 AND 7 CLEAVAGES

The specificity of the enzymes shown in figure 2 indicate that

they convert MEA or MEAP to shorter active forms but do not

inactivate the pentapeptide (HE). Rat brain P2 membranes contain

carboxypeptidases active at pH 7.6 in the metabolism of MEAGL

(cleavage of Gly-Leu) and dynorphin 1-l3 (cleavage of Leu-Lys)

(Benuck et al. 1984; Leslie and Goldstein 1982) (table 4).

Cleavage at the C-terminal of MEAGL to form MEA equaled in rate

that of aminopeptidase at site 1; that of the dynorphin 1-13

exceeded rates for sites 1 and 3 cleavages. Previously, Grynbaum

75
and Marks (1976) found activity toward carboxypeptidase A

(Z-Phe-Leu) and B (Z-Ala-Arg) substrates in purified rat brain P2

fractions and in subfractions. Brain carboxypeptidases active at

physiological pH have neither been purified nor have their actions

toward neuropeptides been established.

Rat brain homogenates and P2 subfractions contain a carboxypeptidase-degrading Z-Glu-Tyr and other N-protected tyrosyl dipeptides

which are active at pH 5.5 and capable of metabolizing angiotensin-II, myelin basic protein peptides, and MEAP with removal of

Phe (rapidly) and Arg (slowly) (Grynbaum and Marks 1976; Marks et

al. 1976, 1981, 1982a, 1982b). Enzyme can be purified several

hundredfold from detergent extracts of particulates and this

allows its properties to be examined. Results indicated that it

resembled cathepsin A or lysosomal carboxypeptidase A. Hook et

al. (1982) and Hook and Loh (1984) showed that secretory granules

of rat pituitary lobes contained a carboxypeptidase B-like enzyme

converting iodinated Met-enkephalin-Arg to Met-enkephalin, or ACTH

1-17 (Gly-Lys-Arg) to ACTH 1-15 (Gly). An enzyme with similar

Properties (inhibited by metal chelating agents and activated by

Co2+) was purified from adrenal chromaffin granules and the pituitary lobe that also converted MEA (see table 5) (Fricker and

Snyder 1982; Supattopone et al. 1984). This enzyme was assayed

with synthetic substrates such as Dns-Phe-Ala-Arg or 3H-benzoyl on

the N-terminal (Stack et al. 1984); activity was highest in pituitary compared to other regions. Carboxypeptidase B-like enzymes,

TABLE 4

Degradation of Dynorphin Fragments and of Met-EnkephalinArg-Gly-Leu by Whole Brain SPM

Rate (nmol/mg protein/min)

Substrate Tyr Tyr-Gly-Gly+ C-terminal C-terminal

dipeptide amino

acid

Dynorphin sequence

1-5 (Leu-enkephalin)

1-6

1-8

8.2 0.7 0.7+(1.4)a

1.9

0.3

0.1 3.5++

4.0 0.5 1.2++

1-13 0.1 0 0.7

1-17 0.2 0.1 0

Met-enkephalin-Arg-Gly-Leu 6.0 0.6 nd 7

+ Measured in the presence of MK 421 and bestatin.

++ Based on formation of dynorphin 1-6 or 1-4.

a Based on release of Tyr-Gly-Gly in the absence of inhibitor.

nd: not determined.

Sequences are based on the structure of dynorphin 1-17: Tyr-GlyGly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln. SPM

containing 50-100 ug of protein was incubated with 10 nmol of peptide in 0.1 ml at 37°C in 20 mM Tris buffer, pH 7.6. Products

were assayed by HPLC as described in the Methods.

76
TABLE 5

Acidic Carboxypeptidases Mediating Conversion or Transformation

Carboxypeptidase A

E.C. 3.4.12A.l

Carboxypeptidase B

Data compiled from Grynbaum and Marks (1976) and Marks et al.

(1976, 1981 , 1982a, 1982b) (carboxypeptidase A); Hook and Loh

(1984), Supattopone et al. (1984), and Stack et al. (1984)

(carboxypeptidase B). GEMSA, guanidoethylmercaptosuccinic acid;

GPSA, guanidopropylsuccinic acid.

active at acid pH having a thiol requirement, have been purified

from spleen and shown to have a broad specificity (McDonald and

Schwabe 1977). Brain enzymes could be involved in trimming of

prohormonal intermediates formed by cleavage at sites adjacent to

basic residues (figure 1).

SITE 3 CLEAVAGE (-Gly-Phe)

Angiotensin Converting Enzyme (ACE, PD-A)

ACE has been the subject of extensive studies largely as a result

of its pivotal role in the renin-angiotensin system and its

vasoactive properties (Soffer 1981). Studies conducted on brain

SPM using enkephalins exclude ACE as a key enzyme for inactivation, but do point to a possible role for conversion of larger

77
forms (Marks et al. 1982a; Benuck et al. 1982b). Brain enzyme

cross-reacts with antibody generated to ACE of peripheral organs,

and is widely distributed in the brain as shown by immunohistochemical work or binding by inhibitors in circumventricular

organs, substantia nigra, and the striatum (Strittmatter et al.

1984; Chevillard and Saavedra 1982). In subcellular fractions,

ACE is particulate bound and found in cellular debris, and in the

P2 fraction and microsomes (Benuck and Marks 1978).

Brain enzyme has been purified from detergent extracts of brain

particulates by ion-exchange, and by affinity procedures using

ricin-Sepharose (Yokosawa et al. 1983a), immunoaffinity to prepare

an immobilized form (Benuck et al. 1981), or the lysyl analog of

MK-421 (MK-521) linked via the epsilon grouping to Sepharose

(El-Dorry et al. 1982; Benuck and Marks, unpublished findings).

Purified enzyme acted on a variety of enkephalins, including ME or

LE, MEAP, and dynorphin 1-8, but not on dynorphin 1-13 and 1-17

(see legend-table 6). The Km for enkephalins was 50 to 100 uM,

yet SPM fractions enriched in ACE from striatun or whole brain at

these concentrations did not act rapidly at site 3 (as confirmed

by use of specific inhibitors). Activity, however, was observed

at higher concentrations of substrate (table 4). The action of

membranes at these concentrations correspond with the specificity

of the purified enzyme (see relative rates, table 6). Reasons for

the discrepancies between the rates for the purified enzyme

(active at lower substrate concentration) and SRI are unclear but

might be related to altered affinities of membrane-bound forms.

ACE has served as a useful model for the design, on a rational

basis, of specific inhibitors (see Cushman and Ondetti 1980; Marks

et al. 1982a; Roques, this volume). Modifications of succinyl

proline to enhance interactions with the metallo- and other subsites led to the synthesis of captopril, which is active at about

Ki 10 nM range. Presence of a free carboxyl group is not mandatory since affinity purified ACE from brain or lung can recognize

the P4-P3 residues of substance P with release of the C-terminal

tripeptidyl amide (Yokosawa et al. 1983b; Cascieri et al. 1984).

The model proposed for the catalytic center will require modification to accommodate the “endopeptidase”-like function of ACE.

This may explain the action of centrally applied ACE inhibitors on

increased levels of iR-substance P in rat brain (Hanson and

Lovenberg 1982) or following intravenous injection on the enhancement of substance P stimulation of rat salivary gland (Cascieri et

al. 1984).

The properties of purified brain ACE as compared to other site 3

enzyme are shown in table 6, which includes activity toward

enkephalin substrates. Conversion of angiotensin-I to -II is

enhanced by high (100 to 150 mM), and inactivation of kinins by

low, anion (Cl-) concentrations; anion levels intracellularly have

been invoked as one mode of regulation. A very large number of

inhibitors have been synthesized and their effects in vivo have

been summarized recently (see Symposium 1984). In view of the

central actions of enkephalin on blood pressure, there is scope

also to examine interaction of ACE inhibitors on enkephalinergic

systems (see Summy-Long et al. 1981; Marks and Benuck 1983).

78
Metalloendopeptidase

Several different lines of evidence showed that the non-ACE pathway acting on site 3 of pentapeptides was a metalloendopeptidase

similar in properties to one purified earlier by Kerr and Kenny

(1974) from kidney brush border. This enzyme has "thermolysin"-

like specificity, although it differs from the bacterial enzyme in

several respects. Clues concerning its presence in SPM were provided in studies conducted with SPM and enkephalin; at low substrate concentrations, cleavage occur-red at Gly-Phe with release

of Tyr-Gly-Gly, and at the C-terminal dipeptide (incubation conditions adjusted to reduce inopeptidase action by addition of

bestatin, or use of D-Ala2 analogs). Initially, this enzyme was

termed a dipeptidyl carboxypeptidase or Tyr-Gly-Gly generating

enzyme (see Schwartz et al. 1981; Marks et al. 1982a). Almenoff

et al. (1981) noted that purified bovine pituitary or brain

metalloendopeptidase (assayed with synthetic substrates, see table

6) also cleaved pentapeptide enkephalins at site 3. Fulcher et

al. (1982), using purified kidney enzyme or SPM, observed that

site 3 cleavage was inhibited by phosphoramidon or Thiorphan.

These data established that site 3 enzyme of SPM was a metalloendopeptidase of the thermolysin type and has received the

designation E.C.3.4.24.11 (Matsas et al. 1983). This enzyme has

been purified fran kidney brush border, intestine, pituitary, and

brain (Fulcher et al. 1983; Almenoff and Orlowski 1983, 1984) and

has been observed in dog pancreatic membranes (Mumford et al.

1980, 1981). It can be purified from detergent extracts by a

number of techniques that include ion-exchange and immunoabsorbent

chromatography using polyvalent or monovalent antibodies (Fulcher

et al. 1983) and affinity chromatography on lentil-lectin columns

(Benuck et al. 1982a). Metalloendopeptidases have a Mr of about

90,000, are glycosylated, and are characterized by phosphoramidon

inhibition.

Purified enzymes are nonspecific, degrade insulin B chain or a

variety of synthetic substrates, and also degrade a number of

neuropeptides at one or more sites. Many of the sites amtain

hydrophobic residues on the amino side of the vulnerable bond,

although there are exceptions. Cleavage at sites adjacent to one

or more basic residues, e.g., dynorphin 1-13 (figure 2), imply a

role in processing for this widely distributed membrane-bound

enzyme.

Aside from phosphoramidon (a microbial sugar-dipeptide), several

groups have synthesized new peptide derivatives active as inhibitors and interacting with the metallosubsite of the enzyme.

Inhibition by metabolic products of enkephalin provided clues that

led to the synthesis of Thiorphan (see Roques, this volume).

Inhibitors provide probes to examine the nature of the active center. Understanding the mechanisms of action of inhibitors is an

important goal and applies equally to other materials acting at

sites 1 or 3.

In studies on structure-activity for different dynorphins, a

relationship was noted between peptide size and rates of breakdown

79
by membrane (table 4) or purified metalloendopeptidase (see table

7). Highest activity was found for the pentapeptide (Km, 20 uM)

and was significantly lower for peptides with 8-17 residues. In

the case of dynorphins 1-l3 and 1-17, incubation with purified

enzyme led to cleavage at two sites, at Gly-Phe and Arg-Ile; incubation with SPM, however, resulted in formation of dynorphin 1-8

as one of products. The metabolism of dynorphin 1-8 (a peptide

with preferential affinity to k-receptors) is complex since it can

serve as a substrate for membrane ACE and metalloendopeptidase.

In studies on the appearance of products with time, SPM degraded

dynorphin 1-8 to release 1-6 and 1-4 by release of C-terminal

dipeptides (Benuck et al. 1984).

Cysteine Proteinase (cathepsin B)

Fusion of primary or secondary lysosomes with synthesizing particles within the RER has focused attention on cathepsin B

(Bienkowski 1983; Docherty et al. 1984). Purified cathepsin B has

exo- and endo-peptidase actions towards polypeptides such as

glucagon and insulin. In recent studies, we showed that it displayed a dipeptidyl carboxypeptidase action toward some enkephalins with C-terminal extensions (table 7). In other cases, it

acted on peptidyl amides C-terminally with removal of the amino

acid amide and may be a factor, therefore, in the metabolism of

neuropeptides having -CONH2 (Marks et al. 1984). The Km for MEAP

was 24 uM, and for MEAGL was 117 uM (C-terminal dipeptide

removal); purified brain cathepsin B acted also on pentapeptides,

but the Km was higher with a value of 470 uM. The low turnover of

enkephalin (kcat 0.89 sec-l) would tend to exclude a role for

cathepsin B in inactivation per se, but the higher values for

hepta-or octa-peptides (25-39) point to a role in processing.

Removal of C-terminal dipeptides can generate smaller active

opioids from precursor forms.

Cathepsin B has been purified from rat brain, utilizing synthetic

substrates (BANA); it was shown to be dependent on -SH and to be

potently inhibited by leupeptin or by an endogenous protein with

1979; Kopitar et al. 1983). Intracellular levels of -SH or enMr 12,500 present in brain in high concentration (Suhar and Marks

dogenous inhibitor thus could provide a mechanism for regulating

peptide processing by cysteine proteinases.

GENERAL COMMENTS ON PROCESSING

Co- or post-translational events are rate limiting, yet little is

known about these processes in the brain. The secretory pathway

for hormones is comparmentalized within the endoplasmic reticulum

(ER) and Golgi complex (Bienkowski 1983). It is established that

most secretory proteins are formed with a hydrophobic leader sequence that becomes detached during translocation of the growing

peptide chain. Recent studies show the presence of a signal

recognition particle on the outer ER surface consisting of a 7s

RNA and several proteins, two of which bind to the Alu transcripts

of the RNA: the leader sequence can engage with a "docking

protein" on the cytoplasmic surface of the ER, forming a pore for

translocation of the polypeptide (Walter and Blobel 1983). Very

80
TABLE 6

Enzymes Cleaving -Gly-Phe (Site -3) of

Pentapeptide Enkephalins

Data derived from Benuck et al. (1982a); Cascieri et al. (1984);

Yokosawa et al. (1983a, 1983b) for ACE. Relative rates for ACE

degradation of enkephalins were Dyn 1-8 (100)) MEAGL, (31)) Dyn 1-6

(27), Dyn 1-5 (18), Dyn 1-13 or 1-17 (0). Metalloendopeptide

data derived from Mumford et al. (1980); Orlowski and Wilk (1981);

Almenoff and Orlowski (1984); Fulcher et al. (1982, 1983). Relative rates found in our studies for enkephalins were Dyn 1-5

(100), 1-6 (71), 1-8 and larger (14), MEAGL (86). (See table 4

for comparison with SPM). Data for cysteine proteinase compiled

from Suhar and Marks (1979); Kopitar et al. (683); Marks et al.,

in press).

81
TABLE 7

Cleavage of Different Enkephalins by Purified Rat

Brain Cathepsin B

Values are expressed relative to substrate 1 (rate was 1.6 umol

per mg protein per min). The substrate concentration used was 0.1

mM and the incubation mixture contained 0.047-0.094 ug of purified

rat brain cathepsin B. Incubations were for 30 min, or in the

case of substrates 5-8, for 240 min. Points of cleavage

little information is available on the nature and specificity of

the signal peptidase other than the fact that some membranes

(pancreatic, pituitary, and liver) contain a detergent extractable

enzyme Which converts some preproproteins (Jackson and Blobel

1980; Mumford et al. 1980). The enzyme cleaves bonds linking the

leader sequence to the prohormone and includes Gly-Lys (PTH),

Ala-Leu (rat GH), Ala-Phe (rat insulin), and Gly-Tyr (porcine

POMC) among others. The rapid action of ER signal peptidase

generally prevents isolation of the preproprotein. Most of the

data on the presequence can be deduced from cloning the appropriate mRNA (Chretien and Seidah 1985).

Proproteins can be processed differently within a given tissue,

dependent on the anatomical regions. One explanation is the

sorting or guiding of prohormones along the ER and Golgi complex

to secretory or storage vesicles, or fusion with primary or

secondary lysosomes (Bienkowski 1983). This might explain the

diversity of products obtainable from POMC in pituitary regions or

proenkephalins in adrenal and various brain areas (Marks 1977;

Seizinger et al. 1984).

A large number of membrane-bound or soluble enzymes are available

for processing at sites adjacent to basic residues (trypticlike

enzymes) or other bonds (reninlike). The strategy of using

secretory vesicles or storage granules as a source of enzyme, combined with RIA for detection of enkephalins, has provided new

information. Enzyme can be extracted from adrenal medullary

chromaffin granules and has been shown to convert ECP’s (enkephalin containing peptides) at pH 8.0 (Lindberg et al. 1982; Mizuno

et al. 1982) or at acidic pH (Troy and Musacchio 1982; Evangelista

et al. 1982). In brain, Devi and Goldstein (1984) observed conversion at pH 7.6 of Dyn-B-29 to form 1-13 by cleavage of the

82
Thr-Arg bond. In pituitary, Loh and Gainer (1982) observed an

acidic enzyme in processing POMC. Wallace et al. (1984) reported

that atrial extracts of Aplysia converted dynorphin 1-13 to 1-8 at

pH 7.6 by cleavage of the Ile-Arg bond. The latter enzyme may be

analogous in specificity to one observed in rat brain SPM converting Dyn 1-13 (Benuck et al. 1984).

Metalloendopeptidase present in membranes and soluble extracts of

brain or pituitary also convert a number of neuropeptides by

cleaving bonds adjacent to one or more basic residues (Chu and

Orlowski 1984; Benuck et al. 1984; see table 8 and figure 2).

Neutral endopeptidases from brain membrane or soluble fractions

have been purified and shown to convert ß-endorphin or smaller

fragments to ME (Koida et al. 1979; Orlowki et al. 1980). Knight

et al. (1982) also purified an enzyme from rat membranes that converted ECP’s (extracted from striatum) to smaller iR-enkephalins.

More recently, brain cathepsin B was shown to convert small

enkephalins with C-terminal extensions to yield ME or MEA (table

7). There is some data suggesting that kallikreins, a group of

serine proteases, play diverse roles in processing. The mRNA of

salivary gland after cloning (mouse) contains sequences that are

homologous with the mouse genome, implying a wide distribution in

tissues of kallikreins (Mason et al. 1983).

A coherent scheme for processing of prohormones cannot be advanced

on the basis of available data. A fully integrated scheme must

take into account any variables (several were cited in

the introduction), not least of which are anatomical factors and

the role of posttranslational modifications, such as glycosylations, N-acetylation, methylation, phosphorylation, sulfation,

formation of pyroGlu on the N-terminal, and CONH2 on the Cterminal. Mechanisms exist for addition of such groups, many of

which are catalyzed by cellular enzymes; but removal of groups

such as N-acetyl or C-terminal amide (rather the removal of the

amino acid itself) is an unresolved area. These groupies modify

neuropeptide actions-- in some cases, activating (a-MSH); in

others, inactivating (endorphins). Since tissues contain a number

of inactive N-acetylated endorphins and other opioid peptides, we

surveyed brain for the presence of enzyme(s), removing specifically the Na-acetyl grouping. Such enzyme(s) were absent in

brain, although the latter contained an enzyme removing N-acetylMet or Ala from di- or tri-peptides,and “acylases” which deacetylated single amino acids (Marks et al. 1983b). N-acetyl endorphins may be inactive forms of opioid peptides or serve as precursors for as yet unidentified peptides with novel properties. The

covalent modifications that affect biological activity of neuropeptides may occur in the processing pathway, or extracellularly.

Despite evidence that insulin exists as a pro- and prepro- form

and the latter were isolated, there has been very little progress

in understanding the manner in which they are processed.

Pituitary AtT-20 cells grown in culture can be induced to process

and secrete insulin after transfection with the proinsulin gene,

demonstrating that they contain the requisite enzymes for processing more than one type of precursor (Moore et al. 1983).

83
Of interest is the finding of Docherty et al. (1984) that insulin

secretory cells contain a cysteine proteinase involved in

conversion of proinsulin. These experimental models together with

availability of opioid peptide intermediates present opportunities

to unravel some of the pathways associated with processing in the

central nervous system.

84
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AUTHORS

Neville Marks, Research Scientist

Myron Bernuck, Research Scientist

Martin J. Berg, Research Technician

Center for Neurohistory

The Nathan S. Kline Institute for Psychiatric Research

Ward's Island

New York, New York 10035

91
Isolation and Identification of Opioid

Peptides

Hisayuki Matsuo, Ph.D.

INTRODUCTION

It is well known that a variety of peptides participate in a

complex network of neural and hormonal communications. In order to

clarify the subtle mechanism of peptidergic communication, it is

essential that yet unidentified peptides be discovered.

Identification of the key peptide leads us to further discoveries

of its related peptides or precursor, with the aid of

immunochemical methods and recombinant DNA techniques.

Radioimmunoassay and immunohistochemical data, as well as

pharmacological findings, will provide further information on the

newly identified peptide.

Within a few years of the discovery of the two enkephalins in

brain (Hughes et al. 1975), an explosive series of related

discoveries have revealed the existence of a vast network of

enkephalin-containing peptides that are produced in many tissues by

the processing of at least three genetically distinct precursors.

Soon after discovery of the Met- and Leu-enkephalins, Metenkephalin sequence was fortunately found in ß-lipotropin(Hughes et

al. 1975) and ß-endorphin(Bradbury et al. 1976; Li et al. 1976).

These facts led to identification of pro-opiomelanocortin

(POMC) (Nakanishi et al. 1979) as a common precursor of ß-endorphin

and adrenocorticotropic hormone (ACTH). To many it seemed obvious

that Met-enkephalin was derived from, POMC via processing through

ß-lipotropin and ß-endorphin. However, POMC does not contain a

Leu-enkephalin unit. Furthermore, although the ß-endorphin sequence

is preceded by a Lys-Arg sequence, a typical processing signal, the

Met-enkephalin sequence is not followed by a recognized signal for

proteolytic processing. This would indicate that the ß-endorphin

sequence is not programed to be a precursor of Met-enkephalin.

Moreover, it had been shown that the tissue distribution of the

enkephalins did not parallel that of ß-endorphin. In order to

92
answer the question as to the nature of the precursors of Met- and

Leu-enkephalins, we carried out a systematic survey for possible

precursors of the two enkephalins in hypothalamic tissue and in

adrenal medulla.

STRATEGY OF OUR SURVEY FOR THE UNIDENTIFIED PEPTIDES

As previously mentioned, it is essential for understanding the

mechanism of peptidergic communication if unidentified peptides are

to be discovered. However, there have been two major obstacles in

the search for the novel peptides in the tissues. One arises from

the unfavorable proteolytic decomposition of the objective peptide

and fragmentation of other higher molecular proteins, which

seriously interfere with the separation and lower the yield of the

peptide. Therefore, tremendous amounts of the tissue must be

collected for the isolation. It is well known that Schally

collected 165,000 pig hypothalami to obtain only 200 μg of the

purified LH-RH(Matsuo et al. 1971). However, about 20,000

hypothalami are sufficient to obtain 200 μg of LH-RH, according to

the actual content of LH-RH determined by radioimmunoassay (RIA).

Such a big difference mainly is a result of proteolytic

decomposition of LH-RH during the extraction. Therefore, methods

should be developed to inactivate the intrinsic protease activity.

After numerous trials, we found that boiling of the tissues for 10

minutes before homogenization is most effective for inactivation of

proteases to minimize the decomposition of the peptide (Kangawa et

a1. 1984). Moreover, recent progress in the sequencing techniques

makes it possible to determine the complete structure at the

sacrifice of the sample of at most 10 nanomoles. Thus, now we are

able to carry out the search for the peptides in the brain of small

laboratory animals. For instance, we successfully purified two

distinct LH-RH-like peptides (Gn-RH I and II), eliciting

gonadotropin (Gn)-releasing activity, from only 2,000 chicken

hypothalami and sequenced them, as shown in figure 1 (Miyamoto et

al. 1983; Miyamoto et al. 1984). In this study, the sensitive assay

using cultured pituitary cell system was effectively used for

detecting Gn-releasing activity. However, such a specific assay

relevant for monitoring the new peptides is not always available.

Thus, the second but most serious obstacle is difficulty in

discovering the appropriate tools for finding undiscovered species

in the tissues. Most of neuropeptides so far identified elicit a

variety of biological and pharmacological actions according to the

method used. For instance, substance P exhibits a wide spectrum of

diverse activities, such as smooth-muscle stimulation, and

hypotensive and sialogogic effects. These facts suggest that

sensitive assay system for the effects on muscle contraction or

blood pressure, even though they are not specific, would be well

suited for detecting unknown bioactive peptides. In this context,

we have been conducting the systematic survey for the unidentified

peptides by utilizing our "nonspecific assay method" for the

effects on contractility of smooth-muscle preparations or blood

pressure.

93
FIGURE 1

Amino Acid Sequences of Chicken Gonadotropin-releasing Hormone

FIGURE 2

Sequences of Endogenous Opioid Peptides

(* indicates the peptides identified by our group)

94
As a matter of fact, α−neo-endorphin was originally discovered in

the fraction exhibiting hypotensive effect of hypothalamic extracts

(Kangawa et al. 1979). The result reveals that our assay method,

even though not specific, is a useful tool for detecting still

undiscovered peptides. Furthermore, our nonspecific assay led us to

the identification of neuromedins K (Kangawa et al. 1983), L

(Minamino et al. 1984) [kassinin-like), B (Minamino et al. 1984),

C (Minamino et al. 1984) [bombesin-like), and N (Minamino et al.

1984) (neurotensin-like) in porcine spinal cord. They were isolated

as the peptides eliciting smooth-muscle stimulant activity.

DISCOVERY OF a-NEO-ENDORPHIN AND "BIG" LEU-ENKEPHALINS RELATED TO

PROENKEPHALIN B

From the earlier stage of our survey, we have utilized a very

simple in vitro assay to determine the effect on the contractility

of various smooth-muscle preparations or on the blood pressure (rat

and rabbit), even though they are not so specific. By the aid of

such a nonspecific assay, α−neo-endorphin was originally isolated

from the fraction eliciting hypotensive activity of porcine

hypothalamic extracts. Even at the time when the peptide was

obtained in a pure state, it was not known that the peptide is the

first form of the long-sought "big" Leu-enkephalin, having a potent

morphinomimetic activity. During the sequencing, we first realized

that the peptide has a Leu-enkephalin unit at its N-terminal

portion, which is followed by a paired basic signal of Arg-Lys.

Then, the peptide was named "neo-endorphin" (Kangawa et al. 1979;

Kangawa et al. 1981). A little later on, dynorphin, which retained

opioid activity even after cyanogen bromide treatment, was purified

as the second form of the "big" Leu-enkephalin by Goldstein and

co-workers (Goldstein et al. 1979; Tachibana et al. 1982). The

discovery of α−neo-endorphin and dynorphin initiated a search for

the unknown Leu-enkephalin precursor. In the attempted survey for

the still unknown "big" enkephalin or proenkephalin,

pharmacological assay for opiate activity or radioreceptor assay

was usually used. However, such methods are not able to

discriminate the objective "big" Leu-enkephalins from the known

opioid peptides, but identification of the key peptides led us to

further discoveries of its related peptides or precursor. When α−

neo-endorphin and dynorphin were identified, it was not so hard to

find their related peptides, indicating a product-precursor

relationship. Since α-neo-endorphin and dynorphin have (Arg6)-Leuenkephalin moiety in common at their N-terminus, which is easily

released by trypsinization, we raised the antiserum specific for

(Arg6

)-Leu-enkephalin and established sensitive radioimmunoassay

for this tryptic fragment (Kangawa et al. 1980). With the aid of the

RIA coupled with trypsinization, two novel "big" Leu-enkephalins

named ß-neo-endorphin (Minamino et al. 1981) and PH-8P (dynorphin(1-8)) (Minamino et al. 1980) were identified (figure 2). Based

on these findings, Numa and co-workers (Kakidani et al. 1982)

elucidated the base sequence of cDNA encoding their common

precursor, named preproenkephalin B, which also contains the third

Leu-enkephalin unit, named Leu-morphin or rimorphin (figure 3).

95
FIGURE 3

Schematic Structures of the Precursors and the Processed Opioid

Peptides. Black and hatched boxes represent Met- and Leuenkephalin units, respectively.

Radioimmnunoassay for (Arg6)-Leu-Enkephalin

The antiserum against (Arg6)-Leu-enkephalin was raised in rabbits

to synthetic (Arg6)-Leu-enkephalin coupled with carbodiimide to

bovine serum albumin. The antiserum was usable at a final dilution

of 1:0,000 for the radioimmunoassay for (Arg6)-Leu-enkephalin,

utilizing 125I-labeled ligand under conditions where 30% to 40% of

ligands were bound to the antiserum. As seen from figure 4,

(Arg6)-Leu-enkephalin is measurable by this method with the

sensitivity of less than 1 pg/tube and the measurable range of the

inhibition curve is 1 pg to 104 pg. The specificity of the

antiserum was evaluated by determining its cross-reactivity with

several peptides. ß-Endorphin does not cross-react with the

antiserum at all in the entire region of concentrations tested and

Met- and Leu-enkephalins do not cross-react appreciably with the

antiserum. This fact verifies that arginyl residue at the carboxyl

terminus of the ligand is necessary for the sensitive recognition

by the antiserum. Thus, the present radioimmunoassay, if used after

trypsinization of sample, makes it possible to detect with good

sensitivity the "big" Leu-enkephalins, such as α-neo-endor hin or

dynorphin in the tissues, which easily releases an (Arg6)-Leuenkephalin from the molecule by trypsinization. The antiserum also

shows some cross-reactivity with (Arg6

)-Met-enkephalin containing

peptides, although sensitivity is of course not so high as in the

case of (Arg6)-Leu-enkephalin (Kangawa et al. 1980).

96
FIGURE 4

Inhibition of binding of 125I-labeled (Arg6)-Leu-enkephalin to the

antiserum raised against (Arg6

)-Leu-enkephalin by serial dilution

of unlabeled ligands: (Arg6)-Leu-enkephalin (Arg6)-Metenkephalin (Arg6

, Lys7)-Leu-enkephalin Leuenkephalin Met-enkephalin and ßh-endorphin

Antiserum used at 1:70,000 dilution.

FIGURE 5

Purification Procedures of "Big" Leu-Enkephalins

from Porcine Hypothalamus

97
FIGURE 6

Separation of ß-Neo-Endorphin(#4) and PH-8P(#7) on Reverse-Phase

HPLC by the Aid of RIA for (Arg6)-Leu-Enkephalin

Hatched bars represent (Arg6)-Leu-enkephalin immunoreactive

peaks(#4 and #7). Column: μ-Bondapak C-18 (Waters). Solvent system:

A linear gradient from (A) to (B). (A) : 0.05M phosphate

buffer(pH2.0): CH3CN = 90:10; (B) : 0.05M phosphate buffer(pH2.0):

CH3CN = 50:50

Purification

On every purification step, pro-Leu-enkephalin containing fractions

were monitored by the use of RIA for (Arg6)-Leu-enkephalin, coupled

with trypsinization. The outline of the purification procedures is

summarized in figure 5. A low-molecular-weight and basic peptide

fraction, obtained from the acid extracts of porcine hypothalami

(30,000 fragments), was chromatographed on SP-Sephadex C-25. In

this chromatographic separation, there were two major

irrununoreactive fractions, designated as fraction G (tubes 115-129)

and fraction I (tubes 149-160), when the latter a-neo-endorphin was

purified. Fraction G yielded a single immunoreactive peak after

the successive chromatographies, as listed in fig. 5. This

immunoreactive material was separated into two immunoreactive peaks

(peaks 4 and 7), as shown in figure 6. Peaks 4 and 7 were further

purified by repeated HPLC. Finally, ß-neo-endorphin (30 nmol) from

peak 4 and PH-8P (20 nmol) from peak 7 were purified, respectively,

to homogeneity (Minamino et al. 1980, 1981)

Structural Analyses

All of

scale.

the structural analyses

Control experiments were

were

made

carried out in a nanomole

in every case under exactly

98
the same conditions except that only the sample peptide was

omitted.

Sequence analyses were performed mainly by the manual dansyl-Edman

procedure. Special caution was exercised to avoid loss of the

peptide on the extraction step by aqueous butyl acetate. Results

thus obtained are summarized in figure 3. Sequence analyses of α−

and ß-neo-endorphin were successfully performed up to the carboxyl

ends. C-Terminal Lys of a-neo-endorphin was also identified by the

3

H-labeling method. In the case of PH-8P, its sequence analysis,

combined with C-terminal analysis by carboxypeptidase A, revealed

the complete structure. Furthermore, tryptic peptides of PH-8P and

ß-neo-endorphin were isolated by HPLC and the amino acid sequence

was determined to corroborate their structures elucidated above.

Consequently, the complete amino acid sequences of PH-8P and ß- and

a-neo-endorphin have been established, as shown in figure 2.

According to the proposed structures, PH-8P, ß- and α-neoendorphin, and their tryptic peptides were newly synthesized. The

structures of α− and ß-neo-endorphin and PH-8P (determined above)

were confirmed in the following way. Upon trypsinization, each

peptide was found to be cleaved specifically at the linkage (6-7)

to yield only two tryptic fragments, which were well separated on

HPLC. The chemical structures of the tryptic peptides separated

above were confirmed by the chromatographical comparison on HPLC

with synthetic peptides. In the case of α−neo-endorphin,

chymotryptic peptides were also compared on HPLC with authentic

specimens. Confirmation of the proposed structures of PH-8P, ßand α−neo-endorphin was provided by comparing natural peptides with

synthetic ones on HPLC.

Thus, α−neo-endorphin was shown to be C-terminally extended peptide

of ß-neo-endorphin. On the other hand, PH-8P was found to be the

N-terminal octapeptide of dynorphin and previously only the

partial sequence (1-13) was known (Minamino et al. 1980, 1981).

"BIG" ENKEPHALINS IN BOVINE ADRENOMEDULLARY GLAND

On the other hand, Udenfriend and his co-workers (Udenfriend et al.

1983) and our group (Mizuno et al. 1980a; Mizuno et al. 1980b;

Mizuno et al. 1981) have identified a variety of enkephalincontaining peptides in adrenomedullary gland, all of which are

known to be processed from the third precursor named

preproenkephalin A. Thus, all the endogenous opioid peptides so far

identified are derived from either of three genetically distinct

precursors: pre-POMC and preproenkephalin A and B.

Since the antiserum used above shows 5% cross-reactivity with

[Arg6)-Met-enkephalin, the radioimmunoassay mentioned above has

proven its capability to detect not only (Arg6)-Leu-enkephalin,

but also (Arg6)-Met-enkephalin with an appreciable sensitivity.

99
Using radioimmunoassay coupled with trypsinization, we sucessfully

purified a dodecapeptide (BAM-12P) as a novel "big" Metenkephalin(Mizuno et al.1980). Furthermore, two novel "big" Metenkephalins, a docosapeptide (BAM-22P) and eicosapeptide (BAM2OP), both of which yield immunoreactive (Arg6

)-Met-enkephalin by

the action of trypsin, were also purified (Mizuno et al. 1980a) from

the side-fractions that were obtained in the purification of BAM12P. Sequence analyses have shown that both peptides contain a

common BAM-12P sequence at their N-terminals with progressive Cterminal extension. The identification of a series of BAM-22P, -2OP,

and -12P in the adrenomedullary gland introduces a new family of

"big" Met-enkephalins into the opioid field, which may be

indicative of the unknown feature of enkephalin biosynthesis. In a

similar manner, BAM-18P (Matsuo et al. 1983) --- which corresponds

to the N-terminal peptide of BAM-2OP and BAM-22P --- and other

types of enkephalin-containing peptide have been identified.

BAM-30P and BAM-77P were found to have a Met-enkephalin unit at

their C-terminal part (Mizuno et al. 1981). On the other hand,

Udenfriend and his associates (Udenfriend et al. 1983) identified a

new series of enkephalin-containing peptides, such as peptides B,

E, F, and I, by the combined treatments of trypsin and

carboxypeptidase B, coupled with a radioreceptor assay for the

released Met-enkephalin. In particular, peptide E, whose N-terminal

part corresponds to BAM-12P, -18P, and -22P, was revealed to

contain Met- and Leu-enkephalin units, implying a possibility that

Met- and Leu-enkephalin may be derived from a common precursor.

Combined results obtained by both groups strongly indicated the

presence of the third enkephalin precursor (proenkephalin A),

containing six Met-enkephalin units and one Leu-enkephalin unit.

These findings afforded the basis for the base sequence analysis of

cDNA (Noda et al. 1982) encoding preproenkephalin A.

Thus, all the endogenous enkephalin-containing peptides so far

identified are known to be derived from either of three distinct

precursors: POMC and preproenkephalin A and B. The recent progress

in recombinant DNA analyses provided precise structure of the

precursors. Most of the enkephalin units are flanked by paired

basic residues composed of Lys and Arg, which are now thought to be

typical processing signals. However, our knowledge of the

processing event intervening from the precursor to the mature

peptides is still very limited (Mizuno et al. 1982). In this

context, the discovery of PH-8P in brain raised a quite

interesting pattern of processing that occurred before Arg-Pro

signal. As clearly seen in the sequence of dynorphin, PH-8P

(dynorphin(1-8)) was located at the N-terminal part of dynorphin,

and followed by Arg-Pro sequence. Since PH-8P is the predominant

opioid in posterior pituitary, it is obvious that PH-8P is

processed out of preproenkephalin B precursor by the specific

cleavage before Arq-Pro linkage, which so far has not been

accepted as the processing site (Mizuno et al. 1984). A similar

processing pattern was also observed in the case of

preproenkephalin A, as shown in the following section.

100
ADRENORPHIN: A C-TERMINALLY AMIDATED OPIOID PEPTIDE

As observed in various peptide hormones, the carboxy terminal amide

structure is a unique feature of peptides exhibiting physiological

activities. Although a variety of opioid peptides have been

identified, such a C-terminally amidated species has never before

been discovered in mammals. Adrenorphin isolated from human

pheochromocytoma tumor derived from adrenal medulla is the first

identification of a peptide with a C-terminal amide structure

(Matsuo et al. 1983). The complete amino acid sequence of

adrenorphin, as determined by microsequencing, is Tyr-Gly-Gly-PheMet-Arg-Arg-Val-NH2 and corresponds to the sequence of the first

eight amino acids of peptide E, which is derived from proenkephalin

A. Adrenorphin exhibits a potent opioid activity in guinea pig

ileum (GPI) assay. Using an antiserum raised against synthetic

adrenorphin, a highly sensitive and specific radioimmunoassay was

developed. The peptide has also been identified in normal human

and bovine adrenal medulla on reverse-phase HPLC, using the RIA for

adrenorphin. The distribution of adrenorphin in rat brain has also

been determined (Miyata et al. 1984).

Adrenorphin was purified as follows. A portion corresponding to the

lower molecular weight (Mr ca. 2,000) that was separated from acid

extracts of tumor tissue by gel filtration on Sephadex G-50 was

treated batchwise with CM-cellulose in a buffer of 10 mM ammonium

formate (pH 6.6). After washing the resin with the same buffer, the

basic peptides adsorbed on the column were eluted with 1M formic

acid and the eluates were then pooled. As shown in figure 7, the

basic peptide pool obtained above was subjected to reverse-phase

HPLC. An aliquot of fractions was trypsinized and then generation

of (Arg6)-enkephalin was analyzed by RIA utilizing anti-(Arg6)-

Leu-enkephalin antiserum, having 5% cross-reactivity with (Arg6)-

Met-enkephalin. As seen in figure 7, five immunoreactive peaks (A

to E) were obtained.

Finally, adrenorphin was purified from the portion of peak B by

successive reverse-phase HPLC (figure 8). Only a tyrosine residue

was identified by dansylation as the amino terminal residue of the

peptide thus purified, confirming its homogeneity. On hydrolysis

with 6N HCl, adrenorphin afforded the following amino acid

composition: Gly 2, Val 1, Met 1, Tyr 1, Phe 1, Arg 2, suggesting

its octapeptide structure. The yield of the peptide was 20 nmol

from 42.6 g of pheochromocytoma tumor. C-Terminal analysis of

adrenorphin by carboxypeptidase method revealed that the C-terminal

is blocked. By the stepwise Edman degradation of the native

peptide, the complete amino acid sequence thus ascertained is TyrGly-Gly-Phe-Met-Arg-Arg-Val-NH2. The presence of C-terminal Val-NH2

was verified as dansyl-valineamide by thermolytic digestion,

followed by dansylation in a manner similar to that described by

Tatemoto and Mutt (1978). For structural confirmation, the

octapeptide amide, according to the adrenorphin sequence determined

above, was synthesized by solid-phase techniques, conducted on a

p-methyl-benzhydrylamineresin. Des-amido-adrenorphin (adrenorphinOH), having a free carboxy terminus, was also synthesized.

101
FIGURE 7

Reverse-phase HPLC of the fraction containing the low-molecularweight and basic peptides obtained from human pheochromocytoma

tumor. A: (Arg6)-Met-enkephalin, B: BAM-12P, Met-enkephalinArg6

-Gly7-Leu8, D:Met-Enkephalin-Arg6-Phe7, E:BAM-18P.

Column: TSK LS-410 ODS SIL(Toyosoda). Solvent system: A linear

gradient from (A) to (B). (80 min): (A) 0.05M phosphate

buffer(pH2.0): CH3CN = 90:10 (v/v); (B) 0.05M phosphate

buffer(pH2.0): CH3CN = 50:50 (v/v)

Natural adrenorphin comigrates with synthetic adrenorphin on

cation-exchange HPLC, and it is well separated from synthetic

adrenorphin-OH. This confirmed that adrenorphin was isolated in an

amide form.

Adrenorphin sequence has a significant feature in its own

biosynthesis. The sequence corresponding to adrenorphin was found

to be present in human (and bovine) preproenkephalin A at positions

210-217, suggesting that adrenorphin is derived from this

precursor. As shown in figure 9, adrenorphin sequence in the

precursor is followed by a glycine residue, serving as a nitrogen

donor to amidate the preceding valine residue (Bradbury et al.

1982). This conversion is thought to be carried out by a specific

amidating enzyme. The glycine residue attached to adrenorphin

sequence connects to the sequence of -Arg-Pro-, which so far has

not been regarded as a typical processing signal. However, a

similar cleavage before Arg-Pro was also observed in the formation

of PH-8P from dynorphin molecule in preproenkephalin B, implying

that the linkage of Arg-Pro is likely a processing signal.

Accordingly, adrenorphin is thought to be programed to be processed

out of the precursor as a C-terminally amidated opioid.

102
FIGURE 8

HPLC of the Purified Adrenorphin Compared With the Synthetic

Adrenorphin and Deamido-adrenorphin (adrenorphin-OH)

a) Final purification of adrenorphin by reverse-phase HPLC (Arrows

indicate the elution positions of synthetic adrenorphin (a) and

BAM-12P(b))

b) Cation-exchange HPLC of natural adrenorphin

c) Cation-exchange HPLC of natural adrenorphin (2) compared with

synthetic adrenorphin-OH(1)

FIGURE 9

Location of Adrenorphin and PH-8P in Their Respective Precursors

Residue numbers are taken from the sequences of human

preproenkephalin A and porcine preproenkephalin B. The sequences

corresponding to adrenorphin and PH-8P are boxed and the flanking

residues are underlined. Natural occurrence of adrenorphin and PH-8P

indicates that a sequence of -Arg-Pro- is likely a processing

signal.

103
It should be noted that Weber et al. (1983) also isolated the identical peptide, named metorphamide, in brain. And they recently

reported the presence of a peptidase activity cleaving dynorphin A

into PH-8P in the atria of Aplysia californica (Weber et al. 1982).

PURIFICATION AND IDENTIFICATION OF "NEUROMEDINS"

In this section, an outline of our refined purification procedure

will be briefly summarized, by which a series of new smooth-muscle

stimulant peptides, named neuromedins have recently been

purified (Kangawa et al. 1983; Minamino et al. 1983; Minamino et

al. 1984a; Kinamino et al. 1984b; Minamino et al. 1984c). As shown

in figure 10, peptides of Mr 700 to 5,000 daltons, prepared from

acid extract of porcine spinal cords (ca. 20 kg), were absorbed on

SP-Sephadex in the presence of 1N AcOH, and then eluted with the

same solution (SP-I), 2M pyridine (SP-II), and 2M pyridine-AcOH (pH

5.0) (SP-III), successively. Fractions SP-II and SP-III thus obtained were the starting materials. After repeated gel-filtrations

of each fraction, remarkable stimulant activity was observed in the

various chromatographic regions. SP-II-D and SP-III-C showed a

potent ileum activity, while SP-III-E and SP-III-F elicited a

uterus activity. These four bioactive fractions were each subjected

to further purification by repeated reverse-phase HPLC or a

combination with ion-exchange HPLC. In this manner, neuromedin K

and L with prominent ileum activity and neuromedin B and C with a

uterus activity were each purified in a homogeneous state. The

structures of four neuromedins were each determined by

microsequencing in a subnanomole level and confirmed by

chromatographic comparison with synthetic specimens that were

FIGURE 10

Purification Scheme of Neuromedins from Porcine Spinal Cord

104
prepared by solid- phase techniques. The complete amino acid

sequences thus determined are listed in figure 11. Neuromedin K

and L have a remarkable sequence homology to the known amphibian

tachykinins, which in common have a C-terminal structure

represented as Phe-X-Gly-Leu-Met-NH2. Such a structural resemblance

of the first peptide to kassinin is a basis for naming it as

neuromedin K, while the second one is designated as neuromedin L--

the next letter in alphabetical sequence. Substance P has hitherto

been thought to be the only tachykinin identified in mammals. Now,

neuromedin K and L are filed as new members of this family. These

two neuromedins, as well as substance P, have been found to share a

common spectrum of tachykinin activity, such as a quick stimulant

action on smooth-muscle and a prompt hypotensive effect. To date,

substance P is well known as a neurotransmitter in the primary

sensory neuron of mammalian spinal cords. The resemblance in

structure as well as in biological actions of neuromedin K and L to

substance P strongly suggests that these new peptides may also

function as a neuromediator in the mammalian neural network.

Another surprising sequence homology was observed between

neuromedin B and C and amphibian bombesin, as shown in figure 11.

Furthermore, neuromedin B and C elicit biological activities

similar to those of bombesin in the contractile reaction of rat

uterus and guinea pig ileum, distinct from those of tachykinins.

Since amphibian bombesin elicits a variety of pharmacological

effects in mammalian central nervous system, neuromedin B and C are

both expected to function in brain as an endogenous bombesin.

Incidentally, it should be noted that neuromedin C is identical to

a C-terminal decapeptide of gastrin-releasing peptide (GRP(18-27)),

which has recently been isolated from canine intestine.

These results revealed that even a nonspecific assay as used in

these works provided an effective too1 for finding hidden

neuropeptides.

FIGURE 11

Sequences of Neuromedins Identified in Porcine Spinal Cord

105
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AUTHOR

Hisayuki Matsuo, Ph.D.

Department of Biochemistry

Miyazaki Medical College

Kiyotake, Miyazaki 889-16, Japan

108
ß-Endorphin: Naturally Occurring or Synthetic

Agonists and Antagonists

Choh Hao Li, Ph.D.

INTRODUCTION

ß-Endorphin (ß-EP) is derived from ß-lipotropin

(ß-LPH) which in turn comes from a large precursor

molecule proopiomelanocortin (POMC) (Eipper and Mains

1980; Chrétien and Seidah 1984). It has been isolated

and sequenced from pituitary glands of various species

(Li 1982), except turkey ß-EP which was deduced from the

structure of ß-LPH (Chang et al. 1980). They all

consist of 31 amino acids, with the Met-enkephalin

sequence at the NH2-terminus. As noted in figure 1,

only the human hormone has glutamic acid at the COOHterminus, while ß-EP from other species has glutamine.

The sequential structure of human ß-EP is remarkably

similar to that of other species. Other than the

position 31, the camel sequence differs only in one

position: His-25 (Tyr); and the ostrich sequence

differs in seven positions: Ser-6 (Thr), Arg-9 (Lys),

Gly-10 (Ser), Arg-11 (Gln), Ala-12 (Thr), Val-23 (Ile),

and Ser-25 (Asn). These differences are comparatively

minor in terms of base pair in the genetic code.

During evolution, the amino acid sequence of ß-EP is

highly conserved.

Synthesis of ß-EP and Analogs

The protocol for solid-phase synthesis of ß-EP and its

analogs was established in an earlier synthesis of

sheep ß-LPH-(42-91), which contained the sequences of

camel ß-EP (Yamashiro and Li 1974). The important

features of the protocol included the use of

symmetrical anhydrides for this coupling amino acid

residues and the use of an appropriate set of sidechain protected groups (see table 1) stable to

trifluoroacetic acid (TFA) and easily removable at the

end of the synthesis by HF. ß-EP and analogs can be

prepared in good yield (10% to 30%) and high purity.

109
FIGURE 1

Amino Acid Sequence of ß-EP from Various Species

One of the ß-EP analogs was synthesized by a new

segment coupling method (Blake 1981; Blake and Li

1981). This involves the use of thiol acids for

coupling segments in aqueous solution:

R1CO-S-+2 Ag++H2N-R2 + R1CONHR2+Ag2S+H+

where Rl and R2 are peptide segments which may be

synthesized by the solid-phase method. The scheme for

the synthesis of [Gly 17]-ßh-EP by this new method is

shown in figure 2.

TABLE 1

Protecting Groups for Amino Acid Side-Chain

Functions in Solid-Phase Peptide Synthesis

Amino Acid Protecting Groupa

Asp

Thr

Ser

Glu

Cys

Met

Tyr

His

Lys

Arg

Trp

Bzl, cyclopentyl

Bzl

Bzl

Bzl, cyclopentyl

3,4-dimethylbenzyl

none or sulfoxide

Z, 2-BrZ, cyclopentyl

Z

2-BrZ, 2-ClZ

tosyl

formyl

a

Z = benzyloxycarbonyl; Bzl = benzyl

110
FIGURE 2

Synthesis [Gly17]-8h-EP by Segment Coupling:

SPPS, Solid-Phase Peptide Synthesis; Cit, Citraconyl; HOSu, N-Hydroxysuccinimide
The purity of the synthetic product should be examined

by both high performance liquid chromatography (HPLC)

and partition chromatography on gels (Yamashiro 1980).

For peptides synthesized by the solid-phase method,

single-deletion peptides are likely to be generated and

extremely difficult to separate from the desired product.

It has been shown that HPLC is inadequate for effecting

these separations (Yamashiro and Li 1981).

Bioassay

The opiate-receptor binding assay with rat brain

membranes was carried out as described by Ferrara et

al. (1979) and Nicolas et al. (1982), using [

3

H2-Tyr2]-

ßh-EP (Houghten and Li 1978) as the primary ligand and

synthetic ßh-EP (Li et al. 1977b) as the standard

competing ligand. The analgesic potency was estimated

by the mouse tail-flick method (Loh et al. 1976).

Naturally Occurring Agonists

ß-EPs isolated from pituitary glands of various species

are naturally occurring analogs of the opioid peptide.

Human (Li et al. 1977b), camel (Li et al. 1976), equine

(Li et al. 1981b), turkey (Yamashiro et al. 1980), and

ostrich (Yamashiro et al. 1982a) ß-EPs have been

synthesized for biological characterization. Table 2

presents potencies of these ß-EPs in the binding and

analgesic assays (Hammonds et al. 1982). Parallelism

of the dose-response curves in both assays indicate

that ß-EPs from different species bind to the same brain

opioid receptors. However, the ratio of binding

activity to analgesic potency varies from 164 to 555

relative to ßh-EP as 100 (see table 2). No correlation

between these two activities exists. It was suggested

that, once bound to the receptor, these naturally

occurring agonists do not possess equal ability or

efficacy to produce analgesia.

TABLE 2

Biological Activities of Human, Camel, Equine,

Turkey, and Ostrich ß-Endorphin

Analgesic Opioid-receptor B/A X 100

ß-EP potency binding activity (Potency

(A) (B) ratio)

Human 100 100 100

Camel 165 270 164

Equine 153 330 216

Turkey 45 96 213

Ostrich 110 610 555

112
Synthetic Agonists

For the last 8 years, over 90 ß-EP analogs have been

synthesized and characterized (Li 1981; Yamashiro and

Li 1984). Some analogs are purer agonists than the

parent peptide as they exhibit higher biological

activities. Five of these analogs are: [Gln8]-ßh-EP

(Li et al. 1981a), [Trp27]-ßh-EP (Li et al. 1982),

[Gln8,31]-ßh-EP (Yamashiro et al. 1982b), (Arg8,Gln31]-

ßh-EP (Yamashiro et al. 1982b), and [Dem1-7]-ßc-EP

(Yamashiro et al. 1983). Their binding activity and

analgesic potency are summarized in table 3. Among

these analogs, [Trp27]-ßh-EP exhibits the lowest

binding/analgesia ratio, indicating that it is a very

efficacious molecule for producing analgesia when it is

bound to the receptor. Residue position 27 in ß-EPs

(see figure 1) is occupied by either His or Tyr. This

represents a marked difference in hydrophobicity when

this property is measured in a two-phase solvent system

(Yamashiro 1980). On the same scale, Trp is the most

hydrophobic amino acid. It is of interest that

[Trp27]-ßh-EP is one of the most potent synthetic

analogs as an analgesic agent (Li et al. 1982).

TABLE 3

Biological Activities of Some Synthetic

ß-EP Agonists

Synthetic

peptides

Analgesic Opioid-receptor B/A X 100

potency binding activity (Potency

(A) (B) ratio)

ßh-EP 100 100 100

[Gln8]-ßh-EP 220 170 77

[Trp27]-ßh-EP 371 68 18

[Gln8,31]-ßh-EP 236 200 93

[Arg8,Gln3l]-ßh-EP 254 150 59

[Dem1-7]-ßh-EP* 440 301 68

*Dem = Dermorphin: H-Tyr-DAla-Phe-Gly-Tyr-Pro-Ser-NH2

There are two Glu residues in the ßh-EP structure at

positions 8 and 31 (see figure 1). Replacement of

Glu3l by Gly does not alter the analgesic potency (Li

et al. 1979) or Tyr (Yamashiro et al. 1982b). However,

replacement of Glu8 by Gln enhances both analgesic

potency and opioid-receptor binding activity. This is

also true for the replacement of Glu8 by Arg (Yamashiro

et al. 1982b). Since alterations of the Met-enkephalin

113
sequence in ß-EP have led to loss of analgesic potency

(Li 1981), it was surprising that the hybrid [Dem1-7]-

ßc-EP is a potent analgesic (Yamashiro et al. 1983).

Dermorphin (Dem) is isolated from the frog skin with

very potent opiatelike activity (Montecucchi et al.

1981). When Dermorphin, the hybrid, and ßh-EP are

assa ed for analgesia (see table 4), Dermorphin and

[Dem1-7]-ßc-EP exhibit nearly identical potency. This

suggests that the 8-31 segment of the hybrid has no

apparent influence on the analgesic actions of the

Dermorphin segment and is in contrast to the analgesic

potency of ß-EP which is dependent on chain length (Li

1981).

TABLE 4

Biological Activities of Dermorphin and

[Dem1,7]-ßc-Endorphin

Analgesic Opioid-receptor B/A X 100

Peptide potency binding activity (Potency

(A) (B) ratio)

ßh-EP 100 100 100

ßh-EP 164 311 190

Dermorphin 450 30 7

[Dem1-

7

]-ßc-EP 440 301 68

Naturally Occurring Antagonist

ß-EP-(1-27) has been isolated and characterized from

pig (Smyth et al. 1978), rat (Zakarian and Smyth 1979),

and horse (Ng et al. 1981) pituitary glands. It is

also shown to occur in rat (Zakarian and Smyth 1979)

and bovine (Ng et al. 1982) brains as well as in human

ectopic ACTH-producing lung cancers (Suda et al. 1982).

Figure 3 presents the protocol for the isolation of

ß-EP-(1-27) from horse pituitary (Ng et al. 1981). The

final steps involved paper electrophoresis and HPLC.

From 300 g of fresh horse glands, 1.2 mg ße-EP-(l-27)

and 3 mg ße-EP were isolated. As shown in figure 4,

only 0.018 mg ße-EP-(1-27) and 0.026 mg ße-EP were

obtained from 16 kg of bovine brains (Ng et al. 1982).

ßh-EP-(1-27) was subsequently synthesized (Zaoral et

al. 1981) for biological investigations.

114
FIGURE 3

Protocol for the Isolation of ß-EP-(1-27)

from Horse Pituitary

FIGURE 4

Protocol for the Isolation of ß-EP-(l-27)

from Bovine Brain

115
Figure 5 presents inhibition of [

3

H-Tyr27]-ßh-EP

binding to rat brain membranes by ßh-EP-(l-27) or

naloxone. Thus, ßh-EP-(1-27) retained 30% of the ßh-EP

potency in displacing tritiated ßh-EP, whereas naloxone

was one-tenth as potent. In the mouse tail-flick

assay, ßh-EP-(1-27) exhibited less than 2% of the ßh-EP

potency in producing analgesia (figure 6). Results are

summarized in table 5. The high ratio of opioidreceptor binding activity to analgesic potency (potency

ratio) predicts ßh-EP-(l-27) as an antagonist to ßh-EPinduced analgesia. This turns out to be the case

(Hammonds et al. 1984).

FIGURE 5

Inhibition of [

3

H-Tyr 27]-ßh-EP Binding to Rat Brain

Membrane by ßh-EP-(1-27) or Naloxone

TABLE 5

Biological Activities of ßh-EP-(l-27) and Naloxone

Analgesic Opioid-receptor B/A

Preparation potency binding activity (Potency

(A) (B) ratio)

A D 5 0

Relative I C5 0

Relative

pmol/ potency nM potency

mouse

ßh-EP 27 100 0.33 100 1

ßh-EP-(l-27) 1500 1.8 1.10 30 17

Naloxone 12.00 2.8

As shown in figure 6, intracerebroventrical (i.c.v.)

injection of various doses of ßh-EP together with a

fixed dose of ßh-EP-(1-27) produced a parallel shift of

the dose-response curve (Hammonds et al. 1984).

116
Similar results were obtained with naloxone. Analyses

of the data are clearly evident that the antagonistic

effect of ßh-EP-(1-27) is competitive in nature and

that the analog is at least 4 times more potent than

naloxone in antagonizing analgesia.

FIGURE 6

Analgesic Effect of ßh-EP and ßh-EP-(1-27)

Synthetic Antagonists

The first synthetic analog of ß-EP shown to be an

antagonist is ßc-EP-(6-31). This analog does not contain the Met-enkephalin segment and appears to possess

measurable analgesic activity (Li et al. 1978). At

high doses,

11 mice,

ßc-EP-(6-31) produces analgesia in 4 out of

and in some mice, inhibition of the tail-flick

response was not blocked by naloxone (Li et al. 1978).

In guinea pig ileum assay, it had about 4% opiate

activity in comparison with ßc-EP (Li et al. 1977a).

At 40 μg but not at 25 μg, ßc-EP-(6-31) was analgesic,

although the main tail-flick latency was not as high as

that with morphine (6 μg) or ßh-EP (6 μg). When 40 μg

ßc-EP-(6-31) was injected i.c.v. together with either

morphine or ßh-EP, it inhibited morphine-induced

analgesia measured 10 minutes later but not 30 minutes

later (Lee et al. 1980). However, the analog appeared

to inhibit significantly ßh-EP-induced analgesia at all

time intervals. When the effect of ßh-EP (6 μg) was at

its peak in 30 minutes, the analog was capable of

antagonizing ßh-EP-induced analgesia in a dose-related

manner with some inhibition at a ßc-EP-(6-3l)/ßh-EP

ratio as low as 2.5 (Lee et al. 1980).

117
We subsequently discovered three synthetic analogs of

ßh-EP with high potency ratio (the ratio of potency in

displacing [

3

H-Tyr27]-ßh-EP to analgesic potency) to be

good inhibitors to ßh-EP-induced analgesia (Nicolas

et al. 1984). These synthetic analogs with their

biological activities are listed in table 6. Lack of

correlation between the opioid-receptor binding

activity and analgesic potency is evident and the

potency ratio is high, indicating that these analogs

may act as antagonists.

Dose-response curves for the analgesic effect produced

by i.c.v. injection of ßh-EP, [Trp27]-ßh-EP, synthetic

analogs, and their combinations are shown in figures 7

and 8. The duration of analgesia for the analogs and

the parent peptide was identical. Dose-response curves

were parallel, suggesting that they produced analgesia

by acting through similar opiate receptors. In

addition, the time for the peak analgesic effect of

ßh-EP and [Trp 27]-ßh-EP either alone or in the presence

of various doses of analogs was the same. There was a

marked decrease in the analgesic response to ßh-EP or

[Trp27]-ßh-EP when small doses of the analogs were coinjected. For each combination of agonist/antagonist

investigated, a parallel shift of the dose-response

curve of the agonist to the right with increasing doses

of antagonist was observed. This parallel shift

indicates competitive inhibition as also shown by

straight lines in the Schild plots. Figure 9 presents

Schild plots for ßh-EP and [Trp27]-ßh-EP as agonists

with various doses of antagonists. From these data,

the apparent antagonist potencies, assuming the potency

of ßh-EP-(1-27 to be 1, were estimated as follows:

[Cys11,26,Phe27,Gly31]-ßb-EP 6; [Arg9,19,24,28,29]- 

ßh-EP, 17; and Gln8,Gly31]-ßh-EP-Gly-Gly-NH2, 48.

Thus, [Gln8

,Gly31]-ßh-EP-Gly-Gly-NH2 is more than 200

times as potent than naloxone in inhibiting ßh-EPinduced analgesia.

Concluding Remarks

Since 1976, ßh-EP has been isolated and identified from

pituitary glands of camel, porcine, human, horse,

ostrich, turkey, bovine, ovine, whale, rat, and mouse.

Camel, human, equine, turkey, and ostrich ß-EP were

synthesized by improved procedure of the solid-phase

method. In the mouse tail-flick assay, analgesic

potency was found in the following order: camel >

equine > ostrich > human > turkey. The opioid-receptor

binding activity order was: ostrich > equine > camel

human > turkey. They are naturally occurring agonists.

118
TABLE 6

Analgesic Potency and Binding Affinity of Synthetic

ßh-EP Analogs with Inhibiting Activity
FIGURE 7

(A) Log-probit dose-response curves for antinociceptive

effect produced by i.c.v. injection of [Trp27]-ßh-EP

alone (o-o) and in the presence of 0.01 μg 0.025

μg 0.04 μg or 0.1 μg of [Gln8

,Gly31]-

ßh-EP-Gly-Gly-NH2. (B) As in figure 7(A), but for

ßh-EP alone or in combination with 0.0045 μg

0.009 μg or 0.02 μg of [Gln8

,Gly311-ßh-EP-GlyGly-NH2. The dose-res onse curve for analgesia

produced by [Gln8

,Gly31]-ßh-EP-Gly-Gly-NH2 alone is

shown in both cases.

120
FIGURE 8

(A) As in figure 7A, [Trp27]-ßh-EP alone or in

combination with 0.010 μg 0.025 μg o r

0.075 μg of [Arg 9,19,24,28,29 ]-ß h -EP. Antinociceptive effect produced by [Arg9,19,24,28,29]-ßh-EP

alone. (B) As in figure 7A, [Trp27]-ßh-EP alone (O-O)

or in combination with 0.05 μg , 0.075 μg or

0.300 μg of [Cys11,Cys26,Phe27,Gly31]-Bh-EP.

Antinociceptive effect elicited by [Cys11,Cys26,Phe27)-

Gly31]-ßh-EP.

121
FIGURE 9

(A) Relationship between dose ratio for analgesia

with [Trp27]-ßh-EP or ßh-EP (O-o) and

corresponding doses of [Gln8,Gly31]-ßh-EP-Gly-GlyNH2. Abscissa: negative logarithm of the molar

dose of antagonist injected per 25 μg of body

weight. Ordinate: log (x-l), where x is the dose

ratio. (B) As in figure 8A, but for [Trp27]-ßh-EP

and corresponding doses of either [Cys11,Cys26,

Phe2 7,Gly3 1]-ßh -EP (A-A) or [Arg 9,19,24,28,29 ] -

ßh-EP

122
The naturally occurring antagonist ß-EP-(1-27) is at

least 4 times more potent than naloxone in antagonizing

analgesia. It was named ß-EP-inhibiting peptide (ßEIP).

Three synthetic analogs of ßh-EP were shown to possess

inhibiting activity to ß-EP-induced analgesia. One of

them, ([Gln8 ,Gly31]-ßh-EP-Gly-Gly-NH2), was estimated to

be more than 200 times more potent than naloxone in the

mouse tail-flick assay.

Among more than 90 synthetic analogs of ß-EP, at least

5 are potent agonists, namely [GLN8-ßh-EP, [Trp27]-ßh--

EP, [Gln8-31]-ßh-EP, [Arg8,Gln8-31]-ßh-EP, and [Dem1-7]-

ßc-EP

In most naturally occurring ß-EP agonists and synthetic

analogs, the opiate-receptor binding activity and

analgesic potency do not correlate. This may be

explained by differences in efficacy of the agonist or

analog once it is bound to the receptor. The ratio of

binding affinity to analgesic potency (potency ratio)

is a measure of the degree of efficacy. Thus, a

naturally occurring or synthetic ß-EP analog with high

potency ratio is expected to be an antagonist to

ß-EP-induced analgesia.

As pointed out earlier, ß-EP-(1-27) with a potency ratio

of 17 is shown to be present in the brain and pituitary

gland. It acts as an inhibitor to ß-EP-induced analgesia

and is 4.5 times more potent than naloxone. This is

highly significant. Inhibition of a peptide hormone by

a naturally occurring segment of the same hormone may

be a general phenomenon in biologically active peptides.

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ACKNOWLEDGMENTS

The experimental work, summarized herein, was derived

from collaborations with my coworkers whose names

appear in the cited publications. This work was

supported in part by National Institute on Drug Abuse

grant DA-03434 and National Institutes of Health grant

GM02907.

126
AUTHOR

Choh Hao Li, Ph.D.

Laboratory of Molecular Endocrinology

University of California, San Francisco

San Francisco, California 94143

127
Enkephalin Degrading Enzyme Inhibitors: A

Physiological Way to New Analgesics and

Psychoactive Agents

Bernard P. Roques and Marie-Claude Fournie-Zaluski

It is now well accepted that the pain suppression effect of morphine is related to the interaction of this alcaloid with binding sites located in the central nervous system (CNS) and more

precisely within structures (e.g., spinal cord, periaqueductal

gray matter, and thalamus) known for their involvement in the

regulation of nociceptive stimuli. Moreover, the wide distribution of opioid receptors in brain probably accounts for the

multiplicity of pharmacological responses (including euphoria)

elicited by administration of morphine (Martin et al. 1976).

In addition to its strong analgesic potency, it must also be

observed that morphine was shown to display anxiolytic and

disinhibitory properties. According to these features, psychic

dependence and respiratory depression-- which are among the

major side effects of narcotics --could be related to overstimulation of brain receptors involved, respectively, in behavioural and bulbar respiratory control (Morin-Surun et al. 1984).

Several thousand compounds were synthesized with the aim to

discard or at least to minimize these major side effects, but

at this time no potent analgesic proved completely devoid of

these serious drawbacks. However, the discovery in the CNS of

the endogenous morphinelike peptides, enkephalins (Hughes et

al. 1975), which interact with multiple opioid receptors (Lord

et al. 1977) and are degraded by recently well-defined metabolic pathways, could allow resolution of the challenging

problem of addiction. In this chapter, we have summarized the

results that we have obtained both in the characterization of

enkephalin degrading enzymes and in the rational design of inhibitors of these various peptidases. These compounds, being able

to prolong the duration of action of the endogenously released

128
enkephalins following nociceptive stimuli, behave as completely

new analgesics acting through a more physiological mechanism.

Owing to the probable role of enkephalins in emotional and

behavioural controls, enkephalin degrading enzyme inhibitors

could occur also as new psychoactive agents.

ENZYMATIC INACTIVATION OF ENKEPHALINS

A weak and transient analgesia was obtained only for high doses

(= 100 μg per mouse) of intracerebroventricularly administered

Met5-enkephalin (Tyr-Gly-Cly-Phe-Met) or Leu5-enkephalin (TyrGly-Gly-Phe-Leu) (Belluzi et al. 1976). These features suggested that, according to their neurotransmitter role, these peptides were quickly removed from the synaptic Cleft. In vitro

incubation of enkephalins with brain tissue has shown that

several peptidases are able to cleave the endogenous pentapeptides into inactive fragments. So, the Tyr-Gly bond can be

hydrolyzed by several membrane-bound aminopeptidases (Hambrook

et al. 1976; Vogel and Altstein 1977; Guyon et al. 1979). As

discussed further, one of these brain enzymes, resembling aminopeptidase M from rabbit kidney (Kerr and Kenny 1974), could be

more specific (Hersch 1981; Shimamura et al. 1983; FourniéZaluski et al., in press [1985a]). Furthermore, a dipeptidylaminopeptidase activity releasing the Tyr-Gly fragment is also

involved in enkephalin degradation (Gorenstein and Snyder 1979).

The putative physiological role of this enzyme will be discussed

in this paper. Finally,

by cleavage of the

the enkephalins are easily metabolized

Gly5-Phe bond under the action of two

enzymes present in brain: the angiotensin converting enzyme

(ACE) (Erdos et al. 1978) and a distinct peptidase, originally

designated enkephalinase (Malfroy et al. 1978). It is now

well established that the brain membrane-bound enkephalinase

is identical (Almenoff and Orlowski 1983; Fulcher et al. 1982)

to the neutral metalloendopeptidase originally isolated by Kerr

and Kenny (1974) from rabbit kidney. The in vitro metabolic

pathways of enkephalins are schematized in figure 1. It is of

major interest to notice that all the enkephalin inactivating

enzymes belong to the group of metallopeptidases offering,

therefore, the possibility to design mixed inhibitors.

129
FIGURE 1

In Vitro Inactivation of the Enkephalins

by Various Enzymes of Brain Tissue

ENKEPHALINASE, DIPEPTIDYLAMINOPEPTIDASE, AND AMINOPEPTIDASB

SOURCES AND ASSAYS OF ENZYME ACTIVITY

Enkephallnase from rat brain and rabbit kidney being identical

(Fulcher et al. 1982; Almenoff and Orlowskl 1983), we obtained

the purified enzyme from the latter. Membrane-bound dipeptidylaminopeptidase activity was purified from rat brain by slight

modification (Bouboutou et al. 1984) of the reported method

(Gorenstein and Snyder 1979). Two membrane-bound amlnopeptldase

activities releasing Tyr from enkephalins were separated from

a particulate fraction (P2) of rat brain using a linear gradient

of NaCl. One peptldase was more sensitive to kelatorphan and

was found to be identical to the amlnopeptldase M from rabbit

kidney. As this enzyme is very likely to be specifically involved in the synaptic degradation of enkephallns, aminopeptidase

M isolated from rabbit kidney was commonly used as the enzyme

source (Fournié-Zaluskl et al., In press [1985a]).

The enkephallnase activity and its inhibition were determined

using either [

3

H] Leu-enkephalln or [

3

H] D-Ala2-Leu-enkephalin

as substrates (Fournié-Zaluskl et al. 1983). The formed [3

H]

Tyr-Gly-Gly metabolite was separated as described (Vogel and

Altstein 1977). Moreover, a fluorimetrlc determination of

130
FIGURE 2

Schematic Representation of the Binding of

or Inhibitors to Thermolysin

enkephalinase activity was also used (Florentin et al. 1984).

Substrates

The formation of [3H] Tyr or [3H] Tyr-Cly by aminopeptidase or

dipeptidylaminopeptidase, activities, respectively, and the

inhibition of these peptidases were evaluated using [3H] Leuenkephalin, as reported by Bouboutou et al. (1984). Determinations of inhibition constants, and kinetic experiments were

computed from regression analysis using a Hewlett Packard

calculator. The biological relevance of the results obtained

with purified enzymes was established by comparison with those

evaluated using membrane preparations of rat brain tissue.

STRUCTURAL AND FUNCTIONAL CHARACTERISTICS OF METALLOPEPTIDASES

As nicely shown from the crystallographic analysis of two metallopeptidases--the carboxypeptidase A (Lipscomb 1980) and the

bacterial endopeptidase thermolysin (Holmes and Matthews 1981;

Monzingo and Matthews 1982), all the zinc metalloproteases have

similarities in their active sites and in their respective

mechanisms of action (Kester and Matthews 1977). As shown in

figure 2, the hydrolysis of a peptide bond could involve: 1) the

coordination of the oxygen of the fragile bond to the Zn atom;

2) the Glu-143 promoted nucleophilic attack of a water molecule

on the carbonyl carbon polarized by the Zn ion; and 3) the

protonation of the nitrogen of the peptide bond to be cleaved

by His-231. In the final step, the bond between the tetrahedral

carbon and nitrogen atoms breaks to yield the two peptide products. Therecent crystallographic study of hydroxamate-thermo131
lysin complexes have suggested that the hydrolysis process

could occur through the initial formation of a pentacoordinate

complex of the metal without displacement of the water molecule

bound to the Zn atom by the oxygen of the fragile bond (Monzingo

and Matthews 1982). On the other hand, a careful analysis of

several complexes between thermolysin and substrates or inhibitors have shown that the specificity of the metallopeptidase

is ensured by: 1) Van der Waals interactions between S1, S1',

and S2' subsites of the enzyme and the lateral chains of the

corresponding P1 , P1', and P2' moieties of the substrate or the

inhibitor; and 2) several well-positioned hydrogen bonds between

donor and acceptor groups of the bound molecule and polar residues of the peptidase, such as Asn-ll2, Arg-203, etc. Owing

to the great similarities in the active site of all metallopeptidases (Kester and Matthews 1977), the knowledge of the

binding mode of inhibitors to thermolysin at the molecular

level allowed us to design potent inhibitors of enkephalin

degrading enzymes in a rational way.

CHARACTERIZATION OF ENKEPHALINASE ACTIVE SITES

Among the various peptidases able to cleave the enkephalins,

in vitro, enkephalinase was the first for which a crucial role

In the biological inactivation of the opioid peptides has been

demonstrated (Roques et al. 1980). This feature is supported

by the strong analgesic potency of enkephalin analogs protected

from enkephalinase degrading activity (Fournié-Zaluski et al.

1979) and by changes in enzymatic activity following chronic

morphine treatment (Roques et al. 1980). Therefore, our first

studies were directed toward the synthesis of enkephallnase

inhibitors. However, the carboxydipeptidase, ACE, also displays

some ability to split the Gly3-Phe4 bond of enkephalins (Erdos

et al. 1978), suggesting the presence of similarities in the

active sites of enkephallnase and ACE. Therefore, given the

involvement of this latter enzyme in blood pressure regulation,

it was very important to develop enkephalinase inhibitors exhibiting both a strong potency and a selectivity as high as possible in anticipation of their eventual clinical use.

PRELIMINARY INVESTIGATIONS OF S1' AND S2' SUBSITES OF

ENKEPHALINASE

sequences X-Ala and Phe-X (X, corresponding to various amino

A careful analysis of a large series of dipeptides with the

132
acids) has shown that the specificity of enkephalinase is

essentially ensured by 1) an S1' subsite which interacts preferentially with aromatic or hydrophobic moiety (Fournié-Zaluski

et al. 1981; Llorens et al. 1980); 2) a moderate but significant

preference of the S2' subsite for short side chains; and 3) an

enhanced affinity for substrates bearing a free COOH-terminal

group (Fournié-Zaluski et al. 1979). Therefore, according to

its specificity toward bonds of the amino side of aromatic or

hydrophobic amino acids, enkephalinase is able to cleave a large

variety of peptides, such as substance P, neurotensin, cholecystokinin, etc. Regarding the differences with ACE, it is of great

importance to notice that the S2' subsite of enkephalinase displays a large aversion for proline (Fournié-Zaluski et al. 1979,

Fournié-Zaluski et al. 1981). To illustrate, compounds bearing

a C-terminal proline such as captopril (Cushman et al. 1977) are

well recognized by the S2' subsite of ACE; whereas, in contrast

to enkephalinase, the S1' subsite of ACE does not exhibit a

significant preference for aromatic or hydrophobic side chains

(Cushman et al. 1977). So, as shown In table 1, In contrast

to Phe-Pro, the dipeptides Phe-Leu and Phe-Ala behave as relatively good enkephalinase inhibitors but are badly recognized by

ACE. Moreover, several interesting binding properties of enkephalinase are reported in table 1. As expected, the replacement

of the natural amino acid L-Phe by its stereoisomer D-Phe leads

to a large loss of inhibitory potency. A similar effect was

obtained by N-methylation of the amide bond of Phe-Ala; whereas,

surprisingly, retroinversion of this bond (change from -CONHto -NHCO-) led only to a tenfold decrease in activity. This

result was interpreted by a topological analogy between the

crucial components (benzyl and methyl chains, NH3

+

, and COOgroups) In the R,R isomer of retro-Phe-Ala and the natural

dipeptide L-Phe-L-Ala (Roques et al. 1983). This was confirmed

by the synthesis of the two enantiomers of retro Phe-Gly (table

1).

This very interesting result indicates that, despite the amide

bond reversal, the oxygen and hydrogen atoms fill similar spatial

positions in both retro and natural amide bonds (Roques et al.

1983). This probably allows the crucially required hydrogen

bond formation of the -NHCO- group within the enkephalinase

active site.

133
TABLE 1

Inhibitory Potency of Natural and Modified Dipeptides on

Enkephalinase and Angiotensin Converting Enzymes (ACE) a

COMPOUNDS IC50 (μM)

ENKASE ACE

L-Phe-L-Leu

L-Phe-L.Ala 1

D.Phe-L.Ala 100

L-Phe-(N-Me)L.Ala

retro-Phe-Ala (R,S + R,R)

retro-Phe-Gly (R)

retro-Phe-Gly (S)

20 700

> 100 > 1000

10

15 NT

100 NT

> 1000

> 1000

NT

L.Phe-L-Pro > 100 NT

a

 The IC50 values (means from five independent experiments with

SEM < 10%) were computed using [3H]-Leu-Enk (20 nM) as substrate

on enkephallnase from mouse striata and ACE from Calbiochem.

RATIONAL DESIGN OF CARBOXY AND THIOL INHIBITORS OF ENKEPHALINASE

According to their selectivity and relatively good potency

against enkephallnase activity, the dipeptides Phe-Ala and Phe-Leu

were selected as starting models. to design highly potent enkeph -

alinase inhibitors by introduction of a Zn-chelating group. As

nicely shown by Cushman et al. (1977) in the synthesis of ACE inhibitors, carboxyl and mercapto groups appeared among the most potent

metal coordinating agents. For the first time, new carboxyalkyl

compounds derived from Phe-Leu were selected as enkephalinase inhibitors (Fournié-Zaluski et al. 1982, Fournié-Zaluski et al. 1983).

The general formula of these molecules is: R-CH(CH2φ)-CONH-CH-

[CH2CH(CH3)2]COOH with R = COOH, 1; R = CH2COOH, 2; R = NH-CH2-

COOH, 3; and R = NH-CH2-CH2-COOH, 4. All these compounds behave

as competitive inhibitors of enkephalinase, with IC values In

the 0.7 to 10 μM range. Similar results were reported for compounds 2 and 3 by Smith and Wilkinson (1982).

134
The most interesting characteristics of this first series of

inhibitors are the following: 1) Whatever the position of the

carboxyl group in the chain, the IC50 values on enkephalinase

remain in the same range, whereas ACE recognition is strongly

modulated by the length of the chain bearing the COOH group.

So, in accordance with ACE inhibitors derived from carboxyalkanoyl-L-proline (Cushman et al. 1977), the most potent blockers

of this latter enzyme are compounds 2 and 3 with IC50 of 20

and 2 μM, respectively.. 2) The enkephalinase specificity of 2

and 3 is tightly decreased as compared to their dipeptide

precursors.

According to the demonstrated selectivity of the S1' subsite of

enkephalinase for aromatic or hydrophobic side chains (FourniéZaluski et al. 1981), various carboxyl inhibitors containing

these moieties as Pl' components were synthesized. Highly potent

enkephalinase inhibitors (ICso of 20 nM) were obtained in two

series of compounds derived from the dipeptlde L-Phe-ß-Ala. The

two series were characterized by introduction on the amino group

of Phe to one of the following chelating residues: φ -CH2-

CH(COOH)- (Berger and Chipkin, personal communication) or φ-CH2-

CH2-CH(COOH)- (Mumford et al. 1982). Interestingly, as compared to 3, introduction of the additional phenyl ring seems to

increase the affinity for enkephalinase. This could indicate

the presence In the enzyme of an S1

subsite interacting favorably

with an aromatic moiety. Moreover, the affinity of these compounds for enkephallnase is modulated by the configuration of

both the a carbon of Phe and the carbon bearing the carboxyl

group. Thus, the S,S isomer is at least 100 times more potent

than the other stereoisomers (R,S; S,R; R,R) (Berger and

Chipkin 1984). In contrast, the inhibitory potency of aompound

3 remains practically identical whatever the configuration of

Phe is. These features can be interpreted by the fact that

binding of the carboxyl group to the metal atom represents the

most important factor for affinity. Therefore, the binding

strength can probably overcome an imperfect fitting of the P1'

moiety in the S1' subsite but is unable to counterbalance an.

additional adverse interaction occurring at the level of the

S1 subsite. On the other hand, the replacement of B-Ala In

the preceding inhibitors by the 2HN-C6H4-COOH group, led also

to efficient enkephallnase blockers (Almenoff and Orlowskl

1983).

135
The second series of enkephalinase inhibitors that we have

designed bears a thiol group as metal chelating agent (Roques et

al. 1980; Fournié-Zaluski et al. 1984b). The general formula

is: R-CH(CH2φ)CONH-CH[CH2-CH(CH3)2]-COOH. In contrast to the

carboxyl inhibitors, the enkephalinase recognition is crucially

dependent on the length of the thiol containing group R. The

most potent compound, with R = CH2SH, exhibits an IC50 of 4.5

nM on enkephalinase but behaves also as a good ACE inhibitor

(IC50 = 55 nM). This unfavorable feature, already observed in

the case of carboxyl inhibitors, could be related to the binding

strength of the coordinating group which minimizes the preferential interaction of the side chains with the specific subsites

of enkephalinase or ACE. Therefore, in order to find a selective enkephalinase inhibitor, various amino acids were used as

P2' components. Increase in the size of the side chain does

not change significantly the IC50 on enkephalinase but enhances

the affinity for ACE. Consequently, the best P2’ moiety for

selective inhibition was shown to be a glycine residue, and the

obtained compound N-[(R,S)-3-mercapto-2-benzylpropanoyl]-glycine

(KI = 2 nM) with a discrimination factor of around 40 was designated thiorphan and used in the first biological studies (Roques

et al. 1980). Interestingly, the reduction of the P1' benzyl

group of thiorphan into a methylene cyclohexyl chain (-CH2-C6H11)

led to a less potent enkephalinase inhibitor (IC50 = 31 nM), but

this change strongly inhibits the binding to ACE (IC50 > 10,000

nM) (Fournié-Zaluski et al. 1984b). Likewise, introduction of

an HS-CH2-CO group on the N-terminal part of L-Phe-L-Leu led to

relatively good enkephalinase inhibitor (IC50 = 70 nM) with

weaker ACE affinity (Altstein et al. 1983).

COMPLETE DIFFERENTIATION BETWEEN ENKEPHALINASE AND ACE INHIBITION BY RETROTHIORPHAN

On the other hand, a large decrease in IC50 on enkephalinase

but not on ACE was observed by N-methylation of the amide bond

of the previous thiol inhibitors (Fournié-Zaluski et al. 1984b).

This finding agrees both with the results obtained at the dipeptide level and with the good affinity for ACE of inhibitors bearing an N-CH3 group or a proline in P2' position (Cushman et al.

1977). So, taking into account that the subsite specificity

of P1’ and P2' residues cannot ensure a complete differentiation

of these two enzymes, the modification of the amide bond was

considered as the most promising discriminating factors. This

136
clearly demonstrated through the synthesis of retrothiorphan,

HS-CH2CH(CH2 φ)NHCO-CH2-COOH; since with a KI = 6 nM, this compound is almost as potent as thiorphan on enkephalinase but

displays a drastic loss of potency on ACE (IC50 > 10,000 nM)

(Roques et al. 1983). Moreover, a stereospecific synthesis of

retrothiorphan showed that the R isomer (KI=2 nM) is much more

potent on enkephalinase than the S isomer (IC50 - 200 nM). In

contrast, the R and S isomers of thiorphan have almost the same

potency (Fournié-Zaluski et al., in press [1985a]). Therefore, the

topological concept of retro-inverso isomers (Goodman and

Chorev 1979) that we have extended for the first time to

enzyme inhibitors could be adapted to other enkephalinase

inhibitors. Preliminary results support this assumption.

Finally, it must be observed that the retro-inversion of an

amide bond in peptides or related compounds bestows to these

molecules an enhanced resistance to proteolytic enzymatic

degradation.

STRUCTURAL CHARACTERISTICS OF THE ACTIVE SITE OF THE NEUTRAL

METALLOENDOPEPTIDASE (EC 3.4.24.11) ENKEPHALINASE AND FUNCTIONAL

IMPLICATIONS

The major structural requirements for selective interaction with

enkephalinase or ACE were schematized in figure 3. Owing to its

endopeptidase activity, enkephalinase might be compared to thermolysin while, in view of its largely preferential exopeptidase

action, ACE could be related to carboxypeptidase A. Therefore,

starting from the crystallographic analysis of the complex of

thermolysin with a thiol (Monzingo and Matthews 1982) or an

hydroxamate inhibitor (Holmes and Matthews 1981), some characteristics of the enkephalinase active site and mechanism of

action can be derived (Fournié-Zaluski et al. 1984b). All of the

Zn metalloenzymes, including ACE and enkephalinase, contain an

essential arginine residue in their active site. In thermolysin,

and possibly in enkephalinase, the oxygen and the hydrogen

atoms of the peptide bonds between the P1' and P2' moieties of

substrates or inhibitors are hydrogen bonded, respectively, to

the guanidinium group of the Arg residue and to the oxygen of

the amide bond of an Asn amino acid. Obviously, this latter

interaction is hindered by N-methylation of the amide bond or

by inclusion of the nitrogen atom in a proline ring accounting

for: 1) the decreased affinity for enkephalinase of inhibitors

with an N-methylated amide group (Fournié-Zaluski et al. 1984b);

2) the severe loss of potency of captopril on enkephalinase

137
FIGURE 3

Schematic drawing showing the main structural requirements

allowing preferential interaction with enkephalinase or ACE

at the level of: the metal atom, the S1' and S2' subsites,

the amide bond.

(IC50 > 10,000 nM) (Roques et al. 1980). On the other hand,

the Arg side chain is located on the "side" of the substrate

or inhibitor in complexed thermolysin (Holmes and Matthews 1981).

Assuming the same kind of disposition for enkephalinase, the somewhat preferential carboxydipeptidase activity of this enzyme

(Florentin et al. 1984) could be due to a larger degree of freedom of the Arg aide chain, allowing the formation of a salt bridge

with the free carboxyl group of enkephalins. Such a process

accounts for the catalytic activity of carboxypeptidase A and

probably of ACE. In this way, it is interesting to note that

enkephalin analos with a CH2OH C-terminal group in place of

COOH are protected from degradation by enkephalinase. The assumed

arginine side-chain flexibility within the active site of enkephalinase probably counteracts the small change in the position

of the oxygen and hydrogen atoms at the retroamide bond as compared to the natural amide bond. This should permit the formation of hydrogen bonds between the retroamide group of retrothiorphan and enkephalinase (Roques et al. 1983). The S1'

hydrophobic pocket of enkephalinase is clearly larger than the

corresponding subsite of thermolysin or ACE, since the former

does not bind compounds with a Trp as P1' component; whereas, a

cyclohexyl ring in this position leads to a complete loss of

affinity for ACE. Therefore, the assumed mobility of a Pl'

residue in the large hydrophobic pocket of enkephalinase could

138
explain the less stringent structural requirement for optimal

binding to the Zn atom in enkephalinase than in ACE (FourniéZaluski et al. 1983). This assumption is supported by the

strong affinity of carboxyl inhibitors bearing a very large

P1' group (p.C6H5-CH2-OC6H4-CH2-) in place of the benzyl residue

(Berger and Chipkin 1984).

BINDENTATE PEPTIDES AS HIGHLY POTENT AND MIXED INHIBITORS OF

ENKEPHALIN DEGRADING ENZYME

As noted in the first section, the enkephalins are cleaved in

vitro by enkephalinase, a dipeptidylaminopeptidase, and a membrane-bound aminopeptidase. Recently, inhibition of this latter

enzymatic activity by bestatin was shown to induce an increase

in brain enkephalin content and subsequently a naloxone reversible analgesia following intracerebroventricular (i.c.v.)

administration (Carenzi et al. 1981; Chaillet et al. 1983).

All the enkephalin degrading enzymes belong to the group of

metalloproteases characterized by a wide specificity. It was,

therefore, theoretically possible to design a compound able to

inhibit the three peptidases, provided that the expected loss

of binding affinity due to a relative inability of the lateral

chains of the inhibitor to fit adequately the respective subsites

of the three different enzymes is counterbalanced by the strength

of coordination to the Zn atom.

As shown by Nishino and Powers (1978), bidentate group such as

hydroxamic, NH(OH)CO, or N-acyl-N-hydroxyamino, -CO-N(OH)- are

able to form strongly stabilized pentacoordinate complexes with

the metal atom of various metallopeptidases. So, the thermolysin

inhibitor, HN(OH)-CO-CH(CH2φ)-Ala-Gly-NH2, behaves also as a

potent enkephalinase inhibitor (Mumford et al. 1981). Likewise,

Blumberg et al. (1981) have shown that amino acid hydroxamates,

such as Z-Phe-NHOH and Z-Leu-NHOH, were able to inhibit enkephalinase and aminopeptidase with IC50 values in the micromolar

range. According to these features, we decided to prepare four

series of inhibitors bearing bidentate groups on structures

related to Phe-Gly or Phe-Ala (Bouboutou et al. 1984; FourniéZaluski et al., in press [1985].

To avoid isomerization of inhibitors belonging to the series

of hydroxamic acids, NH(OH)CO-CH2-CH(CH2φ)CONHCH(R)-COOH, which

may occur during the synthesis by classical methods a new

139
TABLE 2

Inhibitory Potency of Various Hydroxamates and N-hydroxy,

N-acyl Peptides on Enkephalinase and ACE Activity a

a IC50 values (means ± SEM of five determinations) were determined using

[

3H]D-Ala2-Leu-Enk as substrates with pure enkephalinase and ACE.

synthetic procedure was developed (M.C. Fournié-Zaluski et al., patent

and unpublished results). The binding of the bidentate inhibitors is

schematized (table 2) using the currently accepted active site model

(Cushman et al. 1977). As expected, compounds l, 2, and 3 belonging to

three different series of bidentates behave as highly potent enkephalinase inhibitors with IC5O values in the 4 to 15 nM range (table 2).

As for thiol inhibitors (Fournié-Zaluski et al. 1984b), a methylene

spacer separates the benzyl P1' moiety from the metal ion-chelating

group in the most active compounds, 4 and 5. The chromatographic

separation of the two diastereoisomers of 5 led to a pure compound

[(R)-3-(N-hydroxy)-carboxamido-2-benzylpropanoyl]L-alanine, whose

configuration, analogous to that of a natural dipeptide, was established

by NMR spectroscopy (Fournié-Zaluski et al., in press[l985b]).

140
TABLE 3

Influence of the Stereochemistry of Compound 5 on the Binding

Affinity to the Three Enkephalin Degrading Enzymes

On the other hand, among the four series of bidentates, compounds

4 and 5 behave as highly potent and competitive inhibitors of

the dipeptidylaminopeptidase that releases Tyr-Gly from enkephalins. These derivatives are the first described highly potent

inhibitors of this enzyme (Bouboutou et al. 1984).

Very interestingly, compound 5 also interacts with a relatively

good affinity (IC50 = 0.4 μM) to both the aminopeptidase M

isolated from rabbit kidney and a membrane-bound aminopeptidase

partially purified from rat brain (Waksman et al. 1985 ; FourniéZaluski et al., in press [1985a]. The (R) isomer of 5, designated

kelator-phan, behaves therefore as the first fully described

inhibitor of enkephalin metabolism (Fournié-Zaluski et al. 1984b).

More- over, inhibition of the three enkephalin degrading enzymes is

modulated by the stereochemistry of 5 (table 3).

It is interesting to observe that the potency of bestatin to

inhibit the release of tyrosine from enkephalins is almost

identical (IC50 - 0.5 μM) on purified aminopeptidase and on the

set of various aminopeptidases of mouse brain membrane preparation. In contrast, kelatorphan is about fiftyfold less potent

(IC50 - 20 μM) on brain tissue than on pure enzyme (IC50 - 0.4

μM), suggesting that this new inhibitor is more selective than

bestatin on the biologically relevant enkephalin degrading

141
aminopeptidase. As shown in the next section, this assumption

is supported by the similar analgesic effects produced by kelatorphan or by the association of bestatin and thiorphan, since

the efficiency of kelatorphan and thiorphan to inhibit enkephalinase are identical.

IN VITRO AND IN VIVO PROTECTION OF ENKEPBALINS FROM DEGRADING

ENZYMES

As already discussed, in vitro incubation of enkephalins with

rat brain tissue leads to rapid destruction of the peptides.

This feature occurs also after i.c.v. injection of Met- or

Leu-enkephalin. It was, therefore, possible to evaluate the in

vivo protecting ability of a given inhibitor by i.c.v: coadministration in mice of increasing concentrations of this compound

with a fixed dose of [3H] Leu-enkephalin. After 5 minutes, the

mice were killed, and the intact [3H] Leu-enkephalin was determined. In this assay, kelatorphan at 50 μg is able to protect

80% of Leu-enkephalin and this protecting effect is at least as

efficient as that produced by the association of thiorphan (50

μg) and bestatin (50 μg) (Waksman et al. 1985: Waksman et al.,

in press).

Another way of testing the potency of inhibitors to protect the

enkephalins from the various peptidases is to use washed brain

slices. In this condition, more closely related to the actual

physiological situation, the peptide substrate is cleaved only

by membrane-bound enzymes (Patey et al. 1981). As-shown in

figure 4, incubation of a mixture of 20 μM of [3H] Met-Enk with

rat striatal slices leads to the appearance of the tritiated

metabolites Tyr, Tyr-Gly, and Tyr-Gly-Gly, with Tyr as the most

abundant compound. Addition of different inhibitors induces a

decrease in the formed products corresponding to the selectivity

of the inhibitor against each peptidase. In this test, kelatorphan alone was found to be more efficient than thiorphan, bestatin, or the two in combination and occurs as the single compound

to inhibit the Tyr-Gly formation (figure 4) (Waksman et al., in

press).

POTENTIATION OF THE ANALGESIC EFFECT OF ENKEPHALINS EXHIBITING

DIFFERENT SENSITIVITY TO ENKEPHALIN DEGRADING ENZYME

In vivo activity of kelatorphan was compared to that of thiorphan, bestatin, or the two in combination by evaluating the

142
FIGURE 4

Effect of Peptidase Inhibitors on Formation of (

3

H Tyr1 Met ,

5

)

Enkephalin Metabolites by Rat Striatal Slices.

TABLE 4

Analgesic Effects (Hot-Plate Test) of Peptidase Inhibitors

and Potentiation of the Analgesic Effects

of Co administered Opioid Peptide

143
analgesia produced in mice by these inhibitors in the presence

of subanalgesic doses of three enkephalins exhibiting different

sensitivity toward enkephalin degrading enzymes (Fournié-Zaluski

et al. 1984a) (table 4).

When Met-enkephalin, which is sensitive to the three peptidase

was used as analgesic agent, the maximal response was obtained

with 10 μg of kelatorphan (cutoff time, 240 seconds), while the

same effect required the association of bestatin and thiorphan

(10 μg each). Moreover, as judged by its inhibitory potency on

the three enkephalin degrading enzymes, the proper antinociceptive effect of kelatorphan administered i.c.v. was found to be

twofold higher than that of bestatin or thiorphan (table 4, first

column). All inhibitor-induced analgesic responses were prevented by prior administration of naloxone, demonstrating that the

observed effects were due to specific stimulation of opioid

receptors.

Interestingly, naloxone shows pronociceptive effects in these

assays (Jacob et al. 1974; Roques et al. 1980). In contrast,

peptidase inhibitors as well as naloxone are inactive on mouse

tail-flick and mouse tall-withdrawal tests; likely because the

physiological release of enkephalins is not high enough to

lessen the high nociceptive stimuli produced by these latter

tests. So, in strong nociceptive conditions, peptidase inhibitors cannot significantly reduce painful messages, and naloxone

cannot magnify them. Another explanation may be that, in tailflick and tail-withdrawal tests, opioid peptides less sensitive

to enkephalinase, such as dynorphin, should be preferentially

involved in pain regulation. In any case, in the presence of

subanalgesic doses of exogenous enkephalins, peptidase inhibitors

are strongly active on tail-flick and tail-withdrawal tests.

Finally, the protecting activity of the various inhibitors was

compared by measuring on the tail flick-test the apparent analgesic efficiency (ED50) of Met-enkephalin i.c.v. coadministered

with different concentrations of inhibitors. On this test,

kelatorphan was about fivefold more active than bestatin in

combination with thiorphan and was able to decrease 50,000 times

the ED50 of Met-Enk (Fournié-Zaluski et al. 1984b).

144
STUDY OF ENKEPHALIN DEGRADING ENZYMES BY USE OF A TRITIATED

INHIBITOR

The final demonstration of the physiological and selective role

of the three enkephalin degrading enzymes at the level of enkephalinergic transmission requires acute investigations on: 1) the

binding characteristics of these peptidases; 2) a quantitative

determination of their subcellular distribution and localization

in the CNS; and 3) an evaluation of the possible changes in these

parameters under different pharmacological situations (acute and

chronic treatment by inhibitors, opiates, neuroleptics, etc.) or

after lesions of assumed enkephalinergic neuronal pathways.

These studies cannot be done safely without the assistance of a

radiolabeled probe. We have therefore synthesized the inhibitor

4 under its tritiated form: [3H]-[(R,S)-3-(hydroxyamino)carbonyl2-benzyl-1-oxopropyl]-glycine ([3H]-HACBO-Gly; 45 Ci/mmole).

The binding of [3H]-HACBO-Gly to crude rat brain membranes shows

that the inhibitor interacts with two distinct populations of

independent sites with KI values close to 0.3 nM (Bmax = 55 fentomole/mg protein) and 20 nM (Bmax = 600 fentomole/mg protein),

respectively (figure 5).

Inhibition of [3H]-HACBO-Gly binding by different effectors

shows that the high-affinity binding site corresponds to enkephalinase and the low-affinity site very likely to dipeptidylaminopeptidase activity (table 5). Indeed the KI values to the high

affinity site computed from displacement curves were in close

agreement with the KI values obtained from kinetics experiments

performed on both pure and rat brain membrane-bound enkephalinase. Finally, the bidentate HACBO-Gly is poorly recognized by

aminopeptidase (IC50 = 40 μM) and, therefore, the binding of [3H]-

HACBO-Gly to both sites was not inhibited by bestatin (10 μM)

(Waksman et al. 1984, 1985).

VISUALIZATION OF ENKEPHALINASE IN RAT BRAIN

The highly favourable binding characteristics of [3H]-HACBO-Gly

allowed us to perform, for the first time, a direct visualization

of enkephalinase in rat brain by radioautography (Waksman et al.

1984). As shown on figure 6, the selective binding (completely

eliminated in presence of thiorphan 1 μM but unmodified by captopril 1 μM), is especially dense in discrete cerebral regions,

such as globus pallidus, caudate nucleus, putamen, substantia

145
FIGURE 5

Binding Isotherm of [3]HACBO-Gly to Crude

Rat Brain Membranes Preparation (P2Fraction)

The First linear part of the curve corresponds to the

binding with enkephalinase (KD = 0.3 nM) and the second

part to the binding with a dipeptidylaminpeptidase activity.

146
TABLE 5

Inhibitory Potency of Various Peptidase Inhibitors on

the [3H]HACBO-Gly Binding (High Affinity Site Corresponding to

Enkephalinase) to Rat Brain Membranesa

Compounds KI (M)

Thiorphan 3.0 ± 1.0 10-10

Kelatorphan

Phosphoramidon

5.0 ± 0.5 10-10

3.5 ± 0.5 10-10

HS-CH—-CONH-CH2-COOH 6.6 ± 0.4 10-8

C H 2φ

HOOC-CH2-CH—CONH-CH-COOH 5.0 ± 1.2 10-7

CH2φ CH2CH(CH3)2

Captopril > 1 0 - 3

Bestatin 9.0 ± 0.5 10-4

a

 [3H]HACBO-Gly (1 nM), KD = 0.3 nM. KI values were computed

from Cheng-Prusoff equation assuming competitive inhibition.

nigra (SN), olfactory bulb, choroid plexus, and spinal cord.

The density of grains is weaker in hippocampus cortex and cerebellum. Finally, the aspect of the labeling in the striatum and

substantia nigra could indicate the occurrence of enkephalinergic

pathways linking the SN to the putamen and this latter structure

to the caudate nucleus. These assumptions are now being tested

by lesion experiments.

Finally, it must be observed that this preliminary study clearly

indicates that-except for striatum and SN, the distribution of

enkephalinase does not overlap that of ACE (Strittmatter et al.

1984). Likewise the distribution of in vitro enkephalinase

sensitive neuropeptides (cholecystokinin, SP) are not correlated

with that of the enzyme. In contrast, there is a relatively

strong correlation between the radioautographic distribution of

the enzyme labeled with [3H]-HACBO-Gly and its assumed substrates,

enkephalins. However, the very weak labeling of particular

brain areas rich in μ receptors (thalamus, brain stem, etc.), if

confirmed, could be of great physiological interest.

147
FIGURE 6

Autoradiographic Distribution of Enkephalinase

in a Lateral Section of Rat Brain, Labeled with the Tritiated

Inhibitor [3H]HACBO-Gly (3 nM).

The specificity of binding was demonstrated by the complete loss

of [3

H]HACBO-Gly labeling in presence of 1 μM thiorphan (Waksman

et al. 1984).

148
CONCLUSION

The antinociceptive effects elicited by inhibitors of enkephalin

degrading enzymes show clearly the validity of this heuristic

approach. These results were confirmed by various groups

(Chipkin et al. 1982; Greenberg and O’Keefe 1982; Yaksh and

Harty 1982) and several pharmaceutical firms are now working

in this field (Roques and Fournié-Zaluski,in preparation).

As expected, preliminary experiments seem to indicate the lack

of the major side effects of narcotics (tolerance, dependence

and respiratory depression) after chronic treatment by enkephalinase inhibitors (J. Costentin et al., unpublished results).

Nevertheless, the possible clinical use of these compounds

remains conditioned by the strength of the induced analgesia

and by the development of pharmacokinetically acceptable forms.

In any case, inhibitors of enkephalin metabolism behave as useful

tools in the investigation of the physiological role of these

neuromodulators in different brain areas. It is interesting to

note that stereotaxic injection of kelatorphan in rat caudate

nucleus induced behavioural responses similar to those produced

by DTLET, a selective 6 agonist for opioid receptors (Zajac et

al. 1983). Taking into account that the striatal dopamine release seems to be regulated by δ-receptor stimulation (Chesselet

et al. 1982), this feature could suggest the occurrence of an

enkephalinergic tonus in this structure and, therefore, a putative role for enkephalinase inhibitors at this level. Accordingly, these compounds could behave as new psychoactive agents.

The respective roles of the three enkephalin degrading enzymes

in the metabolism of the endogenous peptides remain still to

be specified. It appears clear, nevertheless, that enkephalinase

and an aminopeptidase selectively inhibited by kelatorphan are

involved in enkephalin metabolism. The significant increase in

Tyr formation following selective inhibition of enkephalinase

could suggest, as recently proposed (Carenzi et al. 1983), that

the aminopeptidase activity may increase when the content of

enkephalins reaches too high a concentration. Such a mechanism

should be dependent on both the level and the Km Of enkephalinase

and aminopeptidase, respectively. This model could probably be

investigated using tritiated probes such as HACBO-Gly. Obviously, the thiorphan-induced modulation of the formed tyrosine was

not observed with kelatorphan, since this compound is able to

149
inhibit the three peptidases simultaneously. According to this

property, an analgesia significantly longer than that induced

by thiorphan was observed after administration of kelatorphan

(Fournié-Zaluski et al. 1984a). Finally, the visualization of

enkephalinase in the brain by radioautography strongly supports

a selective role of this enzyme in enkephalin metabolism, but

it would be of great interest to compare by this precise method

the distribution of enkephalinase and that of the various opioid

receptor-subtypes (μ, δ, κ) in different brain areas. Although

the role of the dipeptidylaminopeptidase in enkephalin metabolism

at the synaptic level seems to be not very important, the high

concentration of this enzyme could suggest its involvement in

the regulation of enkephalin levels within the neuron terminals.

This hypothesis would be tested by radioautographic visualization

of this peptidase using [3H]-HACBO-G1y.

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AUTHORS

Bernard P. Roques

Marie-Claude Fournié-Zaluski

Departement de Chimie Organique

U 266 INSERM et UA 498 CNRS

Faculté de Pharmacie

4 Avenue de l’observatoire

75006 Paris, France.

154
Progress in the Characterization of the

Opioid Receptor Subtypes: Peptides as

Probes. Future Directions

Eric J. Simon, Ph.D.

INTRODUCTION

It is my task to review the progress that has been made in the characterization of the

different types and subtypes of opioid receptors. In the framework of the present

conference, I shall try to emphasize those areas of the research in which opioid

peptides have been particularly useful. This does not mean that I plan to ignore

other aspects which are essential for an overview and understanding of this area.

In 1973, three laboratories (Simon et al. 1973; Terenius 1973; Pert and Snyder 1973)

independently reported the existence of stereospecific binding sites for opiates in the

central nervous system of animals and man (Hiller et al. 1973). Since that time, a

large amount of evidence from numerous laboratories has strongly supported the

view that these binding sites represent the recognition portion of functional opioid

receptors.

This discovery was instrumental in the subsequent discovery about 2 years later of

the existence of opioid peptides which appear to be the endogenous ligands of opioid

receptors. Since this entire conference is devoted to these peptides, I shall not

review further their discovery and characterization, except to point out that the

existence of such endogenous ligands lent further confirmation to the functional

importance of opioid binding sites and receptors. It also gave the impetus to the

search for multiple types of receptors, since approximately a dozen opioid peptides

are now known and each could act via its own receptor or receptors.

EARLY EVIDENCE FOR MULTIPLICITY OF OPIOID RECEPTORS

In the mid-196Os, Portoghese (1965) postulated the existence of multiple opioid

receptors based on the relationship between molecular structure of opiate drugs and

their analgesic activity.

The first pharmacological evidence for several classes of receptors was obtained by

Martin and coworkers (Martin et al. 1976; Gilbert and Martin 1976) in experiments

performed in chronic spinal dogs.

Different opiates were found to exhibit differing pharmacological profiles.

Moreover, morphine and the analogues used were unable to substitute for each other

in the prevention of withdrawal symptoms in dogs made dependent on one of the

drugs. Based on these results, Martin postulated the existence of three kinds of

155
opioid receptors which he named for the prototypic drugs used in the study: mu for

morphine, kappa for ketocyclazocine, and sigma for SKF 10,047 (Nallylnormetazocine). As will be seen from the subsequent discussion, these three

types of receptors have withstood the tests of time and of in vitro studies remarkably

well.

After the discovery and characterization of the enkephalins, Hans Kosterlitz and his

group raised the question whether these peptides act via the same receptors as those

through which opiate alkaloids appear to act. To attempt to answer this question,

the Kosterlitz group (Lord et al. 1977) utilized the in vitro bioassay systems it had

used so effectively for many years. They found that opiate alkaloids were more

effective than enkephalins in inhibiting the electrically induced contractions of the

myenteric plexus of the guinea pig ileum. Similar experiments performed with the

vas deferens of the mouse showed that enkephalins exhibit greater potency than

opiates in this system. Furthermore, the inhibition of contraction of the mouse vas

deferens by enkephalins proved to be much less sensitive to reversal by naloxone.

These results were most readily explained by postulating that different classes of

opioid receptors predominate in the two systems. The major receptor present in the

guinea pig ileum was called mu because of its resemblance to Martin’s mu receptor,

while the receptor in the mouse vas deferens was named delta (for deferens).

This group also reported that these two classes of receptors (or rather, binding sites)

could also be demonstrated by binding studies. When competition binding

experiments were carried out in guinea pig brain membrane preparations, it was

observed that enkephalins compete more effectively fo binding against labeled

enkephalins than against labeled opiate alkaloids (e.g., 3H-naloxone), while opiates

compete better against labeled opiates. These results agreed well with those obtained

with the bioassay systems and supported the existence of opiate-preferring (mu) and

enkephalin-preferring (delta) sites. Since then considerable evidence has

accumulated in support of the existence of separate mu and delta sites. The

synthesis of stable analogues of the enkephalins--in particular, [Dala2-

Dleu3[enkephalin (DADLE)--has been very important to the experiments that are

summarized below.

FURTHER EVIDENCE FOR THE EXISTENCE OF MU AND DELTA BINDING

SITES

Protection of Opioid Binding Sites by Selective Ligands

The best way to demonstrate the existence of separate receptors or binding sites is to

be able to inhibit or inactivate one site selectively. Early attempts in our laboratory

to inactivate mu or delta sites selectively using a variety of chemical agents and

enzymes were unsuccessful. Thus, the sulfhydryl group alkylating agent, Nethylmaleimide (NEM), inactivated opiate (naltrexone) and enkephalin (DADLE)

binding to the same extent and with similar kinetics (t l/2 = 8 to 10 min). These

results suggested that differences between mu and delta sites seem to be quite subtle

ones and stimulated us to try selective protection experiments.

We had previously found (Simon and Groth 1975) that opioid receptors could be

protected against inactivation by NEM by the presence of low concentrations of a

ligand. In my laboratory, Dr. Smith (Smith and Simon 1980) carried out experiments

to see whether ligands selective for mu or delta sites gave different degrees of

protection against NEM depending on the type of labeled ligand used. He found

that the inactivation by NEM of 3H-naltrexone binding was prevented more

156
effectively by morphine than by DADLE (figure IA), whereas DADLE was

dramatically more effective than morphine in protecting binding sites for 3Hnaltrexone (figure 1B). At the time these experiments were performed, Robson and

Kosterlitz (1979) carried out very similar protection studies using phenoxybenzamine to inactivate opioid binding sites irreversibly. The results of the two

laboratories were in excellent agreement. These selective protection experiments

furnished strong support for the existence of separate mu and delta opioid binding

sites.

Selective Inhibition of Delta Opioid Binding Sites by Aliphatic Alcohols

More recently, our laboratory has achieved the selective inhibition of delta opioid

binding sites (Hiller et al. 1981, 1984). Ethanol (figure 2) inhibited the binding of

enkephalins and their analogues (in this case, DADLE) at concentrations at which the

binding of 3H-naloxone was unaffected and the binding of dihydromorphine was, in

fact, slightly but consistently stimulated. Inhibition of 3H-DADLE binding was

completely reversible and the potency of the alcohols increased exponentially with

the length of their carbon chain. A number of experiments were done to determine

whether alcohols affect the binding site or the ligand. Space does not permit a

detailed discussion of these studies, but the evidence was consistent with an effect on

the binding site. To cite one study, Scatchard analysis of 3H-DADLE binding in the

presence and absence of n-butanol (0.5%) showed that alcohol decreased the affinity

of binding rather than the number of binding sites (figure 3). Kinetic studies

demonstrated that the inhibition of binding (increase in KD) was the result of an

increase in the rate of dissociation of the ligand-receptor complex. KD’s calculated

from the equilibrium and kinetic experiments were in agreement. These results

support the notion that the inhibition is exerted on the binding site. Moreover, they

indicate that the effect of the alcohols is not due to competitive inhibition of opioid

binding. Competitive inhibition would result in a decrease in the rate of association

of ligand and receptor.

The mechanism of the inhibition is not known. However, we have now tested a

large number of alcohols of varying chain length--primary, secondary, and tertiary--

and have found an excellent correlation between lipid solubility and ability to

disorder cell membranes on the one hand, and potency to inhibit binding to delta

sites on the other (figure 4). We therefore postulate that alcohols increase membrane

fluidity and that delta sites are more sensitive than mu sites to the alteration in their

lipid environment.

Selective Opioid Peptide Ligands for Mu and Delta Sites

The delta opioid binding sites have highest affinity for enkephalins and their

analogues and may be the receptors for endogenous enkephalins. It is therefore not

surprising that a number of highly selective delta ligands recently synthesized are

derivatives of enkephalins. What is surprising is the fact that three of the most

selective ligands currently known for mu sites are also peptides, two of which are

close analogues of enkephalin.

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FIGURE 1

Protection of Opioid Binding From Inactivation by NEM. Membrane preparations

from rat brain were incubated for 10 mins in Tris buffer pH 7.4 with various

concentrations of protecting ligand at 37oC followed by incubation for 20 mins with

NEM (0.5 mM). The reaction was terminated by addition of 0.5 mM glutathione and

the incubation mixture was centrifuged at 20,000 x g for 15 mins followed by two

washings by centrifugation at the same speed. For binding, samples were

resuspended in the original volume of buffer and incubated for 15 mins at 37oC

with 1 nM of either 3H-naltrexone (A) or 3H-DAla2DLeu5 enkephalin (B). The

protecting ligands used were: Morphine

and DAla2Met5enkephalin The data shown are the mean of 5 experiments

(from Smith and Simon 1980).

158
FIGURE 2

Effects of Ethanol on the Binding of 3H-labeled Opioids to Opioid Receptors in Rat

Brain Membrane Preparations. Duplicate 2-ml samples (0.9 to 1.1 mg of protein per

milliliter) in 0.05 M Tris-HCl (pH 7.4) containing 1 mM dipotassium EDTA were

incubated with 1unM 3H-dihydromorphine (specific activity 73.2 Ci/mmole), 3Hnaltrexone (8.5 Ci/mmole), and 3H-DADLE (31.0 Ci/mmole). To assess specific

binding, samples were incubated in the presence or absence of 1 uM unlabeled

ligand. Incubations were followed by cooling in an ice water bath for 10 mins

before filtration. The values represent the means ± standard errors for at least three

experiments (from Hiller et al. 198 1. Copyright 1981, AAAS).

FIGURE 3

Scatchard Analysis of Saturation Curves for the Specific Binding of 3H-DADLE

in Rat Brain Membrane Prep rations in the Presence and Absence o f

0.5% N-butanol. Binding of 3H-DADLE (0.5 to 10 nM) was carried out at 25oC

for 45 mins. Lines of best fit were determined by regression analysis (from

Hiller et al. 1984. Copyright 1984, American Society for Pharmacology and

Experimental Therapeutics),

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FIGURE 4

Correlation between the relative potency of alcohols to inhibit 3H-DADLE specific

binding and (A) their relative membrane-disordering potencies and (B) their

membrane/buffer partition coefficients. The relative inhibitory potency (ethanol =

1) for each alcohol was determined from their IC50 values for the inhibition of 3HDADLE binding. Values for relative membrane-disordering potency and

membrane/buffer coefficient (moles per kilogram of membrane per mole per liter of

water) are from Lyon et al. (1981). Lines of best fit were determined by regression

analysis (from Hiller et al. 1984. Copyright 1984, American Society for

Pharmacology and Experimental Therapeutics).

Synthetic work in the laboratory of Professor B. P. Roques in Paris (Gacel et al.

1980; Zajac et al. 1983) has resulted in two relatively selective ligands for delta

opioid binding sites, D-Tyr-Ser-Gly-Phe-Leu-Thr (DSTLE) and Tyr-DThr-GlyPhe-Leu-Thr (DTLET). More recently, a number of highly selective delta ligands

were synthesized by Mosberg et al. (1983). These are penicillamine analogues of

enkephalin, the most selective of which is [DPen2

, DPen5]enkephalin.

Shaw et al. (1982) have reported on the synthesis of some relatively selective

antagonists for delta receptors which have the disadvantage of possessing low

affinity. More recently, the ICI group (Cotton et al. 1984) has developed an even

more selective and more potent antagonist which has the structure N,N-diAllyl-TyrAIB-AIB-Phe-Leu. Based on the mouse vas deferens assay, this compound is as

potent as naloxone against delta receptors, but is inactive against mu receptors.

For mu binding sites, the most selective ligand is the peptide [DAla2-MePhe4glyol5

]enkephalin (DAGO) synthesized by Handa et al. (1981). An enkephalin analogue

synthesized by Roemer et al. (1977), [DAla2DMePhe4Met(O)5-ol]enkephalin (FK33-824), is also quite mu-selective, though less so than DAGO. It was the first

peptide recognized to have a clear preference for mu receptors. Finally, the peptide

morphiceptin (Chang et al. 1981b) is a highly selective, but rather weak, mu ligand.

It is the amide of the N-terminal tetrapeptide of the heptapeptide casomorphin,

isolated from the milk protein casein (Brantl et al. 1979). There is as yet no mu

160
antagonist derived from a peptide. The best mu antagonists

and naltrexone, though they are not highly selective.

continue to be naloxone

EVIDENCE FOR THE EXISTENCE OF KAPPA AND SIGMA SITES

The other two receptors postulated by Martin, the kappa and sigma receptors, have

also survived remarkably well. Support for the existence of kappa receptors came

from studies in monkeys by Woods and collaborators (1979). These animals reacted

very differently to kappa-type opiates, like ethylketocyclazocine (EKC) or

bremazocine, compared to the way they reacted to morphine. Moreover, morphineaddicted monkeys in abstinence did not show reduction of withdrawal symptoms

when given one of these benzomorphans.

Further support for the existence of separate kappa and/or sigma sites came from

drug discrimination studies. Thus, Shearman and Herz (1981) found that rats trained

to recognize bremazocine were able to generalize to EKC, cyclazocine, and SKF

10,047, but were unable to recognize fentanyl or morphine. These results suggest

that these benzomorphans interact with a receptor different from the receptor for the

mu ligands fentanyl and morphine, which has properties similar to kappa and/or

sigma receptors.

Early attempts to demonstrate separate binding sites for 3H-EKC were unsuccessful

in several laboratories (Hiller and Simon 1980; Harris and Sethy 1980; Pasternak

1980). This was due to two problems not recognized at that time. The rat brain has

the lowest proportion of kappa receptors of any species so far studied. Moreover, it

is now known that EKC and other kappa-type benzomorphans bind almost as well to

mu and delta sites as they do to kappa sites. This mean that a simple investigation

of the distribution and characteristics of the binding of 3H-EKC will give results

which suggest that EKC binds to mu and delta sites, especially in rat brain where

kappa sites represent only 10% to 20% of total opioid binding sites (Maurer 1982).

Kosterlitz and his group (Kosterlitz et al. 1981; Magnan et al. 1982) were able to

demonstrate the existence of separate benzomorphan binding sites with the properties

expected of kappa sites. They accomplished this by the use of guinea pig brain

which is relatively rich in kappa receptors, and by including in the incubation

mixture saturating concentrations of the selective mu ligand DAGO and the deltapreferring ligand DADLE. Sites that show a preference for benzomorphans were

also reported in rats by Chang et al. (1981a), who used high levels of morphiceptin

to block mu sites and of DADLE to block delta sites. Because the remaining sites

bound benzomorphan of both the kappa and sigma type, these researchers preferred

to call them “benzomorphan” sites.

Evidence for the existence of sigma sites was the last to be obtained and is still the

most controversial. Binding sites for phencyclidine (PCP) were first reported to exist

in animal brain by Vincent et al. (1979) and by Zukin and Zukin (1979). In more

recent papers, Zukin and Zukin (1981) and Quirion et al. (1981) reported that opiates

of the sigma type, such as cyclazocine and SKF 10,047, but not the more classical

opiates or the opioid peptides, can displace PCP from its binding site. PCP, in turn,

is able to displace the sigma ligands quite specifically. The authors suggested that

the PCP binding site may also be the sigma opioid binding site. Support for this

intriguing idea came from behavioral experiments by Holtzman (1980). Rats trained

to distinguish PCP from saline were able to generalize the drug cue to cyclazocine

and SKF 10,047 but not to other opiates. Rats trained to recognize cyclazocine were

unable to distinguish this drug from PCP. It has been argued, with considerable

161
justification, that sigma receptors should not be classified as a subclass of opioid

receptors since the sigma-type drugs produce effects most of which are not reversed

by naloxone.

Furthermore, there is now evidence from binding (Itzhak et al. 1985) as well as

pharmacological (Brady et al. 1982) studies that the stereoselectivity of

benzomorphans for sigma sites is opposite to that for more classical opioid sites, i.e.,

the dextrorotatory isomer exhibits higher affinity.

Dynorphin A exhibits high affinity and good selectivity for kappa receptors and it

has been suggested that it may be the endogenous ligand of this receptor type

(Chavkin et al. 1982; Yoshimura et al. 1982). Corbett et al. (1982) have obtained

evidence that the shorter peptides dynorphin 1-8 and dynorphin 1-9 are also rather

selective ligands for kappa binding sites. Other peptides derived from the precursor

prodynorphin, such as dynorphin B (rimorphin) and leumorphin, also seem to bind

preferentially to kappa sites (Rezvani et al. 1983; Suda et al. 1983).

No peptide ligand for sigma receptors is currently known. However, several

laboratories are trying to isolate and characterize putative endogenous ligands for

PCP binding sites. Such a putative endogenous substance has been termed

"angeldustin" by C. Pert.

At present, the dynorphin peptides are the only relatively selective peptide ligands

for kappa receptors. The most selective ligands are two nonpeptide compounds. One

is U-50,488H, a compound synthesized and tested by scientists at the Upjohn

Company (Von Voigtlander et al. 1983). The other is tifluadom, a benzodiazepine

devoid of affinity for benzodiazepine receptors, which was discovered at Sandoz to

be a quite selective kappa ligand (Roemer et al. 1982).

OTHER OPIOID RECEPTOR SUBCLASSES

It should be mentioned that, in addition to the four opioid receptor types (or three,

if sigma is not considered opioid) discussed so far, a number of others have been

proposed. They will be mentioned only briefly since their existence is still very

tenuous.

A receptor found in the rat vas deferens by several laboratories (Lemaire et al. 1978;

Miranda et al. 1979; Schulz et al. 1979) seems to be rather specific for

ß endorphin. It has been named the e receptor. Oka et al. (1981) have reported the

presence in dog and rabbit ileum of a receptor which preferentially binds

enkephalins but seems to be different from delta receptors. They have called this

the iota receptor.

There are also reports of heterogeneity within the major receptor types. Based on

extensive studies with the irreversible opiate ligand naloxazone, Wolozin and

Pasternak (1981) have suggested the existence of two subtypes of mu receptors, mu1

and mu2. Their data suggest that mu1

. is a site to which mu, delta, and kappa

ligands bind with greater affinity than to their "own" type of receptor. The mu site

seems to be identical with the classical mu site. Evidence by Pasternak et al. (1980)

indicates that analgesia may be mediated via mu1 receptors.

Audigier et al. (1982) have reported evidence for two types of kappa receptors,

kappa1 and kappa2. Evidence for subtypes of both mu and kappa receptor classes

162
was also reported by

mouse vas deferens.

Schulz and Wuster (1981) based on cross-tolerance studies in the

DISTRIBUTION OF OPIOID RECEPTOR TYPES IN THE CNS

If the postulated types of opioid receptors represent different entities, it should be

possible to demonstrate their differential distribution.

By in vitro binding and competition experiments, Chang et al. (1979) and our

laboratory (Simon et al. 1980) found differences in the distribution of mu and delta

binding sites in rat brain. Regions were found that were relatively enriched in delta

sites, such as the frontal cortex. However, no brain region has yet been found to

contain pure or nearly pure delta sites. On the other hand, the thalamus seems to be

highly enriched in mu sites. This is evident from the ratio of binding of mu

selective to delta selective ligands (at low concentration, e.g., 0.5 nM) as well as from

competition experiments. While in other brain regions, naloxone competed 6- to 10-

fold more effectively against H-naloxone than against 3H-enkephalin, naloxone is

equally effective against both ligands in rat thalamus. Similar results have been

obtained in bovine brain by Ninkovic et al. (1981) and in our laboratory (Bonnet et

al. 1981) in postmortem human brain tissue (table 1).

TABLE 1

Binding Competition Experiments in Human Brain Regions

Brain

3H-naloxone 3H-DADLE

Region n Naloxone DADLE Naloxone PAPLE

Thalamus

Amygdala

Frontal

cortex

Striatum

5 2.7 ± 0.3 49 ± 4.9 1.4± 0.1 13 ± 2.2

3 2.8 ± 0.2 63 ± 24 14 ± 2.9 3.3 ± 0.7

4 2.7 ± 0.3 57 ± 4.9 12 ± 5.4 2.2 ± 0.5

5 2.6 ± 0.4 58 ± 15 31 ± 5.2 3.8 ± 0.6

n = no. of experiments. Results are expressed as the mean IC (nM) ± standard

error of the mean (from Bonnet et al. 1981. Copyright 1981, Elsevier/North Holland

Biomedical Press).

The differential distribution of mu and delta opioid binding sites has also been

demonstrated by autoradiography of rat brain slices (Goodman et al. 1980). These

data are in good agreement with the results obtained by in vitro binding to

homogenates of brain regions.

The distribution of kappa sites was studied in guinea pig brain regions by

autoradiography (Goodman and Snyder 1982). By binding 3H-EKC or 3Hbremazocine to brain slices in the presence of high concentrations of morphine and

DADLE to block mu and delta sites, it was possible to visualize residual sites

believed to be kappa sites. The highest density of these putative kappa sites was

found in layers V and VI of the cerebral cortex and in the pyriform cortex. Low

levels were seen in the caudate and the nucleus accumbens, while other brain regions

did not have detectable kappa sites.

163
We have studied the distribution of kappa sites in human brain regions (Itzhak et al.

1982a). These experiments were done by measuring the binding of 3H-bremazocine

(1 nM) in both the presence and the absence of mu and delta blockers (100 nM each

of DAGO and DADLE). Most of the regions examined were found to have a high

proportion of kappa sites, ranging from a high of 60% in the hypothalamus to a low

of about 20% in the thalamus. In most regions, the proportion of kappa sites was

about 40% of total bremazocine binding. We have also obtained some evidence that

the bremazocine sites may, in fact, be a mixture of kappa and sigma sites. This is

based on the effectiveness of the sigma ligand SKF 10,047 in displacing bound

bremazocine and, more convincingly, on the ability of the rather selective sigma

ligand 3-hydroxyphencyclidine (Itzhak et al. 1981) to displace a portion of the bound

bremazocine from membranes prepared from human brain regions.

TISSUES ENRICHED IN A PARTICULAR TYPE OF OPIOID BINDING SITE OR

RECEPTOR--AND A METHOD TO ENRICH TISSUES

In order to study a particular type of binding site or receptor, it would be useful to

have tissues that are highly enriched in the receptor in question, or better yet,

contain only a single type. Some useful tissues are known and have been briefly

mentioned previously. They will be summarized here.

For mu binding sites, there is evidence of very high proportions in rat (Chang et al.

1979), bovine (Ninkovic et al. 1981), and human thalamus (Bonnet et al. 1981), and

in rabbit cerebellum (Meunier et al. 1983).

There are some brain regions which are enriched in delta sites, but they all contain

large proportions of other receptor types. The only system known to be highly

enriched in sites essentially indistinguishable from brain delta sites are the

neuroblastoma cells (N4TGl) and neuroblastoma x glioma hybrid cells (NG-108-15)

(Chang and Cuatrecasas 1979) in culture. The mouse vas deferens has long been

known to be enriched in delta receptors but is not very useful for binding studies.

Several tissues have been reported to contain very high proportions of kappa sites.

These include human placenta (Valette et al. 1980), toad brain (Simon et al. 1982),

and guinea pig cerebellum (Robson et al. 1984). No other binding sites have yet

been demonstrated in human placenta. The guinea pig cerebellum could have

somewhere between 85% to 100% kappa sites based on the available evidence. Toad

brain is likely to contain a significant number of mu sites (20% to 30%) but very few

delta sites.

For researchers wishing to study the major types of opioid binding sites in the same

tissue, a method of enrichment has been developed (James and Goldstein 1984;

Goldstein and James 1984). It is based on the selective protection studies (Smith and

Simon 1980) discussed earlier and uses the alkylating opiate, ßCNA, developed by

Portoghese et al. (1979) as the inactivating agent. By adding a relatively selective

ligand for a given type of receptor, this receptor can be selectively protected and

significant enrichment of the tissue achieved.

Finally, it should be noted that selective tolerance to a given receptor type can be

produced in in vitro systems, such as the guinea pig ileum (Schulz et al. 1981b) and

the mouse vas deferens (Schulz et al. 1980). There is also preliminary evidence for

selective tolerance in intact animals (Schulz et al. 1981a). Such a selectively tolerant

animal will be useful for studying the remaining “nontolerant” receptors, since the

164
“tolerant” type or types will be. inactive. It is not yet known whether the

of a receptor by tolerance involves a change in binding, characteristics.

inactivation

THE MOLECULAR BASIS OF OPIOID RECEPTOR HETEROGENEITY

All of the evidence lends rather good support to the existence of heterogeneity

among opioid receptors. The question arises as to the molecular basis of this

observed heterogeneity.

One possibility, which may prove correct for some types or subtypes of receptors, is

that they represent interconvertible conformations of the same receptor. This

possibility has been suggested by Bowen et al. (1981). This theory is unlikely to be

correct for all three types, based on the available evidence. However, in view of the

finding that mu and delta sites often occur in the same regions and even on the same

neurons (Egan and North 1981), the idea that they may be interconvertible forms of

the same binding site is more difficult to rule out, though unlikely in this reviewer’s

opinion.

The other possibility is that all or most of the types of receptors are, in fact,

separate entities. There are again several possibilities if this assumption proves to be

correct.

1. Each may be a distinct polypeptide chain or aggregate of such chains.

2. Two or more types may share polypeptide chains, but differ in one or more

of the polypeptide subunits that make up the active receptor molecule.

3. The different types may consist of identical polypeptides, but differ in their

state of aggregation.

4. They may be identical proteins, but differ in their posttranslational

modifications (e.g., carbohydrate or lipid moieties, phosphorylation,

methylation, etc.).

5. Any of the above differences could occur at the level of the binding site

at the level of proteins and enzymes coupled to the binding site, or both.

or

Detailed answers regarding the exact molecular basis of heterogeneity will have to

await purification and attempted separation of the various receptor types. The

progress made so far in this area of research will be the subject of the next section.

SOLUBILIZATION AND SEPARATION OF TYPES OF OPIOID BINDING SITES

We reported the solubilization of a prebound etorphine-receptor complex in 1975

(Simon et al. 1975). The solubilization of opioid binding sites which retain their

ability to bind opioids in solution was achieved more recently in several laboratories.

Bidlack and Abood (1980) reported solubilization of active sites from rat brain using

Triton X-100. Simonds et al. (1980) used a newly synthesized detergent call CHAPS

to solubilize opioid binding sites from NG 108-15 cultures. This technique was also

applicable to rat brain, though yields of soluble receptors tended to be low.

Our laboratory (Ruegg et al. 1980, 1981) reported the solubilization in good yield

(40% to 50%) of opioid binding sites from the brain of the toad, Bufo marinus, by

the use of digitonin. More recently, we succeeded in adapting this method to

165
mammalian tissues by adding 0.5 M to 1.0 M NaCl during the extraction process

(Howells et al. 1982). Yields of soluble receptor from mammalian brain tend to

range between 20% to 40%.

The solubilized receptors bind antagonists well. However, because of the high level

of sodium and the inhibitory effect of digitonin (even at 0.05% to 0.1%) on agonist

binding (Itzhak et al. 1984b), the soluble receptors bind agonists poorly. It was

therefore difficult to determine which types of receptors had been extracted from

the cell membranes.

This problem was approached in our laboratory (Itzhak et al. 1982b, 1984a) by

sucrose density gradient centrifugation of the extract of guinea pig brain into a

gradient devoid of sodium and containing only a low concentration of digitonin

(0.02%), which was further diluted before binding assays were done on the fractions.

When 3H-bremazocine binding was measured in the fractions, two well-separated

peaks were found, as shown in figure 5. When the bremazocine was bound in the

presence of DAGO and DADLE (100 nM each), the first peak was essentially

unchanged, while the second peak was virtually eliminated. As seen from figure 6,

direct binding st dies on the gradient fractions demonstrated good binding of both

3H-DAGO and 3H-DADLE in peak 2. Competition binding studies provided

further evidence that peak 1 contained predominantly sites of the kappa type, while

peak 2 had the characteristics of a mixture of mu and delta sites. Centrifugation of

receptors solubilized from guinea pig cerebellum on the same gradient (figure 5)

yielded only the first peak of binding which again, as expected, exhibited the

properties of kappa sites. Since this tissue is known to contain mainly kappa sites,

this result validates our method of separation and confirms the finding that the first

peak obtained from guinea pig brain extracts contains largely kappa sites. These

results indicate that kappa sites are separable and seem therefore to be distinct

molecular entities. We have not yet succeeded in separating mu and delta sites from

each other.

Separation of kappa sites from other receptor types has also been reported (Chow

and Zukin 1983) by the use of gel filtration columns on CHAPS extracts of rat brain

membranes. The mu sites (

3H-di-hydromorphine binding) eluted at a position

corresponding to a protein Stokes radius of 70A, while most of the 3H-bremazocine

binding in the presence of 40 nM normorphine and 100 nM DADLE eluted at a

position corresponding to a Stokes radius of 50A. This is in agreement with our

results which showed that the mu-delta peak had a higher apparent molecular weight

than the kappa peak as determined on a calibrated sucrose gradient.

A word should be added about our recent success in the partial purification of

opioid binding sites. Dr. Gioannini in our laboratory (Gioannini et al. 1984)

synthesized a novel derivative of naltrexone, ß-naltrexyl-6-ethylenediamine (NED).

This derivative was found to retain high affinity for opioid binding sites

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FIGURE 5

Bremazocine Binding to Fractions of Solubilized Guinea Pig Brain and Cerebellar

Membranes After Sucrose Gradient Centrifugation. Fractions from brain were

assayed for specific 3H-bremazocine (3 nM) binding in the absence (o) and presence

of DAGO and DADLE (100 nM each). Fractions from cerebellum were assayed

under identical conditions, in the absence of DAGO and DADLE (A). The protein

concentration was assayed for each fraction. Protein markers for the

determination of S20, w were as follows: a) catalase; b) thyroglobulin (monomer); c)

ferritin; and d) thyroglobulin (dimer). Each point for 3H-bremazocine binding is the

mean of at least three assays, which differed from each other by <15% (from Itzhak

et al. 1984a).

(IC50 for competition against 3H-naltrexone (1 nM) was 15 nM). It was coupled to

CH-Sepharose 4B beads which contain a 6-carbon side arm ending in a carboxyl

group (see figure 7). The resulting affinity gel retained solubilized opioid binding

sites from cow, rat, or toad brain very efficiently. Elution of about 20% of the

receptors retained on the column was achieved with micromolar concentrations of

naloxone. In our paper, we reported purifications of 300- to 450-fold. However, in

recent experiments (Gioannini et al., unpublished results) with opioid binding sites

solubilized from cow brain, we found that purification is on the order of 3000- to

The crude digitonin extract was iodinated with 125 5000-fold in a single pass. I and

the prtein eluted from the NED-agarose column was determined by measuring the

total 125I radioactivity in the eluate. Electrophoresis of the eluate on SDSpolyacrylamide gels indicated the presence of only 7 to 8 protein bands. Work is in

progress to determine which bands are derived from opioid binding sites. The

improvement in purification was partly the result of improved washing procedures

prior to elution, but also became manifest because of the improved sensitivity of

protein determination due to the use of radioactivity. It has not yet been possible to

determine the ratio of opioid receptor types eluted from the NED-agarose beads

because all these studies were done in high concentrations of sodium chloride.

167
FIGURE 6

Sucrose Density Gradient Centrifugation of Solubilized Opioid Binding Sites from

Guinea Pig Brain Membranes. Fractions were assayed for the binding of 3Hbremazocine (3.5 nM) in the presence of DAGO and DADLE (100 nM each)

3H-DAGO (7nM) (A), and 3H-DADLE (7 nM) (o). Protein markers for the

estimation of molecular weight are as follows: a) catalase; b) thyroglobulin

(monomer); and c) thyroglobulin (dimer). Each point represents the mean ± SEM of

at least three determinations (from Itzhak et al. 1983. Copyright 1983, Pergamon

Press, Ltd.).

CONCLUDING COMMENTS

The discovery of multiple opioid receptors is of great theoretical and practical

importance. It now becomes important to determine which of the receptors proposed

represent separate molecular entities and which peptides serve as endogenous ligands

for which type of receptor. As stated in this review, one can make the

generalization that peptides derived from proenkephalin generally exhibit highest

affinity to delta receptors, while peptides derived from prodynorphin have a

preference for kappa receptors. There are no known natural opioid peptides which

show preferential binding to mu sites. It is therefore possible that the endogenous

ligands for mu receptors have not yet been discovered. Since the enkephalins have

an affinity for mu only 10- to 20-fold lower than for delta sites, this reviewer feels

that the possibility must be kept in mind that mu and delta represent isoreceptors for

the enkephalins. The suggestion has also been made that mu and delta receptors may

interact allosterically (Rothman and Westfall 1982a, 1982b). Some credence is lent to

this hypothesis by the finding that mu and delta receptors can often be found on the

same neurons (Egan and North 1981).

168
FIGURE 7

Scheme for the Preparation of Affinity Beads for the Purification of Solubilized

Opioid Binding Sites

From a practical point of view, it is evident that the multiplicity of receptor types

could be useful, provided that some of them prove to be functionally distinct. It

would then become possible to design drugs with certain desired properties that have

no undesirable side effects. This could be done by synthesizing highly specific

ligands for the type of opioid receptor that mediates the desired function. Thus, if a

type of opioid receptor were found to mediate analgesia but not physical and psychic

dependence, a more rational approach to the long-sought nonaddictive potent

analgesic becomes possible. It is currently believed that both delta and kappa

receptors may be candidates for such a role, but much more work is required to

confirm or disprove this interesting idea. Such studies require highly selective

ligands as well as highly selective antagonists for each receptor type. We hope that

meetings such as this one will stimulate our organic chemist friends to synthesize

substances which may provide us with the needed specificity.

The isolation and purification of opioid receptors and the separation of all separable,

distinct types is the only way to settle the question of the number of different

binding sites or receptors that really exist. This work will also provide us with

other, very important information, including the chemical composition, subunit

structure, and posttranslational modifications of the molecules that comprise the

binding sites. Similar information is needed for coupling factors and for enzymes

which are coupled to the binding sites and essential for transducing ligand binding

into physiological or pharmacological activity. Once the purified molecules become

available, reconstitution experiments will help us to understand the steps between

binding and pharmacological response. This is currently happening with the

nicotinic cholinergic receptor and one may be optimistic that it will happen in the

not too distant future for opioid receptors as well.

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ACKNOWLEDGMENT

The work in the author’s laboratory was

National Institute on Drug Abuse.

supported by grant DA-00017 from the

AUTHOR

Eric J. Simon, Ph.D.

Professor

Departments of Psychiatry and Pharmacology

New York University Medical Center

550 First Avenue

New York, New York 10016

174
Regulation of Agonist Binding to Opioid

Receptor Types by Sodium and GTP:

Relevance to Receptor Function

Brian M. Cox, Ph.D.; Linda L. Werling, Ph.D.; and

Gary Zarr, M.S., ANC

Measurements of radiolabeled opioid ligand binding to membrane

preparations from mammalian brain tissue (Lord et al. 1977; Chang

and Cuatrecasas 1979; Leslie et al. 1980; James and Goldstein 1984)

have confirmed the apparent presence of heterogeneity in opioid

binding sites analogous to the opioid receptor heterogeneity postulated on the basis of physiological studies (Martin et al. 1976;

Gilbert and Martin 1976) and studies of opiold action in in vivo

bioassays (Lord et al. 1977). The quantitative analysis of opioid

ligand binding to receptor sites is important in the analysis of

opiate drug action because it provides a measure of the critical

primary interaction between the drug or endogenous opioid and the

first in the series of tissue components which mediate the drug or

ligand action. This interaction is a primary determinant of subsequent events initiated by receptor activation. A quantitative

analysis of this process is therefore essential to an understanding

of the events mediating opioid drug or peptide action. It is preferable that the properties of the receptor ligand interaction are

measured under conditions which also permit the observation of

drug-induced responses. However, studies of radiolabeled opioid

binding have generally been conducted under conditions of low

temperature and low salt concentration which preclude the demonstration in the same preparation of any biochemical or physiological consequences of opioid receptor activation (Lord et al.

1977; Chang and Cuatrecasas 1979; Leslie et al. 1980; James and

Goldstein 1984). Furthermore, agonist binding affinity is significantly affected by variations in incubation temperatures (Pert

and Snyder 1974; Simantov et al. 1976) and by the concentration of

sodium and other cations (Simon et al. 1973; Pasternak et al.

1975). It is therefore important to establish that the opiold

receptor types discriminated under nonphysiological conditions

can also be identified by radioligand techniques during incubations

conducted at 37o in the presence of physiological cations.

The immediate biochemical consequences of the activation of each

opioid receptor type in mammalian brain membranes have not been

175
ascertained. Studies of oploid effects in neuroblastoma x glioma

(NG 108-15) hybrid cells have demonstrated that opioid receptors

of the d type provide inhibitory regulation of adenylate cyclase

(Sharma et al. 1975; Traber et al. 1975; Blume et al. 1979). In

recent years, confirmation of the initial report (Collier and Boy

1974) that opioid regulation of adenylate cyclase also occurs In

mammalian brain has appeared (Havemann and Kuschinsky 1978; Walczak

et al. 1979; Law et al. 1981; Cooper et al. 1982). At this time,

the opioid receptor types regulating adenylate cyclase in brain

have not been identified with certainty. It has generally been

difficult to quantify oploid inhibition of brain adenylate cyclase

because of the small fraction of brain cyclase activity that is

sensitive to oploid inhibition.

Electrophysiological studies have suggested that one consequence

of μ- and δ-selective opioid interactions with receptors in the

locus coeruleus and substantia gelatinosa of the rat, myenteric

plexus of the guinea pig, and dorsal root ganglion neurons of the

mouse is an increase in a neuronal potassium conductance,

resulting in an outward flow of potassium ions (Williams et al.

1982; Yoshimura and North 1983; Morita and North 1981; Werz and

Macdonald 1983). In contrast, the k-selective oploid, dynorphin,

apparently has little effect on potassium conductances, but decreases a voltage-dependent calcium conductance in mouse dorsal

root ganglion neurons (Werz and Macdonald 1984). The many types

of potassium and calcium channels in mammalian neurons (Nachshen

and Blaustein 1980; Brown et al. 1982; Petersen and Maruyama 1984),

and their complex regulation by voltage and ion concentrations,

have made it difficult to confirm these observations by direct

measures of opiate-induced alterations in ion fluxes. Furthermore,

it is not presently known if the opioid-induced effects on ion

conductances are mediated through inhibition of adenylate cyclase

or by alternative linking mechanisms.

A secondary consequence of opioid action in the central and peripheral nervous systems is inhibition of transmitter release. The

Inhibition of acetylcholine (ACh) release from guinea pig myenteric

plexus by opioids has been known for many years (Paton 1957; Cox

and Weinstock 1966; Kosterlitz and Watt 1968). Morphine also

inhibits the release of adrenergic neurotransmitter at the cat

nictitating membrane (Trendelenburg 1957; Cairnie et al. 1961) and

in mouse vas deferens (Henderson et al. 1972). Several groups have

reported the inhibition of norepinephrine (NE) release from

cerebral cortex tissue by opiate drugs (Monte1 et al. 1974;

Arbilla and Langer 1978; Hagan and Hughes 1984), and electrophysiological evidence of an opioid-mediated reduction in excitability of noradrenergic terminal fields in rat cerebral cortex

has also been noted (Nakamura et al. 1982). Opioid effects on

transmitter release in striatum are less clear. Loh et al. (1976)

reported that morphine inhibited dopamine (DA) release from rat

striatal slices; this could not be confirmed by Arbilla and

Langer (1978). However, it has recently been reported that the

176
k-selective opioid, dynorphin A, inhibits the potassium-stimulated

release of DA from striatal slices, while [Leu5]enkephalln and

[D-Ala2,D-Leu5]enkephalin (DADLE) had little effect on release

of this amine, but inhibited the release of ACh (Mulder et al.

1984). Using push-pull cannula perfusion of cat caudate nucleus

in vivo, morphine has been reported to stimulate DA release

(Chesselet et al. 1981). It seems possible that this is an

indirect effect resulting from interactions with other neurotransmitters in striatum. An inhibition of DA release by morphine

and enkephalin analogs has been reported, however, in rabbit

retina (Dubocovich and Weiner 1983).

The mechanisms by which opioid receptor activation is translated

into an inhibition of neurotransmitter release are not yet known.

The increase in potassium conductance induced by μ- and δ−

selective opioids (Yoshimura and North 1983; Werz and Macdonald

1984) produces a hyperpolarization of the neuronal membrane that

may be sufficient to inhibit transmitter release from terminals of

that neuron. Likewise, the k agonist-induced reduction in calcium

conductance (Werz and Macdonald 1984) may be of sufficient magnitude to reduce the calcium-dependent release of transmitter.

However, the coupling between opiold receptor activation and

effects on ion conductances is not understood. A role for CAMPdependent phosphorylation in the regulation of potassium ion

channels has been proposed in Aplysia neurons (Kandel and

Schwarz 1982). It is conceivable that opioid-induced inhibition

of adenylate cyclase results in the reduced phosphorylation of a

protein which in its phosphorylated state mediates the closing of

a select set of potassium channels. Clearly, much further work is

required to elucidate the possible role of adenylate cyclase inhibition in opioid-induced increases in potassium conductance and

also in elucidating the possible linkage between k-type opioid

receptors and calcium channels.

The opioid receptor types mediating opiold inhibition of transmitter release in cerebral cortex or other brain structures also

have not been identified with certainty. Recent studies by Hagan

and Hughes (1984) have demonstrated that the μ-selective agonist

[D-Ala2-MePhe4-Gly5]enkephalin-ol (DAGO) was more potent than

DADLE, an agonist with high activity at both δ− and μ-type

receptors, in inhibiting NE release from rat cerebral cortex.

Naloxone Ke values in the range 1 to 4 nM in antagonizing the

effects of both DAGO and DADLE suggest that μ-type receptors

were probably responsible for this action. However, the concentrations (EC50) of DAGO and DADLE needed to produce 50% inhibition

of the maximum inhibition of NE release in this tissue were approximately 100 nM and 500 nM, respectively (table 1). These values

are roughly two orders of magnitude higher than the estimated

affinities of these ligands for μ-type opioid binding sites

measured in TRIS-HCl buffer at reduced temperature, and tenfold to

fiftyfold higher than their receptor affinities measured in the

presence of physiological cations. Other studies have confirmed

the low potencies of opioids in inhibiting transmitter release

177
from brain preparations. Thus, concentrations of opioids in the

range 100 nM to 10 μM have commonly been used to produce

measurable inhibition of transmitter release in studies of this

type (Montel et al. 1974; Loh et al. 1976; Arbilla and Langer

1978). Only in retina were slightly lower concentrations required

for significant inhibition of DA release (Dubocovitch and Weiner

1983) and, even here, effective doses were an order of magnitude

higher than estimated μ receptor affinity.

The reasons for this discrepancy between estimates of affinity and

effective concentrations are not clear. It cannot be accounted

for by the probable existence of "spare" opioid receptors (Cox and

Chavkin 1982) since the greater the receptor reserve, the more the

receptor affinity (KD) should exceed the EC50. Another possible

explanation of the low potencies of opioids in inhibiting transmitter release is degradation by the brain slices during diffusion

through the slice to the receptor sites. This is probably a

significant factor in the low potencies of [Met5]- and [Leu5]-

enkephalin in tissue slices (Loh et al. 1976). However, it is

less likely as an explanation of the discrepancies between EC50

and KD for stable opioids such as morphine, DAGO, and DADLE.

Estimates of binding affinity of stable opioids determined in

brain slice preparations are not very different from binding

affinities determined in homogenates (Davis et al. 1975; Rothman

et al. 1984). It seems more probable that the estimates of agonist

affinity determined in nonphysiological buffers at low temperatures

are a poor reflection of the actual affinities of receptors on

intact neurons at normal body temperature. It remains to be

established that the discrimination of receptor types observed in

ligand binding studies under these nonphysiological conditions

can still be demonstrated in the presence of physiological cations

at 37o. We have therefore set out to determine the characteristics

of opioid binding sites in neural membranes under more appropriate

conditions.

COMPOSITION OF INCUBATION MEDIUM

Opioid binding is regulated by both monovalent and divalent cations

(Simon et al. 1973; Pasternak et al. 1975). An initial decision

must be made as to whether to select conditions comparable to

extra- or intra-cellular ionic composition. Since sodium has a

major effect on the binding of opioids to some receptor types, and

the intracellular sodium concentration is very roughly one-tenth

of the extracellular concentration, the selected sodium concentration will have a major effect on the observed opioid binding

parameters. As a starting point, we have opted to employ a modified Krebs solution (see legend, figure 1) with cation composition comparable to that of extracellular fluid in experiments in

which we sought to confirm the discrimination of μ, δ−, and

k-type binding sites. This solution has the relatively high

ionic strength characteristic of both intra- and extra-cellular

fluid. We assumed that under these conditions the specific inhibitory effect of sodium is maximal. Divalent cations appear to

178
TABLE 1

Effects of Opioid Receptor Activation:

Estimates of Agonist Potency

*In most studies, EC50 concentrations were not determined. The

concentrations noted here are those reported to produce significant effects.

179
have their greatest effect on opioid binding in the regulation

of the interactions of guanine nucleotides with the receptor

(Childers and Snyder 1980). Manganese is probably most active in

reducing the inhibitory effects of guanosine trlphosphate (GTP),

but the concentrations at which this effect is observed are

higher than those present in normal neurons. Magnesium may exert

a similar effect (Charg et al. 1983) and is present in effective

concentrations in physiological fluids. It is therefore appropriate that it should be present in the incubation medium.

Physiological fluids are buffered by bicarbonate, phosphate, and

proteins if they are present. These are inconvenient buffers for

in vitro studies. TRIS-HCl buffers have a long history of use,

but many physiological processes, including neurotransmitter

release, do not function in this buffer. We have therefore used

HEPES buffer, which has good buffering capacity at physiological

pH, and appears compatible with many aspects of cell function.

INCUBATION CONDITIONS

Transmitter release in mammalian neurons does not function efficiently at temperatures much below 30o; most studies of opioid

effects on transmitter release have used 37o incubations (Montel

et al. 1974; Mulder et al. 1984). We have therefore used this

temperature in measurements of opioid binding parameters. These

incubation conditions tend to facilitate the degradation of peptides. In determining the characteristics of each type of opioid

binding site, we have therefore selected relatively stable labeled

and unlabeled ligands. Washed neural membrane preparations

from rat whole brain minus cerebellum or from guinea pig cortex

have been incubated in modified Krebs solution at 37o with selected

radiolabeled ligands. Studies of the rate of ligand association

indicated that equilibrium had been reached by 20 minutes of incubation. Bound and free labeled ligands were separated by rapid

filtration through glass fiber filters, and radioactivity retained

on the filters was measured by liquid scintillation spectrometry.

Binding sites with μ-type characteristics were identified by

[

3H]DAGO; δ−type binding sites by [3H]DADLE binding in the

presence of 10 nM unlabeled DAGO; and k type binding sites by

[

3H]ethylketocyclazocine ([3H]EKC) in the presence of 1 μM

unlabeled DAGO. The concentrations of displacing ligands required

to permit specificity of labeled ligand binding were determined

from displacement curves discussed below. Nonspecific binding was

determined by inclusion in the incubation mixture of 1 μM DAGO,

1 μM [D-Ser2Leu5]enkephalyl-Thr (DSTLE) (David et al. 1982),

or 1 μM EKC for μ-, δ− or k-binding sites, respectively.

Binding site properties were characterized by saturation analysis,

and by displacement studies using selective unlabeled ligands.

Data were analyzed by the nonlinear curve fitting program LIGAND

developed by Munson and Rodbard (1980). In control experiments,

it was confirmed by thin-layer chromatography of extracts of the

incubate that incubation of neural membranes with these labeled

180
ligands for periods as long as 60 minutes did not result in significant degradation of any of the labeled ligands. Incubations of

unlabeled ligands for varying periods of time up to 60 minutes

demonstrated that equilibrium was attained by 20 minutes and that

no reduction of displacement, which might indicate degradation of

displacing ligand, had occurred up to 60 minutes of incubation.

OPIOID BINDING SITE CHARACTERISTICS IN MODIFIED KREBS BUFFER AT 37o

The properties of opioid binding sites in modified Krebs solution

at 37o are similar to those previously identified in medium

depleted of physiological cations. Binding sites labeled with the

selective μ ligand, [

3H]DAGO, are relatively insensitive to

displacement by the δ receptor-selective peptide DSTLE, and by

the k-selective agent, U-50488H (James and Goldstein 1984). Thus,

as observed by other workers (Gillan and Kosterlitz 1982), DAGO

appears to be a very selective ligand for μ-type receptors under

most experimental conditions. This high selectivity of DAGO

makes it very useful in the selective protection of μ sites from

occupation by less selective ligands used for labeling of other

types of sites. Binding sites labeled by [

3H]DADLE appear to

comprise two classes; a fraction of the binding was readily displaced by low concentrations of DAGO, while the remaining binding

was more resistant to displacement by this peptide, but could be

displaced by

effect on [

3H]DADLE binding. Binding sites labeled by [3H]EKC

low concentrations of DSTLE. U-50488H had little

were also readily discriminated into two classes by the use of

DAGO or by U-50488H, which each selectively displaced a fraction

of the [

3H]EKC binding (figure 1). Thus, as previously suggested

b others (Gillan and Kosterlitz 1982; James and Goldstein 1984),

[

3H]DAGO binds almost exclusively to μ-type δ sites. [3H]DADLE

binds to both μ and δ sites, with a slight preference for sites;

however, its affinity for the two types of opioid binding sites is

sufficiently similar in modified Krebs solution at 37o for it

not to be possible to resolve these by saturation analysis. Our

results suggest that it has no more than a fivefold preference

for δ-type sites (Werling et al. 1985a). [3H]EKC binding was

also discriminated into two classes In our studies; it has preferential affinity for k-type sites, and approximately tenfold

lower affinity for μ-type sites. Significant occupation of

δ sites by [

3H]EKC was not observed. Thus, when compared to

previous studies of [

3H]EKC binding using TRIS-HCl buffer at

reduced temperature, where EKC showed relatively poor discrimination between k, μ, and δ sites (Gillan and Kosterlitz 1982),

its selectivity for k sites was increased under our incubation

conditions. Of the displacing ligands, DAGO and U-50488H retained

the μ and k selectivity, respectively, previously reported for

these compounds (James and Goldstein 1984), but DSTLE was less

selective for δ sites under our incubation conditions than in

TRIS-HCl buffer at lower temperature (James and Goldstein 1984).

Under our incubation conditions, this ligand was not sufficiently

selective for δ sites relative to μ sites to be used in the

181
selective masking of these sites. Overall, however, μ, δ,

and k binding sites were still readily identified under our more

physiologic incubation conditions.

EFFECTS OF SODIUM ON THE BIRDING PROPERTIES OF OPIOID RECEPTOR TYPES

The effects of sodium under conditions of constant ionic strength

were determined by the stochiometrlc replacement of the sodium in

modified Krebs solution by potassium. Replacement of sodium by

potassium resulted In a small increase in the affinity of ligands

binding selectively to all three receptor types. A 2.8-fold increase In the affinity of [3H]DAGO at μ sites was observed.

The affinity of [

3H]EKC binding affinity at k sites was increased

2-fold, while the increase in the [

3H]DADLE binding affinity

at δ sites was only 1.2-fold. However, there was also a substantial increase in the number of δ-type binding sites after sodium

replacement, an effect which was not observed at μ and k binding

sites (Werling et al. 1985b). Thus, in these experiments,

δ-type binding sites were substantially more sensitive overall

to the specific inhibitory effects of sodium than the μ and k

sites. This is in contrast to the results of Pfeiffer and Herz

(1982). Using rat brain membranes in TRIS-HCl buffer at reduced

temperature, they found that μ binding sites were more affected

than δ or k sites by the addition of 100 mM sodium to the TRIS

buffer. In these studies, however, the effects of increasing

ionic strength and increasing sodium concentration are confounded.

Our results are consistent with our observation noted above that

DSTLE was less selective for δ binding sites in the modified Krebs

solution than in the TRIS-HCl buffer.

It appears that DSTLE binding to δ sites was reduced more than its

binding to μ sites by the presence of sodium in the incubation

medium. The apparent increase in the number of δ binding sites

when sodium was replaced by potassium (Werling et al. 1985b)

deserves comment. This might indicate that sodium can actually

inactivate δ-type receptors; it seems more probable, however,

that the sites apparently lost in the presence of sodium actually

had an affinity that was too low to be detected under the conditions of that experiment. There is no evidence indicating that

exposure of brain membranes to sodium results in a permanent

depletion of δ binding sites. The magnitude of the sodium effect

observed in these experiments was not large overall, except at

some δ sites.

The effective concentration of sodium in reducing binding to each

type of binding site was determined by measuring the effect of

varying the sodium concentration (at constant ionic strength) on

the binding of standard concentrations of selective labeled opioid

agonists. The effect of reducing the sodium concentration on k

binding was relatively small over the complete concentration range.

Binding to d sites was reduced 50% to 60% at the highest sodium

concentration, relative to the binding in the absence of sodium;

50% of the maximum sodium inhibition was observed at sodium concen182
FIGURE 1

DISPLACEMENT OF SPECIFIC EKC BINDING BY U-50,488H

Increasing concentrations of U-50,488H were incubated

with brain membranes and [

3H]EKC, 2nM for 20 min at

37°. The modified Krebs incubation medium contained:

NaCl, 118 mM; KCl, 4.8 mM; CaC12, 2.5 mM; MgCl2, 1.2 mM;

pH was adjusted to 7.4 with HCl. Bound ligand was separated by rapid filtration through glass fiber fitters,

and radioactivity determined by liquid scintillation

spectrometry. Nonspecific binding was estimated by the

addition of 1 μM unlabelled EKC. In guinea pig cortex

homogenates, U-50,488H showed considerable displacement

of [

3H]EKC binding at 10 nM. In rat brain, the number of

k receptors is relatively low, and U-50,488H displaced

only about 10% of specifically bound [

3H]EKC at this

concentration. Further displacement was not observed

until the U-50,488H concentration exceeded 1 μM.

trations in the range of 10 to 30 mM. Similar results were observed at μ sites. Thus, the effective concentration of sodium

(i.e., its EC50) was much closer to its estimated intracellular

concentration, in the range of 10 to 30 mM (Fozzard and Kipnis

1967), than its extracellular concentration (>120 mM). Preliminary

studies with the sodium-selective ion carrier, monensin (Feinstein

et al. 1977), in intact NG-108 cells have suggested that monensin

can inhibit binding to δ-type receptors by increasing the intracellular concentration of sodium (Puttfarcken et al., unpublished

observations). Since sodium is apparently critical to the inhibition of adenylate cyclase by opioids (Blume et al. 1979), it is

probable that sodium interacts at a common intracellular site

which both facilitates the coupling of the receptor to adenylate

cyclase and also regulates agonist binding properties at δ, and

probably at μ, sites.

EFFECTS OF GTP ON THE BINDING PROPERTIES OF OPIOID RECEPTOR TYPES

Guanine nucleotides regulate the binding of opioids (Blume 1978;

Chang et al. 1983; Rosenbaum and Sadee 1983). We have therefore

also evaluated the effects of GTP on opioid binding under our

experimental conditions. The effects of GTP in the presence of

sodium were more pronounced than the effects of sodium alone in

183
reducing agonist binding. The specific binding of [

3H]DAGO was

almost completely inhibited by 100 μM GTP, resulting in very low

specific binding, with an affinity probably in the micromolar

range. Binding affinities as low as this are not accurately

determined, partly because of the high nonspecific binding at the

labeled ligand concentrations required to characterize these sites,

and partly because the ligand dissociation rate from the receptor

is high, relative to the time needed to separate bound from free

ligand by the filtration technique. However, it is clear that

the μ site affinity for DAGO is low in the presence of GTP, and

now consistent with the concentrations required for inhibition of

adenylate cyclase or transmitter release in rat brain slices.

Binding of [

3H]DADLE at δ sites was also substantially reduced by

100 μM GTP, with a roughly fourfold reduction in KD. In contrast,

[

3H]EKC binding at k sites was only slightly reduced. Scatchard

analysis suggested that GTP reduced the total binding of EKC at ktype sites by 20% to 25% with little effect on the affinity of the

residual sites (figure 2). These results probably indicate that

only a fraction of the k sites was coupled to a GTP binding protein

under the conditions of our experiments. The ligand affinity of

the coupled sites was probably reduced below the measurable range

by GTP, thus reducing the apparent number of total sites while

leaving the remaining sites unaffected by GTP.

Our results thus suggest that GTP substantially lowers the agonist

affinity of opioid binding sites with μ-, δ−, and k-type

selectivity, the difference between the k sites and the μ and

δ sites presumably being related to the extent of coupling of

each receptor type to GTP binding proteins. The significance of

GTP regulation of opioid binding is not clear. It might indicate

that each type of opioid receptor is coupled to adenylate cyclase

through a GTP binding protein, as has been suggested for other

receptor-mediated inhibitions of adenylate cyclase (Rodbell 1980;

Spiegal and Downs 1981). However, GTP binding proteins might

also play a critical role in the coupling of hormone or neurotransmitter receptors to other effector systems (e.g., see Fung et

al. 1981).

The apparent variability in sensitivity to GTP (and hence, presumably in extent of coupling to GTP binding proteins) between

different opioid receptor types also deserves some comment. Bowen

et al. (1981) and Rothman et al. (1984) have characterized binding

sites with δ-type characteristics as Type I or Type II on the

basis of their sensitivity to GTP. By varying treatment conditions, evidence implying a possible shift between Type I and

Type II properties was obtained (Bowen et al. 1981). Thus, some

variability in the extent of coupling, particularly of the δ-type

binding site, appears possible (Rothman et al. 1984). Our results

suggest that a much smaller fraction of k-type sites than μ or

δ sites is coupled to GTP binding proteins under our experimental

conditions. Low fractional coupling might be an artifact of

membrane preparation; however, substantial coupling of μ-type

184
FIGURE 2

INHIBITION OF 3H-DADLE AND 3H-EKC SPECIFIC BINDING BY GTP

Scatchard analysis of the effect of GTP (100 uM) on

the specific binding of [

3H]DADLE to d sites (A), and

[

3HlEKC binding to k sites (B) in guinea pig cortical

membranes. No GTP added; GTP was added to give

a final concentration of 100 μ M at the start of the

incubation with labelled ligand. To correct for

probable GTP hydrolysis, a second addition of an

equal amount of GTP was made after 10 min of incubation at 37o. The reaction was terminated after 20

min incubation, as described in Fig. 1.

sites was observed in the same membrane preparations. Differential

GTP sensitivity between different types of opioid binding site

might therefore indicate a difference in the mechanisms of coupling.

However, GTP sensitivity of opioid agonist binding is also related

to the source of the membranes. Roth and Coscia (1984) have

recently shown that opioid binding sites in microsomal membranes

are not sensitive to GTP regulation, although they continue to

show sodium inhibition of agonist binding. The types of opioid

binding site present in microsomal membranes has not been ascertained as yet. Since microsomal membranes are probably present

in our membrane preparations, our results might be explained if

the proportion of k to μ plus δ sites in microsomal membranes was

higher than in the total crude membrane preparation, but there is

no evidence to suggest such a distribution of binding site types.

Uncoupling of receptors from the catalytic and regulatory components of adenylate cyclase is also a feature of agonist-mediated

desensitization (Law et al. 1983). Thus, an alternative interpretation might be that a significant fraction of k sites is desensitized in guinea pig cortex. Further studies are clearly required

to evaluate these possibilities.

185
SODIUM AND GUANINE NUCLEOTIDES IN THE REGULATION OF OPIOID ACTION

The effect of GTP was to produce an even greater reduction of

agonist affinity than sodium alone. The low affinities induced by

the combined treatment are much closer to the effective concentrations of morphine, DADLE, and DAGO in inhibiting adenylate cyclase

or transmitter release in slices of rat brain cortex or striatum.

Thus, it is not improbable that the active conformations of μ and

δ opioid receptors are regulated by intracellular sodium and

guanine nucleotides. The studies discussed above employed a high

concentration of GTP, comparable to estimates of the intracellular

concentration of GTP in neuroblastoma and glioma cells (0.3 to 0.5

mM) (Franklin and Twose 1977; Cass et al. 1977). However, much

lower concentrations of GTP have been shown to be sufficient to

affect opioid binding (Zukin and Gintzler 1980; Bowen et al. 1981).

Thus, while GTP is probably essential for opioid action through δ

and perhaps μ receptors (Blume et al. 1979), it may not act as a

variable regulator of opioid binding in the intact cell. However,

intracellular compartmentalization of GTP is probable (Cass et

al. 1977; Johnson and Mukka 1979), which would make a regulatory

role for GTP feasible. The critical effective sodium concentration

in regulating binding is close to the intracellular concentration

of this ion (Fozzard and Kipnis 1967). Thus, binding affinities

at opioid receptors might be influenced by physiologic, pharmacologic, or pathologic changes in the local intracellular sodium

concentration.

In several respects, the effects of agonists acting through k-type

receptors appear to differ from those mediated through μ or δ sites.

Electrophysiological studies suggest that while μ and δ agonists

hyperpolarize the membranes of sensitive neurons by increasing

potassium permeability (Williams et al. 1982), k agonists might act

by inhibiting an inward calcium current (Werz and Macdonald 1984).

Agonist binding at μ and δ sites is apparently more sensitive to

inhibition by sodium and GTP than binding at k sites, although

these agents do have some effect on k binding. These results suggest that k receptors may be linked to their effectors in a different manner than μ and δ receptors. To date, the ability of k

agonists, acting through k receptors, to inhibit adenylate cyclase

has not been demonstrated. It is possible that an alternative

transduction system may mediate the consequences of k receptor

activation.

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190
ACKNOWLEDGMENTS

Permission from Stephen Brown and Pamela Puttfarcken for the authors

to report unpublished results is gratefully acknowledged. The experimental work described here was supported by grant DA03102 from

the National Institute on Drug Abuse. The opinions or assertions

contained herein are the private ones of the authors and are not to

be construed as official or reflecting the view of the Department

of Defense or the Uniformed Services University of the Health

Sciences. The experiments reported herein were conducted according

to the principles set forth in the "Guide for the Care and Use of

Laboratory Animals," Institute of Laboratory Animal Resources,

National Research Council (DHEW Publication No. [NIH] 78-23, 1978).

AUTHORS

Brian M. Cox, Ph.D.

Linda L. Werling, Ph.D.

Department of Pharmacology

Uniformed Services University of the Health Sciences

4301 Jones Bridge Road

Bethesda, Maryland 20814

Gary Zarr, M.S., ANC

Department of Anesthesia

Walter Reed Army Medical Center

Washington, D.C. 20307

191
Opioid Receptors for the Dynorphin Peptides

lain F. James, Ph.D.

THE DYNORPHIN/NEOENDORPHIN FAMILY OF PEPTIDES

Since the discovery of the enkephalins in 1975, some 20 other

opioid peptides have been isolated, presenting a confusing picture

of mammalian opioid systems. The confusion was cleared somewhat

by the finding that all the known endogenous mammalian opioids

belong to one of three families: the endorphin family (Nakanishi et

al. 1979), the enkephalin family (Noda et al. 1982; Gubler et al.

1982), or the dynorphin/neoendorphin family (Kakidani et al, 1982).

Each family is coded for by a single gene and is synthesized as a

single precursor protein which is processed by specific enzymes to

produce the biologically active peptides. This paper deals with the

interaction of the dynorphin (dyn) and neoendorphin (neo)

peptides with opioid receptors in mammalian nervous tissue. There

are several reviews dealing with the isolation, distribution, and

possible physiologic effects of dynorphins (Cox 1982; Hollt 1983;

Lee and Smith 1984; Goldstein 1984a, 1984b).

Dynorphin was isolated from porcine pituitary and sequenced

partially in 1979 (Goldstein et al. 1979). The sequence was

completed in 1981 (Goldstein et al. 1981; Tachibana et al. 1982).

Around the same time, the fragment dyn-(1-8) was isolated from

porcine hypothalamus (Minamino et al. 1980). In 1982, a second

dyn was found as part of a larger peptide from porcine pituitary

(Fischli et al. 1982) and independently as a separate peptide from

bovine pituitary (Kilpatrick et al. 1982). This second dyn was

called dyn B (or rimorphin) and the original dyn renamed dyn A.

The sequence of the dyn precursor obtained from the sequence of

the mRNA (Kakidani et al. 1982) showed that two other peptides,

α−neo and ß-neo, were also members of the dyn family. These

peptides had already been isolated from porcine hypothalami

(Kangawa et al. 1981; Minamino et al. 1980). In addition, the

sequence of the precursor revealed a new peptide, an extension of

dyn B comprising 29 amino acid residues. This peptide, known as

dyn B-29 or leumorphin, has since been detected in mammalian

nervous tissue (Nakao et al. 1983).

192
The structures of the dyn and neo peptides have several features

in common and are shown in figure 1. All have the [Leu]

enkephalin sequence at the amino terminus and basic residues at

positions six and seven. Most have another basic residue in

position 10 or 13; the exceptions are dyn A-(1-8) and ß-neo.

These features are important both for receptor selectivity and

affinity.

THE DYNORPHINS AND THE KAPPA OPIOID RECEPTOR

It is now well established that there are at least three and

possibly as many as five different types of opioid receptors.

Several reviews summarize the evidence for this and discuss

properties of the different receptor types (Miller 1982; Wood 1982;

Paterson et al. 1983). Each type of receptor is known by a Greek

letter; hence, the μ opioid receptor is the classical morphine

receptor, enkephalins are somewhat selective for the δ receptor,

benzomorphans and dynorphins for the κ receptor, and

ß-endorphin for the ε receptor (table 1).

TABLE 1

Multiple Opioid Receptors--Summary of Different Types

Receptor

Type

Ligands Tissues

μ morphine, naloxone,

sufentanil

brain

guinea pig ileum

mouse vas deferens

δ enkephalins, DADLE brain

mouse vas deferens

κ benzomorphans,

ethylketocyclazocine

(EKC), dynorphin

brain

guinea pig ileum

mouse vas deferens

rabbit vas deferens

ε ß-endorphin rat vas deferens

brain?

σ benzomorphans?

phencyclidine?

brain

193
FIGURE 1

STRUCTURES OF DYNORPHIN PEPTIDES
There are three ways to approach receptor selectivity. First, the

most simple way is to measure selectivity in a binding assay.

Here, selectivity is expressed simply by the ratios of affinities for

a given ligand at the different receptor types. Though measurement of binding affinity may present technical problems, as

discussed below, the definition of binding selectivity is

straightforward. Affinity does not depend on number of receptors

and is not likely to change from tissue to tissue, as long as

conditions for the binding assay (buffer, temperature, etc.) are

kept the same. One can therefore make general statements about

binding selectivity of a given ligand.

Second, one can determine which type of receptor is activated by

a given agonist in a bioassay after exogenous administration of the

agonist. This is pharmacologic selectivity. The bioassays most

commonly used in opioid research are the guinea pig ileum

longitudinal muscle preparation(GPI) and mouse vas deferens (MVD).

In both bioassays, opiates and opioid peptides act through opioid

receptors to inhibit the electrically stimulated contractions of the

muscle. For pharmacologic selectivity, we are dealing with ratios

of potency at different receptor types. Potency is dependent not

only on affinity for a given receptor, but also on the intrinsic

activity (Ariens et al. 1960) or efficacy (Nickerson 1956) of the

agonist and on the number of receptors in the tissue. Hence, a

compound can be selective for one receptor type in one tissue and

a different type in another tissue. For example, [Leu]enkephalin

works through δ receptors in MVD and μ receptors in GPI (Lord et

al. 1977). It is not possible, therefore, to make general statements

about pharmacologic selectivity. For further discussion of binding

selectivity and pharmacologic selectivity, see James and Goldstein

(1984) and Goldstein and James (1984a, 1984b).

Third, there is the question of physiologic selectivity, i.e., which

receptor mediates the physiologic effects of endogenously released

agonist. Physiologic selectivity will depend on efficacy as well as

affinity, but most importantly it will depend on which receptors

are available at the synapses where the endogenous agonists are

released. Theoretically at least, a ligand may be selective for, say,

κ receptors in binding assays and in pharmacologic assays, but

physiologically may never encounter a κ receptor and may always

be paired with other receptor types. There is as yet very little

information on physiologic selectivity of dynorphins, so this

discussion will be limited to binding and pharmacologic selectivity.

Binding Selectivity

The technical problems associated with binding assays are of two

kinds. First, peptides are often degraded by enzymes present in

the receptor preparations. Second, until recently, the so-called

prototypical radioligands for the different types of opioid receptor

showed rather poor selectivity in binding assays. This is

195
especially true for [

3H] (D-Ala2

, D-Leu5)enkephalin (DADLE), which

is commonly used to label δ binding sites but interacts almost

equally well with μ sites, and for [

3H]ethylketocyclazocine (EKC) or

[

3H]bremazocine, which are used to label κ sites but cross-react

considerably with μ sites (James and Goldstein 1984). We now have

highly selective ligands for μ, δ, and κ binding sites, but only the

poorly selective radioligands were available for most opioid binding

studies until 1984. In the most useful of these studies,

degradation of peptides is controlled and attempts are made to

improve the selectivity of the radioligands by blocking their

binding at secondary sites with unlabeled ligands. This can be

done by adding high concentrations of competing ligands that are

selective for cross-reacting sites along with the radioligands or by

selective alkylation of the cross-reacting sites before addition of

radioligand. Both strategies have been used in the study of dyn

binding.

Corbett et al. (1982) used competitive blocking ligands along with

[

3Hlbremazocine to label κ sites in guinea pig brain. They also

measured binding to μ sites with the selective μ ligand

[

3H](D-Ala2

,MePhe4,Gly5-ol)enkephalin, but used the nonselective

ligand [

3H]DADLE to label δ sites. In competition binding assays

with these radioligands, they estimated a dissociation constant of

0.1 nM for dyn A at κ sites. This was about six fold lower than

the dissociation constant of dyn A at μ sites and about twenty fold

lower than its dissociation constant at δ sites. Dyn A-(1-8) and

α−neo were also slightly selective for κ sites. Quirion and Weiss

(1983) used a similar blocking technique and found that in rat and

guinea pig brain, dyn A, dyn B, and α-neo had the same affinity

for κ sites (5 to 10 nM), but did not differentiate very well

between κ, μ, or δ sites. Strangely, their measurements of κ

binding were made in a different buffer from measurements of μ

and δ binding. For a valid comparison of affinities, measurements

should be made under the same conditions.

In neither of these studies was there adequate control for peptide

degradation. This is particularly important with dynorphins

because some potential degradation products (e.g., [Leu]enkephalin

or [Leu]enkephalin-Arg) would still be active opioids but would

have different receptor selectivities from the longer dynorphins

(see the next section of this chapter). Corbett et al. (1982) ran

their experiments at 0oC, and Quirion and Weiss (1983) added

bacitracin to their assays, but there were no demonstrations that

these precautions inhibited degradation of dynorphins. In our

laboratory, we find that dyn A is degraded even at 0o C or in the

presence of bacitracin (unpublished results).

Pfeiffer et al. (1981) showed by high performance liquid

chromatography and bioassay that dyn A was intact during their

binding assays with human brain. They estimated a dissociation

constant of 0.08 nM for dyn A at sites with properties very similar

196
to those expected for

other receptor types.

κ receptors, but did not measure affinities at

The use of selective alkylation in binding assays was suggested by

some early experiments of Robson and Kosterlitz (1979) and Smith

and Simon (1980). They showed that enkephalins protected their

own binding sites in rat brain from alkylation by

phenoxybenzamine or N-ethyl-maleimide more efficiently than they

protected binding sites for dihydromorphine (DHM) or naltrexone.

We extended these findings using the alkylating agent

ß-chlornaltrexamine (B-CNA), an analog of the antagonist naltrexone,

synthesized by Portoghese et al. (1979). Treatment of membranes

from guinea pig brain with ß-CNA resulted in loss of binding sites

for [

3H]EKC, [3H]DHM and [3H]DADLE. The presence of high

concentrations of dyn A during the reaction with ß-CNA protected

[

3H]EKC sites partially without protecting sites for [3H]DHM or

[

3H]DADLE at all (James et al. 1982). This result suggested that

dyn A was highly selective for κ: over μ and δ receptors.

Furthermore, we could prepare membrane fractions enriched in κ

receptors by treatment with B-CNA in the presence of dyn A,

followed by extensive washing to remove the protecting ligand.

Similarly, we were able to enrich preparations in μ sites by

treatment with ß-CNA in the presence of sufentanil (a μ-selective

agonist) and in δ sites by treatment in the presence of DADLE. We

used these selectively enriched membrane preparations to study

the binding selectivity of a number of opiates (James and Goldstein

1984), including the peptide dyn A-(1-13) amide, chosen because of

its stability in binding assays (Leslie and Goldstein 1982). We

estimated a dissociation constant of 0.02 nM for this peptide at κ

sites and found that it had about 30 times higher affinity at κ

than at μ binding sites and about 80 times higher affinity at κ

than at δ sites (table 2).

TABLE 2

Affinity of Dynorphin and U50,488 at Opioid Receptors in

Guinea Pig Brain

Dynorphin A-(1-13)

amide

Kd (nM) at

μ d k

0.6 1.6 0.02

U50,488 940 2400 0.72

197
Direct assays of dyn binding have been complicated by high non -

specific binding to many different surfaces (Ho et al. 1980).

Recently, however, Young et al. (1983) developed a method for

measuring binding of [

3H]dyn A to sites in guinea pig brain. They

found that both dyn and the kappa ligand UM 1071 were about 200

times more potent than morphine or DADLE in competition with

[

3H]dyn A, a result consistent with binding of [3H]dyn A at κ

sites. Specific binding sites for [

3 H]dyn A, measured by

autoradiography, were localized in the deep layers of guinea pig

cerebral cortex (Lewis et al. 1984), a distribution consistent with

that of κ receptors (Goodman and Snyder 1982).

Binding of [

3H]dyn A-(1-8) to sites in rat and guinea pig brain

was measured by Quirion and Pilapil (1984). In general, ligands

with some selectivity for κ receptors (including EKC, bremazocine,

and dyn A) were more potent in competition with [

3H]dyn A-(1-8)

than ligands selective for μ or δ receptors. Similarly, [

3H] α-neo

appears to bind to κ receptors in rat brain membranes, though it

also labels other types of binding sites (Houghton et al. 1983).

Pharmacologic Selectivity

In pharmacologic assays, the maximum effect of an agonist is often

reached by interaction at its preferred receptor. It may not be

possible to disrupt this interaction and allow measurements of

potency at other receptor types. Hence, there have been no

reliable quantative measurements of pharmacologic selectivity (i.e.,

ratio of potencies at different receptors in the same tissue) for

opioid drugs. Instead, most people make qualitative assessments of

selectivity, limiting their pharmacologic studies to the question of

which receptor type is activated by a given agonist in a given

bioassay. There have been two general approaches to this

question: measurement of sensitivity to antagonism by selective

antagonists, and selective inactivation of receptor types. For

studies with dyn, the antagonists naloxone and MR 2266 (figure 2)

have been most useful.

The most convenient measure of sensitivity to antagonism is the

antagonist Ke, an estimate of the dissociation constant for the

antagonist at the receptor through which the test agonist is

having its effect. Values of Ke are estimated from the shift in

agonist potency when antagonist is present in the medium bathing

the tissue (Kosterlitz and Watt 1968). Naloxone is somewhat

selective for μ receptors, with Ke values around 2 to 3 nM. The

naloxone Ke for κ and δ receptors is 20 to 30 nM (Hutchinson et al.

1975; Lord et al. 1977). MR 2266 is reported to be a selective κ

antagonist (Oka et al. 1982a).

198
FIGURE 2

Structure of MR 2266 and U50,488

In GPI, the naloxone Ke when dyn A-(1-13) is used as test agonist

is significantly higher than that for typical p agonists (Goldstein

et al. 1979; Vaught 1981; Chavkin et al. 1982; Huidobro-Toro et al.

1982) and is the same as that for the κ agonist EKC (Goldstein et

al. 1979; Chavkin et al. 1982; Oka et al. 1982a; Huidobro-Toro et al.

1982). Activity of both dyn A and dyn A-(1-13) is reversed more

easily by MR 2266 than by naloxone (Oka et al. 1982a; Yoshimura et

al. 1982a). Dyn B and α−neo have the same high naloxone Ke

 as

dyn A in GPI (Oka et al. 1982b; Suda et al. 1983; James et al.

1984). There is some disagreement about naloxone Ke values for

dyn B-29 and ß-neo. Oka et al. (1982b) found that ß-neo had the

same Ke as dyn A. Suda et al. (1983) obtained the same result and

also found the same Ke for dyn B-29 and dyn A. In experiments

where all the comparisons of potency and Ke were made relative to

dyn A on the same GPI preparation, we found that naloxone Ke

values for dyn A-(1-8), dyn B-29, and ß-neo were significantly

lower than those for dyn A, but still significantly higher than for

normorphine, a μ agonist in GPI. These intermediate Ke values had

been seen before by Chavkin and Goldstein (1981a) in their study

of structure-function relationships with dyn. Our interpretation is

that an agonist with naloxone Ke intermediate between values

typical of κ and μ receptors is acting simultaneously at both types

of receptor. This is supported by the observation that selective

inactivation of μ receptors in GPI changes the naloxone Ke for dyn

A-(1-8) from the intermediate value to one much closer to that

found for dyn A (James et al. 1984). McKnight et al. (1983) showed

that peptides are degraded during bioassays and that degradation

can affect estimates of potency. Since addition of peptidase

inhibitors to the assay stabilized the peptides but did not change

the intermediate Ke values, these values are not a result of

degradation to fragments with different selectivity from the intact

peptide.

199
The evidence from measurements of antagonist Ke

 values suggests

that, at least in GPI, dyn A, dyn B, and α−neo are selective for κ

receptors. Dyn A-(1-8), dyn B-29, and ß-neo act at both κ and μ

receptors. The structural basis for this loss in selectivity is

discussed in the next section.

TABLE 3

Potency and Naloxone Ke of Dynorphin Peptides in GPI

Peptide ED50 Naloxone Ke

(nM) (nM)

Dynorphin A 0.19 31

Dynorphin B 3.3 33

α−neoendorphin 3.5 22

Dynorphin A-(1-8) 58 15

Dynorphin B-29 2.4 15

ß-neoendorphin 43 1 4

Normorphine 63 3.8

Selective inactivation of receptors can be achieved either by

covalent blocking of a site, as described for binding assays, or by

selective induction of tolerance. In both cases, a decrease in

potency for the test agonist after selective inactivation of a given

type of receptor suggests that the agonist normally works through

that receptor type. In experiments similar to those described

already for binding assays, Chavkin and Goldstein (1981b) showed

that treatment of GPI with ß-CNA caused a decrease in potency for

dyn A-(1-13), [Leu]enkephalin, and normorphine. Treatment in the

presence of dyn A-(1-13) reduced the potency shift for dyn but

not for the other two agonists. Dyn also eliminated the reduction

in EKC potency normally caused by ß-CNA (Chavkin et al. 1982).

Conversely, DADLE present during reaction of the tissue with

ß-CNA reduced the potency shifts for [Leu]enkephalin and

normorphine, but not for dyn and only partially for EKC (Chavkin

and Goldstein 1981b; Chavkin et al. 1982). Other groups found

that inactivation of μ receptors either by induction of tolerance

(Vaught 1981) or by alkylation with ß-funaltrexamine

(Huidobro-Toro et al. 1982) did not alter dyn potency. The

interpretation of these results was that dyn interacts with κ

receptors (hence, the cross-protection of EKC potency) but not μ

receptors in GPI. Reaction of GPI with ß-CNA in the presence of

dyn A also leads to protection of dyn B and α-neo receptors.

Similarly, either dyn B or α-neo will protect receptors for all three

peptides (James et al. 1984). Hence, in GPI, dyn A, dyn B, and

α−neo all act through the same population of receptors.

200
In GPI from animals made tolerant to morphine, fentanyl, or DADLE

(all of which would induce tolerance to μ receptors in this tissue),

the potency of dyn A-(1-13) did not differ from controls.

Induction of tolerance to the κ agonists MR 2034, MRZ 2549 or EKC

also produced tolerance to dyn A-(1-13). Similar results were

obtained in MVD, where tolerance to sufentanil, DADLE, or DADLE

and sufentanil together did not produce cross-tolerance to dyn

A-(1-13), but tolerance to the κ agonists did (Wuster et al. 1981).

MVD from animals tolerant to dyn A or dyn A-(1-13) show

cross-tolerance to EKC and α-neo. Similarly, tissue tolerant to

α−neo was also tolerant to dyn A and EKC. There was no

cross-tolerance between DADLE and dyn A or α-neo (Schultz et al.

1982). Results from cross-tolerance studies support the

measurements of antagonist Ke and the selective alkylation or

protection studies in suggesting that dynorphins act at κ

receptors in GPI or MVD.

A third bioassay used in studying k receptors is the rabbit vas

deferens (RVD). This tissue behaves as though only κ and not μ

or δ receptors were active in inhibiting contraction of the muscle

(Oka et al. 1981). In RVD, dyn A, dyn A(1-8), dyn B, and α−neo

are all potent agonists, whereas [Leu]enkephalin, [Met]enkephalin,

ß-endorphin, and DADLE are inactive even at concentrations in the

micromolar range (Oka et al. 1982a, McKnight et al. 1983; Corbett et

al. 1982; Quirion and Weiss 1983).

In more complex behavioural measurements, there is evidence that

dyn distinguishes between receptor types. There was no

cross-tolerance between sufentanil and dyn A-(1-13) in induction

of catalepsy in rats (Herman and Goldstein 1981). Nor was there

cross-tolerance between morphine and dyn B in measurements of

analgesia after intrathecal administration of the drugs in rats, but

there was cross-tolerance between EKC and dyn B in the same

experiments (Han et al. 1984).

Dynorphins, then, are selective for κ opioid receptors in binding

assays; in three opioid bioassays--GPI, MVD, and RVD; and possibly

also in production of analgesia in spinal cord. It remains to be

seen whether endogenously released dynorphins also act at κ

receptors.

STRUCTURE-ACTIVITY RELATIONSHIPS OF DYNORPHIN PEPTIDES

Dyn A is an extension of the opioid peptide [Leu]enkephalin, yet it

is 700 times more potent than [Leulenkephalin in the GPI assay

and about 3 times more potent in MVD (Goldstein et al. 1979).

Dynorphins are selective for κ receptors while [Leu]enkephalin is

somewhat selective for δ receptors (Lord et al. 1977). What are

the structural features of dyn that cause the increased potency

and the change in receptor selectivity?

201
Removal of the C-terminal four residues has no effect on potency

or selectivity, since dyn A and dyn A-(1-13) have the same

potencies and naloxone Ke values in GPI (Goldstein et al. 1981). In

a series of experiments using peptides obtained by removing one

amino acid residue at a time from the carboxyl terminus of dyn

A-(1-13), Chavkin and Goldstein (1981a) identified three critically

important basic residues, removal of which resulted in loss of

potency in GPI; these were lysine-13, lysine-11, and arginine-7.

Furthermore, removal of lysine-11 caused a drop in naloxone Ke

from relatively high (κ-like) values to values intermediate between

those typical for κ and μ receptors. Removal of arginine-7 caused

a further drop in naloxone Ke to relatively low (μ-like) values.

Hence, lysine-11 and arginine-7 are important not only for the

high potency of dyn A, but also for its κ selectivity. The position

of these basic residues in relation to the enkephalin part of dyn is

also important. Movement of the C-terminal portion of the molecule

one residue to the left, as in (Des-Arg 6)dyn A-(1-13), or one

residue to the right, as in (endo-Gly5a)dyn A-(1-13), causes about

tenfold reduction in potency in GPI (Goldstein et al. 1979).

Substitution of arginine-7 or lysine-11 with alanine also causes a

tenfold to twentyfold drop in potency (Turcotte et al. 1984).

Other groups (Yoshimura et al. 1982b; Corbett et al. 1982;

Sanchez-Blazquez et al. 1984) obtained similar results in their

potency measurements on GPI. Yoshimura et al. (1982b) did not

detect intermediate Ke values; their estimates of naloxone Ke for

dyn A-(1-9) and dyn A-(1-7) were the same as for dyn A. They

did, however, find some degree of cross-tolerance between

morphine and dyn A-(1-7), indicating some interaction of this

shorter dyn peptide at μ receptors.

The receptor selectivity profiles for other members of the dyn/neo

family are consistent with the structure-activity relationships for

dyn A fragments. In general, those endogenous peptides with

basic residues in position 7 and 10 or 11 (dyn A, dyn B, and

α-neo) are κ selective in GPI, whereas those without the basic

residue in position 10 or 11 (dyn A-(1-8) and ß-neo) act through

both k and μ receptors. Movement of the critical basic residue

from position 11 (as in dyn A) to position 10 (as in dyn B and

a-neo) causes a tenfold reduction in potency. Removal of this

residue altogether (as in dyn A-(1-8) and ß-neo) causes a

hundredfold reduction in potency (James et al. 1984).

In the MVD assay, the structure-activity results for shorter dyn

fragments are different from those obtained with GPI. For

example, there is very little difference in the potencies of dyn

A-(1-13) and [Leu]enkephalin (Corbett et al. 1982; Cox and Chavkin

1983; Schultz et al. 1984; Sanchez-Blazquez et al. 1984). When vasa

were made tolerant to DADLE, effectively inactivating δ receptors, a

structure-activity pattern similar to that found in GPI emerged

(Cox and Chavkin 1983; Schultz et al. 1984). In untreated MVD, as

202
dyn is shortened the potency at κ receptors is decreased, but the

shorter peptides can act at δ receptors, resulting in relatively

small net effects. Inactivation of δ receptors reveals the typical κ

structure-activity pattern. In GPI, δ receptors can only be

detected by special treatment of the tissue (Gintzler and Hyde

1984). In untreated tissue, the δ mediated effects are negligible;

hence, the structural requirements for activity at κ receptors are

easily seen.

Measurements of naloxone Ke in MVD also show a slightly different

pattern from GPI, with relatively high Ke (around 30nM) for dyn A,

dropping to intermediate (about 13 nM) for dyn A-(1-8), but rising

again to 30 nM for [Leu]enkephalin (Schultz et al. 1984). The high

Ke for [Leu]enkephalin reflects interaction at δ receptors, which,

like κ receptors, have a relatively low affinity for naloxone (Lord

et al. 1977). Inactivation of δ receptors by induction of tolerance

resulted in a pattern of Ke measurements similar to that found in

GPI, with Ke for [Leulenkephalin being lower than that for dyn

A-(1-8). In MVD, unlike GPI, the intermediate Ke for dyn A-(1-8)

apparently is not caused by cross-reaction at μ receptors.

Inactivation of μ receptors either by selective tolerance or

selective alkylation did not change the Ke. It is possible that in

MVD, dyn A-(1-8) acts through a different type of receptor than

the classical μ, δ or κ types (Schultz et al. 1984).

Changes in the enkephalin part of the molecule also change the

activity of dyn. Substitution of tyrosine-1, glycine-2, or

phenylalanine-4 with alanine in dyn A-(1-13) causes large

decreases in potency in both GPI and MVD (Turcotte et al. 1984).

Substitution of glycine-2 with D-alanine causes a decrease in

potency in GPI for those dyn fragments that show some selectivity

for κ receptors (i.e., are longer than seven residues), but causes

an increase for dyn A-(1-6) and [Leu]enkephalin, both of which act

through μ receptors in GPI (Chavkin and Goldstein 1981a). In

MVD, the D-alanine-2 substitution increases potency of dyn A

fragments up to 10 residues long (Cox and Chavkin 1983),

presumably because of interaction with δ receptors.

Nitration of the phenylalanine residue in position 4 of dyn A-(1-13)

caused about fourfold decrease in potency in GPI but did not

affect potency in MVD. The same modification of [Leu]enkephalin

increased the potency 25-fold in GPI and 40-fold in MVD (Schiller

et al. 1982). The cyclic analog c(D-Cys2,L-Cys5)dyn A-(1-13) was

about five times more potent than the parent compound in GPI,

whereas the same cyclization increased the potency 160-fold for

[Leu]enkephalin. These differential effects on potency of dyn and

enkephalin suggested to Schiller and his colleagues that the

structural requirements for the enkephalin binding domain of the

dyn (κ) receptor are different from the enkephalin receptor (the

μ receptor in GPI). Schiller (1983) extended these observations

and showed by resonance energy transfer fluorescence experiments

203
using the tryptophan-4 analogs of dyn A-(1-13) and

[Leu]enkephalin that the distance between tyrosine-1 and

tryptophan-4 was different in these two peptides. He concluded

that in aqueous solution the predominant conformation of the

N-terminal tetrapeptide in the dyn analog was almost completely

extended, whereas there was considerable folding in the same

segment of the enkephalin analog. He also suggested that there

was no interaction between the N-terminal and C-terminal portions

of dyn A, since he could not measure any energy transfer between

tyrosine-1 and tryptophan-14 in this peptide. These results are

consistent with an earlier study where Maroun and Mattice (1981)

found that circular dichroism spectra of dyn A-(1-13) had the

characteristics of random coil in aqueous solution.

Adopting Schwyzer’s terminology (Schwyzer et al. 1980), dyn A can

be divided into two regions (figure 1): a message comprising the

N-terminal four residues, which says “occupy an opioid receptor”;

and an address comprising residues 5-13, which says “of the κ

type”, As yet, we know of no specific function for residues 14-17,

though they may be involved in control of the processing of

prodynorphin (Devi and Goldstein 1984). The way in which the

address sequence specifies κ receptors is still an open and

fascinating question. Perhaps κ binding sites have negative

charges complementary to the positive charges in positions 7, 11,

and 13 of dyn A, as suggested by Chavkin and Goldstein (1981a).

Alternatively, these residues may act by stabilizing a conformation

of the enkephalin portion that is recognized well by κ receptors

but not by μ or δ, as implied by Schiller (1983). Extended

structures are not absolutely necessary for high affinity and

selectivity for κ receptors. The compound U50,488 (figure 2)

synthesized at the Upjohn Company, has high affinity and is

highly selective for κ receptors (table 2). The conformation of dyn

in its receptor site may well turn out to be very close to the

shape of the U50,488 molecule.

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AUTHOR

Iain F. James, Ph.D

Sandoz Institute for Medical Research

5 Gower Place

London WClE 6BN England

208
Computer Analysis of Radioligand Data:

Advantages, Problems, and Pitfalls

David Rodbard, M.D.; Rudolph A. Lutz, Ph.D.; Ricardo A.

Cruciani, M.D.; Vincenzo Guardabasso, M.D.; Guido O.

Pesce, M.D.; and Peter J. Munson, M.A.

ABSTRACT

Mathematical modeling combined with nonlinear least-squares curve fitting

provides a systematic, objective, reproducible, and consistent method to

aid the interpretation of ligand-binding data. It forces the experimentalist to formulate hypotheses in an unambiguous manner and to consider

alternative, closely related models as plausible counter-hypotheses.

Modeling provides estimates of the "goodness-of-fitw of the theory to the

data and estimates of the minimal uncertainty of the parameters. With the

availability of many programs for micro- and mini-computers, as well as

mainframe computers, these methods are now becoming widely used.

Accordingly, we must emphasize a number of potential problems and

limitations, based on our experience. Interpretation of results of

modeling study should be made, in light of the following points:

a) no amount of computer analysis will compensate for “bad” or

insufficient data, or for poor experimental design;

b) the interpretation of the computer analysis is subject to

the caveat that all underlying assumptions must be satisified;

c) one must examine the data graphically in several coordinate

systems (e.g., "raw data," as well as standardized residuals);

d) one must continuously search for possible systematic biases

or artifacts;

e) one must closely examine the reproducibility of results

between multiple experiments; and

f) one must recognize that all of the “test tubes” in an experiment are

not necessarily “independent observations" in a statistical sense.

In view of these potential problems and limitations, one should always

seek to corroborate results and interpretations of "modelingn studies of

ligand binding by independent biochemical, biophysical, or structural

evidence. In this context, ligand-binding studies, appropriately

analyzed, can play a useful and constructive role.

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INTRODUCTION

Even today, most of the ligand-binding data in the scientific literature

is being analyzed by graphical methods. Graphical methods are not to be

completely condemned: they have served well in enzymology for more than 70

years (e.g., Segel 1975). However, these methods are notoriously

subjective: different users can obtain different "measurementsn from the

same data. Graphical methods usually deal effectively with simple linear

(or linearizable) cases, but not with complex nonlinear ones. With the

growing availability of computer programs for nonlinear least-squares

curve fitting on relatively inexpensive micro- and mini-computers (Munson

and Rodbard 1980; Rodbard 1984; McIntosh 1984; Sacchi-Landriani et al.

1983; Bevington 1969), their use is becoming more widespread in the

biochemical and pharmacological community.

Approaches to computerized analysis of binding data have been

discussed extensively elsewhere, by ourselves and others (Munson and

Rodbard 1980, 1984; Munson 1983a, 1984; Baulieu and Raynaud 1970;

Feldman 1972; Fletcher and Spector 1977; Priore and Rosenthal 1976). In

brief, the "computerizedw approach offers several of the following

advantages:

Ease and speed of data processing;

Freedom from mathematical errors;

Precise formulation of models;

Objective measures of goodness of fit;

Provision for appropriate weighting of data;

Objective criteria for distinguishing models;

Estimation of uncertainty of the parameters of the model;

Optimal estimation of nonspecific binding;

Provision for parameter constraints; and

Ability to pool data,

Computer modeling is not a panacea; careful examination of such

results is mandatory. There are many limitations and pitfalls in

the methodology, perhaps the chief of which is the tendency of the

investigator to believe the results because “the computer said it

was so.” The prudent investigator should consider the following

potential problems: Failure to satisfy the assumptions;

insufficient data to define parameters; failure to replicate

experiments; poor initial estimates or lack of global convergence

of the fitting algorithm; overparameterization; lack of

independence of observations; and failure to use an appropriate

experimental design. We shall discuss each of these potential

advantages and problems in turn.

ADVANTAGES

The advantages of using computerized modeling techniques for ligandbinding data analysis are many. First is the ease and speed of data

processing, potentially allowing the scientist more time for critical

interpretation and evaluation of his or her results. Freedom from

mathematical or computational errors (provided that the data have been

210
accurately entered) is assured. Thus, there is no need to use approximations in the calculations. The computer is equally capable of using the

exact formulation.

Another advantage is the precise formulation of a model, and the ability

to examine other models involving the same number of parameters, as well

as models involving one or two more, or one or two less, parameters. Thus,

a family rather than a single model may be evaluated, along with many

plausible alternative models or counter-hypotheses.

Computerized analysis allows objective measures of goodness-of-fit, e.g.,

the sum of squares of deviations (SS), the mean square deviation (MS), and

the “root mean square error" (RMS error) or standard deviation of a point

around the fitted curve (Draper and Smith 1981). Other tests of

goodness-of-flt examine the randomness of the deviations of the points

around the curve. These include the “runs test” (a nonparametric and

robust test which is very sensitive when dealing with a large number of

residuals, e.g., more than 20, but less sensitive with a small number of

"points"), and the serial correlation of residuals (or equivalently, the

mean square successive difference), which is somewhat more sensitive for

small numbers of observatlons (Bennett and Franklin 1954). Of course,

graphical display of data remains one of the best, if not the best, method

for evaluation of goodness-of-fit. The computer makes it possible to

obtain high resolution graphs, free of manual errors, in several

coordinate systems, e.g., saturation curves, displacement or competition

curves, and Scatchard plots. The utility of these graphs may be improved

when one superimposes the 95% confidence limits for an observation (to

assist detection of outliers) and the standard error (and/or 95%

confidence limits) for the fitted curve(s).

The computer analysis should provide appropriate weighting: an inprecise

data point (say ± 100) should not be counted the same as a more precise

value (± 10 or ± 1). Failure to use weighting, or failure to use proper

weighting, can reduce the performance of the “computer analysis” to

somethlng far worse than simple graphical analysis, where the experimentalist “knows" which portions of the curve are measured precisely and

known reliably, and which portions of the curve are subject to wide

variation. Thus, the computer analysis forces the experimentalist to

state his assumptlons and examine his data regarding its statistical

"error structure” (Rodbard 1984; Rodbard et al. 1976).

The computer permits and facilitates use of objective criteria to

distinguish among several alternative plausible models. Should the data

be described by a model involving one binding site? Two sites? Three

sites? By applying the “extra sum of squares principle,” one can

objectively select among various models of varying degrees of complexity.

The computer analysis provides estimates--usually lower bounds--of the

degree of uncertainty in the parameter values. The true uncertainty is

often much larger because these error estimates are based on the apparent

random variability in the data: they do not protect against systematic

errors (e.g., adsorption of llgand to the test tubes at low

concentrations).

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When dealing with two or more parameters simultaneously, the standard

error of the parameters can be misleading. One must examine the Joint

confidence limits for two parameters considered simultaneously (Munson and

Rodbard 1980, 1984). This is due to the interaction or "covariancen in

the errors in the parameters. For example, an overestimate of the

affinity (K) is usually accompanied by an underestimate of the binding

capacity for that site (R), because K*R--the binding (measured as B/F)

for infinitesimal llgand concentration--is often relatively precisely

determined. An overestimate of nonspecific binding is likely to be

accompanied by an underestimate of the concentration of low affinity

sites, and vice versa.

The computer allows one to estimate nonspecific binding as a fitted

parameter (N). Even when nonspecific binding has been measured repeatedly

and at several concentrations of labeled ligand, it is still subject to

some degree of uncertainty. Subtracting nonspecific binding from the

total binding to obtain specific binding, as is commonly practiced, tends

to propagate and magnify the error of measurement, sometimes leading to

unstable or incorrect results. Considering nonspecific binding as a

"fittedw parameter also gives a more accurate (and usually larger)

estimate of the confidence limits for an observation, for the fitted

curve, and for the estimates of parameters, especially Bmax--albeit at the

cost of an increase in the apparent degree of complexity.

The computer analysis enables us to interject "constraints,n i.e., to test

certain assumptions or to use data from previous experiments or from

independent studies. For example, one can require that a parameter is

fixed or constant, e.g., K11 - 1.3E9. Or, one can force or constrain two

parameters to be equal, i.e. "shared,n but both of them will be fit (e.g.,

R1 - R2, or N1 - N2). Or, shared parameters can be set equal to a

constant (e.g., C1 - C2 - C3 - 1). Use of constraints can be helpful in

simplifying a model to obtain initial or starting estimates of parameters

for the iterative curve fitting procedure.

The computer analysis permits one to pool data from several curves using

the same labeled ligand; from several curves using different labeled

ligands within one experiment (e.g., for a given membrane preparation and

set of conditions); and finally, from multiple experiments. Such pooling

of data is often necessary: a) to test consistency of results; b) to

obtain a sufficient number of observations to permit testing of more

complex models (e.g., involving multiple classes of receptors); and c) to

obtain more reliable estimates of parameters for such complex models. Of

course, if the data are not "homogeneous" --if the qualitative nature of

the system (e.g., cell or membrane preparation) has changed between

experiments--then the loss of information due to heterogeneity and

increased systematic and/or random variability may outweigh the advantages

of the larger amount of data.

LIMITATIONS and PITFALLS

In view of the many advantages, or at least potential advantages, of

computerized mathematical modeling and curve fitting enumerated above, it

is not surprising that many laboratories are in the process of letting up

such facilities. However, computerized curve fitting is not a panacea.

212
It will not change bad data into good. Several of the potential problems

and pitfalls evolve from the inability to simultaneously satisfy all of

the following assumptions of the mass action models.

a) attainment of equilibrium;

b) accurate measurement of bound and/or free ligand concentration

(this implies “perfect separation of bound and free," without

perturbing the preexisting equilibrium);

c) absence of degradation of ligand and/or receptor during the

incubation;

d) absence of cooperative interactions between the receptors,

although cooperativity may also be modeled explicitly:

e) absence of catalytic or enzymatic reactions, and absence of

internalization of receptors or other active transport processes

requiring energy, and hence departure from an equilibrium

situation;

f) accurate and appropriate measurement of nonspecific

binding;

g) chemical identity of the labeled ligand and the corresponding

unlabeled ligand, which requires the absence of an isotope effect,

absence of “damage” during labeling with iodine, accurate

estimation of specific activity (to within a few percent, not

within a factor of two), and homogeneity of labeled ligand

(chemically pure, and not a racemic mixture);

h) absence of extraneous binding proteins (or solubilized receptors)

in the medium;

i) absence of other "special effects", such as ligand induced

conformational changes and induced aggregation or disaggregation.

interaction of receptor with other membrane components

(e.g., guanyl nucleotide binding proteins, phospholipids, and

enzymes);

j) both ligand and receptor are in true solution, i.e., not subject

to unstirred layers or diffusion boundary effects, or to increased

effective concentrations in the vicinity of a solid phase; and

k) if the receptor is membrane bound, there are no membrane ligand

effects due to partitioning of ligand into the membrane, or due to

charge or Van der Waals forces.

The above is only a partial list of the implicit assumptions underlying

most "equilibrium binding analysis" studies. These assumptions are

involved, whether or not the data analysis is performed manually,

graphically, or by computer. Each of these assumptions is vulnerable: in

many systems we have strong evidence that these assumptions are not,

strictly speaking, correct. One can use computer simulation studies to

evaluate the potential qualitative and quantitative problems raised by

failure to meet any one of these assumptions (Buergisser et al. 1981a,

1981b; Rodbard and Catt 1972; Ketelslegers et al. 1975; Munson 1983b;

Krumins and Rodbard, submitted). Further, if any one of these assumptions

is violated, one can potentially utilize a more complex model to fit the

data (DeLean and Rodbard 1980; Ketelslegers et al. 1975) and still obtain

reliable estimates of parameters. Of course, one must pay a price:

usually it becomes necessary to collect substantially more data (e.g., at

several “time points”) to fit the more complex models which usually

involve more parameters (e.g., rate constants, degradation rates, etc.).

For instance, if the "tracer" were damaged or heterogeneous, one can

213
measure its "maximal bindability," which is analogous to the concept of

"immunoreactivity of tracer" in the context of radioimmunoassay, by

progressively increasing the receptor (membrane, cell) concentration and

extrapolating to "infinite" receptor concentration (Krumins and Rodbard,

submitted). Such extrapolations of necessity involve increased random

variability, and it is difficult to protect oneself from systematic errors

as well.

If two or more of the above assumptions have been violated to a

significant or substantial degree, then the necessary models usually

become so complex that they become impractical. For instance, one could

write the equations for a cooperative model involving multiple receptors

under nonequilibrium conditions and in the presence of degradation. With

modern computing facilities, simulation and curve fitting would still be

possible. The problem is that the amount of data required would be

prohibitive, and random errors would have to be reduced to vanishing

levels so that the system would not become "indeterminate" and the

parameters "nonidentifiable" (Carson et al. 1983; DiStefano 1984).

What do we mean when we say that the assumptions must not be violated to

a significant or substantial degree”? This is a subjective matter, a

matter of judgment. This will depend on the system, the conditions, the

magnitude of the errors, the number of sites, the number of parameters,

and the degree of vulnerability of the “other” assumptions.

One of the paradoxes of the use of a statistical approach to the data is

that the better the data (i.e., the more precise the data), the more

vulnerable the results are to minor departures from the assumptions. For

example, in the presence of 10% random errors, one would not be able to

detect the presence of a small (2%) systematic error. However, if by

optimizing experimental conditions, reducing pipetting and counting

errors, and using a high degree of replication (quadruplicates), one can

reduce the random errors to 1%, then a small systematic (2%) error may

appear to be “highly statistically significant." In this sense, the

"sloppy" experimentalist with widely scattered data, while likely to miss

small effects, would be protected from overinterpretation of his/her data:

only very large, dramatic effects would be "statistically significant."

In contrast, the very cautious and quality control minded experimentalist

who reduces his random errors to minimal levels (e.g., ± 1%) has a greater

chance of detecting real but minor phenomena (e.g., a small amount of an

additional class of binding sites). However, this also increases the

chance of being misled by some small systematic error introduced by some

small but definite failure to meet one of the underlying assumptions

(i.e., a small, subtle artifact). It follows that one must not only

reduce random errors as much as possible, but also test all of the

underlying assumptions, and strive to minimize systematic errors insofar

as possible. By providing a measure of random errors, the computer

facilitates the refining of "noisy" ligand-binding systems. Having done

so, one is now faced with the problem that a statistically significant

result is not necessarily "biologically or biochemically significant."

The more precise the data, the greater the likelihood of detecting a minor

artifact as though it were a signal.

214
Failure to meet the assumptions is probably responsible for the greatest

likelihood of misinterpretation of ligand-binding data, again, whether

computerized or not. However, in addition, there are several technical

issues which arise in the curve fitting process and can result in biased

results or misinterpretation. These are briefly discussed below.

Insufficient Data to Define Parameters

The more complex the model, with more fitted parameters, the larger the

number of data points required. For example, for a single class of

binding site, one has a single affinity constant (K) and a single binding

capacity (R). In addition, one probably should regard the nonspecific

binding (N) as a fitted parameter. In contrast, when dealing with 2

classes of binding sites and 2 ligands. one then has 2 x 2 - 4 K values, 2

R values, and 2 N values, or eight parameters altogether. With 3 ligands

and 3 binding sites, one has 3 x 3 - 9 K values, 3 R values, and 3 N

values, or 15 parameters. In order to estimate all parameters simultaneously, one needs a large number of independent observations (e.g., one

might have a displacement curve for each of the three unlabeled llgands

displacing each of the corresponding labeled ligands, or nine curves in

all). Such experiments are necessarily large and may be impractical or

impossible to perform. For instance, one of the three ligands may not be

available in homogeneous form (i.e., a racemic mixture), or in labeled

form with sufficiently high specific activity. One can often still obtain

a reasonable characterization of the system, even when all possible

combinations and permutations of ligands cannot be tested. For example,

if one has a "universal ligand," which appears to label all classes of

sites with equal or nearly equal affinity, then one can use this single

llgand as the “tracer” and displace with the several unlabeled ligands

which display specificity or at least partial specificity or selectivity

for the various classes of sites. This approach has been used successfully by DeLean and Lefkowitz in the characterization of alpha and beta

adrenergic systems (Hancock et al. 1979; Hoffman et al. 1979). Alternatively, one can use perhaps six of the nine possible combinations of

ligands in a cross-displacement study to characterize a ligand-binding

system (Clayton et al. 1979). The more combinations of ligands one uses,

the greater the likelihood of detecting a small, systematic but real

departure from the model. An experimental design using multiple ligands

displaced by the corresponding unlabeled ligands has been employed by

Pfeiffer and Herz (1982) to characterize the mu, delta, and kappa

receptors in rat brain. They succeeded in demonstrating kappa sites with

binding studies using [3H]-ethylketocyclazocine, at a point in time when

other laboratories were having difficulty finding a distinct class of

kappa sites. Loew et al. (1983) have utilized a similar experimental

design to reexamine the mechanisms of the “sodium effect” on opiate

receptors.

The more predictions of the model one tests, the greater one’s confidence

in the model and the more precise the estimates of parameters. Of course,

the larger the experiment, the greater the probability that one will

encounter systematic errors or drift: the human or even the apparatus

(e.g., pipetting machines) are subject to fatigue; the incubation time may

vary; the degree of suction on the filtration machine may vary; the

temperature may vary; the membrane particles may sediment to the bottom of

a tube or flask; and the dryness of the filters and, hence, the efficiency

215
of counting may vary. Thus, the task of accumulating sufficient data to

characterize a complex system becomes progressively more challenging for a

larger model. Often, frozen membrane preparations have different

properties than fresh membranes. Hence, one must use fresh membranes,

prepared every day. Then, the density of the receptors will inevitably

vary from one day to another, even after correcting per gram wet weight of

tissue or per milligram of protein. One can correct for this systematic

error, between experiments, by introducing a scaling or correction factor

for each membrane preparation (designated C) (Munson and Rodbard 1980).

These are additional parameters to be estimated. The C factors can

usually be estimated very precisely, since all of the tubes in an

experiment can be used to help estimate them. Use of the C’s is based on

the assumption that the relative proportions of binding sites must be

constant from one experiment to another. In other words, the "binding

isotherm" (or its Scatchard plot representation) should have a constant

shape. Furthermore, the usual use of C factors involves the assumption

that the specific and nonspecific bindings varies in a proportional manner

between membrane preparations. This is not always the case. Suitable

modification of the model can be made to allow for separate correction or

adjustment factors for the specific and nonspecific binding. Of course,

this further increases the number of parameters, and especially the

uninteresting or extraneous factors (correction factors) which are not

(usually) of biological interest. This results in an "overhead," reducing

the efficiency of experimentation.

It is difficult to give guidelines regarding the number of observations

necessary to estimate a given number of parameters. This will depend on

the precision of the measurements, the concentration range and the

placement of each experimental point, the nature of the "nonuniformity of

variance” (which is usually present), the precision with which one wishes

to estimate selected parameters, the degree of complexity of the model and

the degree of interaction (covariance) of the errors in the model, and the

purposes for which the results are to be used. As a general rule, one

might suggest a ratio of at least five observations per parameter.

However, if one can pool information between experiments, and if one is

willing to do multiple experiments, then one might be able to use

considerably fewer "tubes" within each experiment. The number of

observations needed depends on whether one wants to be able to reject a

given model, to show that a given parameter is nonzero (but perhaps

indeterminate within a factor of 2 or three), or whether one wants to

estimate a given parameter (e.g., binding capacity) within 5% or 10%.

Failure to Test Consistency of Results in Independent Experiments

Use of mathematical modeling and curve fitting enables one to obtain an

estimate of the standard errors of parameters from a single experiment,

These are analogous to the standard errors of the slope and intercept in

standard linear regression. Ideally, these standard errors should predict

the degree of reproducibility of the parameter(s) in future experiments of

the same kind, but this assumes that the future experiments are all

statistically homogeneous, i.e., not subject to additional sources of

variability. However, a within-experiment standard error, even when it is

based on a large number of degrees of freedom. is usually not equivalent

to a between-experiment standard error, due to the unavoidable, additional

sources of between-experiment variation.

216
If one detects a significant “component of variance” between experiments,

one should not be too alarmed or dismayed: this is a common experience and

expected-by any reasonable experimentalist. Often, one finds that the

between-experiment error is two or three times larger than the

within-experiment error. In one of the experiments, due to a small error

in dilution, all of the "points" for one of the competition curves may be

shifted to the right. Another curve may be shifted to the left; another

curve may be shifted slightly upward or downward. If we have an

experimental design involving two labeled ligands and two unlabeled

ligands, we may find that one of the labeled ligands has changed--thus

affecting, not just a single "point," but systematically biasing results

on two of the curves. Likewise, a systematic error in the preparation,

dilution, or purity of one of the unlabeled ligands will affect half of

the data in the experiment. As generally practiced, computer modeling of

the data does not take these systematic errors into account, but treats

each observation as an independent data point. Thus, although we may have

200 observations, their errors may be interacting in a complex and unknown

fashion. This can alter, possibly severely, the probability levels for

tests of goodness-of-fit (e.g., randomness of residuals). Thus, the wise

experimentalist must examine and compare the results from several

experiments. In a multiple binding site model, receptor class number one

(R1) in one experiment might correspond to receptor class number three

(R3) in the next experiment. One can use a graphical method, designated

the “Kd versus Kd plot,” to detect clusters of similar behavior from

different experiments (Munson et al. 1984). Other two-dimensional plots

for pairs of parameters can be useful. If parameters agree closely,

within a few percentage points, then identification of parameters is easy.

However, in complex models, it is common to find that some of the

parameters are poorly determined--perhaps only within an order of

magnitude, if that. Here, one can plot results for log(K) versus log(R),

or log(K11) versus log(K21). etc. Then, one can estimate the betweenexperiment standard errors and confidence regions for pairs of parameters

considered simultaneously.

Failure to Use an Appropriate or Optimal Experimental Design

Another factor that must be considered is the optimal location and spacing

of points. If we know that a relationship is linear, then we can obtain

our most precise estimate of the slope by placing our observations as far

apart as possible: we would have half the observations as far as possible

to the left and half the observations as far as possible to the right.

Likewise, when fitting a relationship which is known to be a perfect

Michaelis-Menten hyperbola or hyperbolic binding isotherm for a single

class of sites, one should attempt to locate about half of the points near

the Km or Kd, and the other half of the points well up on the plateau

corresponding to saturation or "Bmax" (Endrenyl 1981). Thus, the optimal

location of the observations to minimize the errors in one or two

parameters can be quite different from the optimal location of

observations if one wishes to detect deparature of the data from the

model, or to optimize the performance of a test to discriminate between

two different models. Prior information is required in either

event--hence the need for a sequence of experiments to make a series of

successive approximations. This goal (to refine and optimize the

performance of the test) conflicts with the desirability of keeping the

217
experimental design constant for a series of experiments in order to

evaluate between-experiment reproducibility. In principle, one can use

computer simulation and optimization to assist in the selection of

experimental design (Carson et al. 1983; DiStefano 1984; Munson and

Rodbard 1984), although this has yet to be translated into practical terms

for routine application in the context of ligand binding. Next, we

consider a series of issues which, although quite technical, can seriously

influence the interpretation of the computerized analysis.

Initial Estimates of Parameters, and Failure of Convergence

Complex nonlinear models with multiple parameters will often have a

"limited region of convergence." In other words, the program may not find

the true optimal weighted least-squares solution unless the initial

estimates of parameters are reasonably close to this value. Otherwise,

the program may converge to a false “local minimum" or not converge at

all. This might lead the experimentalist to conclude that a given model

has failed, even though it might have been perfectly satisfactory if only

another set of starting estimates had been tested. Given two different

sets of starting estimates, the program may converge to different

solutions corresponding to models which are qualitatively very different.

To avoid (or at least detect) the existence of these problems, one should

attempt repeated curve fitting for complex models, using several different

sets of starting estimates of parameters. At present, fitting complex

models involving more than 9 or 10 parameters is, for practical purposes,

beyond the limits of the personal or desktop microcomputers. On

minicomputers and mainframe computers, one might potentially automate this

process to some extent, e.g., first use a grid-search, then a Nelder-Mead

(simplex) algorithm to select initial starting estimates (Magar 1972), and

then check to see that several different starting estimates lead to the

same solution. Use of constraints (e.g., the parameters must be positive)

and reparameterizing (e.g., use of log(K), log(R), or log(K*R)) can

sometimes lead to better reliabillty of convergence. However, in general,

one cannot guarantee that one has converged to the true optimal solution.

Overparameterization

One should inspect the RMS error; ideally, this should be close to the

“residual error” estimated from the standard deviation of replicate

measurements, For instance, if the measurement error for replicates were

± 2% and the RMS error of the curve fit were ± 10%, one would have a

strong indication of a large and significant degree of lack of fit. On

the other hand, if the measurement error were ± 5% and the RMS error were

± 1%, then something is likely to be wrong. On the average, the fitted

curve should pass no closer to the points than the statistical uncertainty

in the observations. An unusually small RMS may indicate that the model

is overparameterized, involving too many parameters. In effect, the model

is too flexible and is able to explain minor random variation in the

observations. Of course, the measurement error and the RMS error need not

agree perfectly--both are subject to random sampling errors, depending on

their respective numbers of (effective) degrees of freedom. However, the

two measures should be closely comparable (Rodbard 1984; Rodbard et al.

1976).

218
Lack of Independence of Observations

In the least squares curve fitting procedure, we assume that each of the

data points entered into the computer is independent, i.e., that the

errors in the observations are independent and randomly distributed. Even

robust and "nonparametric" methods of analysis retain this requirement.

What constitutes a (reasonably) independent observation? Ideally, each

tube in an experiment would be an independent observation. This would

largely (but not entirely) be true if we could use an automated device for

all mechanical steps (pipetting, shaking, filtering) and if we were to

use randomization. Until the experiment is both fully automated and fully

randomized (and hence, presumably, computer controlled in its entirety),

the various tubes in a rack will not be fully independent. Even then,

neighboring tubes in a rack will have a tendency to give correlated results.

A common, indeed unavoidable question is whether to use each tube as an

independent observation or whether to use the means of duplicates

(triplicates, etc.) as the experimental unit. This somewhat arbitrary

choice can have enormous consequences since it can drastically affect the

apparent number of degrees of freedom and, hence, the possible

significance of “F tests" based on the “extra sum of squares principle.”

It may also affect, or bias, the estimates of the RMS error. For example,

if we have triplicates at each of 10 dose levels, then we may regard the

experiment as providing either 10 or 30 independent observations. If we

are fitting 4 parameters, that might make the difference between 6 and 26

degrees of freedom (number of observations minus number of fitted

parameters). In general, we recommend the conservative approach, where

the dose means is regarded as the independent observation. This should,

if anything, bias the results toward selection of a model with fewer

parameters, i.e., a lesser degree of complexity.

How many replicates should one use? This becomes a critical question. For

a given total number of experimental tubes, the more replicates there are

for each dose, the fewer doses can be used; and the fewer the number of

degrees of freedom, the less likely one will be able to demonstrate and

characterize a complex system. To maximize the number of degrees of

freedom, one would use only singletons at each dose. However, to obtain a

measure of the residual error, within doses, one needs to run at least

duplicates for a fair number of dose levels, and possibly pool this

information from various curves in one or more experiments (Rodbard 1984;

Rodbard et al. 1976). This information is also necessary to decide

whether weighting should be used and, if so, to select an appropriate

weighting model (e.g., constant percentage error) and to estimate the

coefficients of the weighting model (e.g., ± 5% error). With duplicates,

one can begin to detect poor agreement; but identification of an outlier

only becomes possible (usually) when using triplicates or quadruplicates.

219
DISCUSSION AND CONCLUSIONS

The art of hypothesis formulation, experimental design, and data analysis

is partly, but only partly, amenable to assistance from statistical

methods, mathematical modeling, and computerization. These approaches

will not and cannot compensate for any fundamental flaws in the data or

correct for major violations of underlying assumptions. Statistical

analyses protect against random errors but can be completely impotent in

the face of certain types of systematic errors. It is desirable that one

obtain experience with the use of curve fitting programs from a series of

applications of progressive and gradually increasing complexity. Even

this provides no guarantee of protection against erroneous interpretation

or conclusions. A number of arbitrary assumptions and decisions, and

skillful judgment are still required. What then is the role or

contribution of the curve fitting procedure? If implemented properly, it

does provide optimal utilization of the data as well as a systematic

method for formulating and testing hypotheses, analyzing sources of random

errors, and detecting inconsistencies. There are several pitfalls,

including the possibility that one will select an oversimplified model to

minimize mathematical complexity, rather than introduce features which are

needed to realistically represent the system (e.g., degradation,

aggregation of receptors, etc.). Qualitative, intuitive analysis of a

more complex but realistic model may be preferable to a mathematically

exact and statistically optimal analysis of an incorrect model. This

tradeoff between simplicity and realism is one that perpetually confronts

the scientist, with or without computerized modeling. Objective

computerized modeling provides a basis for making an informed compromise

between these two ideals.

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Priore, R.L., and Rosenthal, H.E. A statistical method for the

estimation of binding parameters in a complex system. Anal Biochem

70:231-240, 1976.

Rodbard, D. Lessons from the computerization of radioimmunoassays:

an introduction to the basic principles of modeling. In: Rodbard,

D., and Forti, G., eds. Computers in Endocrinology. New York: Raven

Press, 1984. pp. 75-100.

Rodbard, D., and Catt, K.J. Mathematical theory of radioligand

assays: the kinetics of separation of bound and free. J Steroid

Biochem 3:255-273, 1972.

Rodbard, D.; Lenox, R.H.; Wray, H.L.; and Ramseth, D. Statistical

characterization of the random errors in the radioimmunoassay dose

response variable. Clin Chem 22:350-358, 1976.

Sacchi-Landriani, G.; Guardabaaso, V.; and Rocchetti, M. Nlfit: a

microcomputer program for non-linear fitting. Comput Programs Biomed

16:35-42, 1983.

Segel, I.H. Enzyme Kinetics: Behavior and Analysis of Rapid

Equilibrium and Steady-State Enzyme Systems. New York:

Wiley-Interscience, 1975. 975 pp.

ACKNOWLEDGMENT

Edward Fulton, Jr., assisted in performing several ligand-binding

studies and provided valuable assistance with data analysis.

AUTHORS

David Rodbard, M.D.

Rudolph A. Lutz, Ph.D.

Ricardo A. Cruciani, M.D.

Vincenzo Guardabasso, M.D.

Guido O. Pesce, M.D.

Peter J. Munson, M.A.

Laboratory of Theoretical and Physical Biology

National Institute of Child Health and Human Development

National Institutes of Health

Bethesda, Maryland 20892

222
Recent Developments in Bioassay Using

Selective Ligands and Selective In Vitro

Preparations

Hans W. Kosterlitz, M.D., Ph.D.; Alistair D. Corbett, Ph.D.;

Maureen G. C. Gillan, Ph.D.; Alexander T. McKnight, Ph.D.;

Stewart J. Paterson, B.Sc.; and Linda E. Robson, Ph.D.

INTRODUCTION

In an analysis of the mode of action of endogenous opioids and of

opioid drugs, fundamental problems arise because these ligands bind

to more than one site of the complex macromolecule of the receptor.

Such an interaction may or may not be followed by a biological response of the effector. If the compound is an agonist, the response

may lead either to excitatory or inhibitory activity. On the other

hand, an antagonist compound is able to block such excitatory or inhibitory responses. Therefore, it is important to note that binding

does not decide by itself whether the ligand acts as an agonist or

antagonist or has no pharmacological effect at the receptor.

While responses to opioid peptides or nonpeptides will ultimately

have to be tested in vivo, the pharmacological effects are often

more readily analyzed using excised tissues. Compounds with high

selectivity for each of the three opioid binding sites have been

used in isolated tissue preparations obtained from five different

species (table 1). The receptors in the mouse vas deferens interact with the μ-, δ−, and κ−ligands, for which the receptors have a

high degree of sensitivity. The receptors of the guinea pig ileum

interact with μ- and κ−ligands, those of the rat vas deferens mainly,

but not solely, with μ-ligands, those of the hamster selectively with

δ−ligands, and the receptors of the rabbit vas deferens selectively

with κ−ligands. As far as selectivity of the ligands is concerned,

[D-Ala2,MePhe4,Gly-ol5]enkephalin is suitable for testing the μreceptor in the rat vas deferens, [D-Pen2

,D-Pen5]enkephalin for testing the δ−receptor in the hamster vas deferens, and U-50, 488H for

testing the κ−receptor in the rabbit vas deferens. It is important

to note that some ligands may be agonists in some tissues and antagonists in others. Ethylketazocine and bremazocine are examples of

this behaviour, as they are κ−agonists in the guinea pig ileum and

the vasa deferentia of the mouse and rabbit. In contrast, in the

vas deferens of the rat they are pure μ-antagonists (Gillan et al.

1981) and in the vas deferens of the hamster they are pure δ−

antagonists (McKnight et al. 1984a, in press). These aspects will be

more fully discussed in later sections.

223
TABLE 1

Distribution of Opioid Receptors in the Mouse Vas Deferens (MVD),

Myenteric Plexus of Guinea Pig Ileum (GPI), Rat Vas Deferens

(RVD), Hamster Vas Deferens (HVD), and Rabbit Vas Deferens (LVD)(a)

MVD GPI RVD HVD LVD

μ+δ+κ μ +κ (μ) δ κ

Normorphine

[D-Ala2,MePhe4,Gly-ol5]Enkephalin

ß-Endorphin

[Leu]Enkephalin

[Met]Enkephalin

(D-Pen2,D-Pen5]Enkephalin(b)

Dynorphin A

U-50,488H(C)

Ethylketazocine

Bremazocine

a

0 = no effect, A = agonist, ANT = antagonist. Data are based

on published results (McKnight et al. 1983, 1984a, in press;

Paterson et al. 1984a,b)

b

 [D-ß,ß(CH3)2Cys2,D-ß,ß(CH3)2Cys5]enkephalin (Mosberg et al. 1983)

c

 Trans-3,4-dichloro-N-methyl-N-(2-(pyrrolidinyl)cyclohexyl)-

benzeneacetamine (Piercey et al. 1982)

SELECTIVITY OF OPIOID LIGANDS

The results obtained in this section are summarized in tables 2 to

4. The affinity of a ligand is given as its binding affinity constant, (Ki

, nM)-1, which is the reciprocal of its inhibition constant. In addition, it was found useful to determine the relative

binding affinities by the ratio [Ki

-1 for μ, δ, or κ/ (Ki-1 for μ +

Ki

-1 for δ + Ki-1 for κ)]. The temperature of the binding assays

was 25°C when peptidase resistant compounds were used and 0°C when

peptides that are sensitive to enzyme activity were used.

224
Table 2 gives the results obtained with some of the μ-selective

ligands. The affinity values are around 0.5 nM-1 with the exception of [Met]enkephalyl-Arg-Arg-Val-NH2

 which has a very high

affinity of 8.7 nM-1 The relative affinity of [D-Ala2,MePhe4,

Cly-ol5]enkephalin is 0.99 and that of morphine 0.97; thus, these

compounds are much more μ-selective than any of the other three.

The degree of selectivity is important for agonist compounds but

even more so for antagonists. For instance, naloxone is widely

used as a μ-antagonist, but this is only permissible in low concentrations. The fact has to be remembered that in higher concentrations naloxone also antagonizes the action of compounds having

δ or κ affinities. Unfortunately, it is often not appreciated

sufficiently in in vivo situations that naloxone is a μ-selective

agonist only when used in low concentration. In conclusion, the

most suitable μ-ligands are [D-Ala2,MePhe4,Gly-ol5]enkephalin,

morphine, and also normorphine (which is not included in the

table); naloxone is a selective μ-antagonist if used with care

in low concentrations of up to 15 nM.

TABLE 2

Binding Affinities of μ-Selective Opioids and Their Relative

Binding Affinities for the μ-, δ−, and κ−Sites in Homogenates

of Guinea Pig Brain

μ-Affinity Relative Affinity

(Ki, nM)-1μ δ κ

[Met]EnkephalylArg-Arg-Val-NH2

O°C 8.7 0.66 0.03 0.31

Naloxone 25°C 0.56 0.85 0.06 0.09

Morphine 25°C 0.56 0.97 0.02 0.01

[D-Ala2,MePhe4,Gly-ol5]

Enkephal in

25°C 0.54 0.99 0.01 0

ß-Endorphin 0°C 0.49 0.53 0.42 0.02

The relative binding affinities at the μ-, δ−, and κ−sites are:

Ki for μ-, δ−, or κ/ (Ki

-1 for μ + Ki-1 for δ + Ki-1 for κ).

a

 Paterson et al. 1984a, 1984b; Kosterlitz and Paterson 1985

225
As far as δ−ligands are concerned (table 3), the most selective

peptide analogues are [D-Ser2,L-Leu5]enkephalyl-Thr, [D-Pen2,LPen5

]enkephalin. and [D-Pen2,D-Pen5]enkephalin. They are resistant to peptidase activity and can therefore be used at 25°C in

binding assays and at 37°C in the guinea pig ileum and the mouse

and rat vasa deferentia. While the latter two analogues are

almost fully selective, they are not quite as potent as the first

analogue. It is of interest to note that, while the endogenous

peptides [Leu]enkephalin and [Met]enkephalin have higher affinities to the δ−binding site than the analogues, they are very susceptible to the degrading action of peptidases. The antagonist

ICI 174864 is highly selective for the δ−binding site and is not

affected by peptidases, but it is of low potency.

TABLE 3

Binding Affinities of δ−Selective Opioids and Their Relative

Binding Affinities for the μ-, δ−, and κ−Sites in Homogenates

of Guinea Pig Brain(a)

δ−Affinity Relative Affinity

(Ki, nM)-1μ δ κ

[Met]enkephalin 0°C 1.10 0.09 0.91 0

[Leu]enkephalin 0°C 0.85 0.06 0.94 0

[D-Ser2,L-Leu5]

enkephalyl-Thr

[D-Pen2,D-Pen5]

enkephalin

25°C

25°C

0.56

0.37

0.04 0.96 0

0.004 0.996 0

ICI 174864(b) 25°C 0.005 0.01 0.99 0

The relative binding affinities at the μ-, δ−, and κ−sites are:

Ki for μ, δ, or κ/ (Ki

-1 for μ + Ki-1 for δ + Ki-1 for κ).

a

 Paterson et al. 1984a. 1984b: Kosterlitz and Paterson 1985

b

 N,N-diallyl-Tyr-(α-aminoisobutyric acid)2

-Phe-Leu-OH (Cotton

et al. 1984)

226
With regard to κ−ligands, there are so far no endogenous peptides

or nonpeptide compounds of high potency with a selectivity similar

to those found with μ- and δ−ligands. Fragments of the prodynorphin series are very sensitive to degradation by peptidases

and therefore have to be assayed at 0°C (table 4). The endogenous

peptides dynorphin A and dynorphin B have high affinities (Ki

-1) of

8.7 and 8.5 nM-1, respectively, at the κ−binding site; however, when

the relative affinities were determined, it was found that dynorphins

A and B have values about 0.14 for binding at the μ-site. The endogenous fragment dynorphin A (1-8) has a different binding pattern; its

affinity at the κ−binding site is only 0.75 nM-1 and it has relative

affinities at the μ- and δ−binding sites of 0.22 and 0.16, respectively. The affinity of α−neo-endorphin at the κ−binding site is 5.1

nM-1 and its relative affinities at the μ- and δ−binding sites are

TABLE 4

Binding Affinities of K-Selective Opioids and Their Relative

Binding Affinities for the μ- δ−, and κ−Sites in Homogenates

of Guinea Pig Brain

K-Affinity Relative Affinity

(Ki, nM)-1

μ δ κ

Dynorphin A

Dynorphin B

a-Neo-endorphin

Dynorphin A (1-9)

Dynorphin A (1-8)

(-) -Ethylketazocine

Mr 2266(b)

(-)-Bremazocine

0°C 8.7 0.13 0.04 0.83

0°C 8.5 0.14 0.03 0.83

0°C 5.1 0.10 0.23 0.67

0°C 4.76 0.05 0.06 0.89

0°C 0.75 0.22 0.16 0.62

25°C 1.92 0.32 0.06 0.62

25°C 1.45 0.31 0.07 0.62

25°C 2.44 0.30 0.25 0.45

The relative binding affinities at the μ-, δ−, and κ−sites are:

Ki for μ, δ, or κ/ (Ki

-1 for μ + Ki-1 for δ + Ki-1 for κ).

a

 Paterson et al. 1984a, 1984b; Kosterlitz and Paterson 1985

b

(−)−α-5,9-diethyl-2'-hydroxy-2-(3-furylmethyl)-6,7-benzomorphan

227
0.10 and 0.23, respectively. The nonendogenous peptide dynorphin A

(1-9) has an affinity at the κ−binding site of about 4.8 nM-1, with

relative affinities at the μ- and δ−sites of only 0.05 and 0.06,

respectively. This analogue would be a very suitable κ-ligand if

it were not for the fact that it is readily degraded by peptidase.

Two nonpeptide compounds with affinity to the κ−binding-site are

ethylketazocine and bremazocine, both of which can be assayed at

25°C as they are not subject to enzymatic degradation. Their

affinities at the κ−binding site are 1.9 and 2.4 nM-1 (table 4).

However, their selectivity for an agonist action on the κ−receptor

is not high, as their relative affinities for the μ-binding site

are about 0.31 and the relative affinity of bremazocine for the δ−

binding site is 0.25. In this context, it is noteworthy that both

compounds are μ-antagonists in the rat vas deferens and δ−antagonists in the hamster vas deferens (table 1). Their selectivity for

binding at the κ−site is improved when their effects on the μ- and

δ−binding sites are prevented by the addition of unlabeled μ-ligand

[D-Ala2,MePhe4,Gly-ol5]enkephalin and unlabeled δ−ligand [D-Ala2,-

D-Leu5]enkephalin. Two other nonpeptide compounds which have

affinities at the κ−binding sites are tifluadom (0.25 nM-1) and

U-50, 488H (0.13 nM-1); they are of particular interest because the

relative affinities are 0.84 for tifluadom and 0.99 for U-50, 488H

(Kosterlitz and Paterson 1965).

There is so far no selective κ−antagonist. The best compound is

Mr 2266 which has an affinity of 1.45 nM-1 at the κ−binding site

(table 4). However, its relative affinity is only 0.62 for the

κ−binding site and 0.32 for the μ-binding site; this low selectivity

makes it impossible to identify κ−receptors by the antagonist action

of Mr 2266.

In conclusion, there are so far no κ-agonists or κ−antagonists which

have reached the high standard of potency and selectivity already

achieved for μ- and δ−receptors. It is of particular importance to

search for an antagonist which is potent, and is highly selective

and resistant to degradation by enzymes.

BINDING CHARACTERISTICS AND DEGRADATION PROFILE OF [3H]-DYNORPHIN A

(1-8) AND [

3H]-DYNORPHIN A (1-9)

Dynorphin A (1-8) or [Leu5]enkephalyl-Arg-Arg-Ile, and dynorphin A

(1-9) or [Leu5]enkephalyl-Arg-Arg-Ile-Arg, are of particular interest for two reasons. The octapeptide is endogenous (Weber et al.

1982), while the nonendogenous nonapeptide is the more κ−selective

and more potent of the two peptides (Corbett et al. 1982).

With regard to the possible mode of action of these two peptides,

it is important to note that, in the preparation of the guinea pig

myenteric plexus-longitudinal muscle immersed in Krebs solution,

the onset of their depressant effect is rapid and, after washing

out the peptides, the recovery of the electrically induced twitch

is also not much delayed. Thus, an analysis of the factors involved should give an insight into their mode of action (Corbett

et al. 1982).

228
For this reason, it was decided to investigate binding assays of

[3H]-dynorphin A (1-8) and [

3H]-dynorphin A (1-9) in homogenates

of guinea pig brain and, since both peptides are very sensitive

to the peptidases present in the homogenates, to investigate the

degradation products and the effects of possible peptidase inhibitors (Robson et al. 1983; Gillan et al. 1985, in press).

The degradation products of [

3H]-dynorphin A (1-8) or [3H]-dynorphin A (1-9) were assessed in the supernatant obtained after centrifugation of the homogenates of guinea pig brain previously

incubated at 0°C for 120 minutes or at 25°C for 30 minutes. In

the supernatant, 50% to 60% of the tritium appeared as [

3H]-

tyrosine after incubation at 0°C, whether [

3H]-dynorphin A (1-8)

or [

3H]-dynorphin A (1-9) was used. When bestatin (30 μM) was

present during incubation to prevent aminopeptidase activity, 42%

of the original [

3H]-dynorphin A (1-8) was recovered at 0°C and

4% at 25°C. [

3H]-Dynorphin A (1-9) was even more susceptible to

degradation by peptidases since in the presence of bestatin only

20% was recovered after an incubation at 0°C. A mixture of bestatin (30 μM) and captopril (300 μM) was a more effective

inhibitor of peptidases. The amount of dynorphin A (1-8) recovered

in the supernatant rose to 82% at 0°C and to 37% at 25°C; the amount

of recovered dynorphin A (1-9) was 48% at 0°C (Gillan et al.

1985, in press).

In assays for the determination of κ−binding, μ- and δ−bindings were

suppressed by the necessary amounts of unlabeled [D-A1a2,MePhe4,

Gly-ol5]enkephalin and [D-Ala2,D-Leu5]enkephalin. The κ−binding

of [

3H]-dynorphin A (1-8) was increased by the addition of bestatin

(30 μM) and captopril (300 μM) about 1.4-fold at 0°C and 3.7-fold

at 25°C.

Scatchard plots obtained with [

3H]-dynorphin A (1-8) were found to

be linear at 0°C. Protection from degradation by bestatin (30 μM)

and captopril (300 μM) lowered the KD value, but did not affect the

binding capacity of about 3.5 pmol/g brain; the capuity value is

similar to that obtained with [

3H]-dynorphin A (1-9) and [3H]-(-)-

bremazocine at 0°C. It is also similar to that obtained with

different assay conditions (Quirion and Pilapil 1984).

At 25°C, the maximum binding capacity of [

3H]-dynorphin A (1-8)

varied, as did the Kd values. For this reason, no values are given.

As far as [3H]-dynorphin A (1-9) is concerned, it was found in an

earlier series of experiments (Robson et al. 1983) that the binding

capacity was of the order of 4.9 pmol/g brain:. this value corresponds to the maximum κ−binding capacity of [

3H]-(-)-bremazocine

after suppression of μ- and δ−bindings (5.4 pmol/g brain).

It would therefore appear that the maximum capacity of κ−binding

of [

3H]-dynorphin A (1-9) and [3H]-(-)-bremazocine at 25°C are of

the order of 5 pmol/g guinea pig brain and that both of these compounds may be used for investigations in vitro. However, it has

to be noted that the sensitivity to peptidases of [

3H]-dynorphin A

(1-9) may cause problems which do not arise with [

3H]-(-)-bremazocine. On the other hand, [

3H]-(-)-bremazocine is not κ−selective,

229
as it binds also to μ- and δ−sites. Neither of these two compounds is endogenous; in this respect, dynorphin A (1-8) would be

advantageous, but in inhibitory assays it is less selective and

has a lower affinity to the κ−binding site than dynorphin A (1-9)

(table 4).

THE OPIOID RECEPTORS OF THE HAMSTER VAS DEFERENS ARE OF THE δ−TYPE

Tissues have recently become available which have only one of the

several opioid receptors. The vas deferens of the rabbit contains

only κ−receptors (Oka et al. 1981); that of the rat, almost solely

μ-receptors (Gillan et al. 1981); and that of the hamster, only δ−

receptors (McKnight et al. 1984a, in press).

This section describes the results obtained with the Golden Syrian

hamster. Single vasa deferentia were suspended in 3 ml siliconized

organ baths containing Krebs solution kept at 37°C and aerated with

95% oxygen and 5% CO2. Contractions were induced by electric field

stimulation, by trains consisting of 3 to 4 rectangular biphasic

pulses with intervals of approximately 200 msec; the voltage of each

pulse was supramaximal (50 to 60 mA) and its duration 1 msec. The

rate of trains was 0.1 Hz. The regular contractions caused by

electrical stimulation were of about 0.5 to 1 g tension. Omission

of Mg from the Krebs solution did not significantly affect the

strength of the contraction, in contrast to the finding with the

vas deferens of the mouse (Hughes et al. 1975).

The potencies of the opioid peptides were determined with and

without addition of the peptidase mixture used in this unit

(McKnight et al. 1983), except that the inhibitor thiorphan was

raised to 10 μM.

The first important observation was that neither the selective

μ-receptor agonist [D-Ala2,MePhe4,Gly-ol5]enkephalin nor the

selective κ−receptor agonist U-50, 488H had any significant activity

in the hamster vas deferens. The IC50 values of these compounds

and of normorphine were >l0,000. Thus, the hamster vas deferens

has no significant amounts of μ- and κ−opioid receptors.

In contrast, all the endogenous and exogenous opioid peptides which

interact with the δ−binding site are active in the hamster vas

deferens, either as agonists or antagonists (McKnight et al. 1984a,

in press). When peptidase inhibitors had been added to the solution of

the test medium, the agonist potencies (IC50) of [D-Ala2, D-Leu5]-

enkephalin, [D-Thr2,Leu5]enkephalyl-Thr, and [D-Ser2,Leu5]enkephalylThr were all of the order of 21 nM. These peptides are relatively

selective as δ−ligands; the highly selective [D-Pen2,D-Pen5]-

enkephalin (Mosberg et al. 1983; Corbett et al. 1984) gave an IC50

value of 82 nM, and a similar value was obtained for [D-Pen2,L-Pen5]-

enkephalin. It should be noted that only the two latter peptide

analogues and [D-Ala2,D-Leu5]enkephalin are resistant to peptidases,

whereas the potencies of [D-Thr2,Leu5]enkephalyl-Thr and [D-Ser2,-

Leu5]enkephalyl-Thr are increased Ten-fold when antipeptidases are

added to the bath fluid. [Met5]Enkephalin and [Leu3]enkephalin

are the only endogenous opioid peptides which are active in the

230
hamster vas deferens; they required antipeptidase to measure their

activity.

The most selective δ−antagonist available at present is ICI 174864

(N,N-diallyl-Tyr-[ α−aminoisobutyric acid]2

-Phe-Leu-OH) (Cotton et

al. 1984; Corbctt et al. 1984) with a Ke value of approximately

30 nM; naloxone, which has affinities to the three opioid receptors,

interacts in the hamster vas deferens only at the δ−site, and is an

antagonist with a potency similar to that of ICI 174864. It is of

particular interest that the benzomorphans, bremazocine and ethylketazocine, which have affinities not only to the κ−site but also

at the μ- and δ−sites,are antagonists in the hamster vas deferens.

Bremazocine is a particularly potent δ−antagonist with Ke values of

6.9 nM against [D-Ala2,D-Leu5]enkephalin and 15.8 nM against [D-Ser2,-

Leu5]enkephalyl-Thr as agonists.

In conclusion, the hamster vas deferens is a selective in vitro

preparation for the assay of δ−receptor activities of exogenous

peptides and exogenous peptide analogues and nonpeptide compounds.

It is also very important to realize that the widely used κ−agonists

of the benzomorphan group are antagonists at other receptors in the

nervous system; for instance, ethylketazocine and bremazocine are

antagonists at the μ-receptor in the rat vas deferens and antagonists at the δ−receptor in the hamster vas deferens (Gillan et al.

1981; McKnight et al. 1984a).

TRANSIENT AND PERSISTENT OPIOID EFFECTS OF ß-FUNALTREXAMINE IN THE

MYENTERIC PLEXUS OF THE GUINEA PIG ILEUM

ß-Funaltrexamine (ß-FNA), the fumaramate methyl ester of naltrexone,

is an acute agonist in the isolated myenteric plexus-longitudinal

muscle preparation of the guinea pig, but preincubation with ß-FNA

causes a decrease in the agonist responses to morphine but not to

ethylketazocine. Portoghese and his group found that in acute

experiments these effects are transient. However, after prolonged

exposure of ß-FNA for 30 minutes or more, the depressant effects on

the action of morphine are not reversed by repeated changing of the

bath fluid, but similar effects on the action of ethylketazocine are

reversible (Portoghese et al. 1980; Takemori et al. 1981; Ward et al.

1982). Part of the binding of [

3H]-ß-FNA in homogenates of brains of

guinea pig end mouse is irreversible (Fries et al. 1980; Ward et al.

1980). Preincubation of rat brain with ß-FNA reduced the binding of

[

3H]-naltrexone (Rothman et al. 1983). Finally, evidence has been

presented which supports the view that these findings can be explained

if ß-FNA acts at a site which is not the binding site itself but a

secondary regulatory site (Portoghese and Takemori 1983).

The data presented in this section have the purpose of confirming

the results obtained by Portoghese and his colleagues and extending them by the use of highly selective opioid ligands. The two

approaches (McKnight et al. 1984b; Corbett et al., in press) were binding

assays on homogenates of guinea pig brain and bioassays using preparations of the myenteric plexus-longitudinal muscle of the guinea pig

and the vas deferens of the rabbit as described by Corbett et al.

(1982). The preincubation period was with 100 nM ß-FNA for 30

231
minutes, unless stated otherwise; the tissue was then washed for 60

minutes in order to remove unbound ß-FNA. Antagonist potency of

naloxone (Ke) was determined against [D-Ala2,MePhe4,Gly-ol5]enkephalin in control groups and in preparations pretreated with ß-FNA.

Binding of tritiated ligand was determined by the method of Leslie

and Kosterlitz (1979). Binding was determined with the μ-selective

[

3H]-[D-Ala2

,MePhe4,Gly-ol5]enkephalin (6 nM) and the κ-selective

[

3H]-(-)-bremazocine (0.5 nM) in the presence of 250 nM each of

unlabeled [D-Ala2,MePhe4,Gly-ol5)enkephalin and [D-Ala2,D-Leu5]-

enkephalin to suppress μ- and δ−binding.

In naive preparations of the myenteric plexus, [D-Ala2,MePhe4,-

Gly-ol5]enkephalin is a potent μ-agonist with an IC50 of 4.35 nM,

whereas after pretreatment with 100 nM ß-FNA for 30 minutes, this

value increased to 80 nM; when the concentration of ß-FNA was raised

to 1000 nM, the IC50 of [D-Ala2,MePhe4,Gly-ol5]enkephalin was not

significantly increased to 91 nM. These findings are in agreement

with those of Portoghese and his group, obtained with the less μselective compound morphine. A new important finding is the fact

that the antagonist activity of naloxone is not affected by pretreatment of the preparation with 100 nM ß-FNA for 30 minutes. The

Ke value was 1.9 nM before and 1.8 nM after pretreatment; if the

concentration of ß-FNA was increased to 1000 nM, the Ke value was

raised to 4.7 nM. Thus, pretreatment with 100 nM ß-FNA for 30

minutes irreversibly raises the agonist value of [D-Ala2,MePhe4,-

Gly-ol5]enkephalin about 11-fold without affecting the antagonist

effect of naloxone (McKnight et al. 1984b; Corbett et al., in press).

It was of interest to correlate these observations with data obtained from binding assays. In homogenates of the myenteric plexus ßFNA inhibited the binding of the selective μ-ligand [

3H]-[D-Ala2

,-

MePhe4,Gly-ol5]enkephalin (Ki = 0.8) more potently than the binding

of [

3H]-(-)-bremazocine (Ki

 = 12.1) made selective for κ−binding by

the addition of unlabeled μ- and δ−ligands. Pretreatment with 100

or 1000 nM ß-FNA for 30 or 60 minutes at 37°C did not reduce the

binding of either the μ-ligand (6 nM) or the κ−ligand (0.5 nM) in

the myenteric plexus of the guinea pig ileum. However, when the

pretreatment with ß-FNA was increased to 500 nM for 60 minutes,

in homogenates of guinea pig brain, μ-binding was reduced by about

20% but κ−binding was unaffected (McKnight et al. 1984b; Corbett et

al., in press). This observation was similar to that found with [

3H]-

naloxone binding in rat brain after pretreatment with 1000 nM for

60 minutes (Rothman et al. 1983).

The findings presented in this section support the view first proposed by Portoghese and Takemori (1983) that the irreversible action

of ß-FNA on the μ-receptor is not at the binding site itself, but on

a secondary regulatory site which so far has not been identified

experimentally. As ß-FNA does not affect the response of selective

κ−ligands on the action of κ-receptors in the guinea pig ileum, the

regulatory effect on the coupling between activation of the binding

site and the effector response applies only to the action of μligands but not of κ−ligands. For a further analysis of these

phenomena, it will be necessary to understand the reasons why there

is the absence of an effect of pretreatment by ß-FNA (1) on binding

232
of the selective μ-ligand [D-Ala2

,MePhe4,Gly-ol5]enkephalin at concentrations which depress the response of the myenteric plexus to

electrical stimulation and (2) on the antagonist action of naloxone

on μ-receptors when, at the same time, the agonist action of the

selective μ-ligand is reduced by about 90%.

CONCLUSIONS

1. Agonist and antagonist ligands should have a high degree of

selectivity for one type of opioid receptor. This requirement has

been achieved with the μ-ligand [D-Ala2,MePhe4,Gly-ol5]enkephalin

and with the δ−ligand [D-Pen2,D-Pen5]enkephalin or [D-Pen2,L-Pen5]-

enkephalin. Furthermore, both ligands are peptidase-resistant.

With regard to κ-ligands, the situation is such less satisfactory.

Dynorphin A (1-9) is almost sufficiently selective for κ-binding,

but is readily degraded by peptidases: it is probably possible to

use this fragment of dynorphin A at 0°C. U-50,488H is a highly

selective κ-ligand; it is stable but has a low potency. The

selectivity of tifluadom is almost as high as that of dynorphin A

(1-9), but it has a much lower potency. The benzomorphan analogues

--ethylketazocine, bremazocine, and Mr 2034--are agonists at the κ−

receptor but antagonists at the μ- and δ−receptors. Naloxone is

usually used as a selective μ-antagonist; however, this is only true

in concentrations of not more than 15 nM because in higher concentrations it interacts also at δ− and κ-receptors. The δ−antagonist

ICI 174864 is selective but is of rather low potency. Mr 2266 is an

antagonist at both the κ−receptor and μ-receptor.

2. The tissues which have only one type of opioid receptor are

the hamster vas deferens, which has δ-receptors, and the rabbit vas

deferens, which has κ-receptors. On the other hand, the guinea pig

myenteric plexus-longitudinal muscle has μ- and κ-receptors, the

mouse vas deferens has u-, δ−, and κ-receptors and the rat vas

deferens has μ-receptors and perhaps the putative ε−receptor.

3. While ß-funaltrexamine blocks irreversibly the effect of μagonists on the response to electrical stimulation of the guinea

pig myenteric plexus and the mouse vas deferens, it does not affect

the binding of μ-, δ−, or κ-ligands after pretreatment with up to

1000 nM for 60 minutes. These observations confirm the view proposed by Portoghese and his colleagues that the irreversible action

of ß-funaltrexamine is not at the binding site itself.

4. Finally, it should be noted that several opioid peptides from

which the N-terminal tyrosine has been inactivated or removed may

still have nonopioid biological activity. Such observations are

increasing and may become of importance, e.g., studies by Schweiger

et al. (1982), McCain et al. (1982), Przewlocki et al. (1983), and

Puett et al. (1983).

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235
ACKNOWLEDGMENTS

Supported by grants from the Medical Research Council and the

National Institute on Drug Abuse (DA-00662).

AUTHORS

Hans W. Kosterlitz, M.D., Ph.D.

Alistair D. Corbett, Ph.D.

Maureen G.C. Gillan, Ph.D.

Alexander T. McKnight, Ph.D.

Stewart J. Paterson, B.Sc.

Linda E. Robson, Ph.D.

University of Aberdeen

Unit for Research on Addictive Drugs

Marischal College

Aberdeen, AB9 1AS, Scotland

236
Endorphins and Memory Regulation

Ivan Izquierdo, M.D., Ph.D.; Carlos A. Netto, M.D.; and

Renato D. Dias, M.D.

Memory can only be evaluated by measuring retrieval at

various times from a training experience. The mechanisms underlying memory formation are not known. Changes

in ionic conductance and neurotransmitter release that

may underly changes in neuronal electrical activity

might explain some short-lived memory processes in

invertebrates, but not memory that lasts for days, months,

or years in mammals (Creenough 1984). Hypotheses on the

storage of memory molecules were popular years ago but

have now fallen into discredit, mainly because the halflife of all known molecules, particularly proteins, is

orders of magnitude shorter than the duration of memory

in vertebrates (Greenough 1984). Cajal’s (1911) postulation that memory may involve structural synaptic

changes, including the formation of new synapses, has

received some recent support (Greenough 1984); but it

cannot explain retrieval in seconds or minutes after

training, which is behaviorally indistinguishable from

retrieval at longer periods (same stimuli, same responses) and is thus probably mediated by similar

sensorimotor coupling systems (although it may be modulated by different systems, as discussed below).

We believe that at this stage the most judicious attitude

toward putative memory mechanisms is agnosticism coupled

with an intense search for the mechanisms that modulate

retrieval at different stages in the training-test

process. One reason for this attitude is that it is

inefficient to invest worry, time, and money in a task

for which we have neither the tools nor a good theoretical framework. We do not really know how to look for memory (we can only measure retrieval), or what to look for

(is there an engram? is there a record anywhere in-the

brain of past experiences? what if all neural activity

tends to leave a trace and what we measure as retrieval

is only the result of poor inhibition?). The other

237
reason for the active agnosticism proposed above is

that a large and increasing segment of the population

--the old-- suffers from memory disturbances. Many of

these arc accompanied by cell loss or by a reduction

in the number of synapses in brain regions involved in

memory regulation. These structural losses cannot be

helped by any form of replacement therapy. Even if

brain grafts some day become clinically feasible, it

would be impracticable to implant the brain connections

relevant to a face one has forgotten or a music piece

one does not remember how to play. There is hope for

the alleviation of memory disorders in the search for

drugs active upon the diverse modulatory systems that

appear to be deranged in patients and/or in animals

with age- or disease-related memory disturbances:

the cholinergic (Bartus et al. 1982), central catecholaminergic (Winblad et al. 1985), brain opioid (Gambert

et al. 1980; Jensen et al. 1980, 1981), and pituitary

hormonal systems (Jensen et al. 1981).

This chapter reviews evidence pointing to the involvement of one of the three major opioid systems of the

brain, the hypothalamic beta-endorphin system, in memory

regulation. As will be seen, this system interacts

with the other systems mentioned above.

EFFECTS OF THE POSTTRAINING ADMINISTRATION OF OPIATE

ANTAGONISTS AND OF OPIOID PEPTIDES ON RETRIEVAL

Memory facilitation by the opiate antagonist naloxone

was first described by Gallagher and Kapp (1978) and

Jensen et al. (1978) in an inhibitory avoidance task,

and by Izquierdo (1979) in classical and avoidance

conditioning in a shuttle box and in a habituation

task. Since then, this has become the most confirmed

posttraining pharmacological effect in the memory

literature: it has been observed in 29 aversive and

nonaversive tasks: in 14 different laboratories: and in

6 different species, including humans (Izquierdo et

al. in press, b).

The memory facilitating effect of naloxone has usually

been reported using posttraining injections, but it can

also be observed using pretraining administrations (Izquierdo 1980a). The effect is shared by other opiate

antagonists, whose decreasing order of potency for this

effect roughly parallels their decreasing order of affinity for opiate mu receptors: diprenorphine>naltrexone

> naloxone > levallorphan > dextrallorphan = 0 (Gallagher

238
Posttraining memory facilitation by naloxone suggests

that endogenous opioids may physiologically modulate

memory at that time (Izquierdo 1979). Opiates had been

known for some time to depress memory at low, subanalgesic

doses and to facilitate retention at high, toxic doses

given after training (e.g., 100 mg/kg of morphine, Mondadori and Waser 1979). The effect of these high doses may

be due to a strengthening of the aversive motivation

rather than to a memory effect: animals learn a given

behavior in order to avoid not only the shock, but also

the drug that comes after the shock and makes them feel

sick (Izquierdo 1982). The i.c.v. administration of 200

ug of Met-enkephalin in rats, which is roughly 103 times

the total brain content of that substance, facilitates

the retention of avoidance behavior (Stein and Belluzzi

1978); doses 10,000 to 40,000 times lower have an amnestic effect (Lucion et al. 1982). A major reason to believe

that these effects of toxic doses of opiates or opioids

do not reflect their physiological role in memory is

that, contrarily to all other posttraining facilitatory

drugs known (Gold and McGaugh 1975), naloxone does not

have an inverted U dose-response curve: it facilitates

memory over a very wide dose range (Gallagher 1982).

Subanalgesic doses of opioid peptides given i.p. or

i.c.v. shortly after training inhibit retrieval of

aversive and nonaversive tasks in rats or mice: shuttle

avoidance (Izquierdo et al. 1980a). various forms of inhibitory avoidance (Martinez and Rigter 1980; Izquierdo

and Dias 1983a, 1983b, 1983c, 1983d, in press; Introini

and Baratti 1984; Irquierdo and Netto 1985a, 1985b),

habituation to a tone (Izquierdo et al. 1980a), or to a

new environment (Itquierdo and Netto, in press), etc.

The effect of beta-endorphin can also be observed upon

pretraining administration (Itquierdo 1980a. 1980b), in

which case it can be antagonized by posttraining naloxone (Itquierdo 1980b), which shows that it develops after

rather than during training. Pretraining beta-endorphin

administration disrupts acquisition only at doses 10 or

20 times higher than those which affect retention (Izquierdo 1980b), which stands in contrast to the effects

of the enkephalins. The posttraining i.p. or i.c.v. injection of Met- or Leu-enkephalin also disrupts retrieval

(Izquierdo 1982; Lucion et al. 1982); but their injection

prior to training markedly depresses acquisition at doses

even lower than those that affect retention (Dias et al.

1982), which rules them out as physiological modulators.

If they were released during training, they would make

learning impossible (Dias et al. 1982; Izquierdo 1982).

239
On a molar basis, beta-endorphin is 6 to 10 times more

potent than the enkephalins in producing memory disruption given i.p. (Dias et al. 1982) or i.c.v. (Lucion

et al. 1982). This is further suggestion of an involvement of opiate mu receptors in posttraining memory regulation (Izquierdo and Netto 1985a). In the rat (Izquierdo

et al., in press, a), but not in the mouse (Introini et al..

in press), dynorphin has no effect. The effective dose

range for beta-endorphin is 0.1 to 10.0 ug/kg i.p.

(Izquierdo et al. 1980b; Martinez and Rigter 1980) or

5.0 to 25.0 ng/rat i.c.v. (Lucion et al. 1982).

Naloxone antagonizes the posttraining amnestic effect of

morphine (Izquierdo 1979), levorphanol (Gallagher and

Kapp 1978), the enkephalins (Irquierdo 1982), and betaendorphin (Irquierdo 1982; Irquierdo and Dias 1983a, in

between naloxone and beta-endorphin appears to be

press; Irquierdo and Netto 1985b). At least, the antagonism

competitive (Izquierdo 1982).

The enkephalins morphine, adrenocorticotropin (ACTH),

epinephrine (Carrasco et al. 1982b). and possibly vasopressin (Izquierdo and Dias, in press) release betaendorphin from the rat hypothalamus. This may explain

wholly or in part the amnestic effect of morphine or

the enkephalins at low doses, or of the hormones at high

doses (Gold and Delanoy 1981; Hagan et al. 1982; Izquierdo and Dias 1983a, in press).

BETA-ENDORPHIN IS RELEASED IN THE RAT BRAIN

BY ALL FORMS OF TRAINING STUDIED SO FAR

The biosynthesis of beta-endorphin takes hours (Liotta

et al. 1980) and the ratio of beta-endorphin to its

precursors is high in the hypothalamus and other brain

regions (Rossier et al. 1977). Therefore, if a given

treatment is shown to reduce hypothalamic beta-endorphin

immunoreactivity in a matter of minutes, the effect may

reasonably be attributed to a release of the substance.

All forms of training studied so far cause a quick decrease of rat hypothalamic beta-endorphin immunoreactivity: simple exposure to a training apparatus with no

shocks or any other stimulation during 30 seconds (Perry

et al. 1983; Izquierdo et al. 1984; Izquierdo and Netto

1985a) or 2 minutes (Izquierdo and Netto, in press),

various forms of inhibitory avoidance (Perry et al,

1983; Izquierdo et al. 1984; Izquierdo and Netto 1985a).

a single 5-second inescapable footshock (Izquierdo et

al. 1984). 50 footshocks over 25 minutes (Izquierdo et

al. 1980b, 1981, 1982, 1984), 50 low level tones over 25

minutes (Izquierdo et al. 1984), 50 tone-footshock trials

240
using tone-footshock pairings over 25 minutes (Izquierdo

et al. 1980b, 1981, 1982, 1984), and extinction of the

shuttle avoidance task using tones alone (Izquierdo et

al. 1984).

The effect is observed only when the animals are exposed

to each task for the first time; a repetition of the task

(i.e., a test session) is not accompanied by a brain

beta-endorphin release (Izquierdo et al. 1982, 1984).

The effect obviously does not depend on the duration of

the tasks, on the type of learning, on the presence of

footshocks. on the intensity or the number of footshocks,

or on the degree of stress or arousal presumably involved

in each task (Izquierdo et al. 1984). Therefore, it may

be considered as a result of “novelty” (Izquierdo et al.

1984; Izquierdo and Netto 1985a). The effect is indeed

abolished by prior bilateral transection of the fornix

(Izquierdo and Netto 1985a; Netto et al. 1985), but not

by other hypothalamic deafferentations. The fornix is

the output of the hippocampal system, which has been

proposed to be involved in the recognition of novelty

(Gray 1982). Whether the release of beta-endorphin

caused by training is due just to the recognition of

novelty, or to a reaction to it, or to the inhibition of

that reaction (i.e., habituation) is not known (Izquierdo and Netto, in press).

Depending on how the brain is dissected, the betaendorphin depletion caused by training can also be observed in the rest of the brain excluding the hypothalamus

(Izquierdo et al. 1980b, 1982). The depletion can be

observed as early as 3 minutes (Izquierdo et al. 1984)

or 6 minutes (Izquierdo and Netto 1985a; Netto et al.

1985) after training and lasts for over 2 hours;

recovery is complete at 6 hours from training (Izquierdo

and Netto 1985a).

None of the forms of training listed above, with the

exception of repeated footshocks (Rossier et al. 1977;

Izquierdo et al. 1981). release pituitary beta-endorphin,

as measured either in the gland or in the plasma (Perry

et al. 1983). Repeated footshocks release beta-endorphin

immunoreactive material together with ACTH into the

beta-endorphin is not known.

plasma (Rossier et al. 1977). The role of circulating

In contrast to brain beta-endorphin, brain Met-enkephalin

is unchanged by habituation or shuttle avoidance in

rats (Carrasco et al. 1982c), two tasks whose memory is

strongly affected by pre- or posttraining naloxone

administration (Izquierdo 1979). This, together with the

other reasons given above, rules out the enkephalins from

241
an involvement in posttraining memory modulation. Reasons

for excluding the dynorphin systems (lack of generality

across species, lack of pretest effects) are given elsewhere in detail (Introini et al., in press). Therefore,

evidence points to the brain (but not to the pituitary)

beta-endorphin system as the only major endogenous opioid

system involved in memory regulation; and the influence

of the opioid receptor blockers on retention may be explained by an interference with the normal operation of

this system (Izquierdo 1982; Izquierdo and Netto 1985a).

RELATION BETWEEN THE AMOUNT OF HYPOTHALAMIC

BETA-ENDORPHIK RELEASED BY TRAINING AND

EFFECTIVE DOSES OF THIS SUBSTANCE

All training procedures studied so far reduce brain betaendorphin immunoreactivity by approximately the same

amount, equivalent to 15 to 30 ng of beta-endorphin per

brain (Izquierdo et al. 1984; Izquierdo and Netto, in

press). This amount is similar to doses of the substance

that affect memory when given i.c.v. (5 to 25 ng) (Lucion

et al. 1982; de Almeida and Izquierdo 1984), and corresponds to about 20% of the i.p. ED50 of the drug (Dias et

al. 1982). Approximately 20% of a systemic dose of this

substance enters the cerebrospinal fluid in rabbits

within 1 hour from injection (Houghten et al. 1980).

Therefore, it is reasonable to postulate a role for brain

beta-endorphin in posttraining memory regulation.

Injected beta-endorphin does penetrate the cerebrospinal

fluid in significant amounts, but it is probably quickly

inactivated once in contact with brain tissue by the

removal of N-terminal tyrosine. At 1 hour from injection

of either 125I-labeled (Izquierdo et al. 1980b) or 3HTyrl or 27-labeled beta-endorphin (Houghten et al.

1980). 20% of the label may be found in the brain; but

when the 3H-Tyr label is used, radioactivity corresponds

almost exclusively to free tyrosine, in contrast to

cerebrospinal fluid radioactivity, which was 75% intact

beta-endorphin (Houghten et al. 1980). However, the

report using 3H-Tyr labeling does not mention whether the

label was at position 1 or 27 in the experiment in which

free tyrosine radioactivity was found in brain tissue.

Clearly, interpretations could be very different depending on where the label was.

Anyway, since beta-endorphin has very powerful effects

on behavior when given i.c.v. (Lucion et al. 1982; de

Almeida and Izquierdo 1984). it must be acting on receptors somewhere in the brain; and since it enters the

cerebrospinal fluid when injected, it is likely that

those receptors are near the ventricular walls. The area

241
close to the walls of the third ventricle is particularly rich in opiate receptors and in beta-endorphin

containing fibers originating in the medial basal hypothalamus (Bloom and McGinty 1981). Presumably the

peptide acts on periventricular receptors and is destroyed soon afterwards (Izquierdo and Netto 1985a).

POSTTRAINING MEMORY MODULATION BY ENDOGENOUSLY

RELEASED OR INJECTED BETA-ENDORPHIN: INTERPRETATIONS

Agents that alter retention when given at the posttraining period are usually assumed to affect consolidation (McGaugh and Herz 1972; Gold and McGaugh

1975; Izquierdo 1979; Izquierdo and Dias 1983a); i.e.,

the hypothetical process by which recently acquired

information is transformed, at a loss, from an initially

unstable into a long-lasting stable trace (Izquierdo et

al. 1982).

There are, however, alternative explanations for posttraining drug effects. One, as was mentioned, is that

they may add to the motivational component of the

tasks; for example, by generating an aversive state

that will reinforce behavior in addition to the customary footshocks used during training. Posttraining effects of opioid peptides obtained with i.p. or i.c.v.

doses within the physiological range, or of naloxone,

cannot be explained by motivational effects. These

substances affect retention of aversive and nonaversive

tasks with very different response requirements and very

different motivation in the same way: opioids reduce,

and naloxone enhances, retrieval measured 1 day or more

after training.

These findings also rule out a second possible alternative explanation for posttraining drug effects: that they

may establish long-lasting changes in general activity,

thus leading to an enhancement of response suppression

or of response commission.

There is, however, a third alternative explanation for

posttraining drug effects: that they may add to the

context of the task by setting up a “state” from which

memory may become dependent for retrieval (Zornetzer

1978; Izquierdo 1984). In other words, that they may

establish a form of state dependency (Overton 1974) involving the posttraining period and the state at the

time of testing (Izquierdo and Dias 1983b, 1983c, in

press; Izquierdo 1984). As will be seen below, current

evidence supports this interpretation in the case of

beta-endorphin.

243
BETA-ENDORPHIN INDUCED STATE DEPENDENCY

The first indications that the posttraining deleterious

effect of beta-endorphin on retention could be due to

the establishment of a neurohumoral “asymmetry” (Overton

1974) between the posttraining period and the test session came from various sources. As mentioned above. the

release of brain beta-endorphin is asymmetrical: it occurs after training, but not after test, sessions unless

the task is changed (Izquierdo et al. 1984). In 1978,

de Wied et al. reported that administration of this

substance prior to testing retarded the extinction of a

one-way active avoidance task; effective doses were similar to those that impair retention when given after

training. This was confirmed by Izquierdo (1980a) using

a shuttle avoidance and a habituation task, and by Izquierdo and Dias (1983b, in press) using inhibitory

avoidance conditioning. Pretest retrieval enhancement

can be obtained using 1.0 or 2.0 ug/kg of beta-endorphin

given i.p. or 25 ng/rat given i.c.v. (de Almeida and Izquierdo 1984).

Posttraining naloxone would reduce the asymmetry caused

by the injection or the release of beta-endorphin exclusively after training; and pretest beta-endorphin

administration would also reduce the asymmetry. Thus,

both treatments would be expected to facilitate retrieval,

which is exactly what they do. In contrast, posttraining

beta-endorphin administration would enhance the asymmetry

and would be expected to disrupt retrieval, which is

exactly what it does (Izquierdo and Dias, in press).

The state dependency interpretation is further substantiated by recent observations in animals with bilateral

section of the fornix. This lesion blocks the hypothalamic release of beta-endorphin caused by training

(Netto et al. 1985; Izquierdo and Netto 1985a). In rats

with bilateral section of the fornix, both the posttraining effect of naloxone and the pretest effect of betaendorphin are not seen (Izquierdo and Netto 1985b).

As was mentioned, the hypothalamic release of betaendorphin caused by training may result from the novelty

inherent to each task (Izquierdo et al. 1984). The betaendorphin depletion caused by a training session lasts

about 6 hours (Izquierdo and Netto 1985a). The posttraining effects of naloxone or beta-endorphin, or- the

pretest effects of the latter, on retention do not become

manifest before hypothalamic levels have recovered (Izquierdo and Netto 1985a. 1985b; Izquierdo and McGaugh,

in press). Therefore, there is a period of several hours

after training in which retrieval occurs independently

of this substance.

244
In this respect, it is important to distinguish between

immediate retrieval and retrieval measured 1 to 3 hours

after training. Immediate retrieval (i.e., retrieval

measured 0 hours after training) has been recently

studied in this laboratory in detail. It is better than

retrieval at later times, and it occurs independently of

hormonal or neurohumoral modulation, including betaendorphin modulation; but it depends on the integrity of

the fornix (Izquierdo and Netto 1985b). The role of the

fornix in immediate retrieval is unrelated to its role

in beta-endorphin release, since the effect of the

fornix lesion on immediate retrieval is not mimicked by

naloxonc or reversed by beta-endorphin administration

(Izquierdo and Netto 1985b).

Pre- or posttraining beta-endorphin administration does

not affect retrieval measured 1, 2. or 3 hours after

training (Izquierdo and Netto 1985a; Izquierdo and

McGaugh, in press); its amnestic effect becomes manifest

only 6 hours or more after training. In contrast, the

modulatory influence of the hormones, ACTH and epinephrine, can be seen as early as 1 or 3 hours after

training (Izquierdo and Netto 1985a, 1985b). Apparently,

in the first few hours after training when endogenous

beta-endorphin stores are low because they have been

recently depleted by the training experience, memory is

regulated by other systems, including the hormone systems. Recent findings (Itquierdo and McGaugh, in

press) suggest that beta-endorphin activates a betaadrenergic system which maintains retrieval capability

high for several hours after training.

Thus, the role of beta-endorphin in memory modulation is

limited by the long time of recovery of the system once

it has been activated by appropriately novel experiences.

It may be a “tag” or a “marker” of particularly significant events that the animals recognize as “novel,”

so that these events may be properly recalled only when

new events of similar importance cause a new release of

brain beta-endorphin (Izquierdo and Dias, in press; Izquierdo and Netto 1985a, 1985b). This may be useful to

recall coping strategies, for example. Retrieval of the

“novel” or “significant” events will occur normally and

independently of the beta-endorphin system for a few

hours after each experience, so that animals can react

appropriately to each circumstance. After a few hours,

memory of the events will be side-tracked into a store

from which it can be well retrieved only under the influence of beta-endorphin (Izquierdo et al. 1984; Izquierdo and Netto 1985a, 1985b; Izquierdo and Dias, in

press; Izquierdo and McGaugh, in press).

245
This possible role of beta-endorphin in adaptive behavior is different from, and independent of, those of

the stress hormones ACTH and epinephrine, which are

also released in peculiarly striking events. The release of brain beta-endorphin caused by training is unaffected by treatments that block stress-induced ACTH

release (dexamethasone, anterior hypothalamic deafferentation) or epinephrine release (adenal medullectomy) (Izquierdo and Netto 1985a; Netto et al. 1985). This is so

in spite of the fact that ACTH and epinephrine at high

doses may cause themselves a release of hypothalamic

beta-endorphin (Carrasco et al. 1982b).

MEMORY CONSOLIDATION EFFECTS AND STATE DEPENDENCY

Posttraining memory modulation by beta-endorphin is best

explained by the induction of state dependency; but

other endogenous or exogenous agents acting at the posttraining period may exert their effects through an influence on consolidation (McGaugh and Herz 1972; Izquierdo and Netto 1985b). Posttraining ACTH, epinephrine.

and vasopressin administrations facilitate the late retrieval of inhibitory avoidance tasks acquired with a

low-intensity training footshock (Gold and Delanoy 1981;

Izquierdo and Dias 1983a). Injections of these substances

add to the amounts that are released during training

(Gold and Delanoy 1981); so, there is probably an optimum

circulating level of ACTH, epinephrine. and vasopressin

for consolidation at the posttraining period (Gold and

McGaugh 1975; Gold and Delanoy 1981; Izquierdo and Dias

1983a). Training with low-intensity footshock causes a

low release of endogenous stress hormones and leaves

poor memory which can be boosted with extra amounts of

the hormones given by injection. Training with a strong

footshock releases a larger amount of the hormones and

leaves better memory (Gold and McGaugh 1975; Gold and

Delanoy 1981).

As was mentioned, posttraining memory facilitation by

ACTH and epinephrine can be observed when retrieval is

measured at short periods after training, i.e., when

brain beta-endorphin stores are still low and before the

posttraining effects of naloxone and beta-endorphin can

be observed (Izquierdo and Netto 1985b). In addition,

posttraining facilitation by ACTH and epinephrine is

either unaffected (Izquierdo and Netto 1985b) or enhanced

(Izquierdo and Dias 1983a, in press) by naloxone. Therefore, posttraining facilitation by the stress hormones

is a different phenomenon from posttraining opioid modulation: and, unlike the latter, it may be explained by an

influence on consolidation (Gold and McGaugh 1975; Izquierdo and Dias 1983a. in press).

246
In contrast, pretest retrieval facilitation by ACTH,

epinephrine, and vasopressin seems to depend on brain

beta-endorphin release: it is naloxone-reversible and

mimicked by beta-endorphin (Izquierdo and Netto 1985b;

Izquierdo and Dias, in press). It cannot be detected

less than 6 hours after training, which is typical of

the beta-endorphin effects (Izquierdo and Netto 1985b).

It is possible that the hormones are able to release

beta-endorphin from the brain at the time of testing,

when there is no task-induced depletion, but not right

after training, when the stores are already depleted by

training itself (Izquierdo and Netto 1985a, 1985b; Izquierdo and Dias, in press).

RETROGRADE AMNESIA INDUCED BY ELECTROCONVULSIVE SHOCK

One major side effect of electroconvulsive shock therapy

in humans is retrograde amnesia. This side effect is

widely used in laboratory animals as an experimental

tool. Electroconvulsive shock produces widespread

hormonal and neurohumoral changes (Netto and Izquierdo

1985), among which is a depletion of Met-enkephalin

(Carrasco et al. 1982c) and beta-endorphin (Dias et al.

1981) from the hypothalamus. The depletion of betaendorphin is much larger than that produced by training

(see above). The amnestic effect of electroconvulsive

shock is reversed by naloxone (Carrasco et al. 1982a).

This suggests that it may be due to an exaggeration of

the state dependency inducing effect of the opioids, to

the point where it cannot be readily reversed by a new

training-induced beta-endorphin release, and amnesia

occurs.

The amnestic effect of electroconvulsive shock is

abolished by posterior hypothalamic deafferentation, but

not by anterior hypothalamic deafferentation or by

section of the fornix (Netto and Izquierdo 1985). This

suggests that, if it is an effect mediated by a massive

release of hypothalamic opioids, the brain pathways involved are different from those that are activated by

training (Izquierdo and Netto 1985a). Training is normally not accompanied by a release of brain Met-enkephalin

(Carrasco et al. 1982c).

BETA-ENDORPHIN MEMORY MODULATION AND THE

DESIGN OF COGNITIVE ENHANCING DRUGS

Agents used for the alleviation of human memory disorders

are called cognitive enhancing drugs by the industry.

Many such drugs have been tried (physostigmine, naloxone,

piracetam, codergocrine, vincamine. ACTH fragments,

vasopressin, etc.). None has yet been found to be

universally effective, which was to be expected in view

247
of the many systems involved in memory modulation at

different times, and the search continues. The potential

market is very large: the fast growing aging population

of the world.

It is clear that a hyperfunction of the brain betaendorphin system may result in amnesia; electroconvulsive

shock-induced amnesia may be an example. It is not

clear, however, that electroconvulsive shock-induced

amnesia may be at all a good model for most cases of

human memory dysfunction, such as the Korsakoff or Alzheimer syndromes and senile dementia. In these conditions, the dysfunction is not time-locked to any

particular external event like electroconvulsive shock,

and memory is altered both retro- and pro-actively.

Korsakoff patients are disturbed by their inability to

form new memories; Alzheimer and senile patients are

disturbed mainly by the loss of old but important information (where is the bathroom? how do I get back home?

who is this person who talks to me as if he were my

son?).

Naloxone has recently been tried with apparent success

on several clinical psychometric tests in patients with

senile dementia (Reisberg et al. 1983). The drug would

certainly be expected to facilitate retrieval of information acquired shortly before or after its administration, because it does so in animals both when given

shortly before or after training (Itquierdo 1979, 1980a).

However, in animals treated with posttraining naloxone

(Izquierdo and Netto 1985b; Izquierdo and Dias, in

press) or with a fornix lesion (Izquierdo and Netto

1985a, 1985b), a new release or an injection of betaendorphin, ACTH, or epinephrine will be unable to facilitate retrieval; so it is difficult to expect a beneficial effect of naloxone either in the long run for

most memories or in the short run for some.

Both a reduction in the number of opiate receptor sites

(Jensen et al. 1980) and in the hypothalamic content of

beta-endorphin (Gambert et al. 1980) have been observed

in old rats. Therefore, it is more likely that old age,

or diseases that accelerate, enhance, or mimick the memory deficit caused by old age, may be accompanied by a

hypofunction rather than by a hyperfunction of brain

opioid systems. In view of this, and by virtue of the

physiological role of beta-endorphin in memory discussed

above, possibly this peptide or its analogs or drugs

that may release it (ACTH, vasopressin, epinephrine) may

have a higher therapeutic potential. ACTH, epinephrine,

and vasopressin have additional effects of their own on

consolidation, independent of beta-endorphin (see above).

248
ACTH and vasopressin have been tried clinically with

some degree of success, but their action is too short

and the side effects too many to warrant a more widespread application (Jensen et al. 1981).

Knowledge of the factors that maintain high retrieval

scores shortly after training, independently of ACTH,

epinephrine, or beta-endorphin modulation (Izquierdo and

Netto 1985a, 1985b), may also be important in the search

for cognitive enhancing drugs. Some of these factors

are known: the fornix is necessary for immediate retrieval (Izquierdo and Netto 1985b), and beta-adrenergic

systems, presumably central, are apparently important

in the next 2 or 3 hours (Izquierdo and McGaugh, in

press). Research on these factors is currently in

progress in this and other laboratories.

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ACKNOWLEDGMENTS

This work was supported by Financiadora de Projetos

(FINEP), and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Brazil,

AUTHORS

Ivan Izquierdo, M.D., Ph.D.

Carlos A. Netto, M.D., M.S.

Renato D. Dias, M.D.

Laboratório de Neuroquímica

Departamento de Bioquímica

Instituto de Biociencias

Universidade Federal

do Rio Grande do Sul (UFRGS)

(centro), 90000 Porto Alegre, RS,

Brazil

254
Current Status of RIA Methods for the

Analysis of Enkephalins and Endorphins

R. Wayne Hendren, Ph.D.

INTRODUCTION

Radioimmunoassay has been widely employed as a key analytical

tool in studies of the structure, distribution, and function of

opioid peptides. These peptides normally occur in biological

tissues and fluids in very low concentrations and in the presence

of many other peptides having similar structures, thus making

their analysis a challenging task. Although exquisite sensitivity

and specificity are hallmarks of radioimmunoassay (RIA), this

technique is not always capable of selectively measuring one

opioid peptide in the presence of others. As a result, techniques

for resolving complex mixtures of opioid peptides by gel filtration

and high performance liquid chromatography (HPLC) prior to

quantifying by RIA have been developed and widely adopted. In

addition, a wide variety of antisera has been developed to

selectively recognize structures unique to the families of

opioid peptides derived from the three large precursors: proopiomelanocortin (POMC), proenkephalin, and prodynorphin.

The purpose of this chapter is to review specific applications

of RIA to the measurement of opioid peptides in biological

tissues and fluids. Thorough discussions of the general theory

and practice of RIA are available elsewhere (Chard 1978; Jaffe

and Behrman 1979; Odell and Daughaday 1971; Orth 1975; Van

Vunakis 1980). Practical aspects of measuring endorphins and

enkephalins by RIA have been reviewed by Bayon et al. (1983),

Hong et al. (1983), and Loeber and Verhoef (1981).

ANTISERA

Competition between a radiolabeled antigen, or tracer, and

unlabeled antigen for a limited number of specific antibody

binding sites forms the basis of all radioimmunoassays. The

sensitivity of an RIA is determined by the affinity with which

the antibody reversibly binds the antigenic epitope, or determinant, and by the specific activity of the tracer. The specificity of an antibody results from the recognition of only a

255
portion of the antigen, the determinant, by the antibody binding

sites. Conventional sources of antisera used for RIA are

polyclonal and thus frequently contain multiple populations of

antibodies of differing affinity and specificity. In contrast,

monoclonal antibodies are comprised of a single population with

uniform affinity and selectivity for the antigen.

Numerous antisera have been developed and used for RIA of

opioid peptides. Representative, but far from comprehensive,

listings of antisera against peptides derived from POMC,

proenkephalin, and prodynorphin are provided in tables 1 to 3,

respectively.

Immunogens

Peptides of molecular weight less than 4,000 or 5,000 are

generally only weakly immunogenic unless coupled to a larger

carrier molecule. For this reason, most of the antisera listed

in tables 1 to 3 have been produced by immunizing animals with

a conjugate of the opioid peptide hapten with a carrier protein,

such as bovine serum albumin or thyroglobulin. Although commercial sources of antisera to opioid peptides are included in

these tables, specific information concerning the derivation of

these antisera is often unavailable. Water soluble carbodiimides have been the most commonly used reagents for coupling

opioid peptides to carrier molecules. Carbodiimides promote

amide formation between free amino groups in the hapten and

free carboxyl groups in the carrier, and vice versa (Bauminger

and Wilchek 1980). Many of the antisera produced against

ß-endorphin coupled to carriers with carbodiimides recognize

the middle or carboxyl portions of the hapten, suggesting that

coupling occurs preferentially between the alpha amino terminus

of the hapten and carboxyl groups on the carrier (table 1).

Conversely, antisera directed against the amino end of

ß-endorphin resulted when carbodiimide coupling was forced

through the hapten carboxyl groups by using polylysine as an

intermediate carrier (Gramsch et al. 1983; Meo et al. 1983) or

by acetylating the hapten prior to coupling (Weber et al.

1982c). Alternatively, glutaraldehyde has been widely used to

couple amino groups on opioid peptides to carrier amino groups,

resulting in diimine conjugates. If coupling through hapten

residues other than amino or carboxyl groups is desired, bisdiazotized benzidine or similar reagents may be used to react

with tyrosyl or histidyl residues (Guillemin et al. 1977).

Strategies for influencing antiserum specificity through selection of hapten coupling reagents are discussed further by

Rorstad (1983).

Although most of the polyclonal antisera to opioid peptides

have been produced against conjugates with large carriers,

Bramnert et al. (1982) successfully immunized rabbits with

unconjugated ß-endorphin. Antisera directed against ß-endorphin

have also been produced by immunizing with a crude preparation

of porcine ACTH1 which must have contained substantial quantities of ß-endorphin or its precursors (Yoshimi et al. 1978).

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TABLE 1

Antisera Against Pro-Opiomelanocortin Related Peptidesa
TABLE 1 (continued)
TABLE 1 (continued)
TABLE 1 (continued)
TABLE 1 (continued)
TABLE 2

Antisera Against Proenkephalin-Related Peptidesa
TABLE 2 (continued)
TABLE 2 (continued)
TABLE 2 (continued)
TABLE 2 (continued)

a Unless otherwise noted, antisera were produced in rabbits, and 125I-labeled tracers were used

Sensitivities are those stated in the cited publications. If not stated by the original authors,

the sensitivities (90% B/Bo) have been estimated from the published standard curves. Undefined

immunogenic conjugates are encolosed in parentheses. The subscripts c, h, m, o, p, and r abbreviate

camel, human, mouse, ovine, porcine, and rat, respectively.

b Accurate Chemical and Scientific Corp., Westbury, NY.

c Amersham Corp., Arlington Heights, IL.

d

Immuno Nuclear Corp., Stillwater, MN.

e Karyon Technology Inc., Norwood, MA.

f Miles Scientific, Naperville, IL.

g Contact Dr. Richard L. Hawks, Research Technology Branch, Room 10-A-19, National Institute on

Drug Abuse, 5600 Fishers Lane, Rockville, MD 20857,

h Peninsula Laboratories, Belmont, CA.
TABLE 3

Antisera Against Prodynorphin-Related Peptidesa
TABLE 3 (continued)

a Unless otherwise noted, antisera were produced in rabbits, and 125I-labeled tracers were used

Sensitivities are those stated in the cited publications. If not stated by the original authors,

the sensitivities (90% B/Bo) have been estimated from the published standard curves. Undefined

immunogenic conjugates are encolosed in parentheses. The subscripts c, h, m, o, p, and r abbreviate

camel, human, mouse, ovine, porcine, and rat, respectively.

b Accurate Chemical and Scientific Corp., Westbury, NY.

c Karyon Technology Inc., Norwood, MA.

d Peninsula Laboratories, Belmont, CA.
Occasionally, peptides are inadvertently modified in the course

of conjugation to carrier. For example, Weber et al. (1981)

immunized rabbits with a conjugate made by linking dynorphin(1-

13) to bovine serum albumin (BSA) with a carbodiimide but

obtained antisera directed specifically against α-N-acetyl

dynorphin(1-13). In this case, acetate salts present in the

dynorphin(1-13) preparation were apparently coupled to the free

amino group of the peptide when the carbodiimide was added.

Likewise, antisera directed against the sulfoxide of Metenkephalin are sometimes produced when an immunizing conjugate

of Met-enkephalin is inadvertently oxidized (King and Millar

1980, 1981). The vulnerability of Met-enkephalin to oxidation

has led other investigators to deliberately prepare antisera to

Met(O)-enkephalin and then oxidize all samples prior to assay

(Clement-Jones et al. 1980a). In some cases, an antiserum will

recognize the oxidized and reduced forms of Met-enkephalin

equally well (Liston and Rossier 1984). On the other hand,

Boarder et al. (1982c) found that an antiserum against Met(O)-

enkephalin[Arg6,Phe7] cross-reacted with the reduced form by

only 30%.

Immunization Procedures

Most of the polyclonal antisera for opioid peptides have been

produced in rabbits. The immunogen is usually injected intradermally or subcutaneously at multiple sites in the form of an

emulsion with Freund’s complete adjuvant. Booster immunizations

are usually administered as emulsions of immunogen in Freund’s

incomplete adjuvant at monthly intervals after the primary

immunizations. Each laboratory uses slightly different immunization protocols, and there is little comparative evidence for

evaluating these variations. Detailed procedures for preparing

emulsions of immunogen, protocols for immunization, and procedures for collecting, processing, and storing antisera have

been described by Hurn and Chantler (1980).

Specificity

Development of antisera recognizing only a single opioid

peptide is made difficult by the fact that common amino acid

sequences are shared by peptides derived from the same precursor . The common sequences found among opioid peptides derived

from POMC, proenkephalin, and prodynorphin are shown in figures

1 to 3, respectively. Thus, β-endorphin constitutes the 31

residues at the carboxyl terminus of β-LPH1, and the α-endorphin

sequence is contained within all of the structures shown in

figure 1. Likewise, the Tyr-Gly-Gly-Phe-Met-Lys or Tyr-GlyGly-Phe-Met-Arg sequence is common to the peptides derived from

proenkephalin (figure 2). Furthermore, the Met-enkephalin

structure is common to peptides derived from both POMC and

proenkephalin, and the Leu-enkephalin sequence is common to

peptides derived from both prodynorphin and proenkephalin.

Detailed discussions of opioid peptide structures, biosynthesis,

and nomenclatural conventions are available elsewhere (Burbach

1984; Cox 1982; Morley 1983).

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61 62 63 64 65 66 67 66 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91

ßh-LPH ßh-LPH(1-60) Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-Ile-Ile-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu

ßh-Endorphin Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-Ile-Ile-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu

ßh-Endorphin (1-27) Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-lle-lle-Lys-Asn-Ala-Tyr

Endorphin Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu

α-Endorphin Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr

FIGURE 1

Common Amino Acid Sequences Among Peptides Derived from Proopiomelanocortin.

Numbers refer to the sequence of ß-lipotropin.
Peptide F Tyr-Gly-Gly-Phe-Met-Lys-Lys-Met-Asp-Glu-[-15residues-]-Leu-Gly-Lys-Arg-Tyr-Gly-Gly-Phe-Met

[Met5]-enkephalyl-Lys Tyr-Gly-Gly-Phe-Met-Lys

Peptide I [16 residues] Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-Trp-Trp-Met-Asp-Tyr-Gln-Lys-Arg-Tyr-Gly-Gly-Phe-Leu

Peptide E Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-Trp-Trp-Met-Asp-Tyr-Gln-Lys-Arg-Tyr-Gly-Gly-Phe-Leu

[Leu5]-enkephalin Tyr-Gly-Gly-Phe-Leu

BAM-22P Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-Trp-Trp-Met-Asp-Tyr-Gln-Lys-Arg-Tyr-Gly

BAM-20P Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-Trp-Trp-Met-Asp-Tyr-GIn-Lys-Arg

BAM-12P Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu

Metorphinamide Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-NH2

[Met5]-enkephalyl-Arg-Arg Tyr-Gly-Gly-Phe-Met-Arg-Arg

[Met5]enkephalyl-Arg Tyr-Gly-Gly-Phe-Met-Arg

Peptide B [26 residues] Tyr-Gly-Gly-Phe-Met-Arg-Phe

[Met5]enkephalyl-Arg-Phe Tyr-Gly-Gly-Phe-Met-Arg-Phe

[Met5]-enkephalyl-Arg-Gly-Leu Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu

[Met5]enkephalin Tyr-Gly-Gly-Phe-Met

FIGURE 2

Common Amino Acid Sequences Among Peptides Derived from Proenkephalin
Dynorphin (1-32) Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln-Lys-Arg-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr

Leumorphin Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr

Arg-[15 Residues]

Dynorphin B Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr

Dynorphin A Tyr-Gly-Gly-Phe-Leu-Arg-Arg-lle-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln

Dynorphin A (1-8) Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile

α−Neo-endorphin Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro-Lys

ß-Neo-endorphin Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro

[Leu5]-enkephalin Tyr-Gly-Gly-Phe-Leu

FIGURE 3

Common Amino Acid Sequences Among Peptides Derived from Prodynorphin
Antiserum specificity is evaluated by comparing the concentration of unlabeled hapten required to displace approximately

half of the antibody-bound radiolabeled hapten with the concentrations of potential cross-reactants which produce the same

displacement. Determinants recognized by conventional antisera

to peptides generally contain two to eight amino acid residues.

For example, the “Lucia 9/14” antiserum developed by Ghazarossian

et al. (1980a) recognized the amino acids in positions 5 to 12

of dynorphin(1-13). This antiserum cross-reacted with dynorphin(1-17) and dynorphin(1-32) but failed to recognize α-neoendorphin, ß-neo-endorphin, Met-enkephalin, Leu-enkephalin,

α -endorphin, or ß-endorphin (Ghazarossian et al. 1980a; Zamir

et al. 1983). In contrast, other antisera appear to require

virtually the entire sequence of the hapten for effective

binding (Chang et al. 1979). Thus, the dynorphin(1-13) antiserum developed by Nakao et al. (1981) failed to cross-react

with the hapten fragments containing residues 1 to 5, 4 to 8,

or 8 to 13 and cross-reacted with dynorphin(4-13) by only 1%.

Similarly, Wilkes et al. (1980) found that a highly specific

antiserum against ß-endorphin failed to cross-react with

fragments containing residues 1 to 16, 1 to 17, or 19 to 31 and

concluded that contributions from both the amino and carboxyl

termini of ß-endorphin were required for antibody recognition.

In such cases, it appears that antibodies are developed against

a three-dimensional determinant which may contain residues

quite separated within the primary sequence. These observations

are consistent with evidence that, in the preferred conformation

of ß-endorphin, the hydrophobic Phe18 and Tyr27 residues interact with the Phe4

 and Tyr1 residues (Taylor et al. 1982).

As expected, many ß-endorphin antisera cross-react extensively

with ß-LPH. For example, Guillemin et al. (1977) developed an

antiserum which recognized the carboxyl terminus of ß-endorphin

and cross-reacted completely with ß-LPH and POMC but not at all

with Met-enkephalin. A distinctly different specificity profile

was reported by Bramnert et al. (1982), who produced a ß-endorphin

antiserum which cross-reacted strongly with a fragment containing

residues 1 to 9, required a free alpha amino group, and crossreacted with ß-LPH by only 1.5%. ß-Endorphin generally fails

to cross-react strongly with Met-enkephalin antisera, and vice

versa. Although moderate cross-reaction with Leu-enkephalin is

commonly observed with Met-enkephalin antisera, Miller et al.

(1978) reported assays for both Met- and Leu-enkephalin with

mutual cross-reactions of only 1%.

Complex patterns of cross-reaction may be observed when heterogeneous antibody populations are present (Bayon et al. 1983).

Asymmetric or biphasic curves with full displacement of tracer

may result when separate antibody subpopulations recognize

different antigenic determinants. Impure tracers, e.g., mixtures of mono- and di-iodinated species, can also lead to asymmetric displacement curves. Incomplete displacement of tracer

by fragments of the antigen is observed when a heterogeneous

antiserum recognizes homologous antigens. Ross et al. (1978)

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concluded that an α−endorphin antiserum contained a subpopulation requiring a free alpha amino group, because peptides

lacking this feature displaced the tracer incompletely.

In some cases, the nonspecificity of an antiserum can be

exploited to detect peptides for which a specific RIA may not

be available. Mizuno et al. (1980) used an antiserum against

Leu-enkephalin[Arg 6] to measure BAM-12P1 concentrations in

extracts of bovine adrenal medulla, even though the latter

peptide cross-reacted by only 5%.

The selectivity of an RIA may be improved by physically separating

the analyte from cross-reactants prior to assay. This approach

is discussed in detail in the Sample Preparation section.

Undesired specificities in a heterogeneous antiserum may also

be eliminated by appropriate selection of the tracer. This

approach was illustrated by Akil et al. (1983), who used 125IN-acetyl-ß-endorphin as a tracer to eliminate amino- specific

determinants in an antiserum against N-acetyl-Tyr-Gly-Gly-Phe.

Alternatively, the antibodies may be purified by affinity chromatography, as described by Blake et al. (1982) for a ß-endorphin

antiserum. Others have achieved selectivity by measuring each

sample with two or more antisera having different specificities.

For example, Jeffcoate et al. (1978b) measured concentrations

of ß-endorphin and ß-LPH in human plasma and cerebrospinal fluid

(CSF) with one antiserum recognizing both peptides equally and

with another recognizing only ß-LPH; the difference between the

two immunoreactivities was taken as the concentration of ß-endorphin.

Monoclonal Antibodies

The problems sometimes associated with the use of heterogeneous

antibody populations in RIA may be avoided if monoclonal antibodies of appropriate sensitivity and specificity can be

developed. Monoclonal antibodies directed against the amino

terminus of ß-endorphin have been reported by Gramsch et al.

(1983) and Meo et al. (1983). These antibodies failed to bind

ß-LPH or N-acetyl-ß-endorphin but cross-reacted nearly completely with Met- and Leu-enkephalin. Jones et al. (1983)

described Leu-enkephalin antibodies secreted by two different

hybridoma clones, one of which failed to bind Met-enkephalin.

The antibodies produced by the second clone cross-reacted

completely with Met-enkephalin but not at all with Met(O)-

enkephalin. Monoclonal antibodies which fail to distinguish

Met- and Leu-enkephalin have also been reported by Cuello et

al. (1984). Unfortunately, none of the monoclonal antibodies

produced against endorphins or enkephalins have approached the

sensitivity obtained with polyclonal antibodies, and their

utility for RIA has been somewhat limited.

Sensitivity

A radiolabeled tracer of the highest possible specific activity

is used to maximize sensitivity in RIA. For this reason, most

274
RIAs for opioid peptides incorporate a tracer labeled with

125I, which has a maximum specific activity of 2200 Ci/matom.

Tritium, with a maximum specific activity of 29 Ci/matom, is

sometimes used in enkephalin RIAs, because some enkephalin

antisera do not bind the iodinated tracer as well as the 3Hlabeled hapten.

When 125I tracers are used, the most sensitive antisera can

detect approximately 1 to 5 fmol of POMC-related peptides

(table 1). Similar sensitivities have been achieved with a few

enkephalin antisera and with several antisera raised against

prodynorphin-related peptides (tables 2 and 3). Although RIA

sensitivity is primarily determined by the antibody binding

affinity and the tracer specific activity, additional improvement can sometimes be achieved under nonequilibrium conditions

by delayed addition of tracer.

RADIOLABELED LIGANDS

Radiolabeled ligands for RIA of opioid peptides are commonly

prepared by the chloramine-T catalyzed introduction of 125I,

as first described by Hunter and Greenwood (1962). Applications

of this method to a wide variety of peptides and proteins have

been reviewed by McConahey and Dixon (1980). Alternatively,

some opioid peptides have been radioiodinated by lactoperoxidase

catalysis (Morrison 1980). The specific activity of radioiodinated peptides may be determined by precipitation with

trichloroacetic acid (Chen et al. 1981) or by thin-layer

chromatography (Rosenberg and Teare 1977). The thin-layer

chromatographic method is generally applicable, but some small

peptides, such as Met-enkephalin, are not quantitatively precipitated by trichloroacetic acid. The talc-resin-trichloroacetic

acid test described by Tower et al. (1980) is useful for evaluating individual preparations of radioiodinated peptides that

can be precipitated, e.g., ß-endorphin.

Radioiodinated peptides may be isolated from the crude reaction

mixture by gel filtration. Although these peptides generally

elute prior to radioiodide on gel filtration, 125 I -

Met-enkephalin and 125I-Leu-enkephalin have been observed to

elute after radioiodide due to partial adsorptive retardation

by Sephadex (Khanna and Sharma 1983). Thin-layer chromatography (Gros et al. 1978) and adsorption to octadecylsilylsilica (Clement-Jones et al. 1980b) have also been used to

isolate 125I-Met-enkephalin and to separate the mono- and diiodinated forms.

Radioiodinated opioid peptides may also be purified by HPLC, as

illustrated for 125 I-ß-endorphin and 125 I-Met-enkephalin in

figures 4 and 5, respectively. Introduction of iodine into

ß-endorphin or Met-enkephalin sufficiently alters their physicochemical properties so that the iodinated peptides can be

resolved from the unlabeled starting material by reversed-phase

HPLC. In addition, Met-enkephalin is oxidized to the sulfoxide during

275
FIGURE 4

Purification of 125I-ßh-Endorphin by Reversed-Phase HPLC

Unlabeled ß h -endorphin (A) and 125 I-ß h -endorphin (B) were

eluted from a 3.9 mm x 30 cm μBondapak C18 reversed-phase HPLC

column (Waters Associates, Milford, MA) with a 20-minute linear

gradient from 10% to 60% acetonitrile in 0.08% trifluoroacetic

acid at 1.0 ml/min .

276
FIGURE 5

Purification of 125I-Met-Enkephalin by Reversed-Phase

HPLC

125I-Met-enkephalin was chromatographed as described in the

legend to figure 4 before (A) and after incubation with 0.75 M

dithiothreitol for 16 hr at 37°C (B).

277
the course of chloramine-T catalyzed radioiodination (figure

5A). 125I-Met(O)-enkephalin can be partially reduced to 125IMet-enkephalin by treatment with 0.75 M dithiothreitol for 16

hours at 37oC (figure 5B).

Tritium-labeled peptides are useful for monitoring recovery

from extraction and chromatographic procedures used prior to

RIA. The use of radioiodinated peptides for this purpose is

not strictly valid, given the changes in physical properties

which occur upon iodination. Tritium-labeled enkephalins are

commercially available, and 3H-ß-endorphin and 3H-dynorphin(1-

17) have been prepared by a palladium oxide catalyzed exchange

reaction between tritium gas and synthetic diiodinated derivatives (Houghten and Li 1978; Houghten 1982).

STANDARDS

Quantitative analysis by RIA requires accurate calibration with

standard analyte solutions of known purity and concentration.

Although most of the naturally occurring opioid peptides are

available pure from commercial sources, the purity of primary

standards should be checked periodically.

Reversed-phase HPLC is a powerful analytical tool for resolving

impurities in opioid peptides. The same technique may also be

used preparatively to purify the peptide standard. Opioid

peptides are commonly chromatographed on columns containing

hydrocarbon chains of 8 to 18 carbons covalently bonded to

silica particles. Resolution of peptide mixtures is often

achieved by elution with gradients of acetonitrile or propanol

in aqueous solutions of ion-pairing reagents, such as acetic

acid, trifluoroacetic acid, or heptafluorobutyric acid (Gabriel

et al. 1981; Henderson et al. 1981; Hong et al. 1983; Lewis et

al. 1979; Loeber and Verhoef 1981; McDermott et al. 1981;

Morris et al. 1980). These solvent systems are relatively

transparent in the ultraviolet region and are completely volatile, thus facilitating the detection and recovery of peptides

in a salt-free form suitable for direct measurement by RIA.

Although ultraviolet absorbance detection is most commonly used

for HPLC of opioid peptides, Lewis et al. (1979) have also used

fluorescence detection following post-column derivatization

with fluorescamine.

A peptide’s retention time on reversed-phase HPLC may be estimated from its amino acid composition by an empirical method

described by Meek (1980). With the exceptions of α−endorphin

and ß-endorphin(77-91), the observed relative retention times

of the peptides shown in figure 6 are consistent with those

predicted from the amino acid compositions.

Preparation of standard solutions from weighed portions of dry

peptide is unreliable, because water and salts frequently

constitute a significant fraction of the mass. The absolute

278
FIGURE 6.

Separation of Opioid Peptides by Reversed-Phase HPLC.

A mixture of Met(O)-enkephalin (1), ß-LPH 77-91 (2), Metenkephalin (3), α-endorphin (4), Leu-enkephalin (5), γ−endorphin

(6), ßc-endorphin (7), ßh-endorphin (8), and [Leu5]-Bh-endorphin

(9) was resolved by reversed-phase HPLC as described in the

legend to figure 4.

279
concentration of a standard peptide solution is best determined

by quantitative amino acid analysis following acid hydrolysis

(Hong et al. 1983). Alternatively, the concentration may be

calculated from ultraviolet absorbance measurements, provided

the extinction coefficient is accurately known. Peptide standards will generally be most stable when stored dry under an

inert gas.

SAMPLE PREPARATION

Extraction and purification steps are often required before RIA

can be used to measure opioid peptides in biological tissues

and fluids. Opioid peptides are widely distributed in the

central nervous system, gut, peripheral circulation, and endocrine tissues. Tissue-, cell-, and species-specific variations

in processing of the precursors POMC, proenkephalin, and prodynorphin determine the characteristic and distinct distribution

patterns observed for the various opioid peptides (for reviews,

see Adler 1980; Bouras et al. 1984; Burbach 1984; Clement-Jones

and Besser 1983, 1984; Cox and Baizman 1982; Cuello 1983;

Goodman et al. 1983; Gramsch et al. 1982; Liotta and Krieger

1983; Miller et al. 1979; Rossier and Bloom 1982; Simon 1982;

Smyth 1983; Terenius 1982; Yang et al. 1980; Zakarian and Smyth

1982). Depending on the tissue or fluid to be analyzed, different sample preparation problems are encountered.

Tissue Extraction

Conditions for extracting opioid peptides from biological

tissues are chosen to maximize peptide recovery and to minimize

degradation by endogenous proteases. Enkephalins are best

extracted from tissue with 0.1 N HCl or 2 N acetic acid (Miller

et al. 1979). Ghazarossian et al. (1980a) found that dynorphin

could be reliably extracted from porcine pituitaries only when

0.1 N HCl/methanol (50:50) was used at 70°C. In order to avoid

losses of sample and tracer, these investigators also diluted

the tissue extract and standards for the dynorphin RIA in the

same HCl/methanol mixture and included 17% methanol, 0.1% BSA,

and 0.1% Triton X-100 in the final assay mixture. Likewise,

Guillemin et al. (1977) used an assay buffer containing 0.15 M

NaCl, 0.1% gelatin, and 0.01% BSA and diluted the tracer in the

same buffer containing 0.1% Triton X-100 to prevent adsorptive

losses of 125I-ß-endorphin. Detailed procedures for extracting

endorphins and enkephalins from biological tissues have been

described by Hong et al. (1983).

When easily oxidized peptides such as Met-enkephalin are to be

analyzed, some investigators deliberately convert all of the

extracted peptide to the sulfoxide with hydrogen peroxide,

thereby avoiding complications of variable recoveries among

samples. The sensitivity of methionine to oxidation is illustrated by Huang et al. (1979), who first reported the existence

of Met-enkephalin[Arg6] based on the isolation of the sulfoxide

280
form. Using a somewhat analogous approach, Boarder et al.

(1983) measured relative concentrations of acetylated and

nonacetylated endorphins in tissue extracts by assaying samples

before and after treatment with acetic anhydride.

Many opioid peptides are present in biological tissues in

concentrations of pmol/g and occasionally nmol/g, levels which

are easily measured with RIAs capable of detecting fmol quantities. For example, ß-endorphin concentrations of 45 nmol/g

and 40 pmol/g were found in human anterior pituitary and hypothalamus) respectively (Gramsch et al. 1980). Similarly, Emson

et al. (1984) measured a Met-enkephalin concentration of 905

pmol/g in the globus pallidus region of human brain, and Zhu

et al. (1983) reported dynorphin concentrations of 252 and 37.4

pmol/g in rat anterior pituitary and hypothalamus, respectively.

Chromatographic Separation

Mixtures of opioid peptides obtained from extracts of biological

tissues may be further separated and purified prior to RIA by

gel filtration chromatography and/or HPLC. Gel filtration

chromatography is often used to separate opioid peptides from

the larger precursors. Thus, ß-endorphin and ß-LPH in tissue

or plasma extracts are readily separated by chromatography on

Sephadex G-50 or Bio-Gel P60 in a solvent such as 0.1 N acetic

acid containing 0.1% BSA (Hollt et al. 1979a, 1979b; Nakao et

al. 1978; Wardlaw and Frantz 1979). Ion exchange chromatography,

for example with SP-Sephadex, may be used in conjunction with

gel filtration chromatography to separate N-acetylated peptides

from the nonacetylated forms. This technique was used by Suda

et al. (1982) to detect ß-endorphin(1-31), ß-endorphin(1-27),

N-acetyl-ß-endorphin(1-31), and N-acetyl-ß-endorphin(1-27) in

plasma and pituitary tumor tissue from patients with ectopic

ACTH-producing tumors. Similar techniques were employed by

Vuolteenaho et al. (1983) and Zakarian and Smyth (1979) in

studies of acetylated and nonacetylated peptides derived from

POMC in rat and human pituitaries. Various combinations of gel

filtration media and solvents suitable for chromatography of

opioid peptides have been summarized by Zakarian and Smyth

(1982).

Caution is required when gel filtration chromatography alone is

used to identify an opioid peptide, because some peptides are

retarded by adsorptive processes as well as by size exclusion.

Thus, Zhu et al. (1983) observed that leumorphin, or dynorphin

B-29, was retarded much more than expected by Sephadex G-50.

The resolving power of reversed-phase HPLC for separating

mixtures of opioid peptides is illustrated in figure 6. Reversedphase HPLC has been applied in combination with gel filtration

and/or ion exchange chromatography to resolve complex mixtures

of opioid peptides in extracts of biological tissues and fluids

prior to detection by RIA. For example, Cone et al. (1983)

281
fractionated extracts of rat brain, spinal cord, and pituitary

gland on a column of Sephadex G-50 eluted with 0.1 M acetic

acid containing 0.15 M NaCl and 0.1% Triton X-100, then further

chromatographed the effluent on a μBondapak C18 reversed-phase

HPLC column in an acetonitrile gradient in 5 mM trifluoroacetic

acid, and finally assayed each fraction with 5 different antisera against α−neo-endorphin, ß-neo-endorphin, dynorphin-A(1-

8), dynorphin A(1-17), and dynorphin B. Similar techniques

have been applied to identify the peptides derived from POMC

(Burbach and Wiegant 1984; Margioris et al. 1983; Weber et al.

1982c), proenkephalin (Stern et al. 1982) and prodynorphin

(Bergstrom et al. 1983; Minamino et al. 1981; Przewlocki et al.

1983; Seizinger et al. 1984; Suda et al. 1984; Tozawa et al.

1984; Weber et al. 1982a, 1982b) present in extracts of various

biological tissues and fluids. Applications of HPLC and RIA to

the measurement of endorphins and enkephalins in tissue extracts

have been reviewed by Loeber and Verhoef (1981). Nyberg et al.

(1983) have extended the combination of separation and detection

techniques to include gel filtration chromatography, agarose

electrophoresis, HPLC, radioreceptor assay, and RIA in characterizing the opioid peptides in pooled human CSF.

Plasma and CSF

Most investigators have extracted opioid peptides from plasma

prior to RIA in order to eliminate nonspecific interference by

plasma proteins. This interference is usually manifested by

decreased binding of tracer to antiserum and increased nonspecific binding of tracer. The problems associated with

direct RIA for ß-endorphin in plasma have been discussed by

Lindall et al. (1979). Silicic acid has been commonly used to

extract ß-endorphin and ß-LPH from plasma (Angwin and Barchas

1982; Bramnert et al. 1982; Facchinetti and Genazzani 1979;

Ghazarossian et al. 1980b; Hollt et al. 1979a; Petrucha et al.

1983). The adsorbed ß-endorphin may then be recovered by

elution with solvent mixtures such as 1% acetic acid in 40%

acetone (Bramnert et al. 1982) or 2 N HCl/acetone (20:80)

(Facchinetti and Genazzani 1979). Reversed-phase Sep-Pak C18

cartridges (Waters Associates, Milford, MA) have also been used

to extract FOMC-related peptides (Cahill et al. 1983) and Metenkephalin (Clement-Jones et al. 1980b) from plasma. Gay and

Lahti (1981) have described procedures for fractionating mixtures of endorphins and enkephalins by adsorption and sequential

elution from Sep-Pak C18 cartridges. Likewise, Inturrisi et

al. (1982) and Wardlaw and Frantz (1979) extracted ß-endorphin

and ß-LPH from human plasma with talc. Alternatively, immobilized antibodies have been used to extract and separate

ß-endorphin and ß-LPH in plasma samples to be assayed by RIA

(Aronin and Krieger 1983; Lindall et al. 1979; Yamaguchi et al.

1980). Enkephalins may be extracted from plasma with a porous

copolymer of styrene and ethylvinylbenzene cross-linked with

divinylbenzene (Vogel and Altstein 1977) or with XAD-2 resin

(Ryder and Eng 1981) and subsequently eluted with 50% to 60%

ethanol or methanol. To compensate for variable oxidation

282
of Met-enkephalin during plasma extraction, Clement-Jones et

al. (1980a, 1980b) and Ring and Millar (1980, 1981) routinely

oxidized all plasma extracts with H202 prior to analysis with a

Met(O)-enkephalin RIA. Similarly, Boarder et al. (1982c)

oxidized extracts containing Met-enkephalin[Arg6, Phe7] prior to

incubation with an antiserum directed against the corresponding

sulfoxide.

Several groups have reported direct RIA measurements of ß-endorphin in unextracted rat (Akil et al. 1979), horse (Bossut et

al. 1983), and human (Akil et al. 1979; Hindmarsh et al. 1983;

Wiedemann et al. 1979; Wilkes et al. 1980) plasma. Wilkes et

al. (1980) found comparable values for plasma ß-endorphin in

normal subjects by direct assay and after extraction with

silicic acid. Nevertheless, the values reported by Wilkes et

al. (1980), 33 +/- 2.6 fmol/ml for men (n=27) and 32 +/- 1.4

fmol/ml for women (n=25), are higher than the values for normal

subjects reported by others. Likewise, Hindmarsh et al. (1983)

reported concentrations of immunoreactive ß-endorphin in normal

human subjects of 43 to 55 fmol/ml, based on measurements with

a commercially available RIA kit on unextracted plasma. In

contrast, Wiedemann et al. (1979) found ß-endorphin concentrations of <2.9 fmol/ml in 7 of 14 normal subjects and concentrations of 4.6 to 13 fmol/ml in the remaining 7 subjects, also

using unextracted plasma for the measurements.

A wide variety of opioid peptides have been detected in plasma

from normal human subjects, e.g., ß-endorphin, several forms of

immunoreactive dynorphin, and many opiate active forms, including

some which become opiate active only after trypsin treatment

(Boarder et a.l. 1982a, 1982b). Many investigators have reported

very low concentrations of ß-endorphin in plasma from normal

human subjects when samples are extracted prior to assay. For

example, Bramnert et al. (1982) reported ß-endorphin concentrations of <3 fmol/ml in 26 of 27 normal subjects, and Yamaguchi

et al. (1980) found concentrations of <1.5 fmol/ml in 7 of 19

subjects and values of 2.3 +/- 0.2 fmol/ml in the remaining 12.

Likewise, Aronin and Krieger (1983) and Cahill et al. (1983)

both reported a ß-endorphin concentration of 2.3 fmol/ml in

normal subjects; Goland et al. (1981) found 1.3 fmol/ml; Nakao

et al. (1978, 1979) reported values from <0.9 to 1.7 fmol/ml;

and Suda et al. (1978) found normal ß-endorphin levels to be

<0.48 fmol/ml. Other groups have reported a range of values

between 4 and 12 fmol/ml for ß-endorphin in normal human plasma

(Akil et al. 1979; Atkinson et al. 1983; Bertagna et al. 1981;

Facchinetti et al. 1984; Genazzani et al. 1983a; Ghazarossian et

al. 1980b; Hollt et al. 1979b; Wardlaw and Frantz 1979). Reported concentrations of ß-LPH in normal human plasma have

varied between 3.5 and 12.9 fmol/ml (Aronin and Krieger 1983;

Facchinetti et al. 1984; Genazzani et al. 1983a; Ghazarossian et

al. 1980b; Goland et al. 1981; Hollt et al. 1979b; Nakao et al.

1980a, 1978; Wardlaw and Frantz 1979; Yamaguchi et al. 1980).

283
Increases in plasma concentrations of ß-endorphin and ß-LPH

are associated with a variety of factors, including insulininduced hypoglycemia (Nakao et al. 1979; Smith et al. 1981),

stress, physical exercise, and labor (Genazzani et al. 1984).

Marked elevations in plasma concentrations of ß-endorphin and

ß-LPH have also been observed in cases of adrenal or pituitary

disorders, such as Addison’s disease, Nelson’s syndrome, Cushing’s

disease, and ectopic ACTH production, which result in pituitary

hyperfunction (Bramnert et al. 1982; Bertagna et al. 1981;

Hollt et al 1979b; Krieger et al. 1979, 1980). Diurnal

variations in ß-endorphin and ß-LPH concentrations in normal

human subjects have been noted by Facchinetti et al. (1984) and

Jeffcoate et al. (1978a); minimum concentrations were observed

at 8:00 to 11:00 p.m., and maximum concentrations at 8:00 to

9:00 a.m. Human plasma concentrations of ß-endorphin and

ß-LPH increase during prepuberty and reach adult levels by

adolescence, but no sex-related differences in these levels

have been detected (Genazzani et al. 1983b).

Normal plasma concentrations of ß-endorphin in some other

species appear to be substantially higher than in humans. Thus,

ß-endorphin levels of 75 +/- 15 fmol/ml in rats (Akil et al.

1979) and 72 to 205 fmol/ml in horses (Bossut et al. 1983) have

been reported.

Concentrations of 24 to 244 fmol/ml Met-enkephalin (ClementJones et al. 1980a, 1980b) and 97 +/- 18 fmol/ml Leu-enkephalin

(Ryder and Eng 1981) have been reported in plasma from normal

human subjects.

Procedures for extracting Met-enkephalin from CSF with octadecylsilyl-silica followed by elution with 1% formic acid in 80%

methanol have been reported by Clement-Jones et al. (1980b).

Akil et al. (1978b) also extracted Met-enkephalin from acidified

CSF with Biobeads SM-2 and eluted the adsorbed material with

methanol.

Concentrations of 5.1 to 144 fmol/ml ß-endorphin (Clement-Jones

et al. 1980c. Hosobuchi et al. 1979; Nakao et al. 1980a), 9.1

to 122 fmol/ml ß-LPH (Clement-Jones et al. 1980c; Jeffcoate et

al. 1978b; Nakao et al. 1980a, 1980b), and 14 +/- 0.9 fmol/ml

dynorphin(1-17) (Tozawa et al. 1984) have been measured in

human CSF. Met-enkephalin levels in human CSF have been variously reported as 9 to 51 fmol/ml (Clement-Jones et al. 1980c)

and 3 pmol/ml (Akil et al. 1978b; Sullivan et al. 1977).

Acupuncture has been reported to increase CSF concentrations of

Met-enkephalin without affecting the CSF level of ß-endorphin

or the plasma levels of either ß-endorphin or Met-enkephalin

(Clement-Jones et al. 1979). Denko et al. (1981) have reported

apparent ß-endorphin concentrations of 110 to 136 fmol/ml in

human urine, but the immunoreactive material was not characterized physically.

284
SEPARATION OF BOUND AND FREE LIGAND

Once the binding of radioligand and unlabeled analyte to

antibody has reached equilibrium, the antibody-bound fraction

of radioligand is measured following physical separation from

the unbound fraction. Dextran-coated charcoal has been widely

used to adsorb radiolabeled opioid peptides not bound by antibody and thus effect a separation of the bound and free fractions of tracer. Following centrifugation to pellet the charcoal, the supernatant containing the antibody-bound radioligand

is decanted into a second tube for counting. Detailed procedures for the preparation and use of charcoal as a separating

reagent for RIA have been described by Odell (1980).

Antibodies directed against the nonbinding regions of the

primary antibodies, so-called second antibodies, are also

commonly used to separate the bound and free fractions of

tracer in RIAs for opioid peptides. If the antibody recognizing

an opioid peptide determinant is obtained from rabbits, then a

goat antirabbit gamma globulin might be used as the second

antibody. In this case, the primary antibodies and bound

fraction of radioligand will be precipitated from solution by

the second antibody, provided that sufficient nonimmune rabbit

serum is added as a carrier. After the precipitated antibodies

are pelleted by centrifugation, the supernatant containing the

fraction of radioligand not bound to antibody is decanted and

discarded. When large numbers of samples must be assayed, the

second antibody separation technique is more convenient than

the charcoal method, because each tube need not be individually

decanted into a second tube prior to counting. Although the

second antibody technique can be very tie-consuming when a

second incubation is included (Guillemin et al. 1977), this

potential disadvantage can be eliminated by adding the second

antibody in a pre-precipitated form with nonimmune rabbit

serum. Methods for using the second antibody technique, including the pre-precipitation method, have been reviewed by Midgley

and Hepburn (1980).

The antibody-bound fraction of radioligand may also be precipitated by adding polyethylene glycol (Ghazarossian et al.

1980b; Miller et al. 1978; Ross et al. 1978) or ammonium

sulfate (Weissman and Gershon 1976) along with carrier serum.

The use of these reagents for separating antigen from antigenantibody complexes has been reviewed by Chard (1980).

CALCULATIONS

Measurements of radioligand displacement allow the concentration

of an opioid peptide in an unknown sample to be determined by

RIA through interpolation from a standard displacement curve

relating levels of antibody-bound radioligand to increasing

quantities of unlabeled analyte. The standard displacement

curve may be described by the four-parameter logistic formulation shown in equation 1 (Rodbard and Hutt 1974).

285
Y=[(a - d)/(1 + (X/c)b

)] + d (1)

In this equation, Y is the level of radioligand (cpm) bound to

antibody in the presence of a quantity X of analyte, a is the

level of radioligand bound to antibody in the absence of competing analyte, d is the level of radioligand bound nonspecifically in the absence of antibody, c is the quantity of analyte

required to displace 50% of the radioligand bound to antibody

in the absence of analyte, and b is a slope factor which is

equal to 1 under ideal circumstances. The four parameters in

equation 1 may be calculated by the weighted iterative nonlinear

regression analysis described by Rodbard and Hutt (1974), using

a computer program available through the National Technical

Information Service (Rodbard et al. 1975). Alternatively, the

standard displacement curve may be linearized by plotting the

logit function of %B/B0 versus the natural logarithm of the

analyte quantity (Rodbard et al. 1969). When defined in terms

of the parameters used in equation 1, the logit function can be

derived in the form shown in equation 2.

logit (B/B0) = ln[(B/B0)/(1 - (B/B0))] = ln(X/c)-b (2)

Thus, logit (B/B0) is a linear function of ln X, where B/B0 =

(Y - c)/(a - d) is the fraction of radioligand remaining bound

to antibody in the presence of quantity X of analyte, after

correction for nonspecific binding.

VALIDATION

Measurements of opioid peptide concentrations by RIA must be

validated for each sample type or application. Any factor

which interferes with the specific binding of radiolabeled

ligand to antibody will simulate analyte and lead to falsely

elevated estimates of peptide concentration. Such effects may

result from adsorption of tracer to sample proteins or to glass

or plastic surfaces, from proteolytic degradation of tracer, or

from cross-reaction with shared determinants in related or

unrelated peptides. The impact of these factors on the validity

of RIAs for opioid peptides has been discussed by Bayon et al.

(1983), Rorstad (1983), and Vuolteenaho et al. (1981).

Parallel Displacement Curves

Displacement of antibody-bound radioligand by increasing

quantities of an opioid peptide will parallel the displacement

produced by a substance having an identical determinant, such

as a common sequence of amino acids. Unfortunately, this is

not a very sensitive test of immunological equivalence because

ligands with tenfold differences in affinity constants may

yield essentially parallel displacement curves (Bayon et al.

1983). Thus, displacement of a radioligand by a biological

tissue or fluid extract in parallel with an unlabeled opioid

peptide is necessary but not sufficient to attribute the extract

immunoreactivity to the peptide.

286
Dose-Response Linearity

When an extract of a biological tissue or fluid is assayed at

different dilutions, the measured peptide concentrations should

vary linearly with sample dilution and should extrapolate to a

zero intercept at infinite dilution. If low and constant

levels of nonspecific interference are encountered, measured

concentrations may be corrected by concurrently analyzing

appropriate blank or analyte-free extracts (Hong et al. 1983).

Akil et al. (1979) measured plasma levels of ß-endorphin using

standards prepared in pooled hypophysectomized plasma, while

Inturrisi et al. (1982) dissolved standards in talc-treated

plasma. Unfortunately, an appropriate blank may be difficult

to prepare because the technique used to extract the antigen

from a pooled sample may also remove some of the interfering

substances. When biological fluids contain peptides derived

from multiple tissues, simple surgical procedures may not

produce analyte-free fluid. For example, Smith et al. (1981)

reported that adrenalectomized patients with Cushing’s disease

have normal plasma levels of Met-enkephalin, suggesting a

source other than the adrenal gland for the circulating peptide

Likewise, ß-endorphin is still detectable in CSF in cases of

hypopituitarism (Jeffcoate et al. 1978b) and following hypophysectomy (Schlachter et al. 1983).

Tracer Degradation

Apparent immunoreactivity will be detected if the radiolabeled

ligand is degraded by proteases in the biological tissue or

fluid extract being assayed. The radiolabeled products of

tracer degradation will not be bound by antibody and will thus

appear to have been displaced by antigen. In some cases, the

false immunoreactivity caused by tracer degradation even parallels the displacement curve generated by authentic analyte.

For example, Di Augustine et al. (1980) found proteolytic

tracer degradation to be responsible for all of the apparent

ß-endorphin immunoreactivity in rat mast cells. Conversely,

Cone and Goldstein (1982) extracted 125I-dynorphin B from an

RIA mixture containing a brain extract and analyzed the recovered tracer by HPLC to demonstrate its stability during the

incubation. Protease activity may be destroyed by boiling

samples prior to assay or, in some instances, by adding specific

inhibitors such as aprotinin (Zyznar 1981).

Interspecies Comparisons

Although the enkephalin sequences are common to all species

studied, the structures of the larger opioid peptides such as

ß-endorphin display species-dependent variation (Burbach

1984). As shown in figure 7, the structures of human, bovine,

ovine, camel, porcine, and rat ß-endorphin are identical except

for variations at positions 23, 27, and 31. Conflicting reports

have been published concerning the structure of rat ß-endorphin.

Although Rubinstein et al. (1977) found purified rat ß-endorphin

287
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 26 29 30 31

Human Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-lle-lle-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu

Bovine, ovine, camel Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-Ile-Ile-Lys-Asn-Ala-His-Lys-Lys-Gly-Gln

Porcine Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-lle-Val-Lys-Asn-Ala-His-Lys-Lys-Gly-Gln

Rat Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-lle-lle-Lys-Asn(Val)His-Lys-Lys-Gly-Gln

FIGURE 7

Species Variation in ß-Endorphin Structure

See text for discussion of uncertainty in the rat structure.
FIGURE 8

Specificity Profile of an Antiserum Directed Against the

Carboxyl Terminus of ßh-Endorphin

Displacement of 125I-ßh-endorphin from a ßh-endorphin antiserum

(New England Nuclear, Boston, MA) was determined at various

concentrations of unlabeled ßh-endorphin [Leu5]-ß h -

endorphin γ−endorphin α−endorphin

[des-Tyr1]-ßh-endorphin and Met-enkephalin

289
to be identical with the ovine and camel forms by tryptic

mapping, Drouin and Goodman (1980) concluded from analysis of

the sequence of a cloned rat POMC gene that a valine residue

occupies position 26 instead of alanine. Caution must be

exercised when an antiserum produced against ßh-endorphin is

applied to the RIA of samples derived from other species.

Thus, an antiserum directed against a determinant in the carboxyl terminus of ßh-endorphin, such as the one illustrated in

figure 8, may not recognize the bovine, ovine, camel, porcine,

and rat forms equally well.

Physical Characterization of Analyte

Physical isolation, purification, and characterization of

analyte affords the most definitive identification of immunoreactivity in biological tissues or fluids. Techniques such as gel

filtration chromatography and HPLC, as described in the Sample

Preparation section, may be applied along with electrophoresis,

tryptic mapping, or partial sequence analysis to isolate and

characterize opioid peptides. Purification and characterization

of immunoreactive substances can detect unexpected crossreactions. For example, when Julliard et. al. (1980) purified

the ß-endorphin immunoreactivity in extracts of human placenta

and analyzed the material by electrophoresis, they found that

the immunoreactivity could be attributed to the heavy chain of

immunoglobulin G. Other instances of unexpected crossreactions have been documented, including the cross-reaction of

myelin basic protein with ß-endorphin (Bayon et al. 1983).

FOOTNOTES

1The following abbreviations are used: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (CDI), immunoglobulin G (IgG), lipotropin (LPH), thyroglobulin (Tg), bovine adrenal medulla

peptide 12 (BAM-12P), adrenocorticotropic hormone (ACTH).

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302
ACKNOWLEDGMENTS

Portions of this work were supported by National Institute on Drug Abuse

contract numbers 271-79-3627 and 271-81-3801.

AUTHOR

R. Wayne Hendren, Ph.D.

Chemistry and Life Sciences Unit

Research Triangle Institute

P.O. Box 12194

Research Triangle Park, North Carolina 27709

303
The Analysis of Endogenous Opioid Peptides

With HPLC, Radioreceptor Assay, Radioimmunoassay, and Mass Spectrometry

Dominic M. Desiderio, Ph.D.; Hisayoshi Takeshita, M.D.,

Ph.D.; Hiroshi Onishi, M.D., Ph.D.; Genevieve Fridland, B.S.;

Francis S. Tanzer, Ph.D., D.D.S.; Claire Wakelyn, B.S.; and

Chhabil Dass, Ph.D.

INTRODUCTION

Since the discovery of the endogenous opioid peptidergic pathways,

it has been the goal of many scientists to understand and

rationalize the metabolic roles that these peptidergic pathways

and their corresponding peptides may play. One focus for this

type of research activity that is of great interest is the

participation of the peptidergic pathways in pain mechanisms.

Because each individual peptidergic pathway is comprised of a

larger precursor that is the result of gene expression, several

potential intermediate larger precursors, the working peptide

itself, and several inactive metabolites, it is crucial to employ

state-of-the-art analytical methodologies to efficiently prepare

purified chromatographic fractions and to measure the endogenous

amounts of these peptides. The molecular specificity of the

measurement process must be unambiguous (or nearly so) so that the

analyst has confidence that the measurement that is being made

reflects with high fidelity the structure of the target compound.

This objective of unambiguous molecular specificity is a crucial

aspect of this type of research because many conclusions are made

from that analytical measurement, many other laboratories around

the world will try to reproduce or refute that measurement, and in

some cases, some decisions on the clinical course of management of

a patient will be involved. For all of these reasons, it is of

paramount importance to know the structure of the compound which

is being measured.

This chapter describes the use of a unique combination of several

state-of-the-art analytical assay methodologies for the study of

the role that endogenous opioid peptides play in nociception.

That overall objective of this work has five specific aims:

1. Describe accurately the precise molecular events which are

involved in nociception processes.

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2. Quantify endogenous opioid peptides in biological extracts

with a minimum of structural ambiguity in that measurement. While

minimum structural ambiguity is generally assumed during the

measurement of an endogenous peptide, it is rarely proven

(Desiderio 1984). Stated simply, do we know the structure of

the peptide which is being measured?

3. Utilize this analytical methodology to monitor four

peptidergic pathways, namely, endorphinergic, enkephalinergic,

dynorphinergic, and substance P-ergic pathways. (Hughes 1983;

Comb et al. 1982; Kirkpatrick et al. 1981; Gubler et al. 1982;

Noda et al. 1982; Nawa et al. 1983).

4. Utilize gradient elution, reversed-phase high performance

liquid chromatography (RP-HPLC) as a prepurification step for the

peptide to be measured (Desiderio et al. 1984a).

5. Following RP-HPLC, utilize a series of eight different

detectors to establish the identity of that peptide which is being

measured. For radioreceptor assay (RRA), utilize four ligands

(etorphine, dihydromorphine, DADL, ethylketoazocine) to focus on

four different receptors; for radioimmunoassay (RIA), utilize

antibodies that are directed toward leucine enkephalin (LE) and

methionine enkephalin (ME); and utilize several mass spectrometric

(MS) methods [field desorption (FD), fast atom bombardment (FAB)+

18O peptide internal standards, protonated molecular ions -(M+H),

and amino acid sequence-determining ions].

To demonstrate this type of novel measurement procedure, the

peptide-rich fraction that is derived from the canine pituitary

is analyzed. Gradient RP-HPLC separates a broad range of

hydrophobicity which includes a number of endogenous peptides. A

standard mixture of synthetic peptides is used to define the

retention time for those peptides. Individual fractions are

collected and analyzed with RRA. Selected enkephalin fractions

are analyzed by RIA and FAB MS.

MATERIALS AND METHODS

Synthetic Peptides

Dynorphin 1-7, dynorphin 1-8, dynorphin 1-12, dynorphin 1-13,

dynorphin 1-17, dynorphin 8, big dynorphin, substance P,

beta-endorphin, alpha-neo-endorphin, beta-neo-endorphin, ME-Lys,

and ME-Lys-Arg are purchased from Peninsula Laboratories (Belmont,

CA). ME, LE, ME-Arg-Gly-Leu, and ME-Arg-Phe are purchased from

Sigma Chemical Company (St. Louis, MO). ME-sulfoxide is

synthesized from ME using H2O2. ME-sulfoxide elutes on the

RP-HPLC before ME.

Tissue Procurement

Biologic tissue is obtained from two sources, dogs and humans. From

the canine model, cerebrospinal fluid (CSF), the four canine teeth,

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and several brain tissues (pituitary, hypothalamus, caudate nucleus,

thalamus, etc.) are obtained. From the human, CSF and teeth are

obtained. Tissue acquisition is rapid (within minutes) to

minimize any chemical or enzymatic interconversions. Tissue is

stored at -70" under which biochemical extractions are begun.

Extraction Procedures

The tissue is homogenized (Polytron, setting 3, 15 seconds, 4

times) in acetic acid (3 ml, 1N). Proteins are precipitated with

acidified acetone (10 volumes, 80:20 = acetone: 0.1 N HCl). The

solution is centrifuged (27,000 x g, 20 minutes) and the

supernatant is dried under a stream of nitrogen. The residue is

resuspended and injected onto a prewashed Sep-Pak. The peptiderich fraction is eluted with acetonitrile: trifluoroacetic acid

(80:20; 0.5% TFA) (Desiderio and Yamada 1983).

Reversed-Phase HPLC

Advantage is taken of the recent developments in analytical

RP-HPLC separations of peptide mixtures. Previous workers have

shown that a buffer: peptide ion-pair possesses a sufficient level

of hydrophobicity to participate effectively in RP chromatographic

separation mechanisms. These types of RP analytical columns have

been used in many laboratories for rapid, efficient, and high

recovery separations of mixtures of peptides.

A Varian RP-HPLC instrument, coupled to a microcomputer, and a

C-18 analytical column (10 micron pore diameter) are utilized.

Acetonitrile organic modifier and a volatile triethylamine

formate (TEAF) buffer (pH 3.1) are used. The flow rate is

1.5 ml/min-1; separations are done at ambient temperature; and a

fraction collector is utilized to collect fractions every minute.

The gradient developed for this work is: 10% acetonitrile at the

beginning of the elution; 22% at 48 minutes; 30% at 60 minutes;

and 100% at 65 minutes. This gradient efficiently separates most,

but not all, of the peptides in a standard synthetic mixture. It

is important to realize the fact that it is possible to

experimentally increase the chromatographic resolution of any

RP-HPLC process. Experimental parameters that can be readily

modified include the column temperature, flow rate, buffer,

percentage of organic modifier, type of organic modifier, ionic

strength, and several other experimental parameters. Much time

can be invested in this optimization procedure and excellent

RP-HPLC chromatograms can be produced for mixtures of synthetic

compounds and even in some cases, for biologic extracts.

However, because of the high molecular specificity of one of the

detectors that is utilized in this study, namely, mass

spectrometry, this additional work to improve chromatographic

resolution is not necessary.

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While it is understood that RIA and RRA assays demand purified

chromatographic fractions before analysis, this goal is

extremely difficult (if ever possible) to attain and, indeed, in

our laboratory, we have noticed spurious results that result

from incomplete chromatographic resolution. However, because of

the availability of a mass spectrometer and the presence of

molecular ion information and amino acid sequencing-determining

information that can be provided by this type of detector, the

maximum amount of chromatographic resolution is not required.

This resolution versus specificity phenomenon is widespread and is

further rationalized by considering the fact that no matter what

mixture of synthetic peptides may be the target of the study,

the next biologic extract to be separated may provide a new

peptide that coelutes with a peptide which has been studied by

RIA, RRA, or mass spectrometry.

Radioimmunoassay

Because a majority of laboratories around the world utilize RIA

kits for the measurement of biologically important peptides,

commercially available (Immunonuclear, Stillwater, MS) RIA kits

were used to measure ME and LE in a study comparing our results

with those obtained in other laboratories.

Radioreceptor Assay

Again, because several laboratories utilize RRA for the

measurement of peptides, and because it is extremely useful to

have a "screen" for a gradient HPLC which can be used to detect

peptides that bind to an opiate receptor, a novel type of RRA was

developed.

The canine animal model is utilized to provide the limbic system

synaptosomes for RRA (Desiderio et al., submitted). The canine

model is utilized because it is used for our tooth pulp studies

(Tanzer et al. 1981) and because, in an evolutionary sense, the

dog is closer to the human than the other types of laboratory

animals that are commonly utilized. The limbic system is

considered to include the cingulate and parahippocampal gyri, the

hippocampus, the septum, the amygdaloid body, and the hypothalamus

(Heimer 1983). The limbic system contains a high concentration of

opioid receptors (Atweh and Kuhar 1983). Presumably, these

opiate/opioid receptors are located on the synaptosome surfaces

and, therefore, a synaptosomal-enriched, fraction is produced from

the canine limbic system. The four ligands, etorphine,

dihydromorphine, DADL, and ethylketazocine, are utilized for RRA

because of their relative specificities for the four receptors,

sigma, mu, delta, and kappa, respectively. While these four

radiolabeled synthetic ligands are used for competitive

displacement studies, it is known that each ligand does not have a

specific and exclusive interaction with only one of the four

receptors, but rather some overlap occurs amongst the different

ligand specificities for the receptors.

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Mass Spectrometry

A Finnigan MAT 731 (Bremen, FRG) double-focusing forward geometry

(E,B = electric sector before magnetic sector) MS that is

outfitted with a combination El-FI-FD-FAB ion source is utilized.

Fast atoms are produced by an Ion Tech (Teddington, UK) gun, and

fast atoms that have translational energies up to 8 kilovolts are

utilized. A fast atom bombardment probe tip that is beveled at an

appropriate angle of incidence (60") is used: a methanolic

solution of the peptide or an HPLC-purified fraction is placed

onto the FAB tip: and the methanol is evaporated. Glycerol (0.3

ml) is utilized as the liquid matrix for FAB as a means to provide

a continuously replenished surface of molecules and to prevent

sample burning (Martin et al. 1982). Collision activated

dissociations (CAD) occur in a 134 microliter volume which is

located immediately after the MS ion source acceleration plates.

Either unimolecular dissociations will occur, or the

collisionally activated ions will decompose, in the first

field-free region of the mass spectrometer. The products of those

dissociations are collected and analyzed by combining the electric

and magnetic fields in a linked-field scan, keeping the B/E ratio

constant. A B/E linked-field scan collects all of the product

ions that derive from a selected precursor ion, as opposed to

another type of linked-field scan, B2/E, that will collect all of

the precursor ions that lead to a selected product ion.

While any type of compound may be used for an internal standard

for the measurement of peptides, an internal standard that has the

same chemical structure as the target compound is the most

desirable type of internal standard. Toward this end, the direct

chemical incorporation of a stable isotope into the peptide

produces a compound which possesses very similar, if not

identical, physical and chemical properties. For example, a

stable isotope-incorporated peptide internal standard will

virtually coelute from a RP-HPLC column with the target peptide,

and the mass spectrometric behavior (ionization, evaporization,

fragmentation, etc.) will also be similar. The experimental

benefit to using a stable isotope-incorporated peptide internal

standard for the measurement of endogenous peptides is that the

mass spectra, while being similar in general features, will

contain unique ions that will also shift the appropriate number of

masses to reflect the number of stable isotopes which have been

incorporated.

Peptide internal standards are synthesized in our laboratory by

incorporating 18O atoms specifically and only into the carboxy

terminus of the peptides. The source of the stable isotope (18O)

that is used in this study is preferentially enriched H2

18O.

(Desiderio and Kai 1983).

An Apple II microcomputer is interfaced to the source and detector

regions of the mass spectrometer and is used to effectively

308
acquire the analog data from the mass spectrometer and to digitize

the data (Desiderio et al. 1984b). The use of a microcomputer

for the acquisition and reduction of the mass spectrometric data

extends the number of measurements which can be made, effectively

reduces operator fatigue and errors, and, because of the increased

amount of data that can be acquired, also enhances the

signal-to-noise ratio.

RESULTS AND DISCUSSION

Recovery Studies

In the past, we have shown that approximately 26% of the

radioactively labeled LE, when added early in the extraction

scheme, is collected following HPLC. The early addition of

radioactively labeled LE provides sufficient time for the

exogenously added peptide to equilibrate with the endogenous

enkephalin. This permits time for the exogenous peptide to

penetrate into subcellular structures, and for the equilibration

of peptide:peptide or peptide:protein, or other types of

interactions to occur.

Reversed-Phase HPLC Chromatograms

Figure 1 contains the RP-HPLC chromatogram of the peptide-rich

fraction that is extracted from a canine pituitary. A gradient

of the concentration of the organic modifier (acetonitrile) is

utilized in order to cover a wide elution range of peptides, from

amino acids and dipeptides, through the pentapeptides, up to,

perhaps, precursors. Several detectors have been used for this

RP-HPLC separation.

One of the detectors that is used is the RRA which uses

etorphine as the displaced ligand. This ligand is chosen

because it is displaced from several types of receptors and thus

serves as an efficient detector having a broader range of

specificity. This RP-HPLC chromatogram demonstrates the screening

aspect of using an etorphine-based RRA. RRA may also be used to

semiquantitate endogenous peptides (Desiderio et al., submitted).

It must be strictly remembered, however, that etorphine is

displaced from the receptor and that no peptide structure can be

assigned to any of the observed RP-HPLC peaks.

A second detector is a UV detector. The UV absorption profile

at 200 nm is superimposed on the chromatogram. Along the top of

this chromatogram, indicated in figure 1 by the arrows, are those

retention times where the synthetic peptides elute. Coelution of

a peak at that arrow does not substantiate the structure of a

peptide. That type of structural information can only be provided

by appropriate amino acid sequence-determination data.

A third detector used in this study is MS, and this type of

analytical data is shown below.

309
FIGURE 1

RR-HPLC chromatogram utilizing a detector that is based on a synaptosomal receptor

and displacement of the ligand tritiated etorphine. UV absorption, gradient, and

elution times of synthetic peptides are shown.
Radioimmunoassay

Table 1 contains the analytical measurement of enkephalins that

are obtained by utilizing commercial RIA antibodies. Peptides

have been measured in a variety of tissues and fluids (Tanzer et

al. 1984).

The data in table 1 are divided into two groups, controls and

treated. The controls are those patients whose teeth are removed

with no preliminary orthodontic treatment (Walker et al.,

submitted). It can be seen that the RIA data range in value from

1.3 to 1,138 ng of enkephalin g

-1 wet weight of tooth pulp tissue.

On the other hand, the treated population is comprised of those

patients who, before their four canine teeth were extracted, had

the two upper (and two lower) teeth connected together, and a

force applied between those two teeth. The amount of endogenous

enkephalin is significantly reduced when stress is applied to

human teeth.

Radioreceptor Assay

Table 1 also contains analytical measurements that utilize the

canine limbic system synataptosomal RRA preparation and

etorphine as the displaced ligand. Again, the data in table 1

indicate that, for the controls, the measured amounts range from

47 to 6,726 ng of enkephalin g-1 wet weight of tissue, and that

the treated data are reduced.

It is interesting to note that the two sets of data parallel

each other, but that the RRA assay data are numerically higher

than RIA data.

The difference in RIA and RRA data is a reflection of several

experimental factors. The RIA antibody is raised by a host

animal's immune system against an immunogenic peptide: carrier

protein complex, and not necessarily against the pentapeptide.

The RRA preparation contains several types of receptors. Each

radioligand has its own specific affinity toward its preferred

receptor and a reduced affinity toward the other receptors. The

binding constants of each ligand-receptor pair are also different.

For these reasons, among other experimental reasons, it is

relatively difficult to directly compare RRA results, either to

other RRA-ligand receptor pairs or to radioimmunoassay. At best,

general trends can be observed in the data.

These two sets of data indicate that stress effectively

mobilizes the enkephalinergic peptidergic pathway (and perhaps

also others) either in an overall fashion (amongst the

individual family members of the peptide pathways), or-that one

target peptide that is being measured in this study (ME and LE)

is preferentially metabolized.

311
TABLE 1

Opioid Concentrations Measured as Ng Enkephalin

Equivalents g

-1 Wet Weight Tissue in a Radioreceptor Assay,

which is Based on the Displacement of 3H-Dihydromorphine,

and in a Radioimmunoassay

(a) HPLC fraction collected at synthetic ME (YGGFM) retention

time.

(b) HPLC fraction collected at synthetic LE (YGGFL) retention

time.

* first tooth extracted

** sample used for MS corroboration

n.d. = not detected

Patients TH, SG, and SS were fitted with springs.

312
Mass Spectrometry--Molecular Ion Determination

The other analytical methods that were employed above (HPLC,

RIA, and RRA) for peptide measurement cannot convey any primary

structural information in the analytical measurement. Only when

a firm structural parameter (mass of the entire molecule or a

subfragment of the structure) is measured can we confidently state

that the peptide we think we are measuring is actually the one

that is being measured. While mass spectrometry has been utilized

for many years to quantify a great number of other types of

compounds, peptides have been rigorously avoided for MS

measurements because of their low volatility.

However, with the recent development of FAB MS, these experimental

problems have been overcome. Indeed, recent work demonstrates

that MS may even be the method of choice for measuring endogenous

peptides, or at least, it can now be used to calibrate the other

available analytical methods. FAB MS is ideally suited for

peptide studies because a protonated molecular ion may be formed

for the entire structure of the compound or, as described below,

specialized MS techniques can produce amino acid sequencedetermining ions.

As indicated above in the RP-HPLC example, FAB MS is utilized to

determine the molecular ion of several of the RRA-responding peaks

that are shown in figure 1. In the area surrounding fraction 17,

a protonated molecular ion is found that corresponds to a

molecular ion of 1.259. It is known that alpha-neoendorphin,

which elutes in this area, has a molecular weight of 1,229. While

the structure of this particular compound is not yet known, by

utilizing MS peptide standards it is estimated that the peak at

1,259 mass units corresponds to 3 to 4 micrograms of peptide

material. This peptide will be further characterized by utilizing

a linked-field (B/E) scan to elucidate the amino acid

sequence-determining information that derives only from the

molecular ion.

In a second area of the chromatogram in figure 1 (fractions 21 and

22), a protonated molecular ion corresponding to ME is observed at

574, and its intensity correlates semiquantitatively to

approximately 600 ng of material. A more intense peak is also

observed at 453, and a less intense peak at 485. The latter two

ions are presumed to be protonated molecular ions. The peak at

453 is also found in several of the HPLC fractions, and it may be

due to chromatographic background material.

This set of three molecular ions (544, 485, and 453) in a region

of a chromatogram, which may erroneously be thought to correspond

to only an HPLC-pure ME peak, is an effective example that clearly

demonstrates the need for the judicious use of a combination of

detection systems in peptide research. One cannot depend upon

313
only one detection system to measure a peptide because of the

limited molecular specificity which is inherent in most detection

systems, even if extensive HPLC purification is done and

appropriate calibration curves are drawn. It is interesting to

note that the RRA analysis of this selected peak (fractions 21 and

22) in a large number of human tooth pulp extracts does yield

highly variable results, This may be due to the variable

proportions of these peptides, whose molecular ions are noted

above, and also due to the peptide interactions with the RRA but

not necessarily with the RIA antibody.

Mass spectrometric analysis of a third (fractions 36 and 37)

region of the RP-HPLC chromatogram in figure 1 indicates the

presence of a protonated molecular ion at mass 900, a mass which

corresponds to the molecular weight of the proenkephalin

molecule, Tyr-Gly-Gly-Phe-Met-Arg-Phe. No amino acid sequencedetermining information has been determined yet for this peak.

The amount of material in this fraction approximates 50 ng. FAB

MS does not indicate the presence of dynorphin-1-17, a peptide

which elutes before proenkephalin under these RP-HPLC conditions

and which has a molecular weight of 1,571 mass units. However,

a very small {but real) peak is observed instead at 1,755 mass

units.

FAB-CAD-B/E,B'/E'-SIM-Microcomputer Measurements

FAB MS very effectively produces the protonated molecular ion

(M+H)+ of a peptide. Collision activated dissociations (CAD) may

cause the fragmentation of that protonated molecular ion into

its constituent amino acid sequence-determining ion fragments.

On the other hand, spontaneous unimolecular decompositions may

occur. Linked-field scanning (B/E) can be used to select a unique

amino acid sequence-determining ion that can form the basis of the

analytical measurement. A B/E scan collects all of the product

ions that arise from a selected precursor ion. 10O-incorporated

peptide internal standards can be used to shift appropriate

monitored masses by several mass units and also to provide an

excellent internal standard, because a stable isotope-incorporated

peptide possesses equivalent phvsicochemical properties during

homogenization, equilibration, Sep-Pak, RP-HPLC, and MS processes.

A microcomputer effectively extends the number of analytical

measurements beyond that number which can be made manually.

The data in table 2 indicate that there is sufficient level of

detection sensitivity in the MS method to measure endogenous

amounts of LE and ME in the extracts from the anterior and

posterior pituitary, the caudate nucleus, and tooth pulp. A

previous method that used field desorption mass spectrometry (FDMS) has been used to measure enkephalins in the hypothalamus and

CSF. At the current level of sensitivity with this MS

instrumentation, approximately 20 ng of enkephalin g-1 wet weight

of tissue can be measured.

314
TABLE 2

Mass Spectrometric Measurement of Enkephalins

in Canine Tissue Extracts (ng g-1) and CSF (ng ml-1)

SOURCE ENKEPHALIN AMOUNT METHOD*

Hypothalamus LE 170 1

Cerebrospinal fluid LE 44 1

Pituitary

Anterior LE 70 2

ME 2,950 2

Posterior LE 2 2

ME 3,760 2

Caudate nucleus LE 1,500 2

Tooth pulp** LE 30 2

ME 179 2

Tooth pulp

Control LE 20 2

ME 487 2

Electrostimulated LE 45 2

ME 390 2

From Desiderio 1984.

*Method 1: FDMS-SIM-Microcomputer;

2: FAB-CAD-B/E-B'/E'-SIM-Microcomputer

**Unstimulated pooled tissue from five dogs

As indicated below, some recent instrumental developments indicate

that the subnanogram level of sensitivity will soon be attained.

The data in table 2 demonstrate the ability of mass spectrometry

to quantify the endogenous amount of a peptide in a biologic

extract. These types of analytical measurement data represent,

for the first time, the kinds of analytical measurement that can

now be made for peptides by utilizing novel MS processes and

that can greatly extend the molecular specificity of the

measurement itself.

This extension of the molecular specificity of peptide

measurements by MS methods can be readily rationalized by

considering the following experimental parameters: a peptide-rich

fraction is extracted from a Sep-Pak; RP-HPLC will provide a

"purified" peptide fraction (however, note the limitations to this

statement concerning purity in the previous section); FAB MS will

produce a protonated molecular ion of a peptide (or peptides) in

315
an HPLC fraction; a B/E linked-field scan will select the amino

acid sequence-determining information from a selected unique

protonated molecular ion; and, an 18O-incorporated peptide

internal standard will provide the best internal standard for

quantification of the peptide. This combination of unique

instrumental developments, as stated above, greatly extends the

confidence that we are quantifying the peptide that we think we

are.

CONCLUSIONS

The data described in this manuscript demonstrate a novel,

multifaceted approach to the analytical measurement of endogenous

opioid peptides in biological extracts. This type of a

multifaceted approach is obligatory for today's research problems

into opioid neuropeptides because of the inherent complexity of

the biologic extracts, because of the numerous peptides which can

coelute irrespective of the gradient that is developed for HPLC,

and because of the great importance that is attached to obtaining

an accurate understanding of endogenous opioid peptidergic

pathways.

Each analytical approach that is utilized in this and other

studies possesses a different molecular specificity which,

according to increasing molecular specificity, may be ranked in

the following order: HPLC, bioassay (BA), RRA, RIA, FAB MS, and

the combination FAB-CAD-B/E-SIM-μCOM. Each analytical method has

its own unique mode of interaction with an opioid peptide. For

example, HPLC can measure several secondary structural

characteristics of a molecule, including electroactivity,

fluorescence, ultraviolet absorption, and refractive index, or it can

be used online with a mass spectrometer (LC-MS) (Desiderio and

Fridland 1984). A bioassay using the response of biological

tissues, such as the vas deferens or guinea pig ileum, measures the

biologic effect that results from the interaction of a peptide

with its biologic receptor on those tissues. RRA monitors the

interaction of several different types of opioid receptors (mu,

delta, sigma, and kappa) with a variety of synthetic ligands. RIA

may be relatively specific, but it must be remembered that

generally a small peptide such as an enkephalin must be chemically

conjugated to a larger protein carrier molecule and that this

peptide: carrier protein antigenic complex is the molecule(s) that

is injected into an animal, where the host animal's immune system

raises an antibody to that complex. The antibody may or may not

be entirely specific toward the structure of the entire original

small peptide target molecule. By analyzing these measurement

methods in such a critical, structural manner, it can be seen that

they cannot determine the structure of the entire target molecule.

FAB MS can produce a protonated molecular ion of a peptide to

substantiate the presence of that peptide in an HPLC fraction-(but

it must be remembered that a specific amino acid sequence is not

necessarily specified), or an amino acid sequence-determining ion

may be selected and analyzed by linked-field scan processes. By

using an 18O peptide internal standard, quantification may be

performed in the latter measurement mode. It must be remembered

316
that, even with MS producing a protonated molecular ion, several

amino acid sequences may produce exactly the same nominal and

accurate mass. For example, for a pentapeptide, 51 or 120

different amino acid sequences are possible.

Finally, several future directions are clear from this type of work.

On one hand, we are aiming toward analyzing the larger peptide

precursors by enzymatically excising appropriate target peptides,

utilizing a combination of trypsin and carboxypeptidase.

Furthermore, the intact larger peptide precursors will be analyzed

by utilizing HPLC protein columns, where those columns have pore

sizes that range up to 300 angstroms. Lastly, newer mass

spectrometry instrumentation will be used in these studies. For

example, a VG 7070E-HF mass spectrometer has been installed in our

laboratory. This newer MS instrumentation is capable of analyzing

peptides that have molecular weights which range up to 15,000 mass

units--a mass range that will encompass the larger peptide

precursors. Both positive and negative ions may also be studied

in our continuing effort to develop several different molecular

specificities and/or sensitivities. We will target our

measurement sensitivities toward the subnanogram amount for

quantification of endogenous peptides.

REFERENCES

Atweh, S.F., and Kuhar, M.J. Distribution and physiological

significance of opioid receptors in the brain. Br Med Bull

39:47-52, 1983.

Comb, M.; Seeburg, P.H.; Adelman, J.; Eiden, L.; and Herbert, E.

Primary structure of the human met- and leu-enkephalin

precursor and its RNA. Nature 295:663-666, 1982.

Desiderio, D.M. Analysis Neuropeptides by Liquid Chromatography

and Mass Spectrometry. Amsterdam: Elsevier Science Publishers,

1984, 235 pp.

Desiderio, D.M.; Kai, M.; Tanzer, F.S.; Trimble, J.; and

Wakelyn, C. Measurement of enkephalin peptides in canine

brain regions, teeth, and CSF with HPLC and mass

spectrometry. J Chromatogr 342: 245-260, 1984a.

Desiderio, D.M.; Laughter, J.; Katakuse, I.; Kai, M.; and

Trimble, J. A microcomputer data acquisition system for mass

spectrometry. Comp Enhanc Spectros, 2:21-31, 1984b.

Desiderio, D.M., and Fridland, G. A review of combined liquid

chromatography and mass spectrometry. J Lig Chromatogr 7(S-2):

317-351, 1984.

Desiderio, D.M., and Yamada, S. Field desorption mass

spectrometric measurement of picomole amounts of leucine

enkephalin in canine spinal cord tissue. Biomed Mass Spectrom

10:358-362, 1983.

Desiderio, D.M., and Kai, M. Preparation of stable isotopeincorporated peptide internal standards for field desorption

mass spectrometry quantification of peptides in biologic tissue.

Biomed Mass Spectrom 10:471-479, 1983.

317
Desiderio, D.M.; Onishi, H.; Takeshita, H.; Tanzer, F.S.; Wakelyn,

C.; Walker, J.A., Jr.; and Fridland, G. Measurement of opioid

peptides in human tooth pulp with a limbic synaptosomal

radioreceptor assay. J Chromatogr - Biomed Appl, submitted.

Gubler, U.; Seeburg, P.; Hoffman, B.J.; Gage, L.P.; and Udenfriend,

S. Molecular cloning establishes proenkephalin as precursor of

enkephalin-containing peptides. Nature 295:206-208, 1982.

Heimer, L. The Human Brain and Spinal Cord. New York:

Springer-Verlag, 1983.

Hughes, J. Biogenesis, release, and inactivation of enkephalins and

dynorphins. Br Med Bull 39:17-24, 1983.

Kirkpatrick, D.L.; Taniguchi, T.; Jones, B.N.; Stern, A.S.; Shively,

J.E.; Hulliha, J.; Kimura, S.; Stein, S.; and Udenfriend, S.

A. highly potent 3200-dalton adrenal opioid peptide that contains

both a [Met]- and [Leu]-enkephalin sequence. Proc Natl Acad Sci

USA 78:3265-3268, 1981.

Martin, S.A.; Costello, C.E.; and Biemann, K. Optimization of

experimental procedures for FAB MS. Anal Chem 54:2362-2368,

1982.

Nawa, H.; Hirose, T.; Takeshima, H.; Inayama, S.; and Nakanishi, S.

Nucleotide sequences of clones cDNAs for two types of bovine

brain substance P precursor. Nature 306:32-36, 1983.

Noda, M.; Furutani, Y.; Takahashi, H.; Toyosato, M.; Hirose, T.;

Inayama, S.; Nakanishi, S.; and Numa, S. Cloning and sequence

analysis of DNA for bovine adrenal preproenkephalin. Nature

295:202-205, 1982.

Tanzer, F.S.; Desiderio, D.M.; and Yamada, S. HPLC isolation and

FD-MS quantification of picomole amounts of methionineenkephalin in canine tooth pulp. In: Rich, D.H., and Gross, E.,

Pierce Chemical Co., 1981. pp. 761-764.

eds. Peptides: Synthesis-Structure-Function. Rockford, IL:

Walker, J.A. Jr.; Tanzer, F.S.; Harris, E.F.; Wakelyn, C.; and

Desiderio, D.M. Stress decreases endogenous methionine

enkephalin in human teeth. Am J Orthod, submitted.

ACKNOWLEDGMENT

Support from the National Institutes of Health (GM-26666 and

GM-28611) is acknowledged.

AUTHORS

Dominic M. Desiderio, Ph.D.

Department of Neurology

Director, Stout Neuroscience

Francis S. Tanzer, Ph.D., D.D.S.

College of Dentistry

Laboratory

Hisayoshi Takeshita, M.D., Ph.D. Claire Wakelyn, B.S.

Hiroshi Onishi, M.D., Ph.D. Chhabil Dass, Ph.D.

Genevieve Fridland, B.S.

The Charles B. Stout Neuroscience Mass Spectrometry Laboratory

The University of Tennessee Center for the Health Sciences

959 Court Avenue, Memphis, Tennessee 38163

318
Reverse Phase HPLC of Peptides:

Application to the Opioid Peptides

Tatsuhiko Kanmera, Ph.D., and Reginald P. Sequeira, Ph.D.

High performance liquid chromatography (HPLC) is probably the

most versatile method for the rapid and effective separation of

peptides and proteins by using reverse phase, gel permeation,

and ion exchange columns (Hearn 1980; Dizdaroglu 1984). The

factor contributing most to the increased use of HPLC for

analysis of peptides in biological specimens is the development

of reverse phase (RP) columns. These columns containing a

nonpolar support are used with an organic solvent in aqueous

buffer as the mobile phase. The control of separation selectivity via manipulation of the mobile phase composition provides a

powerful means of achieving rapid analytical separation. The

tremendous progress made during the past decade in the area of

peptides in general and especially in the opioid peptide research

could partly be attributed to availability of the RP-HPLC

technique. It has provided a means for the separation and the

isolation of a variety of opioid peptides derived from their

precursors. While a number of studies have shown the separation

conditions for different opioid peptides, the elution conditions

required to obtain an optimum resolution have to be defined for a

particular set of opioid peptides. The objective of this chapter

is to provide some general principles of the RP-HPLC method of

peptide separation and its application to the opioid peptides.

PRINCIPLE

Reverse phase liquid chromatography predominantly depends upon

the hydrophobic interaction between a nonpolar stationary phase

on a silica support and hydrophobic groups of the solute

molecules, and the concomitant expulsion of hydrophobic groups to

the polar mobile phase. The solutes are eluted by increasing the

concentration of the organic solvent in the mobile phase

(decreasing the polarity of the mobile phase). The hydrophobicity of a peptide depends upon its amino acid composition and,

hence, peptides containing hydrophobic amino acids, such as Trp,

Phe, Leu, Ile, Tyr, Val, etc., are retained strongly on the

319
column and elute later. Indeed, the relationship between the

retention time and the amino acid composition of a peptide could

be statistically analyzed. The retention times have been successfully predicted for relatively small peptides (Sasagawa et

al. 1982; Meek and Rossetti 1981). Despite the predominant

hydrophobic interaction, other interactions (such as ionic interactions) also occur between the stationary phase and solutes.

Such interaction properties vary among each set of columns and

elution conditions. Therefore, it is important to optimize the

separation condition for a particular peptide mixture.

COLUMN

A variety of reverse phase columns are commercially available,

e.g., μBondapak (Waters), LiChrosorb (E. Merck), Ultrasphere

(Altex), MicroPak (Varian), Partisil (Whatman), Zorbax (DuPont),

Nucleosil (Macherey-Nagel), Vydac (Vydac), Supelcosil (Supelco),

TSK (Toyo Soda), etc. There are several types of reverse phase

columns having different pore sizes, bonded phases, and column

dimensions. The elution patterns of peptides sometimes are

affected significantly by these factors. It would be helpful to

know their effects on elution of peptides. Most commercially

available packing materials have alkyl chains ranging from c-3 to

c-18. It has been observed by many workers that the retention

times of a peptide on c-8 and c-18 (or ODS) columns, which are

most commonly used for peptide separation, are virtually similar

(Wilson et al. 1981). A bonded phase with a shorter alkyl chain

retains peptides less strongly, and hence, the peptides tend to

elute earlier. However, we have observed most peptides are

retained even on C-3 or cyanopropyl columns strongly enough to

yield reasonable resolution. For larger peptides and proteins,

alkyl chain length is less important. It has been shown that

alkyl chain length does not affect the retention times of

proteins (Lewis and DeWald 1982). Recent, a number of reverse

phase columns with large pore sizes (>300A) have become commercially available. These columns are designed particularly for

the separation of large peptides and proteins to yield better

resolution and recoveries. While relatively small folded

proteins could be separated effectively on a small pore size

column (Chaiken et al. 1984), the we of large pore size columns

may help to achieve better separation of unfolded large peptides

(>30 residues) such as protein fragments (Pearson et al. 1981).

As shown in figure 1, the wide pore columns also could effectively separate small peptides. However, since they have less

surface area, the column capacity might be less than that for

small pore size columns. Reverse phase columns of 15 to 25 cm

length are commonly used. These columns separate up to 1 mg of

peptides without significant loss of resolution.

Resolution and peak shapes of peptides are not greatly affected

by the size of columns when a gradient elution is employed. As

shown in figure 1, 2 cm columns separated enkephalins and

dynorphin peptides as effectively as the 25 cm column with the

same packing material (figure 2, panel B). However, the column

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FIGURE 1

Separation of

Dynorphin Related Peptides on

Short Reverse Phase Columns

with Different Pore Sizes.

Samples containing ug each

of dynorphin peptides are

eluted on short reverse phase

columns. (2 cm) with a linear

gradient of 0.1% TFA (a) -

0.1% TFA/CH3CN (b) [10-20%

(b) in 10 min and then 20-

30% (b) in 20 min].

Flow rate, 0.8 ml/min.

Ambient temperature.

Structures of the dynorphin

peptides are listed in the

legend for figure 2.

size directly affects its capacity. Although the shorter columns

are not useful for a large-scale preparation, they are relatively

inexpensive. We are routinely using 2 cm guard columns with the

same packing material as used for analytical columns to analyze

peptide samples. It might also be suggested to use such small

columns for routine purification of radioactive peptides used for

a radioimmunoassay. Sincethetypes of silica, coating, and

packing process vary with manufacturers, the resulting elution

patterns and resolution could be different among various columns

(Pearson et al. 1981; figure 2, panels A and B). Endcapping,

which often appears in manufacturers' specifications, is a

process to inactivate free silanol groups. The silanol groups

cause one of the most unfavorable ionic interactions with basic

groups of peptides. Resides, the endcapping greatly extends the

column lifespan.

MOBILE PHASE

Aqueous Buffer

The selection of a mobile phase is one of the most important

factors for the effective separation of peptides. Optimization

of the elution condition might be achieved by appropriate

selection of a buffer (or acid) and an organic solvent, and

sometimes by the addition of ion pairing reagents. The use of

321
aqueous acid or buffer is essential to obtain effective

resolution and peak shapes. The most commonly used aqueous

buffers for RP-HPLC of peptides are phosphate (or phosphoric

acid), trifluoroacetic acid (TFA), formate, and acetate.

Phosphate. The phosphate buffer is superior in its transparency

to other buffer systems, being transparent in the UV range up to

200 nm. Peptide elution with a phosphate buffer system containing a transparent organic solvent is usually monitored at 210 to

220 ml. While the phosphate buffers can be used in a wide range

of pH, highly acidic pH (2.0 to 3.0) would be appropriate for

peptide separation. Higher column efficiency for peptides was

observed at the acidic pH than at pH 3 to 7 (Rivier 1978; Hearn

et al. 1979). In general, the phosphate buffer appears to yield

a little better resolution of peptides than acetate or formate

(Hearn 1980; figure 2). Anionic ion pair reagents, such as hexane

sulfonate or SDS, are sometimes added to the buffer. These

reagents with negative charges form ion pairs with protonated

basic groups, such as α−amino, ε−amino, and arginine guanidyl

groups; consequently the retention times of peptides increase

(Hancock et al. 1978). Addition of anionic ion pairing reagents

is particularly useful for separation of polar peptides which

otherwise are not retained on the reverse phase column. Cationic

ion pair reagents, such as alkylamines, can also be added to alter

elution profiles (Hancock et al. 1979). In particular, the high

recoveries and resolution of peptides and proteins were obtained

with triethylammonium phosphate (Rivier 1978). This may be due

to the effect of triethylammonium ion to block the interaction

between the silanol on silica and the peptides. A drawback of

the phosphate buffers is that since they are not volatile, a

desalting process is required to obtain salt-free peptides. The

phosphate buffers coupled with UV detection at 210 to 220 nm

would be particularly useful for the sensitive analysis of a

peptide mixture.

TFA. TFA may be the most popular solvent for the purification of

aides because of its complete volatility. It has the

advantage of low absorbance compared with acetic acid and formic

acid, which permits the highly sensitive detection at 210 to 220

nm. The concentrations between 0.05 to 0.1 % are commonly used.

Its high dissolving power has made it more popular than dilute

phosphoric acid for the separation of peptides. In order to

eliminate the change of the baseline absorbance in a gradient

elution, equivalent amounts of TFA should be added to the organic

solvent. Similarly, more hydrophobic perfluorinated acids, such

as pentafluoropropanoic acid and heptafluorobutyric acid, can be

used for peptide separations. Since the perfluorinated acids

form ion pairs with basic groups of peptides, the retention time

increase with the hydrophobicity of the counter ions. Increase

in the retention time is most apparent with basic peptides

(Bennett et al. 1980). A better recovery of ß-endorphin with 0.1%

TFA than with dilute formic acid was observed (Dunlap et al.

1978). However, we have observed that the ionic strength of 0.1%

TFA is not high enough to block unfavorable nonspecific

interactions between very basic peptides and silica supports.

322
Formate and acetate. Although these solvent systems have the

disadvantage of higher absorbance at wavelengths below 220 nm,

peptides can be detected at 210 to 220 nm at a lower absorbance

full scale unit. As with the TFA system, it would be recommended

to add the acid to the organic solvent to minimize the baseline

absorbance change upon gradient elution. Because of the volatility, these buffers are frequently used in the form of ammonium

or pyridinium, for isolation and analysis of peptides in conjuction with radioimmunoassay of peptides. Probably ammomonium

acetate is the most commonly used buffer for the separation of

endogenous opioid peptides. Pyridine acetate is sometimes used

with fluorescence detection of postcolumn derivatized peptides.

Although 10 mM ammonium acetate is commonly used, concentrations

more than 50 mM might be required for some peptides to avoid

nonspecific electrostatic interactions.

Organic Solvent

Methanol, acetonitrile, and propanol are frequently used as the

organic modifier for the reverse phase HPLC of peptides. The

relative elution power of these solvents is in the following

order: propan-1-ol > propan-2-ol > acetonitrile >> methanol

(Wilson et al. 1981). Among these solvents, acetonitrile is most

transparent, permitting detection up to 200 nm, and has very low

viscosity. Gradient elutions, therefore, can be monitored

without significant change of the baseline absorbance. In both

methanol and acetonitrile systems, peptide elutions are usually

monitored at 210 to 220 nm. Propanol has a disadvantage of high

viscosity which causes high back pressure and slower mass

transfer of large peptides and proteins. Such problems may be

solved by increasing the temperature of the propanol system. Due

to its high elution power, propanol is better suited for elution

of very hydrophobic peptides and proteins.

DETECTION

UV Detection

Most HPLC systems are equipped with a UV detector. Since peptide

bonds strongly absorb UV light at wavelengths below 220 nm, all

peptides can be detected at the wavelength unless UV absorbing

mobile phase is used. When pure transparent buffer systems such

as dilute TFA and phosphate are used, 10 to 100 ng of peptides

can be detected at 210 to 220 nm. Since many organic compounds

also absorb UV light strongly at the same wavelength, it is not

possible to discriminate peptide peaks from those of other

organic compounds. In order to obtain a flat baseline at higher

sensitivity, it is important to use pure mobile phases. Peptides

containing Tyr or Trp residues can be detected at 280 nm. By

comparing the elution profile at 280 nm with that at 210 to 220

nm, it can be judged whether the peptide contains these aromatic

residues. The detection limits at 280 nm would be subnanomoles.

323
The advanced computerized detectors currently available provide

UV spectra of eluate which would give structural information of

the eluted components, including the types of aromatic residues.

Fluorescence Detection

For peptides containing Tyr or Trp residues, elutions can be

monitored online with a fluorescence detector; fortunately, all

enkephalin containing peptides have Tyr residues. The detection

limit for peptides is 6 to 10 pmol (Schlabach and Wehr 1982). If

the HPLC system is equipped with a postcolumn derivatization

system and a fluorescence detector, eluted peptides are monitored

after modification with fluorescamine (Stein 1981) or o-phthalaldehyde (Schlabach and Wehr 1982). Fluorescamine reacts with α−

and ε−amino groups; therefore, the peptides must contain at least

one free amino group to react. Since fluorescamine reacts only

with primary amino groups, this method has the advantage of less

interference by nonpeptide substances than that for ultraviolet

detection. In contrast, the application of o-phthalaldehyde is

limited to Lys-containing peptides, since it reacts only with ε−

amino groups of lysine residues. The detection limit of these

methods is 10 pmol of amino groups. The disadvantage of these

methods is that the eluted peptides are destroyed by the derivatisation. For preparative purpose, the eluent has to be split

so that only an aliquot is taken for fluorescence detection.

Details of the instrumentation have been described by Bohlen et

al. (1975).

Other Detection Methods

Electrochemical detection of enkephalin-related peptides has been

reported (Mousa and Couri 1983). Subnanogram amounts of ßendorphin was detected by this method. The electrochemical

detection, however, is applicable to the peptides with oxidizable

residues (DiBussolo 1984). Mass spectrometry technique was used

to quantitate endogenous Leu-enkephalin in the HPLC fraction of a

tissue extract (Desiderio and Yamada 1982). The application of

this method to other peptides with higher molecular weight is

yet to be developed. Radioimmunoassay (RIA) is the most sensitive detection method. Despite its cumbersome experimental

procedure, it has been widely used in biological studies because

of its unique selectivity and extremely high sensitivity. This

method will be discussed below. The receptor binding assay of

enkephalin has been wed for the detection of enkephalin-containing peptides after the digestion of the HPLC fractionated

peptides with trypsin and carboxypeptidase B (Stein and

Udenfriend 1984).

RP-HPLC AND RADIOIMMUNOASSAY OF OPIOID PEPTIDES

By using a combination of HPLC to effect physicochemical

separation and RIA for measurement, it is often possible to

achieve a specificity and sensitivity unsurpassed by any other

analytical techniques. Since a variety of structurally related

324
peptides are derived in different sites, RIA of the whole extract

does not necessarily provide sufficient information regarding

their molecular forms and identity. Specific determination of a

particular opioid peptide in a biological sample requires its

chromatographic separation from related peptides and other

interfering substances. This can generally be achieved by HPLC

which, although too insensitive on its own for this purpose for

determining the amounts of opioid peptides in most biological

specimens, can be linked to an RIA which acts as an exquisite

detection system for both the peptide under investigation and its

other immunoreactive forms (precursor/metabolite). These can be

quantitated accurately provided a suitable standard is employed

in the assay. Otherwise, it is reported as "equivalent immunoreactivity."

The RIA of HPLC fractions of various opioid peptides has been

reviewed earlier (Loeber and Verhoef 1981; Hong et al. 1983) and

also in this monograph. In general, volatile buffers such as

ammonium acetate or pyridine acetate and TFA are preferred in

those situations where RIA detection is employed. Conversely, if

nonvolatile buffers are to be used, adjustment of the sample pH

and salt concentration to those of the standard is necessary. In

view of extremely high sensitivity of RIA, special care has to be

exercised to prevent contamination. The recovery of injected

peptides is sometimes fairly low due to nonspecific interactions

between the packing material and peptides. Thus, the possibility

of immunoreactive material eluting from the column has to be

considered, especially after eluting the standard peptides on the

column for calibration. The injector loop and syringes also have

to be cleaned properly. It is strongly recommended to wash the

column extensively and run a blank to ensure no background

immunoreactive material is eluting from the columns.

OTHER CHROMATOGRAPHIC CONDITIONS

Since peptides elute at a specific concentration of the organic

solvent in a gradient, sample volume, when dissolved in a weaker

solvent does not affect the elution profile significantly. If

the sample volume is more than the capacity of the injection

loop, it is possible to inject repeatedly before starting a gradient elution. Such multiple injection should not be made for

isocratic elutions. Column temperature is usually not a very

important parameter for seperation of peptides. Most peptide

elutions are carried out at 20 to 45 °C. Increasing the temperature is expected to facilitate the mass transfer of solute and

resolution (DiBussolo 1984). However, a poor resolution has also

been observed at an elevated column temperature (Rivier 1978).

Operations at an elevated temperature may improve the resolution

when a viscous organic solvent, such as propanol, is used.

The flow rates of 0.5 to 2 ml/min have been employed for the RPHPLC of peptides. However, flow rates of 0.5 to 1 ml/min have

been observed to yield better resolution for large peptides and

proteins because of their relatively slower diffusion (Jones et

325
al. 1980). Use of a guard column is strongly recommended since

column damage often occurs at the top of column due to coating

and breaking of the packing material and gradual accumulation of

insoluble material from samples. Such damage not only ruins the

resolution, but also yields irregular peak shapes. By placing a

guard column between the injector and the analytical column, the

column lifespan greatly increases. This is especially important

when complex mixtures, such as crude tissue extracts, are injected

onto the column.

APPLICATION TO OPIOID PEPTIDES

The structures of proenkephalin, prodynorphin, and proopiomelanocortin containing Met- or Leu-enkephalin sequences have been

demonstrated by recombinant DNA techniques. The post-translational processing mechanisms of the precursors and the distribution of each form of the enkephalin-containing peptides appear to

vary among biological tissues (Akil et al. 1984). Defining the

molecular forms of the opioid peptides in a given tissue would

provide an important clue to the function of the peptides. The

development of the RP-HPLC techniques has made a great contribution to the studies in this area. The RP-HPLC is also an important technique for the studies of the processing mechanisms and

metabolism of the opioid peptides.

A number of studies on the separation of synthetic and natural

opioid peptides have been reported (e.g., Hong et al. 1983;

Loeber and Verhoef 1981; Lewis et al. 1979). In table 1, conditions for the RP-HPLC separation of opioid peptides are listed.

Most of these elution conditions are used for the isolation of

the peptides or for the identification of the peptides in the

tissue extracts. Although μBondapak C-18 and Ultrasphere ODS

have been used frequently for opioid peptides, they are not

necessarily the best columns currently available since the

quality of the columns is being improved rapidly. The separation

of biologically active peptides, including opioid peptides, on

commercially available columns were compared using dilute TFA as

the mobile phase (Tan 1983). As listed in table 1, most

frequently used aqueous buffers are ammonium acetate (or formate)

and TFA. Acetonitrile (CH3CN) and methanol are the common

organic solvents. Effects of aqueous buffer on the separation of

dynorphin peptides are shown in figure 1. When 0.1% TFAacetonitrile was used as the mobile phase, only Met- and Leuenkephalins eluted from the LiChrosorb column. Although the

dynorphin peptides with at least two arginine residues could be

eluted at higher concentrations of acetonitrile, trailing was

noticed. Similarly, a poor resolution pattern was observed with

10 mM ammonium acetate, pH 4.1, which had been sometimes employed

for the separation of opioid peptides. Similar profiles were

observed for the Zorbax ODS and MicroPak columns. On the

contrary, Supelcosil LC-8-DB, which is claimed to be deactivated

for basic compounds, yielded a reasonable elution profile.

Ultrapore ODS also gave a good resolution for the peptides. The

elution profiles for the LiChrosorb and other columns were

326
FIGURE 2

(following page)

Effects of Mobile Phase on RP-HPLC

Separation of Dynorphin Peptides

Six dynorphin peptides were eluted on Supelcosil LC-8-DB (0.46 x 25 cm) and

Lichrosorb RP-8 (0.46 x 25 cm) with the following linear gradients:

Panels A and B, 0.1% TFA (a)-0.1% TFA/CH3 CN (b), 25%-30% b in 20 min

and then 30%-60% b in 10 min, 0.1 AFU at 215 nm, mu-g each peptide;

Panels C and D, 50 mM triethylammonium phosphate (TEAP, pH 3.0) (a) -

CH3CN (b), 15%-30% b in 30 min, 0.1 AFU at 215 nm, mu-g each peptide;

and

Panels E and F, 50 mM ammonium acetate (AcONH4, pH 5.0) (a) - CH3CN (b),

-2 mu-g each peptide, 0.2 AFU at 220 nm.

Flow rate, 0.8 ml/min. Room temperature.

Amino acid sequences of the peptides are:

Met-EK, Tyr-Gly-Gly-Phe-Met; Leu-EK, Tyr-Gly-Gly-Phe-Leu;

Dyn (1-8), Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile;

Dyn (1-9), Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg;

Dyn (1-13), Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys; and

Dyn B, Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr.

327
FIGURE 2

328
TABLE 1

Separation of Various Opioid Peptides by RP-HPLC:

Comparison of the Column, Solvent System, and Detection Method Employed(a)
TABLE 1 (continued)
TABLE 1 (continued)
TABLE 1 (continued)
greatly improved when the concentration of TFA (for example, 0.15%

TFA) or ammonium acetate was increased. Use of TFA at the high

concentration is not recommended because the concomitant decrease

of pH may ruin the column. Much better resolution was observed

with 50 mM ammonium acetate, pH 5.0 (figure 2, panels E and F).

These data suggest that the poor resolution of the dynorphin

peptides is due to the nonspecific interactions of the basic

peptides with the silica support and such interactions are

suppressed by increasing the acid or salt concentration. The

best resolution and peak shapes were observed for the elution

with a triethylammonium phosphate buffer (TEAP). In the system,

the elution profiles of the dynorphin peptides on the Supelcosil

and LiChrosorb columns are very similar. This may suggest that

most of the ionic interactions are blocked by triethylammonium

phosphate. In general, amines are considered to block interactions between silanols and peptides (Hancock and Harding 1984).

In the separation of the dynorphins, both columns gave reasonable

separation when 50 mM triethylammonium phosphate was employed.

Despite the fact that phosphate is an excellent buffer, it is not

frequently used for the studies of opioid peptides probably due

to its nonvolatility. Although only two columns are compared

here, the data revealed the possible differences in interaction

properties among various columns and that the problems might be

solved by optimizing the elution conditions.

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AUTHORS

Tatsuhiko Kanmera, Ph.D.

Laboratory of Biochemistry, Department of Chemistry

Faculty of Science

Kyushu University 33

Fukuoka 812, Japan

Reginald P. Sequeira, Ph.D.

Department of Pharmacology

Kasturba Medical College

P.O. Manipal 576119

Karnataka, India

336
Peptides as Drugs in the Treatment of Opiate

Addiction

Hemendra N. Bhargava, Ph.D.

To date, morphine and its derivatives are still the most widely used drugs

for the relief of pain of moderate to severe intensity, such as the pain of

terminal cancer, the traumatic pain, or the postoperative pain. However,

these drugs suffer from the disadvantage that on repeated administration

tolerance develops to many of their pharmacological actions, particularly

to the analgesic effect. This then leads to the use of increasing amounts

of the drug to provide the same degree of pain relief, The diminished

effect of opiates is attributed to pharmacodynamic tolerance, although in

some cases dispositional tolerance (hepatic microsomal enzyme induction)

may be a contributing factor (Masten et al, 1974; Liu et al. 1978). In

addition, prolonged use of narcotic drug leads to the development of

physical dependence as evidenced by the appearance of a constellation of

distressing withdrawal symptoms and by their immediate suppression

following readministration of the drug. Furthermore, chronic use of this

class of drugs leads to the development of emotional dependence. The

tolerance, physical and emotional dependence, and the drug-seeking

behavior are termed the drug addiction process.

In spite of great advances made in understanding the mechanisms by

which tolerance to and physical dependence on opiates are produced,

these processes are still poorly understood (Way 1980). Obviously, the

treatment modalities are not available. Much of the research has

concentrated on the role of brain neurotransmitter systems in the

development of opiate-induced tolerance-dependence processes.

Pharmacological alteration in the activity of brain chemical substances

indicate that cyclic 3’,5’-adenosine monophosphate (cAMP) (Ho et al.

1973a), gamma aminobutyric acid (GABA) (Ho et al. 1973b), and 5-

hydroxytryptamine (5-HT) (Shen et al. 1970) may be involved in the

genesis of tolerance and physical dependence phenomena. Administration

of cAMP, GABA, and agents known to increase 5-HT activity enhance the

development of tolerance to and physical dependence on opiates.

However, evidence against the role of central 5-HT also exists in the

literature (Algeri and Costa 1971; Marshall and Grahame-Smith 1971;

Bhargava and Matwyshyn 1977; Bhargava 1979). The development of some

337
of the symptoms of narcotic withdrawal syndrome appears to be

associated with a hypoactivity of cholinergic systems (Bhargava et al.

1974; Bhargava and Way 1975, 1976) and hyperactivity of dopaminergic

systems (Iwamoto et al. 1973) in the brain. Cholinergic agonists and

dopaminergic antagonists inhibit the symptoms of opiate abstinence

syndrome, indicating a cholinergic-dopaminergic imbalance in the

production of abstinence symptoms.

Acute and chronic treatment with narcotic drugs affects the activity of

the pituitary hormones (George 1971). Narcotic analgesics, particularly

morphine, influence pituitary function by their action on the central

nervous system (CNS). The hypothalamus acts as the final common

pathway for the integration of all neuronal activity that affects the

anterior pituitary activity. It is also the principal central anatomical site

through which narcotic analgesics exert their effects on the pituitary. In

general, acute administration of narcotic drugs stimulates the pituitary

(increases the release of pituitary hormones) except for pituitary

gonadotropin secretion, whereas, in nearly all cases, chronic

administration of these drugs depresses pituitary function (George 1971).

Although the mechanism(s) by which narcotics influence hypothalamic

pituitary function are not clear, two possibilities exist. The hypothalamus

contains neurosecretory cells which secrete releasing or release inhibiting

factors (peptide hormones) for the control of pituitary activity. It is

plausible that narcotic analgesics exert their effect on the pituitary

through direct actions on the neurosecretory cells by interfering with the

synthesis and/or release of these peptide hormones. The second

possibility involves the presence and role of cholinergic and

monoaminergic pathways in the CNS (Shute and Lewis 1967) and the

transmitters that are released at their terminals. The hypothalamus

contains varying concentrations of acetylcholine (ACh), norepinephrine

(NE), dopamine (DA), and 5-HT. If one assumes that the neurosecretory

cells, which contain the releasing and release inhibiting factors, are in a

postsynaptic position, then any change in the synthesis, release, or

degradation of transmitter substance in the presynaptic neurons

innervating these cells could influence the synthesis and/or release of the

cell contents. Changes in turnover rates and concentrations of the

neurotransmitter substances in the brain have been associated with

alterations in the anterior pituitary functions (Corrodi et al. 1968; de

Schaepdryver et al. 1969).

The hormones of the mammalian posterior pituitary, vasopressin and

oxytocin (figure 1), elicit both endocrine (Knobil and Sawyer 1975) and

extra-endocrine effects (Delanoy et al. 1979; Legros et al, 1978; van Ree

et al. 1978). The extra-endocrine activities include centrally mediated

effects on learning and memory, which have been interpreted as

modification of consolidation or retrieval of information (de Wied 1973;

Walter et al. 1975, 1978a). In addition, the neurohypophyseal hormones

can induce specific behavioral patterns in rodents (Kruse et al. 1977).

It is known that certain pituitary hormones can influence the behavior of

animals when injected peripherally. For instance, vasopressin and its

analogs facilitate retention of conditioned avoidance behavior in

hypophysectomized (Lande et al. 1971) and intact (King and de Wied 1974)

338
rats and increase resistance to extinction of active and passive avoidance

behavior (de Wied 1971; Ader and de Wied 1972). These observations

suggest that vasopressin and its analogs are involved in learning and

memory processes. Since development of tolerance to opiates is thought

by some (Smith et al. 1966; Cohen et al. 1965) to be a learning process,

Krivoy et al. (1974) reported that a naturally occurring vasopressin

analog, desglycinamide9-lysine vasopressin (Lande et al. 1972), which has

essentially a behavioral profile similar to vasopressin but which is

endocrinologically rather inert (de Wied et al. 1972), facilitated the

development of tolerance to the analgesic effect of morphine in mice only

if morphine was administered before the peptide. The dose of the peptide

used was 50 μg/mouse subcutaneously (s.c.). The tolerance was induced

by multiple injections of morphine and the analgesia was measured by the

hot-plate test. Desglycinamide9-1ysine vasopressin did not affect the

analgesic response to an acute injection of morphine.

Cys - Tyr - Ile - Gln - Asn - Cys - Pro - Leu - Gly - NH2

Oxytocin

Cys-Tyr-Ile-Gln-Asn-Cys-NH2

Tocinamide

Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2

Arginine Vasopressin (AVP)

Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-OH

Desglycinamide Arginine Vasopressin (DG-AVP)

Cys-Tyr-Phe-Gln-Asn-Cys-NH2

Pressinamide

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Arg-Gly-NH2

Arginine Vasotocin (AVT)

FIGURE 1: Structures of Posterior Pituitary Hormones and Their Analogs

339
Consistent with this finding, it was demonstrated that Brattleboro rats

with hereditary diabetes insipidus which lack the ability to synthesize

vasopressin exhibited resistance to develop tolerance to the analgesic

action of narcotics (de Wied and Gispen 1976). These authors suggested

that vasopressin plays an important role in the development of tolerance

to the actions of narcotic analgesics and that its mechanism of action is

dissociated from its endocrine effect and resembles that of its known

influence on memory consolidation. In addition to vasopressin and its

analogs, it was found that oxytocin also facilitates the development of

tolerance to and physical dependence on morphine in the rat (van Ree and

de Wied 1976). Studies by Schmidt et al. (1978), however, indicate that

oxytocin and vasopressin antagonized neither the analgesia induced by

morphine nor the development of tolerance to the analgesic effect of

morphine in mice and rats.

While the hormones from the pituitary are well characterized, oxytocin

has also been shown to serve as a precursor for a peptide, Pro-Leu-GlyNH2

 (melanotropin-release inhibiting factor or MIF), with divergent

biological activities. This COOH-terminal tripeptide of oxytocin, which

can be released enzymatically from the hormone by a membrane-bound

hypothalamic enzyme (Celis and Taleisnik 1971; Walter et al. 1973), was

originally proposed to be the natural factor that inhibits the release of

melanocyte-stimulating hormone (MSH) from the pituitary (Celis et al.

1971). MIF was isolated from bovine hypothalamic tissue and was shown

to inhibit MSH release, both in vitro and in vivo (Celis et aL 1971; Nair

et al. 1971). As yet, the role MIF as the MSH-release inhibiting

hormone as well as its formation from oxytocin is not universally

accepted, and its efficacy in humans has not been demonstrated (Kastin et

al. 1979). However, this peptide has attracted a great deal of attention

for its action on the CNS. In animals, MIF potentiates the behavioral

effects of small amounts of L-dopa and reverses both the tremor induced

by oxotremorine and the sedation caused by deserpidine (Kastin et al.

1975). These behavioral responses to MIF could be demonstrated in

hypophysectomized rodents (Kastin et al. 1975), indicating a direct action

in the CNS. MIF has also been shown, at least in some Parkinsonian

patients, to ameliorate the symptoms of rigidity and tremor (Barbeau

1979).

Effects of COOH-Terminal Fragments of Neurohypophyseal Hormones on

the Central Nervous System

Although oxytocin was not active, the COOH-terminal tripeptide of

oxytocin, MIF, and peptides related structurally to MIF (figure 2), such as

N-carbobenzyloxy-MIF (Z-MIF), Leu-Gly-NH2, D-Leu-Gly-NH2,

cyclo(Leu-Gly), all attenuated puromycin-induced amnesia. This is also

true of Pro-Lys-Gly-NH2, the COOH-terminal peptide of lysine

vasopressin (figure 3).

The effect of vasopressin on memory-related processes has a longer

duration of action (de Wied 1973) in spite of its short biological half-life

(Lauson 1974). It is, therefore, possible that either the hormone can

trigger an event that results in long-term changes or it is converted into

an active metabolite with long biological half-life.

340
Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2

Oxytocin

Pro-Leu-Gly-NH2 (MIF)

N-Carbobenzoxy-Pro-Leu-Gly-NH2(Z-MIF)

Leu-Gly-NH2

D-Leu-Gly-NH2

Cyclo(Leu-Gly)

FIGURE 2: Structures of Oxytocin, MIF, and Analogs of MIF

Arginine and lysine vasopressin, as well as cyclo(Leu-Gly) (CLG), were

effective when given up to 24 hours before training (Flexner et al. 1978).

Interestingly, Z-MIF was active even if given 5 days before training

(Flexner et al. 1978). It appears, therefore, that not only the.

neurohypophyseal hormones but also their COOH-terminal peptides had

long duration of action.

Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2

Arginine Vasopressin

Pro-Arg-Gly-NH2

Cyclo(Arg-Gly)

Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH2

Lysine Vasopressin

Pro-Lys-Gly-NH2

Z-Pro-Lys-Gly-NH2

FIGURE 3: Structures of Arginine and Lysine Vasopressin and Their

COOH-Terminal Fragments

The effect of C-terminal peptides of neurohypophyseal hormones on the

development of tolerance to and physical dependence on opiates has been

studied. Studies by van Ree and de Wied (1976) indicated that not only

are oxytocin and 8-arginine vasotocin more potent than 8-arginine

vasopressin, but that their C-terminal tripeptide, MIF, and its analog,

CLG, were as effective as oxytocin in facilitating the development of

tolerance and physical dependence in the rat. MIF and CLG were

administered s.c. in doses of 1 μg per rat. Tolerance and physical

dependence were measured by analgesia and changes in body weight and

temperature, respectively.

341
Z-Pro-Arg-Gly-NH2

Arg-Gly-NH2

Z-Pro-Leu-Gly-NH2

Z-Pro-Lys-Gly-NH2

Cyclo(Arg-Gly)

Cyclo(Leu-Gly)

Cyclo(Lys-Gly)

FIGURE 4: Structures of COOH-Terminal Peptides of Arginine

Vasopressin, Lysine Vasopressin, Oxytocin, and Their Cyclic Analogs

Since de Wied and coworkers had observed facilitation in the development

of tolerance to and dependence on morphine (van Ree and de Wied 1976)

and on beta-endorphin (van Ree et al. 1976), it was decided to examine

some analogs of MIF which could perhaps inhibit the genesis of narcotic

tolerance and dependence. Replacement of an L-residue in a peptide

hormone or in an agonistic analog by D-isomer has been reported to result

in certain instances in a competitive inhibitor or a partial agonist. These

findings might be explained by steric misplacement of an active element

from its preferred orientation in the “active site,” such that intrinsic

activity is decreased or even lost with the retention of receptor affinity

(Walter 1977). Based on this assumption, the effect of Z-Pro-D-Leu on

the development of tolerance to and physical dependence on morphine was

determined in the mouse (Walter et al. 1978b). Various strains of mice

were made tolerant to and dependent on morphine by s.c. implantation of

morphine pellets for 3 days. Control animals were implanted with placebo

pellets. Tolerance to the analgesic effects of morphine was determined

by measuring the jump threshhold to an increasing electric current;

tolerance to the hypothermic effects was determined by measuring body

temperature after intraventricular (i.v.t.) injection or morphine. The

degree of physical dependence development was quantitated after either

abrupt or naloxone-precipitated withdrawal symptoms such as changes in

body weight, body temperature, and stereotyped jumping behavior. When

Z-Pro-D-Leu was injected once a day during the exposure to morphine,

the tolerance to the analgesic and hyperthermic effects of morphine did

not develop. Similarly, the withdrawal-induced hypothermic response in

morphine-dependent mice was blocked by Z-Pro-D-Leu. These studies

have been confirmed by Kovacs et al. (1981).

Further studies revealed that the stereochemistry of Z-Pro-D-Leu was not

important since all four stereoisomers-Pro-Leu, D-Pro-Leu, Pro-D-Leu,

and D-Pro-D-Leu-were active (Walter et al. 1979). In addition, it was

found that even MB?, CLG, and a number of analogs of MIF (figure 5) were

active in inhibiting the development of tolerance to and physical

dependence on morphine in mice (Walter et al. 1979; Bhargava et al. 1980).

Structure-activity relationship studies with MIF in inhibiting naloxoneprecipitated withdrawal revealed that MIF was very effective when

injected daily at a dose of 50 μg per mouse. The addition of a Nbenzyloxycarbonyl(Z) group apparently did not alter the activity, but the

addition of Z-Gly or substitution of pyro-Glu (pGlu) for the NH2-terminal

proline produced analogs with reduced activity. Replacement of the

proline residue by 3,4,-dehydroproline (Δ

3

-Pro), deletion of the proline

342
moiety, dimethylation of the primary carboxamide group, or replacement

of the glycinamide moiety by glycine resulted in inactive analogs of MIF

(figure 5). The substitution of Gln, Met, or Tyr for Leu in Z-Pro-Leu gave

potent analogs, but the substitution of either Ser or ΔPhe produced

peptides with reduced activity. The peptides discussed above can be

considered to be analogs of MIF. In addition to the above peptides, some

cyclic peptides like cyclo(Leu-Gly) and cyclo(Phe-Pro) exhibited activity.

Since the initial studies with MIF and its derivatives on the development

of tolerance to and physical dependence on morphine in the mouse

provided encouraging results, these studies were further expanded.

Chronic administration of opiates to mice results in the development of

tolerance to the analgesic and hypothermic effect (Bhargava 1981a), to

locomotor depressant and stimulant activities (Goldstein and Sheehan

1969; Eidelberg and Erspamer 1975; Matwyshyn and Bhargava 1980), and to

the inhibitory effect on gastrointestinal transit (Pillai and Bhargava

1984a). In the rat, chronic treatment with opiates results in the

development of tolerance to their analgesic, hypothermic, hyperthermic,

cataleptic, locomotor activity (Bhargava, 1980c, 1980d, 1981b 198lc,

1981d; Bhargava and Kim 1982), and antidiuretic activity (Huidobro 1978;

Pillai and Bhargava 1984b). The following section describes studies

conducted in this laboratory on the effects of MIF and its analogs on the

development of tolerance to and physical dependence on morphine in the

mouse and on morphine, ß-endorphin, and buprenorphine in the rat. The

mechanism of action of those peptides is also examined. The effect of

another peptide, (thyrotropin releasing hormone or TRH) pGlu-His-ProNH2

, on morphine tolerance, dependence, and withdrawal syndrome is

described. In addition, the effects of MSH and protein synthesis inhibitor,

dactinomycin, on opiate-induced tolerance and dependence in rodents is

described.

Active Analogs

Pro-Leu-Gly-NH2 MIF)

Z-Pro-Leu-Gly-NH2 (Z-MIF)

Z-Gly-Pro-Leu-Gly-COOH

Glu-Leu-Gly-NH2

Pro-Leu

Z-Pro-Leu

Z-D-Pro-D-L eu

Z-Pro-D-L eu

Z-D-Pro-Leu

Z-Pro-Gln

Z-Pro-Ser

Z-Pro-Met

Z-Pro-D Phe

Z-Pro-Tyr

Cyclo(Leu-Gly)

Cyclo(Pro-Phe)

*Dicyclohexylamine

Inactive Analogs

Δ

3

-Pro-Leu-Gly-NH2

Z-Pro-Leu-Gly-N(CH3)2

Z-Pro-Leu-Gly-COOH

Z-Leu-Gly-NH2

Z-Pro-Leu-NH2

Z-Pro-Gly

Z-Pro-Ala

Z-Pro-D-Ala DCHA*

Z-Pro-Ile

Z-Pro-Val

Z-Pro-Glu

Z-Pro-Phe

Z-Ala-Pro

Cyclo(Leu-Ala)

Cyclo(Pro-D-Leu)

FIGURE 5: Structures of Active and Inactive Analogs of Pro-Leu-GlyNH2

 (MIF) in Blocking Naloxone-Precipitated Withdrawal in Mice

343
MATERIALS AND METHOD

Animals

Male Swiss Webster mice weighing 25 to 30 g (Scientific Small Animals

Inc., Arlington Heights, IL) and male Sprague-Dawley rats weighing 200 to

250 g (King Animal Co., Oregon, WI) were acclimated for at least 4 days

prior to being used in a room with controlled temperature (23 ± 1°C),

humidity (65 ± 2%), and light (L 600 to 1800 h). The animals had free

access to food and tap water.

Chemicals

Cyclo(Leu-Gly) was synthesized according to the method of Fischer (1906).

MIF and related peptides were dissolved in distilled deionized water and

injected S.C. in a volume such that each mouse or the rat received 1 ml/kg

of the drug solution. Morphine sulfate was dissolved in physiological

saline and injected intraperitoneally (i.p.) in 1 ml/kg volume. Human ß-

endorphin was dissolved in physiological saline and injected in volume of

10 μl via the indwelling cannulae implanted stereotaxically in the lateral

ventricle of the rat. Buprenorphine was dissolved in distilled deionized

water and injected s.c.

STUDIES WITH MORPHINE IN MICE

Induction and Assessment of Tolerance to Morphine

Each mouse was rendered tolerant to morphine by s.c. implantation of a

morphine pellet containing 75 mg of morphine base (Way et al. l969;

Bhargava 1978a). The effects of MIF and its theoretically derived analog

CLG on tolerance to the analgesic, locomotor stimulant, and depressant

actions of morphine were investigated in mice. Nice were divided into

three groups and were injected with peptide-vehicle (water), MIF, and

CLG (2 mg/kg each), respectively. Two hours later, mice from each of

the three groups were further divided into two subgroups. One subgroup

was implanted with placebo pellets (one each) and the other subgroup was

implanted with morphine pellets. The injections of water and the peptide

were repeated twice, 24 hours apart, in their respective groups. The

pellets were removed 70 hours after their implantation. Six hours after

pellet removal, tolerance to morphine was assessed as described below.

Assessment of Tolerance to the Analgesic Effect of Morphine

The degree of tolerance to the analgesic effect of morphine was

determined by measuring the analgesic response to a challenge dose of

morphine using the tail-flick procedure. The placebo and morphine pellet

implanted mice were challenged with 4 mg/kg and 10 mg/kg of morphine,

respectively. The tail-flick latencies to thermal stimulation were

determined prior to (To) and at “t” minutes (Tt) after morphine injection.

The basal latencies were found to be 1.3±0.2 sec. A “cut off” time of 10

seconds was used to avoid damage to the tail. The percent anaIgesia was

calculated by using the formula: (Tt-To)/(10-To) × 100. Percent

analgesic response was calculated for each mouse and results were

expressed as mean percent analgesic response ± S.E.M. Nine mice were

used for each treatment group. The differences in the means were

analyzed by the Student’s t-test.

344
Assessment of Tolerance to the Locomotor Effects of Morphine

Preliminary studies indicated that a 10 mg/kg dose of morphine depressed

locomotor activity, whereas an 80 mg/kg dose increased the activity of

the mice. These results are consistent with the studies of Eidelberg and

Erspamer (1975). In subsequent studies, these two doses of morphine were

used to measure tolerance to the locomotor activity effects. Mice were

treated with water or the peptide and implanted with placebo or morphine

pellets as described above. Six hours later, the locomotor response was

measured by means of circular activity cages as described by Bhargava

(1978b). Each cage measured 35 cm in diameter and 20 cm in height and

was equipped with six light sources and six photo cells placed just above

the floor level. The lights were placed orthogonally to each other so that

the light beams crossed in the center of the cage. Activity was measured

by the number of times the light beam was broken within a specified

period of time. The activity counts were recorded automatically on a

digital electronic counter.

Each mouse was placed in the activity cage for a 5-minute acclimation

period that was followed by activity recording for 30 minutes. These

constituted the control or basal counts. The mouse was taken out of the

cage and, after a rest period of 35 minutes, injected with an appropriate

dose of morphine. It was then placed in the activity cage and, after a 5-

minute acclimation, the activity was again recorded for 30 minutes. The

activity of the morphine-treated group was expressed as percent of

control or basal activity. The results were expressed as mean percent

activity + S.E.M.

STUDIES WITH MORPHINE IN THE RAT

The effects of MIF and several of its analogs on the development of

tolerance to its analgesic, hypothermic, hyperthermic, locomotor

depressant, and cataleptic effects of morphine were investigated in the

rat. The rats were made tolerant to and physically dependent on

morphine by implantation of four morphine pellets during a 3-day period

(Bhargava 1977a, 1978c). Rats were injected with peptide-vehicle (water)

or an appropriate peptide. This was followed 2 hours later by

implantation of placebo or morphine pellets. The vehicle and the peptide

injections were repeated two more times 24 hours apart. The pellets were

removed 70 hours after their first implantation. The pellet implantation

and removal were done under light ether anesthesia The development of

tolerance to the analgesic, locomotor depressant, and hyperthermic

effects of morphine was determined at 6 hours after pellet removal, and

tolerance to hyperthermic and cataleptic effects was determined at 24

hours after pellet removal. These time intervals were used as a matter of

convenience to allow the handling of a large number of rats. The daily

doses of peptides used were as follows: MIF (2 mg/kg, 7 μmoles/kg), CLG

(2 mg/kg, 11 μmoles/kg), Pro-ILeu-Gly-NH2 (3.2 mg/kg, 10 μmoles/kg),

and Leu-Gly-NH2 (2.2 mg/kg, 10 μmoles/kg)

345
Assessment of Tolerance to the Analgesic Effect of Morphine

The effect of single and multiple administration of MIF and CLG and of

various doses (dose-response) of the peptides on the development of

tolerance to the analgesic effect of morphine was determined. In single

administration studies, the peptides were given 2 hours before the first

pellet implantation, whereas; in multiple injection studies, the peptides

Were given daily for 3 days, Tolerance to the analgesic effect of

morphine was measured as described above for mice,

Assessment of Tolerance to the Locomotor Depressant Action of

Morphine

The locomotor activity was measured as described above for mice.

Preliminary studies indicated that morphine at an 8 mg/kg dose produced

an 80% decrease in motor activity and this dose was used in subsequent

tolerance studies.

Assessment of Tolerance to Hypothermic and Hyperthermic Effects of

Morphine

Preliminary studies indicated that morphine at 50 mg/kg produced

hypothermia and at 8 mg/kg produced hyperthermia. The rats were

treated with vehicle or peptides and implanted with placebo or morphine

pellets as described above. The effects of 8 mg/kg and 50 mg/kg of

morphine on rectal temperature of rats from all treatment groups were

determined at 6 and 24 hours, respectively, after pellet removal. The

rectal temperature of each rat was measured prior to and 30 minutes

after morphine injection using a telethermometer (Yellow Springs

Instruments Co., Yellow Springs, OH) model no. 73 and probe type 423.

The difference in the rectal temperature of each rat before and at 30

minutes after morphine administration was calculated for each treatment

group. The data were expressed as mean change in temperature+ S.E.M.

Assessment of Tolerance to the Cataleptic Effect of Morphine

Twenty-four hours after pellet removal, morphin-induced catalepsy was

measured by using a bar test (Costall and Naylor 1974). Each rat was

injected with morphine (50 mg/kg) and 30 minutes later the intensity of

the cataleptic effect was determined. The forepaws of the rat were

gently placed across a bar which was held 10 cm above floor level by two

iron stands. If the rat did not step off the bar within 60 seconds, it was

removed from the bar and was considered to have a 100% catalepsy. If

the rat stayed on the bar for less that 60 seconds, then the percent

catalepsy was calculated using 60 seconds as 100% response. The

catalepsy was expressed as mean response ± S.E.M.

STUDIES WITH HUMAN ß-ENDORPHIN IN THE RAT

The effects of MIF and CLG on the development of tolerance to the

analgesic, cataleptic, and hypothermic effects of ß-endorphin were

investigated in the rat. Tolerance to ß-endorphin was induced by i.v.t.

346
injections of 15 μg of ß-endorphin given every 8 hours for 3 days

(Bhargava 1981d). Control rats were injected with i.v.t. saline. To study

the effect of peptides, rats were injected with vehicle, MIF, or CLG (2

mg/kg). They were then divided into two subgroups, one of which was

injected with saline i.v.t. and the other with ß-endorphin (15 μg). The

injections of vehicle, MIF, and CLG were repeated two more times 24

hours apart. To follow the course of development of tolerance to ß -

endorphin, the analgesia, body temperature, and catalepsy were measured

1 hour after ß-endorphin injection in rats given various number of

injections. At the time of the eighth injection, rats from all six groups

were injected with ß-endorphin; and 1 hour later, analgesia, body

temperature, and catalepsy were measured as described above.

STUDIES WITH BUPRENORPHINE IN THE RAT

The effects of MIF and CLG on the development of tolerance to the

analgesic and the hyperthermic effects of buprenorphine, a mixed opiate

agonist-antagonist analgesic, were determined. Tolerance to

buprenorphine was developed by twice daily injections of 0.5 mg/kg s.c.

for 4 days (Bhargava 1982). The peptides were given in daily doses of 2

mg/kg. The degree of tolerance developed to buprenorphine was tested on

day 5.

STUDIES ON THE MECHANISM OF ACTION OF PEPTIDES INHIBlTING

OPIATE-INDUCED TOLERANCE

Determination of Brain and Plasma Concentration of Morphine

To ascertain whether treatment with MIF or its analogs altered the

distribution of morphine, the brain and plasma were analyzed for

morphine. The rats were treated with vehicle or peptide and implanted

with placebo or morphine pellets as described above. Twenty-four hours

after the pellet removal, rats from all treatment groups were injected

with morphine (50 mg/kg) and were sacrificed 1 hour later. Brain and

plasma samples were collected and stored at -20°C until analyzed for

morphine by the fluorometric procedure (Kupferberg et al. 1964;

Bhargava 1977b).

Assessment of Dopaminergic Receptor Function in Rats Treated

Chronically with Morphine or ß-Endorphin

Dopamine (DA) receptor function was assessed by both behavioral and

biochemical methods. Behaviorally, DA receptor sensitivity was assessed

by measuring the locomotor activity and body temperature responses to

apomorphine, a DA agonist (Ernst 1967). Biochemically, the receptors

labeled with 3H-spiroperidol were characterized in morphine naive and

morphine-dependent rats (Bhargava 1983a). Rats were made tolerantdependent by S.C. implantation of four morphine pellets during a 3-day

period and were treated with peptides as described above. Twenty-four

hours after the removal of the pellets, the binding of 3H-spiroperidol to

striatal membranes was determined. In order to study a direct action of

the peptides on dopamine receptors, their effects on the binding of 3H347
spiroperidol and 3H-apomorphine to striatal and hypothalamic membranes

of rats were determined (Bhargava 1983b).

RESULTS

The Effects of MIF and CLG on Development of Tolerance to Morphine in

Mice

Administration of MIF or CLG prior to and during chronic morphinization

inhibited the development of tolerance to the analgesic effect of

morphine. A dose (4 mg/kg) of morphine produced 22.5% analgesia 30

minutes after its administration to mice implanted with placebo pellets

and pretreated with water. The analgesic response to morphine in mice

implanted with placebo pellets was not altered by MIF or CLG. Tolerance

to the analgesic effect of morphine developed as a result of pellet

implantation, and it was inhibited by both MIF and CLG treatment as

evidenced by a greater degree of analgesia in peptide-treated, morphine

tolerant mice.

Depending upon the dose employed, morphine produced either

locomotor depressant or locomotor stimulant effects in mice. The

locomotor activity rates of mice following 10, 20, and 80 mg/kg were

57.5 ± 14.8%, 97.6 ± 8.2%, and 209.0 ± 9.0%. respectively, of their own

control values, Thus, a low dose of morphine decreased motor activity

and a high dose stimulated it.

Tolerance developed to both the locomotor stimulant and depressant

effects of morphine. A dose of morphine (80 mg/kg) which stimulated

motor activity (213% of controls) failed to increase it in morphine

tolerant mice (115 ± 26%). Treatment with MIF or CLG did not modify

the development of tolerance to the stimulant action of morphine.

Administration of MIF or CLG inhibited the development of tolerance to

the locomotor depressant effect of morphine. Morphine (10 mg/kg)

decreased the activity of mice implanted with placebo pellets to 27.5% of

their own control, and this was not modified by MIF or CLG pretreatment.

In morphine tolerant mice, the same dose of morphine (10 mg/kg) reduced

the motor activity to 86.8% of controls, indicating the development of

tolerance. Mice pretreated with MIF or CLG showed 23.5% and 32.2%

activity, respectively, of their control values. These values were virtually

identical to those obtained for mice from the placebo pellet implanted

group.

Effects of MIF and Its Analogs on Tolerance to Morphine in the Rat

Administration of MIF and CLG inhibited the development of tolerance to

the analgesic effect of morphine in the rat. Multiple injections of MIF

had no effect on morphine-induced (2 mg/kg) analgesia in rats implanted

with placebo pellets. Tolerance developed as a result of pellet

implantation. A dose of 8 mg/kg of morphine produced 36.5% analgesia

in morphine tolerant rats pretreated with water (vehicle). In contrast,

morphine tolerant rats treated with 2 and 4 mg/kg of MIF daily exhibited

respectively 74.0% and 64.9% analgesia. Dose-response relationship

348
studies established that the minimum daily dose of MlF necessary to show

significant inhibition in the development of tolerance to morphine was 0.5

mg/kg.

Administration of CLG (2 and 4 mg/kg) also inhibited the development of

tolerance to morphine. In chronic saline-injected rats implanted with

placebo pellets, morphine at a dose of 1 mg/kg produced significant

analgesia at 30, 60, and 90 minutes after its administration, which did not

differ [F(2,5) = 0.78; p = 0.49] from that observed in CLG-treated rats.

In morphine pellet implanted rats, morphine (5 mg/kg) produced an 18%

analgesic response, whereas rats injected with CLG exhibited an 86%

analgesic response at 30 minutes after morphine injection. Dose-response

relationship studies indicated that the minimum daily dose of CLG to

show a significant inhibitory effect was 0.5 mg/kg. The effects of single

injections of MIF and CLG on development of tolerance to the analgesic

effect of morphine in the rat revealed that the minimum doses of CLG

and MIF were 4 and 8 mg/kg, respectively.

Administration of not only MIP but also its analogs-CLG, Pro-ILeu-GlyNH2

 and Leu-Gly-NH2-inhibited the development of tolerance to

locomotor depressant, hyperthermic, hypothermic, and cataleptic effects

of morphine in the rat. Dose-response relationship studies indicated that

in the rat, morphine at 2, 4, and 8 mg/kg decreased the locomotor activity

by 35.6%, 60.8%, and 80.5%. Multiple injections of MIF or CLG to

placebo pellet implanted rats did not alter morphine-induced depression of

locomotor activity. An 8 mg/kg dose of morphine, which produced a

78.5% decrease in activity in placebo pellet implanted rats, produced only

a 29.3% decrease in morphine pellet implanted rats. However, in rats

pretreated with MIF or CLG and implanted with morphine pellets, the

decrease in motor activity was similar to that in placebo pellet implanted

rats. Similar effects were produced by Leu-Gly-NH2 and Pro-ILeu-GlyNH2

.

Administration of morphine to rats produced either hyperthermia (8

mg/kg) or hypothermia (50 mg/kg). As a result of pellet implantation,

tolerance developed to both the hyperthermic and hypothermic effects of

morphine. In placebo pellet implanted rats, injection of morphine (8

mg/kg) increased the body temperature by 0.9°C, whereas, in morphine

pellet implanted rats, only a 0.2°C rise was noted. All four daily

administered peptides blocked the development of tolerance to the

hyperthermic effect of morphine. A similar inhibitory effect on the

development of tolerance to the hypothermic effect of morphine was

produced by all four peptides.

Administration of morphine at a 50 mg/kg dose also produced catalepsy

which lasted for 50 to 60 seconds. The peak cataleptic response was

noted at 30 minutes after morphine injection. A 78.5% catalepsy (as

defined in the Materials and Method section) was observed in rats

implanted with placebo pellets. In contrast, in rats implanted with

morphine pellets, only a 26.5% cataleptic response was noted. Treatment

with MIF and its analogs did not modify morphine-induced catalepsy in

morphine naive rats, but inhibited the development of tolerance to

morphine-induced catalepsy.

349
Effects of MIF and Its Analogs on Brain Uptake of Morphine in Placebo

and Morphine Pellet Implanted Rats

Chronic administration of MIF or its analogs to rats implanted with

placebo or morphine pellets did not alter the uptake of morphine in the

brain. Sixty minutes after an injection of morphine (59 mg/kg), the brain

and plasma concentrations of morphine were approximately 400 ng/g and

1000 ng/ml, respectively, regardless of prior pretreatment.

Effects of MIF and CLG on the Development of Tolerance to ß-Endorphin

in the Rat

Chronic administration of ß-endorphin to the rat resulted in the

development of tolerance to its analgesic, cataleptic, and hypothermic

effects. Daily injections of MIF or CLG inhibited the development of

tolerance to the above pharmacological effects. At the time of the

seventh injection, the analgesic response to ß-endorphin (15 μg) was only

4% in comparison to 16.3% following the first injection. Administration

of MIF or CLG (2 mg/kg/day) blocked the development of tolerance to the

analgesic effect of ß-endorphin as evidenced by a 24.3% and 24.5%

analgesic response to ß-endorphin. At the time of eighth injection of ßendorphin, complete tolerance to its analgesic effect was observed, since

only 1.8% analgesia was seen. The analgesic response to ß-endorphin in

MIF or CLG treated rats given ß-endorphin chronically was

indistinguishable from the chronic saline injected rats.

Chronic administration of ß-endorphin (15 μg) to rats also resulted in the

development of tolerance to its cataleptic effect and it was blocked by

MlF or CLG treatment. At the time of the first and seventh injections,

the cataleptic response to ß-endorphin was 61.2% and 28.1%,

respectively. However, in MIF or CLG pretreated rats, the catalepsy was

maintained at 67.5% and 61.0%, respectively. At the time of the eighth

injection, all the treatment groups had a catalepsy score of 50% except

those injected chronically with ß-endorphin and having a 24% response.

Acute administration of ß-endorphin produced a hypothermic effect. The

body temperature decreased from 37.3 to 36.7°C (p < 0.05). Tolerance to

the hyperthermic effect of ß-endorphin developed very rapidly. Even

the second injection of ß-endorphin did not alter body temperature of

the rats. On repeated injections, instead of hypothermia, a hyperthermic

effect was observed (37.2 to 39.1°C). The hyperthermic response was

attenuated by treatment with MIF and CLG.

Effects of MIF and CLG on the Development of Tolerance to

Buprenorphine in the Rat

Administration of buprenorphine to rats produced dose-dependent

analgesia and hyperthermia. Chronic treatment with buprenorphine

resulted in the development of tolerance to its analgesic and

hyperthermic effects, Concurrent administration of MIF or CLG inhibited

the development of tolerance to both the pharmacological effects of

buprenorphine (Bhargava 1982).

350
Effects of MIF and CLG on Dopamine Receptor Sensitivity in Rats

Treated Chronically with Morphine or ß-Endorphin

Chronic administration of morphine by morphine pellet implantation or of

ß-endorphin by repeated i.v.t. injections resulted in the development of

supersensitivity of brain dopaminergic (DA) receptors, and the latter were

blocked by MIF or CLG treatment. The development of DA receptor

supersensitivity was evidenced by an enhanced hypothermic or locomotor

activity response to apomorphine. Administration of apomorphine (2

mg/kg i.p.) to rats implanted with placebo pellets and given peptidevehicle (water) decreased the body temperature from 38.0 to 36.9°C,

whereas in rats implanted with morphine pellets the body temperature

decreased to 36.2°C. In rats implanted with morphine pellets, and given

MIF or CLG injections, the decrease in body temperature after

apomorphine injections was similar to that observed in placebo pellet

implanted rats (Bhargava 1981b). In addition, administration of

apomorphine produced a greater increase in locomotor activity in

morphine pellet implanted rats when compared with placebo pellet

implanted rats. The enhanced response to apomorphine was blocked by

CLG (2 and 4 mg/kg). The effects of two doses of CLG did not differ.

Similarly, enhanced locomotor activity response was seen in chronic ßendorphin treated rats and it was blocked by MIF or CLG treatment

(Bhargava 1981f).

The biochemical studies revealed that chronic administration of morphine

was associated with enhanced activity of striatal dopaminergic systems.

This was evidenced by a decrease in the Kd value of 3H-spiroperidol

binding in morphine tolerant-dependent rats compared with placebo pellet

implanted rats. However, the Bmax values in the two groups did not

differ (Bhargava 1983a). Concurrent administration of MIF or CLG

antagonized the decrease in Kd value of 3H-spiroperidol induced by

chronic morphine treatment.

Direct interaction studies revealed that neither MIF nor CLG affected the

binding of 3H-spiroperidol in the striatum, but they did enhance the

binding of 3H-apomorphine to striatal and hypothalamic membranes. The

enhancement in binding was not due to changes in Bmax values, but due to

a decrease in the Kd value (Bhargava 1983b).

DISCUSSION

The present studies indicate that the hypothalamically derived peptide

factor MIF and several of its analogs can inhibit the development of

tolerance to various pharmacological effects of morphine, ß-endorphin,

and buprenorphine in rodents.

In mice, some of the morphine-induced responses include analgesia,

hypothermia, locomotor depression, and locomotor stimulation. Tolerance

developed to all these effects on chronic administration of morphine. The

development of tolerance to the analgesic and locomotor depressant

effect was blocked by MIF and CLG, but the tolerance to the locomotor

stimulant effect was not modified by these peptides. In our earlier study

(Walter et al. 1978), it was demonstrated that the development of

351
tolerance to the analgesic and hypothermic effect of morphine was

inhibited by carbobenzyloxy-Pro-D-Leu (Z-Pro-D-Leu), an analog of MIF.

In rats, the most prominent effects of morphine are analgesia,

hyperthermia, hypothermia, catalepsy, and locomotor depression, and

tolerance developed to all these effects on chronic administration. The

development of tolerance to the above mentioned pharmacological effects

of morphine was blocked not only by MIF, but also by several of its

analogs. Thus, Pro-ILeu-Gly-NH2, Leu-Gly-NH2, and CLG were all

active. This indicates that substitution of ILeu- in place of Leu- does not

alter the activity of MIF. The plasma half-life of MIF was found to be 9

minutes (Redding et al. 1973), and yet in these studies, MIF was active

when given in single administration or multiple administration 24 hours

apart. This indicates that MIF may be producing its actions by conversion

to an active metabolite with, perhaps, a long biological half-life. The

studies on metabolism of MIF revealed that it is primarily metabolized by

the cleavage of the Pro-Leu-bond with the formation of proline and Leu

Gly-NH2 (Redding et al. 1973). The present studies demonstrate that

Leu-Gly-NH2, which is the major metabolite of MIF, is also an effective

inhibitor of the development of tolerance to opiates.

The present studies also indicate that repeated intraventricular

administration of ß-endorphin to rats results in the development of

tolerance to its pharmacological effects. These results are in agreement

with previous studies (Tseng et al. 1977; van Loon et al. 1978).

Administration of MIF or CLG also inhibited the development of tolerance

to ß-endorphin. These studies thus provide additional evidence for the

similarities between the exogenous and endogenous opiates.

The development of tolerance to the analgesic and hyperthermic effects

of buprenorphine was also antagonized by MIF and CLG. All these studies

show the similar effects of MIF and its analog CLG in inhibiting the

development to tolerance of some actions of the endogenous and

exogenous opiates, particularly to their analgesic activity.

The development of physical dependence to morphine was also inhibited

by MIF and CLG as evidenced by antagonism of withdrawal-induced

hypothermic response (Walter et al. 1979). However, other signs of

withdrawal like body weight loss and stereotyped jumping response were

unaffected by the two peptides (Bhargava et al. 1980).

The mechanism(s) by which MIF and its analogs inhibit the development of

tolerance to the pharmacological effects of exogenous and endogenous

opiates remains to be delineated. It is possible that these peptides may be

acting as endogenous opiate antagonists. Opiate antagonists like

naltrexone have been shown to inhibit the development of dependence on

morphine (Bhargava 1978a). These peptides, however, do not affect

morphine-induced analgesia or displace the 3H-ligands for μ, δ, or

κ opiate receptors (Bhargava et al. 1983a), suggesting thereby that these

peptides do not interact at opiate receptors. The development of

tolerance to morphine is associated with enhanced sensitivity of brain DA

receptors (Lal 1925; Ritzmann et al. 1979; Bhargava 1980c, 1981b).

Similarly, chronic administration of ß-endorphin results in increases in

352
striatal homovanillic acid concentration suggesting an increase in DA

turnover (van Loon et al. 1978). Preliminary work in this laboratory

indicates that chronic administration of ß-endorphin to rats is associated

with enhanced sensitivity to apomorphine (Bhargava 1981f). Christie and

Overstreet (1979), using tritiated spiroperidol as ligand, observed that

morphine tolerant rats exhibited supersensitivity of DA receptors.

However, rats withdrawn from morphine exhibited subsensitivity of DA

receptors. Our studies indicate that morphine- or ß-endorphin-induced

supersensitivity of brain DA receptors is blocked by MIF and CLG

(Ritzmann et al. 1979; Bhargava 1980c, 1981b, 1981f). Studies in our

laboratory indicate that in morphine tolerance-dependent rats, the binding

of 3H-spiroperidol to striatal DA receptors is enhanced because of

increased affinity. The number of binding sites do not appear to change.

These changes are inhibited by both MIF and CLG (Bhargava 1983a). This

suggests. the existence of an interaction between MlF and brain

dopaminergic systems. In fact, MIF has been shown to potentiate the

behavioral effects of L-dopa (Barbeau 1973; Plotnikoff et al. 1971;

Plotnikoff and Kastin 1974a) and of apomorphine (Kostrzewa et al. 1978).

Our results also indicate that MIF and CLG enhance the binding of 3Hapomorphine to striatal and hypothalamic DA receptors by enhancing the

affinity of the ligand to its receptors (Bhargava 1983b). Evidence for the

existence of binding sites for MIF in the brain has been provided recently

(Chiu et al. 1980). The binding of MIF to brain tissue was shown to be

inhibited by CLG, indicating that CLG may be interacting with the MIF

receptors or similar receptor sites. These studies thus indicate that MIF

and its analogs may be enhancing dopaminergic neurotransmission in the

brain.

Peptides Other than MIF and Its Analogs which Inhibit the Development of

Tolerance to and Dependence on Opiates and the Withdrawal Symptoms

The following section, describes the effects of peptides other than MIF

and its analogs on the addiction to opiates. They include α-melanocyte

stimulating hormone (α-MSH), actinomycin D (dactinomycin), and

thyrotropin releasing hormone (TRH).

α−Melanocyte-stimulating hormone. For the past several years, our

hypothesis has been that if there are mechanisms present in the body to

induce tolerance and dependence to endogenous and exogenous opiates,

then there must also be present systems or substances which could

prevent or retard the development of the addictive states. Studies

indicate that an adrenocorticotrophic hormone (ACH)-secreting mouse

pituitary tumor cells synthesize a large glycoprotein molecule (ProACTH/endorphin) which serves as a precursor for smaller molecular forms

of ATCH and also for ß-lipotropin (Mains et al. 1977; Eipper and Mains

1978). ACTH is cleaved further to a-MSH and corticotropinlike

intermediate lobe peptide (CLIP) in cell suspensions of intermediate lobe

of the rat. ß-Lipotropin and ACTH and their fragments ß-endorphin, γ−

lipotropin, α−MSH, CLIP, and α-MSH are released at the same time by the

pituitary.

The effects of some of these fragments on tolerance to and dependence

on morphine was determined (Szekely et al. 1979). The tolerance was

353
induced in the rat by twice daily injections of morphine in increasing

doses of 4.35 to 14.5 mg/kg S.C. during a 4-day period. Tolerance

developed to the analgesic effect of morphine as measured by the tailflick test. Administration of 0.1 mg/kg s.c. of α-MSH immediately prior

to each morphine injection inhibited the development of tolerance to the

analgesic effect of morphine. The development of dependence on

morphine was studied in the mouse. α−MSH treatment was shown to

inhibit naloxone-precipitated loss in body weight, but the naloxoneprecipitated withdrawal jumping was not modified by α−MSH. On the

other hand, administration of CLIP 1 mg/kg S.C. had no effect on

morphine-induced tolerance and dependence development (Szekely et al.

1979).

Actinomycin D. It is envisioned that the development of tolerance to

morphine may be related to an increase in the synthesis of receptor

proteins in the CNS. Cohen et al. (1965) showed that actinomycin D

administered to mice did not produce analgesia or affect morphine

induced analgesia. However, when given i.p. (0.03 to 8.0 μg per mouse),

it produced a dose-dependent inhibition in the development of tolerance

to morphine. Similar effect was demonstrated in the rat. It was

concluded that the inhibition of tolerance may be related to the

suppression of the synthesis of new RNA and consequently of proteins or

peptides which decrease the effects of morphine. Because widespread

metabolic disturbances were noted after prolonged treatment with

inhibitors of protein synthesis (Young et al. 1963), Cox et al. (1968)

studied the effect of ectinomycin D on the development of tolerance to

morphine in the rat, induced by continuous intravenous infusion of

morphine for 4 hours. Infusion of actinomycin D at a rate of 10 μg/kg/hr

blocked the development of tolerance to the analgesic effect of not only

morphine but also of diamorphine, etorphine, and pethidine, and confirmed

the finding of Cohen et al. (1965). The interpretation of the inhibitory

effect of actinomycin D on opiate tolerance was confounded by the fact

that Loh et al. (1971) showed that actinomycin D increased the uptake of

morphine in the brain, perhaps by modifying the blood-brain barrier.

Thyrotropin releasing hormone and analogs. In addition to MIF, studies in

our laboratory have explored the effect of other hypothalamic peptides,

particularly the TRH. TRH is a tripeptide (pGlu-His-Pro-NH2) which

releases thyrotropin and prolactin from the anterior pituitary (Bowers et

al. 1971). It is distributed ubiquitously in the CNS and is concentrated in

the nerve terminals (Brownstein et al. 1974; Hokfelt et al. 1975). Besides

its endocrine activity, TRH has been shown to have direct effects on the

CNS since they can be observed in hypophysectomized rodents. TRH

enhances the stimulant action of L-dopa in pargyline-pretreated

hypophysectomized and thyroidectomized rodents (Plotnikoff et al. 1974),

and antagonizes the CNS depressant drugs like barbiturates and alcohol

(Plotnikoff et al. 1974), tetrahydrocannabinol (Bhargava 1980b; Bhargava

and Matwyshyn 1980), ketamine (Bhargava 1981g), and morphine

(Bhargava et al. 1982).

The mechanisms by which TRH produces its effect on the CNS or modifies

effects of other drugs have not yet been elucidated. It is possible that it

may serve as a neurotransmitter or a neuromodulator since it is

354
concentrated in the nerve terminals. A TRH uptake process sharing

properties of a high-affinity transport system, like saturation kinetics,

high-affinity kinetic constants, and temperative and sodium dependency,

has been shown to exist in the rat cerebellar slices (Pacheo et al. 1981).

The specific receptors for TRH have been characterized in the brain by

radioligand binding studies using 3H-TRH (Burt and Snyder 1975; Burt and

Taylor 1980) and by using 3H-(3-MeHis2) TRH (Simasko and Horita 1982).

The half-life of TRH in plasma has been found to be 4 minutes (Redding

and Schally 1972), yet its pharmacological activities indicate that it has a

much longer duration of action, It is thus possible that TRH produces its

action via an active metabolite or by modifying other neurotransmitter

systems. Studies show that TRH undergoes metabolic changes by two

pathways. The first involves a specific TRH amidase which hydrolyzes

the terminal amide of the molecule to produce pyroglytamylhisidyl proline

(TRH acid). The second pathway involves a nonspecific pyroglytamyl

peptidase which cleaves pGlu-His-bond to yield His-Pro-NH2. The latter

is rather unstable and readily cyclizes to form histidyl-proline

diketopiperazine, which is also referred to as cyclo(His-Pro) (Prasad et al.

1977). TRH acid does not appear to be pharmacologically active (Prange

et al. 1975; Prasad et al. 1977), whereas cyclo(His-Pro) has been found to

be active and may be responsible for some of the CNS actions of TRH

(Bhargava 1980a, 1981a; Bhargava and Matwyshyn 1980; Prasad et al.

1977; Yanagisawa et al. 1979). However, it is not a prerequisite for

TRH-like agents to form a cyclic analog to exhibit its pharmacological

effects, since SAR studies indicate that TRH analogs which could not

form the diketopiperazine structure were able to antagonize the effects

of morphine just like the TRH did (Bhargava et al. 1982). Further support

against a role of cyclo(His-Pro) in mediating TRH effects stems from the

studies showing the absence of the binding sites for 3H-cyclo(His-Pro) in

the brain (Koch et al. 1982) and that cyclo(His-Pro) does not displace 3HTRH from its binding sites in the brain (Burt and Taylor 1980).

TRH is a weakly basic tripeptide. Its structural requirements to elicit

TRH-like endocrine activity are rather rigid The TSH releasing activity

increases only by methylation in the 3-position of histidine ring.

Structural modifications have yielded compounds which are only resistant

to degradation by the enzyme (Brewster et al. 1980; Brewster and Rance

1980; Miyamoto et al. 1981; Porter et al. 1977; Bhargava et al. 1982), but

they are not more active than TRH at its binding sites in the brain

(Simasko and Horita 1982).

The studies involving the effect of TRH on opiate addiction in our

laboratory were prompted by the previous reports on the interactions of

TRH with endogenous and exogenous opiates, mostly involving single

administration of an opiate. Many of these findings have recently been

reviewed (Bhargava et al. 1983b), and the reader may wish to refer to this

article. In summary, TRH by itself is devoid of analgesic activity and

does not modify analgesia induced by morphine or ß-endorphin (Martin et

al. 1977; Holaday et al. 1978; Bhargava 1981d). Similarly, the

stereospecific binding of 3H-dihydromorphine (Martin et al. 1977; Holaday

et al. 1978) or of 3H-naloxone (Tache et al. 1977) to brain homogenates is

not affected by TRH. TRH antagonizes the catalepsy induced by ß -

endorphin in the rat (Holaday et al. 1978), respiratory depression induced

355
by morphine in the rat (Horita et al. 1976), depressant and hypothermia

effects of morphine in the mouse (Bhargava et al. 1982) and of ßendorphin in the rat (Cache et al. 1977; Holaday et al. 1978).

The following section describes the experiments carried out in this

laboratory with TRH and its analogs on the development of tolerance to

and dependence on morphine and also on the morphine abstinence

syndrome.

The effect of TRH on the development of tolerance to the analgesic,

hypothermic, constipating, and urinary retention activities of morphine

were investigated in rodents. Tolerance to morphine was induced in mice

by pellet implantation procedure as described previously. TRH

administered subcutaneously in a dose of 4 mg/kg twice a day for 3 days

inhibited the development of tolerance to the analgesic effect of

morphine as evidenced by increased analgesic response to a challenge dose

of morphine in TRH treated morphine tolerant mice as compared to

vehicle treated morphine tolerant mice. The inhibition of tolerance to

the analgesic effect of morphine was produced without altering the

disposition of morphine in brain and plasma, indicating that TRH may be

interacting directly in the CNS with processes responsible for the genesis

of tolerance phenomenon (Bhargava 1961a). Chronic administration of

morphine resulted in the development of tolerance to its hypothermic

effect. However, this tolerance was not modified by TRH treatment

(Bhargava 1981a). A single injection of morphine was found to inhibit

gastrointestinal transit in the mouse in a dose-dependent fashion. Chronic

administration of morphine resulted in the development of tolerance to

the inhibitory effect on gastrointestinal transit which was not affected by

daily injections of TRH (Pillai and Bhargava 1984a).

Administration of morphine also caused a dose-dependent decrease in

urinary output in the rat, Tolerance developed to this effect and it was

not affected by chronic treatment with TRH given in a dose of 10 mg/kg

twice a day for 3 days (Pillai and Bhargava 1984b).

Thus, TRH blocks tolerance to the analgesic effect of opiates selectively

and does not modify the tolerance to constipating, hypothermic, and

urinary retention activities of opiates. These findings suggest that

different mechanisms may be involved in the development of tolerance to

various pharmacological effects of morphine. Furthermore, selective

blockade by TRH of the tolerance to the analgesic effect of morphine

may represent a clinically beneficial effect.

Chronic administration of TRH inhibits the development of physical

dependence on morphine in mice (Bhargava 1980a). The physical

dependence on morphine was induced by pellet implantation as described

for tolerance studies. The degree of physical dependence development

was assessed by monitoring the intensity of withdrawal symptoms such as

body weight loss, hypothermia during abrupt and antagonist-induced

abstinence, and the stereotyped jumping response after administration of

an antagonist like naloxone (Bhargava 1977a; Way et al. 1969). The

greater the intensity of these signs, the greater is the degree of

356
dependence. A single injection of TRH (4, 8, or 16 mg/kg s.c.) given prior

to morphine pellet implantation did not affect the development of

dependence on morphine. However, the same doses given prior to and

during the course of development of dependence inhibited the

development of withdrawal-induced hypothermia. The intensity of

naloxone-induced withdrawal jumping syndrome measured 18 to 18 hours

after the last injection of TRH was unaffected. Similarly, the

withdrawal-induced body weight loss was unaffected by TRH treatment.

The inability of TRH to modify body weight loss may be related to its

suppressive effect on food intake (Morley and Levine 1980) and increased

gastrointestinal activity (Morley et al. 1979). Studies in our laboratory,

however, indicate that in the mouse, TRH administered centrally causes

inhibition of the gastrointestinal transit and this effect is mediated via

stereospecific opiate receptors (Bhargava and Pillai 1984, 1985).

The effects of TRH on the withdrawal symptoms observed after

termination of morphine treatment were also investigated in the mouse.

Mice were made physically dependent on morphine as described before.

Morphine pellets were removed 72 hours after their implantation and at

various times thereafter the intensity of withdrawal symptoms was

assessed in vehicle and TRH injected animals. Intracerebral

administration of TRH (1-25 μg per mouse) prevented the hypothermic

response observed during abrupt and naloxone-precipitated withdrawal.

TRH also inhibited the naloxone-induced withdrawal jumping response in a

dose-related manner (Bhargava 1980a). These studies were consistent

with the findings of Morley et al. (1980) who showed that naloxone

induced withdrawal in morphine-dependent rats is associated with

lowering of serum TRH concentration,

The abstinence syndrome in morphine-dependent mice was inhibited not

only by TRH, but also by its postulated metabolite cyclo(His-Pro)

(Bhargava 1981e). Intracerebral administration of cyclo(His-Pro) (1 or

5 μg per mouse) inhibited both the naloxone-induced stereotyped jumping

response as well as the withdrawal-induced hypothermia. In addition, the

analogs of TRH, namely L-N-(2-oxopiperidin-6-yl-carbonyl)-L-histidyl-Lthiazolidine-4-carboxamide (MK-771) and γ−butyrolactone-4-carboxylhistidylprolineamide (DN-1417) also inhibited the intensity of morphine

abstinence syndrome (Matwyshyn and Bhargava 1984). Studies are ongoing

in these laboratories to delineate the mechanisms by which TRH and its

analogs modify the development of tolerance to and physical dependence

on opiates.

In summary, peptides derived from the mammalian systems and their

analogs may find their applications in the management of withdrawal

syndrome of opiates, to antagonize certain side effects of opiates and as

prophylactics in the development of addiction to opiates.

357
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ACKNOWLEDGMENT

The studies described here were supported in part by USPHS grant DA02598 from the National Institute on Drug Abuse.

365
AUTHOR

Hemendra N. Bhargava, Ph.D.

Professor of Pharmacology

Department of Pharmacodynamics

College of Pharmacy

University of Illinois at Chicago

Health Sciences Center

Chicago, Illinois 60612

366
Progress in the Potential Use of Enkephalin

Analogs

Robert C.A. Frederickson, Ph.D.

With the discovery of the first endogenous opioid peptides a decade

ago came the speculation that the elusive analgesic without abuse

potential was finally within reach. This speculation was tempered

by the more pessimistic predictions that these materials are

probably artifacts of some sort; that, even if they are real, they

may have nothing to do with analgesia; and, finally, that, even if

they are real and have analgesic properties, their exploitation will

merely result in the redesign of morphine. Ten years of research

and development now give us a little more basis for reasoned

evaluation of the various speculations. The natural pentapeptide

enkephalins were quickly realized to have no therapeutic utility

since they are almost immediately degraded upon injection into the

whole animal. Thus, a number of industrial concerns embarked upon

analog synthesis programs to develop an enkephalin-related structure

that would resist enzymatic attack and allow the study of their

pharmacology in the whole animal. Various endorphins and analogs

have been evaluated for a number of uses besides analgesia, and

these various studies will be reviewed briefly. The work of the

past 10 years has already demonstrated that the endogenous opioids

are indeed real and that at least some of them are involved in one

way or another in pain/analgesia mechanisms. Whether they might

provide the long-sought nonaddicting analgesic remains to be

demonstrated. Of the various analogs synthesized, one in particular, metkephamid, remains under clinical evaluation and will be

discussed in some detail. The future of this area of research will

rely on the development of receptor-specific structures and the

improvement of bioavailability. The methodology and progress in

these efforts will be critically reviewed.

STRATEGIES FOR THE THERAPEUTIC EXPLOITATION OF ENDOGENOUS OPIOIDS

With the recognition and acceptance of the existence of the

endogenous opioids, several possible ways (table 1) to manipulate

these systems for experimental and possible therapeutic purposes

were considered. The small enkephalin pentapeptides promised

little potential in the unmodified state since they are almost

367
TABLE 1

Strategies for Exploiting the Therapeutic

Possibilities of the Endogenous Opioid Systems

1. Development of B-endorphin and analogs.

2. Development of releasers of endogenous opioids.

3.

4.

Development of inhibitors of specific inactivating enzymes.

Development of enkephalin analogs.

immediately degraded upon systemic administration. Since

B-endorphin is less susceptible to enzymatic degradation than the

smaller enkephalins, a number of studies were undertaken to evaluate

the effect of direct administration of this substance on pain and

various mental disorders (Oyama et al. 1980; Kline et al. 1977;

Berger et al. 1980; Marx 1981). B-endorpbin produces potent,

long-lasting analgesia when injected directly into spinal fluid

(Oyama et al. 1980); and careful, controlled studies in schizophrenic

patients have indicated statistically, although not clinically,

significant beneficial effects (Berger et al. 1980). B-endorphin

has limited access to the brain, however, end the cost for sufficient

quantities of this large peptide is still prohibitive. The potential

clinical utility of this substance seems limited at this time.

Other approaches include development of agents which will release

the endogenous substances or protect them from degradation by the

various inactivating enzymes. The latter approach is particularly

interesting and has received much attention. Extensive efforts

have delineated the enzymes which inactivate the enkephalins (see

Marks, this volume; Roques, this volume). The neutral metalloendopeptidase, enkephalinase A, appears to be important in synaptic

inactivation of enkephalins, and a selective inhibitor of this

enzyme, thiorphan, has been synthesized (Roques et al. 1980). This

agent has been demonstrated to potentiate the analgesic activity of

various enkephalin analogs and may produce analgesia by itself

under certain conditions (Roques et al. 1980; Frederickson 1984).

Thiorphan does not appear likely to have clinical utility since its

bioavailability by systemic routes of administration appears

limited and the analgesic activity it exerts when given alone is

not remarkable. This relative lack of direct analgesic activity

may be due to a very low basal activity of the enkephalinergic

systems. If clinical pain were to activate these systems, however,

such a compound might have clinical utility not predicted by

standard analgesic model systems. Efforts are in progress in a

number of laboratories to produce successors to thiorphan which may

have clinical utility. Much of these efforts are being directed at

improved bioavailability and a broader spectrum of inhibition of

the various enkephalin-degrading enzymes (see Roques, this

volume). Although an intact and active endogenous opioid system

would be necessary to achieve utility with the enzyme inhibitors,

this will not be a requirement for a direct-acting opioid receptor

ligand. The rationale and efforts at developing better enkephalin

analogs are-discussed in the next two sections.

368
RATIONALE AND MECHANISM FOR ENKEPHALIN ANALOGS

The multiple opioid receptor concept provides a strong theoretical

rationale for the development of receptor-selective analgesic

structures. This in turn offers promise for reduction of physical

dependence or abuse potential, respiratory depression, and

psychotomimetic or other unwanted side effects. Since the natural

opioids are too labile to have therapeutic utility, the research

challenge is to develop analogs with desirable receptor selectivity

and which are able to achieve effective concentrations at the

appropriate receptors in the brain after systemic administration.

Since high activity resides in the enkephalinlike pentapeptide and

tetrapeptide sequences, the preparation of analogs of these or even

smaller fragments is more feasible and cost-efficient than

analogs of the larger endorphins. Many hundreds of such analogs

have been synthesized, and considerable structure activity data are

discussed in this volume and elsewhere (Frederickson 1977;

Morley 1980). The basic mechanistic model upon which the enkephalin

analog and enzyme inhibitor approaches are based is illustrated in

figure 1.

Two analogs of Met-enkephalin are of particular interest because

they have progressed to the clinic. These are FK 33-824 and

metkephamid (LY127623), which are shown in figure 2. The work with

these two enkephalin analogs has confirmed the important concept

that appropriate modification of the natural enkephalin structure

can produce systemically active analgesic agents. The present

status of these two new compounds is discussed in the next section.

STATUS OF ENKEPHALIN ANALOGS IN PRECLINICAL AND CLINICAL TRIALS

FK 33-824

Preclinical. This compound showed high activity at opioid

receptors in vitro and demonstrated preference for the μ-receptor

compared with the δ−receptor (Kosterlitz et al. 1980). It was

100 to 1,000 times more potent than morphine as an analgesic when given

by the intraventricular route and was active also after systemic

administration (Römer et al. 1977). Naloxone precipitated a marked

withdrawal syndrome in monkeys self-administering this compound

(Römer et al. 1977).

Clinical. Von Graffenried et al. (1978) examined FK 33-824 in

normal male volunteers. After 0.1 to 1.2 mg administered intramuscularly, all subjects in this study experienced a “feeling of

heaviness in all the muscles of the body, often combined with a

feeling of oppression in the chest or tightness in the throat which

induced a certain amount of anxiety.” Other symptoms noted were a

marked increase in bowel sounds, redness of face, injection of the

conjunctiva, chemosis, whole body flush, rhinitis vasomotorica, and

a flare reaction after intradermal injection. Plasma prolactin and

growth hormone were increased. Expected morphine-like effects, such

as changes in emotional behavior or mental alertness, formication,

and nausea, were not observed; and the unexpected signs were not

blocked by opioid, histamine, serotonin, or cholinergic antagonists.

The lack of blockade with nalorphine, a mixed agonist-antagonist,

369
FIGURE 1

Neuronal model for mechanisms for producing analgesia by compounds

interacting with enkephalinergic systems. The natural enkephalins

are not analgesics, but rather neurotransmitters sending brief

neuronal messages. The message is brief because the enkephalins

are rapidly inactivated by the enzymes as shown. Beta-endorphin,

reaching these receptors as a circulating neurohormone, on the

other hand, might be able to provide a message sustained enough to

be perceived as analgesia since it is less susceptible than shorter

enkephalins to these degradative enzymes. One approach to developing analgesics based on the enkephalins is to modify the structure

to provide protection from these enzymes, so that when they are

applied exogenously they produce analgesia as discussed above for

beta-endorphin. Another approach would be to develop inhibitors of

the degradative enzymes so that the activity of the natural

enkephalins would be prolonged sufficiently to provide an analgesic

effect.

suggested to the authors that the effects were not mediated by

opioid receptors, but a subsequent report (Stubbs et al. 1978)

claimed that the hormonal effects and other side effects could be

blocked by the pure opioid antagonist naloxone.

FK 33-824, administered intramuscularly (i.m.), was reported to

have analgesic activity against experimental pain in man (Stacher

et al. 1979). A single i.m. dose of 1 mg produced a significant

increase in tolerance to electrically evoked pain, but caused no

change in threshold to pain. FW 34-569, an (N-Me)Tyr1 analog of

FK 33-824, administered i.m. at 0.5 and 1.0 mg, stimulated growth

hormone and prolactio release and inhibited the release of cortisol

and LH in human volunteers, but neither dose influenced pain

threshold to a hot-plate analgesimeter (Lindenburg et al. 1981).

There have been no reports of the evaluation of FK 33-824 in

pathological pain after systemic administration, but it did have an

analgesic effect after epidural administration (0.02 to 0.5 mg) in

subjects with postoperative pain. The effect, however, was rated as

370
FK 33-824 (Sandoz)

LY127623 (Metkephamid, Lilly)

FIGURE 2

Structures of the two Met5-enkephalin analogs, FK33-824 and LY127623,

which have been evaluated in clinical studies as potential analgesics.

unpredictable and dose-independent, and the investigators saw no

advantages over epidural morphine (Andersen et al. 1982). This

compound is apparently no longer being pursued clinically as an

analgesic candidate.

Metkephamid

Preclinical. Metkephamid is an analog of Met-enkephalin with

several simple modifications, as shown in figure 2. Like FK 33-824,

it competes with labeled opioid ligands for binding in brain

homogenate and produces potent naloxone-reversible depression of

the electrically induced twitch of both mouse vas deferens (MVD)

and guinea pig ileum (GPI), with IC50 values in the nanomolar

range (Frederickson et al. 1981, 1982). In the MVD preparation,

pA2 values for naloxone versus metkephamid, normorphine, and

Met-enkephalin were determined to be 7.60, 0.32, and 7.54,

respectively. These data suggested that metkephamid and Metenkephalin share preference for a similar receptor, presumably the

δ−receptor. This differs from normorphine, which prefers the

μ-receptor. This was corroborated by the GPI:MVD ratios which were

about 4.1 for metkephamid and 0.25 for morphine, indicating a

371
sixteenfold greater δ−selectivity for metkephamid compared to

morphine. This contrasted with the ratios for competing with

(

3H)Nx

 versus (

3H)DADL binding in brain homogenates, which were 0.6

and 0.1, respectively, for metkephamid and morphine (Frederickson

et al. 1981, 1982). These latter data indicated that, although

metkephamid had a sixfold greater preference for the δ−receptor

than did morphine in the binding studies, it still had a slightly

higher affinity for the μ-receptor than for the δ−receptor.

Metkephamid has little or no affinity for the K-receptor.

The difference between the μ:δ binding ratios and the ratios in the

isolated muscle preparations suggests that either metkephamid has

considerably greater efficacy at the δ-receptor than at the

μ-receptor or that the δ−receptors in MVD differ from the

δ−receptors in brain. Support for the latter suggestion has been

recently reported (Brantl et al. 1982). Indeed, both factors most

likely contribute to the lack of complete correlation in general

between brain binding activity and activity on the isolated muscle

preparations.

TABLE 2

In vivo - in vitro correlation: Metkephamid is about 70 times

more potent than morphine as an analgesic after administration

into the lateral ventricle. This correlates best with the

relative activity of these compounds on the MVD which reflects

mainly activity at the delta receptor.

1. Pleasures of μ-Receptor Activity

IC50 Values (nM) Ratio

Morphine Metkephamid MOR/MET

3H-Naloxone binding 6.5 2.5 2.6

guinea pig ileum 100 21 4.8

2. Measures of d-Receptor Activity

3H-DADL binding

mouse vas deferens

75

390

3. Analgesia (mouse hot plate)

4.4 17.0

5.4 72.2

ED50 Values

(Nanograms/mouse, ICV)

103 1.5 68.7

Metkephamid demonstrated distinct antinociceptive activity when it

was administered by systemic routes of administration (Frederickson

et al. 1981, 1982), being anywhere from one-third to 10 times as

372
potent as morphine, depending on the test system and the route of

administration. When given by the intraventricular route,

metkephamid was almost a hundredfold more potent than morphine. The

relative analgesic potencies of metkephamid and morphine by this

route correlated much better with their relative potencies on the

MVD preparation than on the GPI preparation (table 2). This

suggested that metkephamid produced its greater analgesic activity

by action on the δ−receptor and, indeed, data to be discussed later

confirm a correlation between analgesic activity and activity at

the δ−receptor. An attractive possibility is that such δ−mediated

analgesia may be associated with less physical dependence or abuse

potential than is the more conventional μ-mediated analgesia.

tletkephamid produced little stimulation of locomotor activity or

naloxone-precipitated withdrawal jumping in mice compared with

morphine. Chronic treatment of rats with wtkephamid, furthermore,

produced only slightly more physical dependence than did saline,

unlike other drugs similarly tested such as morphine, meperidine,

and pentazocine (Frederickson et al. 1981, 1982). Metkephamid was

also reported to have a lesser depressant effect than morphine on

respiration in a rodent model.

The ability of metkephamid to cross the placental barrier was

assessed by measuring the maternal and fetal serum levels in rats

and sheep (table 3) at various times after i.m. injection

of metkephamid (Frederickson et al. 1982). In the rat, the fetal:

maternal ratio of metkephamid in blood at 1 hour after injection

was about 1:60 compared with 1:1.8 for meperidine. In sheep, the

fetal:maternal ratio for metkephamid was less than 1:200 compared

with 1:1 or 2 reported for meperidine. This indicates a remarkable

advantage of metkephamid over meperidine for use in obstetric

analgesia.

TABLE 3

Maternal and Fetal Serum Levels (μg/ml) of Hetkephamid

and Heperidine at Various Times after Administration* to

Sheep (Intramuscular) and Rats (Subcutaneous)

Species Time (min)

Metkephamid

Maternal Fetal

Meperidine

Maternal Fetal

Sheep

Rat

Rat

0 0.0 0.0

10 9.46±3.01 0.0

20 9.43±2.6 0.0

45 8.54±2.6 0.0

60 7.17±0.28 0.12±0.04

45 3.73±0.89 2.02±0.23

*The dose of metkephamid administered to the sheep was 5 mg/kg.

The rats received 50 mg/Kg of metkephamid or meperidine (table

modified from Frederickson et al. 1982).

373
The preclinical profile of metkephamid described above demonstrated

that metkephamid was different enough from standard analgesics and

promised enough therapeutic advantage to warrant entering clinical

trial.

Clinical. In initial safety studies, metkephamid was administered

to normal male volunteers in single i.m. doses ranging from 0.5 to

150 mg (Frederickson et al. 1980). No clinically relevant effects

were seen by routine clinical chemistry, electrolytes, urinalysis,

hemograms, EKG, blood pressure, or heart rate. At doses greater

than 12.5 mg, subjects reported a mild retro-orbital burning that

progressed to nasal congestion and dry mouth. A heavy sensation in

the extremities, emotional detachment, and conjunctival injection

were also reported, but no flushing or changes in bowel sounds were

noted and a flare formation did not occur after intradermal

administration. Serum prolactin was increased, but no change in

serum growth hormone was observed after 75 mg.

Clinical tests in postoperative pain have demonstrated metkephamid

to be efficacious as an analgesic in man (Calimlim et al. 1982;

Bloomfield et al. 1983). In one controlled double-blind clinical

trial by Calimlim et al., setkephamid at a single parenteral dose

of 70 mg was compared with meperidine at 100 mg and placebo in

30 patients with severe postoperative pain. All measures indicated

that the analgesic activity of metkephamid 70 mg was significantly

greater than placebo and not less than that of meperidine 100 mg.

The duration of activity was about 4 hours, and up to the 4-hour

point, metkephamid 70 mg appeared more efficacious than did

meperidine 100 mg (figure 3). The frequency of remedication with

metkephamid was also less than with meperidine or placebo. In a

second controlled double-blind study (Bloomfield 1983), metkephamid

at 70 mg and 140 mg i.m. was compared with meperidine at 100 mg and

placebo in 60 postpartum women with severe pain after episiotomy.

Using subjective reports as indices of response, patients rated

pain intensity, pain relief, and side effects at periodic intervals

for 6 hours. Metkephamid at the 140-mg dose was rated most

effective, followed in order by meperidine (100 mg), metkephamid

(70 mg), and placebo. Only metkephamid at 140 mg and meperidine at

100 mg showed statistically significant superiority over placebo.

Both treatments took effect within 1⁄2 hour, peaked at 1 to 2 hours;

and, with 140 mg metkephamid, maximum analgesia was sustained for 6

hours, i.e., 2 hours longer than with meperidine.

There was a higher incidence of minor side effects with metkephamid

than with the other treatments in these studies, but these effects

were relatively transient and were not distressing to the patients.

The side effects peculiar to metkephamid were a sensation of heavy

limbs, dry mouth, eye redness, and nasal stuffiness. The spectrum

of these side effects suggested that the pharmacological properties

of metkephamid are different from those of standard narcotic

analgesics. It has been suggested that this might be due to

greater utilization of the δ−receptor than is the case with standard

analgesics (Bloomfield et al. 1983). Metkephamid has high affinity

for both the μ- and δ−receptors, but little or no affinity for the

374
κ−receptor. This unique receptor preference of metkephamid may

also contribute to its apparently lesser potential for physical

dependence and respiratory depression. It is not yet clear whether

more selective δ−activity or some combination of μ- and δ−activity

FIGURE 3

Time-effect curves of the analgesic activity of metkephamid (70 mg,

i.m.) compared with placebo and meperidine (100 mg, i.m.). Pain

was assessed subjectively at each interview by: (a) a reported

pain score on an ordinal scale of 0 (no pain) to 4 (“terrible”

pain); (b) a reported score for pain relief compared with premedication pain level on an ordinal scale of 0 (no relief) to 4

(complete relief); and (c) an analog scale of pain consisting of a

20-cm line marked 0 (“no pain”) at one end and 100 (“worst pain I

have ever felt”) at the other end. From these observations, the

mean pain intensity scores, mean pain relief scores, and mean pain

analog scores were calculated for each observation time and plotted

as shown. The placebo generally had no effect on pain. Metkephamid and meperidine began to reduce pain by 1⁄2 hr, with peak

analgesic effect usually at 1 hr; the analgesic effect was

considerably diminished by 4 hrs. Up to the 4-hr point, metkephamid

(70 mg) appeared more effective than meperidine (100 mg).

Modified from Calimlim et al. Copyright 1982, Lancet.

375
is most desirable for the best analgesia versus adverse effect

profile. Finally, if the transfer across the placenta seen in rats

and sheep proves to be the case in humans as well, metkephamid

could be an important advance for obstetric analgesia. This latter

question is under investigation.

FUTURE GOALS AND DIRECTIONS

An evaluation of future prospects first requires consideration of

some dogma which seem far too widely accepted. These include the

beliefs that 1) opioid analgesia is μ-receptor, and not δ-receptor,

mediated; 2) that endogenous opioids and analogs produce physical

dependence and, therefore, offer no advantage as potential new

analgesics; and 3) that endorphins do not cross the blood-brain

barrier (B-B-B). Certainly, if these or even some of these beliefs

are true, then the future looks bleak for the possibility that

enkephalin analogs will provide any therapeutic advances. In this

section, these dogma will be critically examined and the major

goals and prospects for future development of this area will be

explored.

μ-Receptors Versus d-Receptors in Analgesia

Several lines of evidence support the existence of separate μ- and

δ−receptors (Goodman et al. 1980; see also chapters by Simon,

Portoghese, and Kosterlitz, this volume). The prevalent opinion

has been, however, that the μ-receptor rather than the δ−receptor

mediates analgesia. Roques and colleagues (Gacel et al. 1981;

Chaillet et al. 1984) have reported that the analgesic activity of

a series of enkephalin analogs correlated better with activity on

the GPI than on the MVD. They concluded, therefore, that analgesia

correlates with μ-activity and not with δ−activity.

A number of other investigators, on the other hand, have reported

strong evidence for the existence of δ−mediated analgesia. We, for

example, have observed that the analgesic ED50’s in the mouse

hot-plate test after intraventricular administration of a series of

opioids of widely differing μ- versus δ−activities correlated very

well with IC50 values on the MVD and K values for inhibition of

3H-DADL binding (both preferentially δ1 measures), but less well

with measures of μ-activity (GPI and 3H-naloxone binding). A

portion of this data is shown in table 4. A number of confounding

factors may contribute to the differences between these results and

those discussed above. For example, δ−preferring analogs are not

generally modified at the C-terminal, while μ-preferring analogs

are, and generally in ways which provide enzymatic stability and

better bioavailability. Even as simple a modification as amidation

of the C-terminal acid increases both μ-preference and bioavailability to brain receptors. Thus, μ-preferring analogs may appear

more potent for reasons not related to their μ-preference. This

is not adequately compensated by utilization of the intraventricular route of administration because the brain possesses

C-terminal inactivating enzymes.

376
TABLE 4

Correlation between the activity on isolated tissues and analgesic

activity for several opioids with widely differing δ− versus

μ-receptor activity. The analgesic activity appears to correlate

best with δ−receptor activation as represented by IC50 on MVD.

IC50 (nM) Analgesic

GPI ED5 0 Compound MVD GPI MVD Preference (ng/mouse, ICV)

Tyr-D-Ser-Gly-Phe-Leu-Thr-OH 0.63 65 105 d 1.2

Tyr-D-Ala-Gly-Phe-(Me)Met-NH2 5.6 21 3.9 d 2.2

Morphine 390 100 0.26 μ 82.7

(Me)Tyr-D-Ala-Gly-(Et)Phe- 507 13 0.03 μ 144.5

Ch2-N(Me)2

There are other experimental means besides in vivo/in vitro

structure activity correlation studies for evaluating the roles of

different receptor types in pharmacologically induced behavioral

changes. These include cross-tolerance studies and studies with

selective antagonists.

Cross-tolerance between metkephamid and morphine was assessed in

the mouse writhing test for analgesia (Hynes and Frederickson

1982). The mice were treated chronically with either saline or

morphine on a 5-day schedule with four doses per day. Sixteen to

twenty hours after the last injection of morphine, dose-response

curves were generated to morphine and metkephamid. The doseresponse curve for morphine was shifted to the right in the

morphine-treated animals, resulting in a 3.5-fold increase in the

ED50 value for morphine. A similar shift to the right in the

dose-response curve was not observed for metkephamid in the

morphine-tolerant mice (figure 4). Only at the higher end of the

dose-response curve was there some reduction in the analgesic

activity of metkephamid in the morphine-tolerant mice. These

results were interpreted to indicate that a δ−receptor-mediated

mechanism contributes to the analgesia produced by metkephamid,

although a μ-mechanism appears to become more prominent at the

higher doses. Yaksh and colleagues (1984) have reported similar

findings of a relative lack of tolerance to metkephamid in

morphine-tolerant animals, using both the shock titration model

with the monkey and the hot-plate model with the rat.

Further support for the concept that metkephamid produces

analgesia by action on δ−receptors was provided by studies with

naloxazone (Hynes and Frederickson 1982). It was possible to

determine a dose of this irreversible antagonist which, when given

20 hours earlier, would selectively antagonize morphine-induced

analgesia without affecting analgesia produced by metkephamid

(figure 5). The above studies suggest that metkephamid is

producing analgesia by some mechanism or receptor type not utilized

by morphine under the conditions of the experiments described.

377
FIGURE 4

Inhibition of writhing by morphine (circles) and metkephamid

(squares) in morphine-tolerant mice (filled symbols) and salinetreated mice (open symbols). The morphine dose-response curve is

shifted to the right in the chronic morphine-treated mice, but the

metkephamid curve is not (modified from Hynes and Frederickson

1982).

This different mechanism is presumed to be δ−mediated because

metkephamid does not recognize the K-receptor adequately for this

to provide an explanation.

There are other lines of evidence for δ−mediated analgesia,

particularly at the spinal level. Hylden and Wilcox (1983)

reported evidence that antinociceptive activity induced in mice by

intrathecal opioids was mediated by both μ- and δ−receptors, but

not by K-receptors. Tung and Yaksh (1982) reported identical

findings for opioid-mediated analgesia in the rat spinal cord.

Tseng (1982) utilized cross-tolerance studies to differentiate

morphine and D-Ala2, D-Leu5-enkephalin and provided evidence for

both μ- and δ-receptors mediating analgesia in the spinal cord.

As a final note here, it should be recognized that demonstrating

that the analgesic activity seen in a given species in a given test

system under a given set of conditions, which appears to be

mediated by a particular set of receptors, does not imply that a

different receptor type cannot contribute in a different species in

a different test system and/or under a different set of test

conditions.

Physical Dependence

A general conclusion from the above discussion would be that

analgesia can be induced by actions on both μ- and δ−receptors. A

more pertinent question now concerns whether there is any

difference in the activity profile of the two receptors such that,

378
for example, δ−mediated analgesia is associated with less physical

dependence or abuse potential than is μ-mediated analgesia.

Certainly, the data presented for metkephamid imply lesser dependence potential for a given degree of analgesic activity consequent

upon activation of δ−receptors compared to μ-receptors. A number

of investigators, however, have reported the development of

physical dependence consequent upon prolonged exposure to

enkephalin analogs and implied a close correlation between

analgesia and physical dependence (Pert et al. 1976; Wei and Loh

1976; Römer et al. 1977; Miglecz et al. 1979; Wei 1981). None of

these studies focused carefully on potential differences depending

on receptor selectivity, however; they merely demonstrated that

opioid peptides, like opioid alkaloids, can produce physical

dependence. Since none of the peptides tested was devoid of

μ-activity, and since the protocol for such studies is to expose

the organism to increasing levels of a drug until dependence is

produced, none of these studies answers whether δ−receptor

activation may be associated with similar or less physical

dependence than is μ-receptor activation. Careful studies

comparing in a quantitative dose-related fashion the analgesic

versus physical dependence-producing properties after intraventricular administration of highly μ- and δ−selective enkephalin

analogs, taking into account their relative bioavailabilities and

enzymatic susceptibilities, will be required to answer the

important question here.

FIGURE 5

Analgesic dose-response curves for morphine (circles) and

metkephamid (squares) in the mouse hot-plate test. The filled

symbols represent data generated in mice pretreated 20 hrs. earlier

with saline, subcutaneous (s.c.). The open symbols refer to data

generated in mice pretreated 20 hrs. earlier with naloxazone, 50

mg/kg, s.c. The morphine dose-response curve was shifted to the

right after naloxaxone pretreatment, but the metkephamid curve

was not.

379
Therapeutic Potential Other Than Analgesia

The dramatic proliferation of investigations inspired by the

discovery of opioid receptors and their endogenous ligands has

revealed a multitude of other possible physiological functions and,

thus, potential therapeutic utilities for the opioids besides

analgesia. There is, for example, evidence for opioid modulation,

which would suggest potential opioid utility, in neuroendocrine

disorders (Meites et al. 1979), sexual disorders (Meyerson and

Terenius 1977), food intake and obesity (McKay et al. 1981),

cardiovascular disorders (Holaday and Faden 1978; Lang et al.

1982), convulsive disorders (Frenk et al. 1978; Frenk 1983), and

mental disorders (Verebey 1982; Shah and Donald 1982; DeWied

1980). For detailed discussions of the evidences for an opioid

excess or an opioid deficiency in the etiology of schizophrenia, see

Watson et al. (1979) and Berger et al. (1980, 1981). The specific

receptor types involved in each of the proposed roles for opioids

are not yet fully established, but efforts are increasing to

elucidate such questions. Presently, there is evidence that σ−

and/or K-receptors are involved in psychotomimetic activity,

K-receptors in appetite suppressant activity, and δ−receptors in

hypotensive and anticonvulsant activities. More definite assignment of receptor type to function will be served by development of

ligands with more selective agonist and antagonist activity for

each subset of opioid receptor. If such agents can be delivered to

the brain by systemic administration, their evaluation in animal

models of disease states and in the clinic, when justified, may

provide some significant therapeutic advances.

Peptide Drug Delivery

It is well recognized that there is a significant B-B-B to

peptides, and this definitely includes the opioid peptides. The

B-B-B to peptides has been the subject of two recent reviews

(Pardridge 1983; Meisenberg and Simmons 1983). The B-B-B consists

of tight junctions between adjacent endothelial cells, and most

peptides diffuse very slowly through this barrier. While there are

transport systems for amino acids and dipeptides, there does not

appear to be any such system for the enkephalins. Brain capillaries have a high aminopeptidase activity and rapidly degrade

enkephalins. Modification of the P-position of enkephalins, such

as by replacement of Gly2 by D-Ala2, does improve penetration,

however; and there are brain structures with diminished barriers,

such as the circumventricular organs and the choroid plexus. Thus,

enkephalin analogs can penetrate the B-B-B sufficiently after

systemic administration to have behavioral effects. This is

exemplified, for example, by metkephamid which is very potent

indeed after intravenous administration.

Thus, while B-B-B passage of peptides is limited, this is not the

limiting factor in the potential therapeutic use of enkephalin

analogs. The major limitation to realization of the therapeutic

potential of peptides including enkephalin analogs is the lack

of sufficient oral bioavailability. These peptides suffer

dramatic degradation in the gut, and the small proportion of a dose

delivered to the gut which reaches the circulation suffers

380
further substantial degradation in first pass through the liver.

Due to the variability of oral absorption, a parameter of major

importance for peptide delivery is the oral to intravenous

delivery ratio. Increasing the absolute potency of a peptide or

its penetration of the B-B-B will increase its apparent oral

potency, but may decrease its safety as a drug by increasing the

chance of an overdose after oral delivery. The major focus should

be on increasing peptide survival through the gut and liver or on

finding ways to bypass the gut and liver. The ways of attacking

this problem include chemical modification to improve lipid

solubility and enzymatic stability (which have the possible

drawback of also improving B-B-B penetration) and/or exploring new

pharmaceutical preparations and alternate routes of noninvasive

administration, such as sublingual, transdermal, and intranasal.

CONCLUSIONS

A new opioid receptor recognized subsequent to the discovery of

the endogenous opioids is the δ−receptor. There is reason to

believe that activation of this receptor, like activation of the

u-receptor, will produce analgesia. The possibility that

δ−mediated analgesia is associated with less physical dependence

and abuse potential than is μ-mediated analgesia requires

verification and is worth pursuing. There are a number of

potential therapeutic utilities for the opioids besides

analgesia. The realization of this potential will require the

development of more selective agonists and antagonists at each

of the receptors and the improvement of the oral bioavailability

of these peptides. Nonpeptide opioids might achieve this end, but

the greatest receptor selectivity achieved to date has been with

peptide analogs. And, it must not be forgotten that the world

has had thousands of years of experience with nonpeptide opioids

without achieving the elusive goal.

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enkephalin analogue in normal man. Lancet 2:1225-1227, 1978.

Tseng, L.F. Tolerance and cross-tolerance to morphine after chronic

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Tung, A.S., and Yaksh, T.L. In vivo evidence for multiple opiate

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Wei, E.T. Enkephalin analogs and physical dependence. J PET

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Director, Central Nervous System

Research

Health Care Division

Monsanto Company

Life Sciences Research Center

700 Chesterfield Village Parkway

St. Louis, Missouri 63198

384
Opioid Peptides as Drug Products: FDA

Regulatory Requirements

Charles P. Hoiberg, Ph.D., and Rao S. Rapaka, Ph.D.

Recent progress in opioid peptides research has resulted in the

development of potentially beneficial drugs. The beneficial

effects of these peptides have yet to be demonstrated in humans

with clinical trials. Following successful clinical trials,

these drugs may be approved by the Food and Drug Administration

(FDA) based on the demonstrated therapeutic effectiveness and

safety. Since it iS expected that many opioid peptide drugs

will undergo human clinical trials in the near future, it

appears to be the appropriate time to acquaint researchers with

the regulatory process involved in drug approval, particularly

since the regulations concerning the submission of drug

applications have been recently revised. This chapter will

address the regulatory requirements in general wlth particular

emphasis on the manufacturing and control requirements. For

specific regulatory information concerning neuropeptides. the

chapter by Gueriguian and Chlu should be consulted.

FDA LEGISLATIVE HISTORY

The passage of the Import Drugs Act in 1848 marked the beginning

of Federal regulatory legislation. This statute was passed when

quinine used by American troops in Mexico to treat malaria was

found to be adulterated. In 1906, the original Food and Drug

Act was passed. mandating drug standards for strength, quality.

and purity. The use of toxic preservatives and dyes in foods

and "cure-all" claims for worthless and dangerous patent

medicines led to the enactment of this law. In 1938, the

Federal Food, Drug and Cosmetic (FD&C) Act was enacted after

over 100 deaths resulted from a marketed elixir of sulfanilamide

which contained diethylene glycol. This legislation required

predistribution clearance for safety of new drugs prior to

interstate shipment. Following the thalidomide tragedy in

Europe in 1962, the Kefauver-Harris amendments were passed.

These amendments required that the effectiveness of a drug

product, as well as its safety, be demonstrated prior to

approval and that the FDA be notified when a drug is to be

tested in humans.

385
INVESTIGATIONAL NEW DRUG APPLICATIONS

Under the current FD&C Act, a manufacturer of a new molecular

entity must have on file with the FDA an approved application

(e.g., a new drug application [NDA]) prior to interstate

distribution. However, in order to give a researcher the

opportunity to investigate the safety and efficacy of a compound

which has potential therapeutic activity, an exemption to the

FD&C Act may first be granted. This is accomplished by filing

with the FDA a "Notice of Claimed Investigational Exemption for

a New Drug," commonly referred to as an "Investigational New

Drug (IND) Application". During the IND phase, human clinical

trials and additional animal testing are to be performed under

carefully controlled conditions. The objectives of the FDA

reviews at the IND stage are to assure (1) that safety standards

and the rights of the test subjects are observed, and (2) that

the quality of scientific evaluation is adequate to permit a

proper assessment of the effectiveness and safety of the drug.

On June 9, 1983, the FDA published (Federal Register, Vol. 48,

No. 112, p. 26720) a proposal to revise the current regulations

governing the review of investigational drugs. These regulations would apply to new drugs, antibiotic drugs, and

biological drugs, including biological products that will be

used in vitro for diagnostic purposes. Under the present and

the proposed regulations, the following information should be

included in an IND application:

1. A complete list of all components present in the drug

substance and drug product, using the best available

descriptive names.

2. The quantitative composition of the investigational

drug product, including inactive components.

3. The name(s) and address of the manufacturer(s) of

the drug substance and the drug product.

4. A description of the methods of preparation of the drug

substance and the drug product. Detailed information

should be provided regarding the extraction, isolation,

synthesis, and/or purification procedures.

5. Information regarding the analytical methods and

release specifications used to assure the identity,

strength, quality, and purlty of the drug. In the

initial phases of an IND study, greater emphasis is

generally placed by the FDA on the identification and

control of the raw materials and the new drug substance

than on the final dosage form.

386
6. Test results of all preclinical investigations, such as

animal toxicology/pharmacology data, as well as a

summary of previous human experience, if any, with the

investigational drug (e.g., published material). Additional animal studies may be performed concurrently

with the proposed clinical trials. The data submitted

should adequately demonstrate that the described drug

product is reasonably safe to warrant investigation in

human subjects.

7. A detailed clinical protocol which describes the

rationale and objectives of the proposed multiphased

investigation. As described in the pending IND

Rewrite, the progressive three phases of an IND study

proposed by a pharmaceutical company or research center

would be as follows:

a. Phase 1 is to include the initial

introduction of an investigational new

drug into humans. These studies should

be closely monitored and be conducted

in patients or normal volunteer

subjects. This phase should be

designed to determine the metabolism

and pharmacologic actions of the drug

in humans, the side effects associated

with increasing doses, and, if

possible, early evidence of

effectiveness. During this phase,

sufficient information about the

pharmacokinetics and pharmacological

effects of the drug should be obtained

to permit the design of

well-controlled, scientifically valid,

Phase 2 studies.

Phase 1 should also include studies of drug

metabolism, structure-activity relationships, and

mechanism of action in humans, as well as studies

in which investigational drugs are used, as

research tools to explore biological phenomena or

disease processes.

The total number of subjects and patients included

in Phase 1 studies will vary with the drug, but is

generally in the range of 20 to 80.

b. Phase 2 is to include the controlled clinical

studies conducted to evaluate the effectiveness of

the drug for particular indications in patients

with the disease or condition under study and to

determine the common short-term side effects and

risks associated with the drug. These studies are

387
typically well controlled, closely monitored, and

conducted in a relatively small number of

patients, usually involving no more than several

hundred subjects.

c. Phase 3 studies are expanded controlled and

uncontrolled trials. They are performed after

preliminary evidence of effectiveness of the drug

has been established, and they are intended to

gather the additional information about

effectiveness and safety that is needed to

evaluate the overall benefit-risk relationship of

the drug and to provide an adequate basis for

physician labellng. Phase 3 studies usually

include from several hundred to several thousand

subjects.

Much simpler protocols are permitted by the agency for

"sponsor-investigator INDs" (e.g., an individual

researcher associated with an academic institution) or

for "treatment INDs" (e.g., a request by a practicing

physician to administer an unapproved drug primarily

for treatment purposes within the investigational

context).

8. Information regarding the scientific training and

experience of each investigator.

9. Copies of all informational material, including all

labels and labeling which will be utilized by each

investigator. The immediate package of an

investigational new drug must bear a label with the

statement "Caution: New Drug - Limited by Federal (or

United States) law to investigational use." The

immediate container label should also indicate the nonproprietary or established name; the dosage form (e.g.,

capsule, injection, etc.); the strength of the drug per

dosage unit (e.g., mg/ml); the route of administration;

the name and address of the investigator; and the lot

number.

10. A statement that each investigator will obtain prior

permission (informed consent) from each human subject

who is involved in the study. The patient should be

provided with all pertinent information, such as the

nature of the study, the potential hazards, and the

possible beneficial effects.

11. A statement that the FDA and all investigators will be

notified immediately if any adverse effects arise

during the animal or human testing, or if the study is

discontinued. The sponsor is also to submit, at

intervals of 1 year after the date of submission of the

IND, annual reports which describe the progress of the

investigation.

388
After submitting an IND, the sponsor must wait 30 days after the

date of the FDA receipt of the application before the clinical

studies may be initiated. This 30-day delay is utilized by the

agency to determine whether the investigational drug and the

protocol are reasonably safe to justify investigation with human

subjects. In general, this safety evaluation is performed by a

review team consisting of a medical officer, a chemist, and a

pharmacologist, and for certain drug products, a microbiologist. If the IND is not placed on "clinical hold" or if

the 30-day delay is waived by the agency, the human testing may

proceed. However, if the FDA notifies the sponsor that the

proposed study is subject to a clinical hold, the clinical

testing may not be initiated until the cited deficiencies are

corrected to the agency's satisfaction. Examples of deficiencies cited for the imposition of a clinical hold are as

follows:

1. Unreasonable and significant risk of illness or injury

to the human subject.

2. Insufficient information provided so that a proper

assessment of the risk of the study could not be

determined. such as inadequate animal testing data,

lack of assurance that an injectable drug is sterile

and pyrogen-free, etc.

3. The clinical investigator is not adequately qualified

to perform the investigation.

4. A misleading or erroneous investigation brochure.

Under the proposed IND regulations, the director of a review

division will also have clear authority to impose a clinical

hold at Phases 2 and 3 of the clinical trials. In addition, the

FDA may communicate in writing or orally with the sponsor at any

time during the course of the investigation concerning other

deficiencies or recommendations. Such comments or advice,

however, are considered to be solely advisory. To aid the

sponsor in meeting the FDA requirements. the agency has

published and makes available to all investigators various

guidelines and instructional brochures. The agency also

encourages sponsors to contact the review division prior to and

during the clinical trials for guidance.

Once the sponsor feels that the results of the animal and human

testing have adequately demonstrated the safety and effectiveness of the investigational drug, a new drug application may be

filed to permit the marketing of the drug product.

NEW DRUG APPLICATIONS

On February 22, 1985, the FDA published (Federal Register, Vol.

50, No. 36, p. 7542) a comprehensive revision of the Code of

389
Federal Regulations pertaining to the procedures and requirements for the submission, review, and approval of new drugs and

antibiotics for human use. The final rule, commonly referred to

as the "NDA Rewrite," went into effect on May 23, 1985, except

for the postmarketing reporting requirements for adverse drug

experience; these were delayed until August 22, 1985. The

agency, however, will accept applications that are in the format

prescribed under the previous regulations up to February 24,

1986.

To reduce duplication, an applicant may by reference incorporate

information that has previously been submitted to the agency in

drug master files (DMFs), INDs, and other new drug applications. The identlty of the reference file should be fully

described, such as the file name, the reference number, the

submission date, and the volume number. If the reference

document was submitted by a party other than the applicant, a

Letter of Authorization from such party must be filed.

To facilitate the drug approval process, the applicant is

required to submit two copies of an application: an archival

copy and a review copy.

Archival Copy

The archival copy of an NDA is to be a complete application. It

is retained as the sole file copy after the approval. Prior to

approval, it serves as a reference source for information not

contained in the technical sections of the segmented review

copy, which will be discussed later.

The archival copy includes the following: (1) an application

form; (2) an index; (3) a summary; (4) five technical sections

(six for anti-infectives); (5) drug samples and labeling; and

(6) case report forms and tabulations. These sections are

described below:

1.

2.

Application Form: This form serves as a cover sheet

and contains basic identifying information regarding

the applicant and the proposed drug product. Examples

of the type of information to be provided are: the

name and address of the applicant; the application

date; the name of the drug product; a checklist

identifying the enclosures filed; and a commitment to

comply with all applicable laws and regulations.

Index: A comprehensive index is to be provided in the

archival copy; it should indicate the volume number and

the page numbers where the summary, the technical

sections, and other supporting information can be

located.

390
3. Summary: The archival copy should contain an overall

summary of the entire application. In addition, each

of the segmented copies of the review copy is to

contain this overall summary, so that each of the FDA

review disciplines will have a general understanding of

the data and information submitted in the complete

application. The summary should also contain an

annotated copy of the proposed labeling, a discussion

of the benefits and risks of the drug product, a brief

description of any marketing history of the drug

outside the United States, and a synopsis of each

technical section.

The summary should be approximately 50 to 200 pages

long, depending on the nature of the drug and on the

degree of information available; it should be written

in the same detail and meet the editorial standards

required for publication in refereed scientific and

medical journals. When feasible, the data presented in

the summary should be in tabular and graphic forms.

Since the FDA intends to use the overall summary to

prepare the Summary Basis for Approval document,

updated summaries should be filed with major resubmissions. The agency is currently preparing

guidelines for the format and content of the overall

summary.

The following information should be included in the

summary of the chemistry, manufacturing, and controls

technical section.

a. Drug Substance

(1)

(2)

(3)

(4)

(5)

Description (names, physical properties,

chemical properties, and stability);

Manufacturers (names and addresses);

Methods of manufacture (synthesis and

purification);

Process controls at each stage of

manufacturing and packaging; and

Specifications and analytical methods.

b. Drug Product

(1) Composition and dosage form (capsule, tablet,

aerosol, etc.);

(2) Manufacturers (names and addresses);

391
(3) Methods of manufacture (manufacturing process

for the finished dosage form);

(4) Specifications and analytical methods;

(5) Container/closure systems;

(6) Stability (data summary, expiration dating

period, and recommended storage conditions);

and

(7) Test formulations (composition and lot

numbers of the drug products used in the

clinical trials, toxicology studies, etc.).

4. Technical Sections: The five technical sections (six

with anti-infectives) of the application are each to

contain all the specific information needed by the

review disciplines to make a knowledgeable and thorough

review of the proposed drug product.

The required technical sections for a new drug

application are as follows:

(a) Chemistry, Manufacturing, and Controls Section;

(b) Nonclinical Pharmacology and Toxicology Section;

(c) Human Pharmacokinetics and Bioavailability Section;

(d) Microbiology Section (anti-infective drugs only);

(e) Clinical Data Section; and

(f) Statistical Section.

The content and format of the Chemistry, Manufacturing,

and Controls Section will be expanded in the discussion

of the review copy.

5. Samples and Labeling: When requested, the applicant

will be required to provide representative samples of

the drug substance, the drug product, and the reference

standards directly to the FDA laboratories so that the

regulatory suitability of the proposed analytical

methods may be determined. The review chemist may also

request samples of the finished market package in order

to perform a visual examination of the drug product,

the container/closure system, and the placement of the

immediate container label.

The methods validation package should consist of copies

of the appropriate pages contained in the chemistry

technical section, so that additional review will not

392
be required. The agency has prepared and is distributing detailed guidelines describing the

preparation of the requisite method validation packages

and the submission of the drug samples.

This section of the application is also to include

copies of the proposed labels and all other labeling

for the drug product. Should the labeling not contain

an established name for the active component present in

the finished dosage form, the name proposed to the

United States Adopted Name Council should be stated.

6. Case Report Forms and Tabulations: The last section of

the archival copy is to contain the case report tabulations for each adequate and well-controlled study,

as well as the case report forms for each patient who

died during a clinical study or who did not complete

the study due to adverse events. Additional case

report forms may be requested by the agency should it

be felt that ancillary data are necessary to conduct a

proper review of the application.

Applicants are encouraged to meet with the agency prior

to submitting an application to discuss the extent of

the information that must be provided in this section.

Review Copy

The second copy of an application to be filed with the agency is

the review copy. This submission is to consist of 5 or 6

detailed technical sections, each containing an overall summary

and an application form. These separately bound sections are to

contain the technical and scientific information required for

approval by each of the following review disciplines: clinical;

pharmacology: chemistry; statistics; biopharmaceutlcs; and

microbiology (anti-infective drugs only). The filing of this

segmented review copy permits the various scientific reviewers

of the agency to evaluate the applications concurrently, thereby

expediting the approval process. Guidelines have been prepared

by the agency setting forth the extent and nature of the information that should be provided in each of the technical

sections.

Since the chemistry section of the review copy for all NDAs may

be submitted 90 to 120 days prior to the submission of the

archival copy, this section should include, although not

required, all requisite material needed to facilitate review,

such as: an index; copies of all labellng; appropriate

references (INDs, DMFs, etc.); Letters of Authorization; and the

overall summary which should include information regarding the

composition and method of manufacture of each investigational

formulation.

393
The chemistry technical section should fully describe the

composition, the synthesis, the manufacture, the stability, the

specifications, and the control procedures for the drug

substance, the drug product, and the components used in

preparing the drug product. The following requirements should

be addressed using the same or similar format to expedite review:

1. Drug Substance (active ingredient)

a. Description Including Physical Properties,

Chemical Properties, and Stability:

1.

2.

3.

4.

5.

Names: A listing of the names used for the

drug substance should be provided including,

where appropriate, the established name, the

proprietary name, and the chemical name, the

code designations.

Formulas: The chemical structural formula,

the molecular formula, and the molecular

weight should be indicated, if known.

Physical and Chemical Properties: The

pertinent physicochemical characteristics

should be described, including appearance,

solubility properties, pH and pKa values,

melting and boiling points, isomeric and

polymorphic forms, and chemical stability

(e.g., potential degradation products).

Proof of Structure: A full technical

description and interpretation of the data

obtained and the reference standards employed

in the structure elucidation of the drug

substance should be provided.

Stability: A complete description and

interpretation of the studies performed and

of the data collected should be submitted,

including validation data demonstrating the

suitability of the analytical methods

utilized (e.g., stability indicating

capability). The stability samples should be

evaluated in containers that approximate the

containers in which the drug substance is to

be stored and shipped.

b. Manufacturers: The name and address of each

manufacturer that will perform any part of the

synthesis, extraction, isolation. and/or

purification of the drug substance should be

stated and its responsibilities designated.

394
c. Methods of Manufacture: A full description should

be provided of the acceptance tests and

specifications for the raw materials, as well as

of the methods and components used in the

synthesis, preparation, and purification of the

drug substances; the description should be in

detail equivalent to that used in scientific

journals. Any alternative methods or variations

in the synthesis should be noted, with an

explanation of the circumstances under which they

would be used and of the effect they would have on

the purity and stability of the drug product. If

the drug substance is prepared by fermentation or

by extraction from natural sources (plant or

animal), a full description of each step of the

process should be provided.

d. Process Controls: A full description of the

control procedures performed at each stage of the

manufacturing, processing. and packaging-of the

drug substance should be provided, including the

specification and test procedures for pivotal and

key/critical intermediates.

e. Specifications and Analytical Methods: A full

description of the acceptance specifications and

the analytical methods used to assure the

identity, strength, quality, and purity of the

drug substance should be provided. Actual and

potential impuritles, such as by-products,

degradation products, isomeric and polymorphic

components, heavy metal contaminants, and residual

solvents should be indicated.

2. Drug Product (finished dosage form)

a. Components: A list of all components used in the

manufacture of the drug product, regardless of

whether they undergo chemical change or are

removed during manufacture. should be included.

Each component should be identified by its

established name, if any, or by its complete

chemical name, using structural formulas when

necessary for specific identification. If any

proprietary preparation or other mixtures are used

as components, their identity should be fully

described, including a complete statement of

composition and of any other information that will

properly identify the material. Proposed alternatives for any listed component should be fully

justified.

395
b. Composition: A statement of the quantitative

composition of the drug product should be provided, specifying the name and amount of each

active and inactive ingredient contained in a

stated quantity of the drug product. A batch

formula should be included which is representative

of the one to be employed in the manufacture of

the finished dosage form. Any calculated excess

for an ingredient over the label declaration

should be designated as such and the percent

excess shown.

c. Specifications and Analytical Methods for

Components: A full description of the acceptance

specifications and the test methods used to assure

the identity, strength, quality, and purity of

each inactive ingredient should be submitted.

Alternate release methods should be demonstrated

to be equivalent to, or better than, the proposed

regulatory method.

d. Manufacturers: The name and address of each

manufacturer that performs the manufacturing,

processing, packaging, labeling, or control

operations of the drug product should be listed

and its responsibilities described.

e. Methods of Manufacture, Packaginq Procedures, and

In-Process Controls: A detailed description of

the manufacturing and packaging procedures should

be included which specifies the facilities, the

materials flow plan, the equipment used, and the

various sampling points. In this regard, the

submission of a schematic diagram of the production process may be helpful. A description of

the in-process controls, including analytical

tests and appropriate data to support the

specifications, should be included.

f. Specifications and Analytical Methods For Drug

Product: A full description should be provided of

the sampling plans, the release specifications.

and the analytical methods which will be implemented to assure the identity, strength, quality,

purity. and bioavailablllty of the drug product.

The accuracy, sensitivity, specificity, and

reproducibility of the proposed test methods

should be established and documented. All

alternate release tests should be demonstrated to

be equivalent to, or better than, the proposed

regulatory method.

g. Container-Closure Systems: Full information

should be submitted regarding the physical,

396
chemical, and biological characteristics of the

container-closure or other component parts of the drug

product package to assure its suitability for the

intended use; the test methods employed should be

specified and the manufacturing process described. A

description of the entire packaging operation and

relevant in-process controls should also be Included.

h. Stability: A complete and detailed description

of, and data derived from. studies of the stability of the drug product should be submitted,

including information showing the suitability of

the sampling plans, the analytical methods

employed, and the stability protocol. Any additional stability studies under way or contemplated should be indicated. Stability data should

be submitted for the finished dosage form in the

container-closure systems in which it is to be

marketed. If the drug product is to be reconstituted at the time of dispensing, stability data

should also be included for the solution prepared

as directed. The expiration dating period should

be clearly specified.

3. Environmental Impact Analysis Report:

An environmental impact analysis report should be filed

in accordance with 21 CFR 25. The environmental impact

of the manufacturing process and of the ultimate use of

the drug product should be described as set forth in

the Federal Register published April 26, 1985.

Information regarding administrative policies and procedures for

the drug approval process, such as time frames, action letters,

and supplements to an approved application can be obtained from

the FDA consumer safety officers and/or the Federal Register

notice published on February 22, 1985.

An abbreviated organization chart is provided in figure 1 to

Identify the various units involved in the drug approval process.

BIOAVAILABILITY/PHARMACOKINETIC AND BIOEQUIVALENCE STUDIES

For a long time, it was believed that if the dosage form

contained the stated amount of an active ingredient, the patient

is assured of full availability of-the medication for the stated

therapeutic purpose; it was also assumed that dosage forms

containing equal amounts of the active ingredient are equipotent. (Bioavailability indicates the rate and relative

amount of drug that reaches the circulation. Two drug products

are termed bioequivalent if both of them are approximately equal

in their bioavailable dose and rate of supply.) Soon it was

realized that dosage forms containing the same amount of active

ingredient need not necessarily be bioavailable to the same

397
FIGURE 1

Organizational Chart of the Food and Drug Administration
extent, as the bioavailability is influenced by several factors,

such as particle size of the drug, polymorphic forms of the

drug, presence of different excipients, differences in

formulation, the physiologic state of the individual utilizing

medication, etc.

An organization chart of the Office of Drug Standards is

presented in figure 2. Realizing the importance of bioavailability/bioequivalence of drug products, on January 7,

1977, the FDA published a final order entitled "Bioavailability

and Bioequivalence Requirements." These were published in the

Federal Register (Vol. 42[5], pp. 1624-1653). These requirements took effect in July 1977 and were published as two

separate regulations to deal more effectively with the issues of

drug bioequivalence of multisource generic drugs and the issues

of drug-to-drug bioavailabllity involving new drug entities. In

this regard, pharmacokinetics (involving the study of kinetics

of absorption, distributlon, metabolism, and excretion of drugs

and their pharmacologic response) should be fully utilized in

the early developmental stage of the drug product to ensure an

adequate delivery of the drug to the site of action; examination

of bioavailabillty at a later stage may require reformulation

due to poor absorption as well as potential by giving rise to

questions of the validity of the results of "pivotal" clinical

studies used to support the submission.

The following sections should be included for all INDs and NDAs

submitted to the Agency requiring a bioavailability review:

background, chemistry, metabolism, pharmacology, statistics, and

pharmacokinetics. Draft guidelines are available from the FDA

and these guidelines are appropriate to identify pivotal studies

as well as addltlonal studies that should be considered with the

IND/NDA phase of drug development. Some of these are:

1 . Bioavailability Study(ies)

Proposed marketed dosage form(s) compared to a reference(s), e.g., comparison of a solid dosage form to a

reference solution and/or to a different route of

administration.

2. Dose Proportionality Study(ies)

a. A dose proportionality study over the dosing range

proposed in the labeling, used in Phase 2 and 3

clinical trials; or

b. A dose proportionality study to the highest tolerated single dose.

3. Bioequivalence Study(ies)

a. An in vivo comparison of dosage form(s) used in

399
FIGURE 2

Organizational Chart of the Office of Drug Standards
pivotal clinical study(ies) to the proposed marketed

dosage form(s).

b. An in vivo comparison of the proposed marketed

dosage form(s), e.g., multiple strengths if the

drug/excipient ratio varies.

4. Dissolution of Solid Oral Dosage Forms

a. Dissolution profiles of all solid dosage forms

used in pivotal clinical studies.

b. Dissolution profiles of all solid dosage strengths

and forms proposed for marketing.

c. Evaluation of a) and b) above with respect to

similarity and/or deviations from the proposed

marketed product.

5. Multiple Dose Studies

To demonstrate that steady-state plasma and/or other

biologic fluid concentration can be predicted from

single dose.

6 .

7.

Define Michaelis-Henten Kinetics

To more fully describe the effects of Michaelis-Menten

Kinetics on the body at steady-state if single dose

studies indicate the possibility of nonlinear kinetics.

Specific Patient Populations and Diseased States

Drugs which are targeted for a particular patient

population, whether a disease state or an age group,

e.g., pediatrics, should be evaluated within that

group. For instance, the effect of renal disease, i.e.,

reduced G.F.R., should always be performed for drugs

extensively cleared by the kidney. Although it can be

anticipated that failure of the main organ to metabolize

a drug will alter the clearance and half-life, the

compensatory pathways of elimination cannot necessarily

be predicted. Therefore, drugs extensively cleared or

bound by any organ should be studied in patients with

disease of the organ in order to quantitatively define

the effects of various levels of disease. The most

important organs in drug elimination and binding are the

kidney, liver, and plasma (proteins). Disease or

alteration of function in the kidney in particular is

not only disease dependent but also age dependent.

Drugs extensively eliminated by the kidney should always

be studied for effects of renal disease and age. The

degree of liver disease is more difficult to define for

401
8.

9.

drugs extensively metabolized by the liver; if identifiable subpopulations of patients exist with the

indicated use and liver disease, they should be

studied. Similarly, for drugs extensively protein

bound, studies of the effect of derangements in the

plasma proteins should be conducted if there is a

likelihood of significant plasma protein changes in some

patients for whom the drug is indicated.

Effect of Food

Drugs labeled to be administered with food should be so

investigated to support labeling. Because of the

potential for alteration in drug absorption when

administered with food, the effect of food on bioavailability, especially those critical drugs, i.e.,

drugs with a narrow therapeutic ratio or drugs requiring

close patient titration, should be investigated and

labeled appropriately.

Combination Drug Products-Compatibility with Respect to

Pharmacokinetics

10. Drug-Drug Interaction

Drugs indicated to be coadministered with specific drugs

should be investigated for evidence of interaction.

In addition, for good submission. an overall report should

be prepared containing all the pertinent information with

appropriate references to the individual study reports. The

abbreviated Summary and Recommended Format for the Biopharmaceutics Report is expected to contain the following

information:

1 .

2.

3.

4.

A summary of the mode of action and therapeutic index,

where applicable and if known, of the drug and a listing

of other chemically or pharmacologically related active

substances.

A summary of the absorption, distribution, metabolism,

and elimination of the drug, including references of

supportive studies.

A summary of the drug protein binding, pKa, pharmacoklnetlc data such as Ka, Kel, T 1/2, relative area

under the curve and/or absolute bioavailability, dose

proportionality, and therapeutic dose range where

appropriate.

A summary of the pivotal bioavailability studies which

establish the bioavailability/pharmacokinetics and

metabolism of the drug, including data on the proposed

402
dosage form, and basic study design with appropriate

statistical summary.

5. In the case of products containing new chemical

entitles, an evaluation of potential biovailability/bioequivalence problems of the dosage form

and/or the drug based on medical, physiochemical, and

pharmacoklnetlc criteria or on information to permit

waiver of evidence of bioavailability where appropriate.

6. A summary of the pivotal studies which support bioavailability and/or pharmacokinetic statements in the

labeling, with an explanation of the basic study design,

reference drug (product) selection, and statistical

analysis.

7. A listing of all human bioavailability/pharmacokinetic

studies pertaining to the drug which were performed by

the firm or its agent, with appropriate referencing.

8. Summary documentation of the analytical methodologies

used in pivotal bioavailability/pharmacokinetic studies

for the drug and/or metabolite(s), at the concentration

of the drug (metabolite(s)) in physiological fluid

documenting the specificity, linearity, sensitivity,

limit of detection, and reproducibility.

9. A full statement of the composition of the drug product.

10. Summary of the content uniformity data. Where

applicable, the solubility profile of the drug as a

function of pH should also be provided.

11. In vitro dissolution data on all proposed dosage forms,

including instrumentation, media, agitation,

temperature, sampling times, sink conditions, and

infinity value* (if less than percent in 1 hour).

*Defined as same reading at 150 rpm (paddle/basket) at

three successive 10-minute intervals.

CONCLUSION

The ultimate goal of a medicinal chemist is to have his or her

discovery successfully utilized in the treatment and/or

management of a disease state. The path to success, however, is

often long and tortuous. Although many years of research may

have been invested in the isolation, identification, and/or

synthesis of a pharmacologically active compound, the drug

product cannot be routinely used in the practice of medicine

until extensive animal and human testing is completed. The

results of this testing, plus information regarding manufacturing and control procedures, must be evaluated by the Food

and Drug Administration prior to the marketing of a drug product.

403
The time interval between the discovery of a potentially

therapeutic compound and its general use in the medical

community may involve many scientific and regulatory hurdles.

However, this delay is usually offset by the assurance that the

prescribed drug products are safe and effective.

AUTHORS

Charles P. Hoiberg*, Ph.D.

Food and Drug Administration

Rockville, Maryland 20857

Rao S. Rapaka**, Ph.D.

National Institute on Drug Abuse

Rockville, Maryland 20857

*Please note that the interpretation of the agency's policies

represents the opinion of the author, not necessarily those of

FDA.

**Please note that the views expressed are those of the

author, and not necessarily those of NIDA.

404
A Few Thoughts on the Development and

Regulation of Neuropeptides

John L. Gueriguian, Ph.D., and Yuan-Yuan H. Chiu, Ph.D.

INTRODUCTION

In the past decade, scientific and technical progress has facilitated the

rapid identification and study of many new neuropeptides. Immunocytochemistry, radioimmunoassays, automated solid-phase synthesis, high

resolution purification procedures, and, most importantly, genetic

engineering and in-situ hybridization techniques have been extremely

contributory to this field as they have in others. Neuropeptides may be

neurotransmitters, neuromodulators, or neurohormones and thus possess a

wide range of activities and effects. These new substances, and their

synthetic analogs, hold considerable promise toward the development of

many potent drugs. Of course, the traditional aspects of drug

development and regulation apply here as they would in any other area.

And yet, this extremely original field also requires novel considerations

and approaches dictated by its particular merits.

THE ORIGINAL CAST

Hypothalamic neurohormones

Many neurohormonal analogs are currently being studied in humans, some

of them very actively. Luteinizing-hormone releasing hormone (LHRH)

and its analogs are clinically investigated as stimulators and inhibitors of

the male and female pituitary-gonadal axis. An LHRH analog has already

been approved by the Food and Drug Administration (FDA) for the

palliative treatment of hormone-responsive prostatic cancer. The

potential of growth-hormone releasing factor (GRF) and its analogs to

beneficially affect general growth parameters is also explored at

present. Sufficient practical knowledge and experience have already been

developed in these and other areas -- thyrotropin releasing hormone

(TRH), corticotropin releasing factor (CRF), etc. -- to benefit adjacent

and related fields.

Opioid peptides and hormonal activities

Longer length peptides of various opioid families, e.g., dynorphin,

beta-neoendorphin, and beta-endorphin, have been detected in the brain

405
and the pituitary (Goldstein and Ghazarossian 1980; Goldstein et al.

1981); and usually carry hormonal modulation functions, as opposed to

the neurotransmitter function of other opioid peptides. These peptides

act through a tonic inhibitory effect on corticotropin and gonadotropins

via the inhibition of CRF and LHRH, respectively. In addition, opioid

peptides modulate the release of adrenocorticotropic hormone (ACTH)

during stress and stimulate the release of prolactin, growth hormone,

and thyroid-stimulating hormone (TSH).

Other neuropeptides

Substance P was the first neuropeptide discovered more than 50 years

ego (von Euler and Gaddum 1931), but it was not until 40 years later

that its chemical structure was determined (Chang et al. 1971). Several

other molecules have been discovered and characterized since. These

peptides are neurotransmitters or neuromodulators, and have effects on

the brain development, thermoregulation, cardiovascular and sleep

regulation, feeling (pain, analgesia, euphoria), or behavior (memory,

learning, feeding).

SECOND GENERATION PRODUCTS

Development of analogs

The automation of solid-phase peptide synthesis and the availability of

the sophisticated contemporary purification and analytical tools have

provided us with an excellent opportunity to seriously study the

therapeutic applications of neuropeptide analogs. The development of

agonistic and antagonistic analogs is of both scientific and economic

significance.

Medical advantages

Since neuropeptides are usually multifunctional, analogs may be designed

to increase the therapeutic index of the drug class intended for use in a

specific indication. Analogs may be more resistant to metabolic

degradation and thus become more available at the site of action. They

may be designed to pass the blood-brain barrier more effectively than

the natural ones. Finally, one may develop dosage forms which are more

acceptable to the patient than the conventional parenterals (e.g., nasal

sprays, transdermal patches, or depot microspheres). A number of analogs

of vesopressin, sometostetin, LHRH, and GRF have already undergone

relatively extensive clinical investigations.

Economic advantages

In most circumstances, natural products are not patentable, and

economic considerations dictate that nonpatentable drugs are usually

unable to make it in the marketplace. Analogs, on the other hand, can

and, in point of fact, should be patented, and are therefore able to

sometimes yield sizeable returns on research and development

investments. Also, analogs may contain fewer amino acid residues and

are usually more potent than the natural product, even though careful

406
studies are needed to define the minimal sequence consistent with

reasonable pharmacodynamic activity (Baskin et al. 1984). Such built-in

advantages will increase the probability of the eventual marketing of a

given product.

Special requirements

Analogs also deserve special attention and handling, particularly from

the point of view of unsuspected toxicities. Antagonistic LHRH analogs,

for example, possess structures drifting farther and farther away from

the original molecule while exhibiting lowered relative potencies. Thus,

experts were not surprised when some highly hydrophobic

arginine-containing antagonists were shown to produce untoward effects

in certain animal species.

SOME BASlC FACTS AND THEIR PRACTICAL CONSEQUENCES

Chemistry

The approximately 50 neuropeptides discovered so far have many common

characteristics. They generally are small peptides and very few of them

contain more than 50 amino acid residues. The smaller neuropeptides,

e.g., TRH and enkephalins, contain only three and five amino acid

residues, respectively. Even the relatively larger ones, such as CRF and

GRF, contain only 41 and 43 amino acid residues, respectively. All this

is, of course, good news for the organic chemists. But the implications

for the physiopharmacologist are more uncertain. Indeed, most

neuropeptides have quite flexible tertiary and, in some cases, even

secondary structures. The fact that many of these peptides are usually

linear and contain none or only one disulfide bond further compounds

this characteristic. Thus, the conformation of these peptides may be

greatly influenced by their environment. In other words, the structure

adopted in the aqueous solution may be quite different from that found

in the nonaqueous phase. Structure-activity relationship studies are

already inherently complex; and, given the relative greater flexibility of

neuropeptides (as opposed to steroids, for example), their experimental

behavior becomes even more difficult to understand and to interpret.

Any hypothesis as to the efficacy and safety of a given analog has to be

carefully tested during animal and, later, human studies. During such

studies, we may find that a desired pharmacodynemic effect is

accidentally lost in a first analog, while a second one causes some

totally unexpected side effects, even though it retains the expected

therapeutic activity.

Structural studies will nevertheless be pursued and may, at times,

provide helpful hints end ideas. The crystal structure of Leu-enkephelin

(Tyr-Gly-Gly-Phe-Leu) recently published by Camerman et al. (1983),

serves es en excellent example. These authors found that the side chains

of the four independent Leu-enkephalin molecules in the same crystal

lattice assume different orientations. In addition, the fully extended

polypeptide backbones of two molecules have a slightly more puckered

conformation that that of the remaining two. Moreover, the two glycine

residues (Gly2 and Gly3) in two of the four Leu-enkephalin molecules

adopt a conformation closer to that of D-amino acid than to L-amino

407
acid. In fact, an analog with a D-Ala substitution for GIy2 was found to

be biologically active, whereas an analog with a D-Phe substitution for

Leu5 was inactive. This may help explain why D-amino acid analogs of

many other peptides are not only biologically active, but sometimes more

potent.

Biochemical data

We know that neuropeptides can be present at several sites in the

central nervous system, with distinctly different functions at each site;

also, several peptides may be present at a given site. Through DNA

cloning, many of these neuropeptides were found to be synthesized in

the form of precursors, which are biologically inactive large polypeptides.

The precursors usually contain several small biologically active peptides,

which are flanked at both ends by pairs of basic amino acids, lysine and

arginine, and liberated by the action of trypsinlike proteolytic enzyme,

followed by carboxypeptidase B-mediated cleavage. The liberation of the

small peptides is tissue specific, i.e., different sets of peptides with

distinct physiological functions may be formed from the same precursor,

depending on the local tissue abilities and needs. The processing of

proopiomelanocortin (Nakanishi et al. 1979, 1960) illustrates well some of

these complications. In the anterior pituitary lobe, proopiomelanocortin

is processed to produce ACTH and beta-lipotropin (LPH), and some of

the beta-LPH is further split into beta-endorphin and gamma-LPH. In the

intermediate lobe, ACTH and beta-LPH are no longer present, because

ACTH is further cleaved to form alpha-melanostimulating hormone (MSH)

and corticotropin like intermediate lobe peptide, and beta-LPH into

gamma-LPH and beta-endorphin completely. The processing of the initial

precursor in the hypothalamus and other sites of the brain may be

different from the particular ones described above. The processing of the

precursor can also occur at the RNA level. For instance, the RNA

transcribed from the calcitonin gene is processed into an mRNA in the

brain different from that in thyroid cells (Rosenfeld et al. 1983). It then

leads the synthesis of two different precursor polypeptides which yield

calcitonin in the thyroid and the so-called calcitonin gene-related

peptide whose function in neural tissues is at present unknown.

A precursor may contain several copies of the same peptide, and one

peptide may also be present in two different precursors. The precursor

preproenkephalin (Comb et al. 1982; Gubler et al. 1982; Noda et al.

19828, 1982b), for example, contains four copies of Met-enkephelin

molecule, one copy of Leu-enkephalin, and other peptides. On the other

hand, Leu-enkephelin was also found to be present in a second precursor,

beta-neoendorphin/dynorphin (Kakidani et al. 1982). Moreover, many

peptides encoded in a gene family can act in close coordination to cause

behavioral and physiological changes. The study on the sea slug Aplysia

showed (Scheller et al. 1983, 1984) that multiple peptides coded by

genes in a five-membered gene family together orchestrate the complex

behavior repertoire of egg-laying (Kandel 1979).

Since many neuropeptides act at the membranes where receptors reside,

the conformation of the peptides while binding to receptor molecules

may be quite different from that found in the simple aqueous phase.

Kaiser and Kezdy (1984) have suggested that an amphophilic structural

408
site of the neuropeptide may be present to accommodate the amphophilic

environment of the membrane lipid bilayer. By examining the amino acid

sequence of GRF, they found that the first 29 amino acid segment could

form an amphophilic alpha-helix, which may explain why the shortest

biologically active GRF agonist is, precisely, the fragment spanning

residues 1-29.

Again, the above considerations indicate the complexity of the processes

that we are proposing to deal with. They underline the potential for

progress while also emphasizing the need for imeginative yet careful

investigation.

CHEMISTRY AND MANUFACTURING CONTROLS

The general FDA requirements in chemistry and manufacturing controls

for drug substances (pure peptides) and drug products (formulated dosage

forms) for human trials emphasize the structural identity, the chemical

purity and stability, and the biological potency of the active moiety, and

also the lot-to-lot consistency of both the active principle and its

contaminants. With the currently available technologies, the chemical

purity of a small peptide prepared on a small scale can reach 99% or

greater without major or unsurmountable difficulties. The important issue

is whether this purity level can be achieved in the scaled-up production

lots. If not, the materials to be used in preclinical and clinical studies

should preferably be prepared at a purity level which can best be

achieved at the production scale. Chemical structure should be firmly

determined by suitable chemical and physical methods; thus, the amino

acid sequence, the disulfide linkage, if any, and the substitution with

D-amino acid residue and other functional groups in the peptide molecule

must be established. The biological potency of an agonist should be

compared to its natural product by a suitable bioassay, whereas the

biological potency of an antagonist may be measured by using a suitable

inhibitory bioassay and a proper reference standard. When the biological

potency of a lot is found to fall within the established acceptable range,

dosing can be prescribed in terms of the weight of the peptide

calculated as the free base (excluding the content of moisture and salt,

e.g., acetate). The stability data should be collected to support the

intended shelf life of the lots used in clinical trials and the

to-be-marketed production lots. The identity, toxicity, and biological

activity of the major contaminants present in the drug substance and of

the major degradation products present in the dosage form after storage

should be documented.

ANIMAL STUDIES

Potency determinations

The biological potency of a given compound should be established against

a reference standard and expressed in terms of its specific activity.

Experts should determine, if they have not already done so, the

appropriate chemical reference standards for each class of activity. The

rat, obviously a perennially popular animal for research studies, is

acceptable to define potency; but its use for other preclinical studies of

LHRH analogs, for example, has been discouraged because its endocrine

physiology radically differs from that of the human,

409
Clinical studies using peptidic drugs

Peptidic drugs pose some interesting problems of their own that have to

be satisfactorily resolved before clinical trials are allowed to proceed.

For example, their manufacturing methodologies might create special

regulatory situations, some of which have already been substantially

addressed (Gueriguian et al. 1981). Their widespread use requires precise

efforts of definition and standardization (Gueriguian et al. 1982). Many

natural peptides and their analogs are also somewhat different from

ordinary drugs in that they are usually highly potent substances with

relatively few side effects. Certainly, this is generally true for LHRH

analogs (Gueriguian et al. 1984). Under these circumstances, one is

compelled to handle them somewhat differently from ordinary drugs.

General regulatory requirements for peptidic drugs

Phase 1 studies using natural peptides and their analogs may commence

after the drug has been well defined chemically, and after preliminary

end well-conducted animal studies have not raised any significant safety

issue. The required animal studies are usually performed using two

species, one of them preferably a nonrodent, from 2 weeks to 3

months, depending on the situation. The FDA pharmacologists in charge

would, of course, make specific recommendations based on the merits of

each case. Before phase II and III studies are permitted, additional

requirements must be met: (1) tightened physicochemical specifications

consistent with a prolonged and enlarged use of the drug; and, (2) if

warranted by pertinent observations during phase I studies, additional

animal studies to further support the safety of the drug. Pivotal studies

must be double-blinded and well-controlled against either a

placebo-treated group or an established medical therapy, as the situation

requires. Efficacy of treatment has to be clinically defined, and

so-called biochemical efficacies alone, e.g., the fall in blood

testosterone to castrate levels, are not sufficient by themselves. The

efficacy of a drug will depend on the demonstration of objective and

subjective proof of reversal, or at least stabilization, of a well-defined

disease process. Prior to any therapy, the diagnosis must have been

confirmed and, if at all possible, objectively recorded.

Specific requirements

If uncommon or unusual administration routes are chosen, e.g., the

intranasal, that administration form should be tested for bioequivalency

with a parenteral form. Regardless of the form of administration,

adequate tests must be performed to detect any peptidic antigenicity. If

and when immunogenicity is detected, additional clinical and laboratory

scrutiny is warranted (particularly of liver, kidney, and bone marrow

functions) to exclude any evidence that neutralizing antibodies are

present, or that an immune-related disorder is developing. In certain

cases, one may have to use a number of sophisticated methods, e.g.,

measurements of complementemia and detection of specific antibodies in

urinary casts. Children may be treated, but under stricter guidelines, and

only after the tested drug has previously been safely used in adults.

410
Pharmacological studies

The pharmacokinetic parameters of analogs should be determined,

preferably in a primate species, and, if at all possible, through direct

measurement of drug blood levels. General pharmacological and

toxicological requirements may be initially satisfied by animal studies in

two species (one of which may be a rodent), at two reasonable multiples

of the intended maximal human dose, and for 2 to 4 weeks. If proof of

intended efficacy is already on hand, and the toxicological studies have

not suspected or documented adverse effects, open label clinical studies

may begin in the form of a single dose study. Such preliminary studies in

humans allow the retention of promising products for further studies

while the less promising moieties are readily and quickly rejected.

SOME USEFUL CONCEPTS IN DRUG DEVELOPMENT AND REGULATION

Shuttling

After relatively limited animal studies, initial human trials were

permitted to gauge the clinical usefulness of certain LHRH agonistic

analogs (Gueriguian et al. 1984). Under the circumstances, unpromising

or ineffective drugs would have been quickly detected and discarded. At

this point, the FDA mandated additional animal studies to more firmly

establish the general safety of the more promising compounds. Additional

ad hoc animal studies would certainly be required in the future if

clinical toxicity were suspected. We refer to these back-and-forth

maneuvers between animal and human studies as “shuttling,” an

extremely useful technique that has helped us to safely and rapidly

develop LHRH analogs for at least certain clinical indications. Shuttling

may be useful in the development of therapeutic neuropeptides as well.

Developmental algorithm

During the continuing development of these same LHRH analogs, we

found it extremely important to create and to try to adhere to a

rational algorithm for drug development. LHRH itself was first used as a

diagnostic tool. This was fortunate in that it allowed any number of safe

studies, using an endogenous compound at slightly supraphysiological

doses, to determine the effects of a natural peptide permitted to

wander, so to speak, outside its physiological compartments. After

considerable scrutiny, the FDA and industry eventually agreed that

LHRH analogs should next be administered to patients with

hormone-responsive metastatic prostatic cancer. Various reasons

militated in favor of this choice: (1) this indication was based on a sound

scientific rationale; (2) it had a high estimated probability of therapeutic

success; (3) its benefit versus risk ratio appeared to be as high as

attainable at the time; (4) it would allow the safe use of relatively

higher doses of the compounds for relatively longer periods of time; and,

finally, (5) such studies would allow for the careful collection of

considerable toxicological data in the human. From that point on, and

after a suitable lag period, the analogs were tried in the treatment of

other pathological conditions, the moment their benefit versus risk ratios

411
were found to be acceptable. The above considerations may usefully

guide the clinical studies of any novel class of drugs, particularly the

related group of neuropeptides, even though it is well understood that

each case has to be judged on its own merits.

Lead population samples

LHRH analogs have so far been shown to be generally safe and effective

in the treatment of true precocious puberty. Initially, however, many

hypothetical safety questions loomed very large, e.g., the potential

immunogenicity of peptide analogs and the eventual pathological

consequences of such antigenicity. To this day, the reversibility of

long-term inhibition of the pituitary-gonadal axis can only be proven by

actual experimentation. To met these challenges, academic investigators

and regulators from the FDA defined and applied the concept of a “lead

population sample,” i.e., a small group of patients enrolled in a clinical

study that fully meets general criteria of safety and effectiveness,

during which a very careful and precise monitoring is in force, so that

one may be able to eventually detect hypothetical side effects, if and

when they occur. As time passes by and safety is better established,

other patients may be enrolled in the study. If the hypothesized event,

or any other untoward event, is picked up in the lead sample, protective

measures may be immediately taken to insulate the larger but lagging

populations from such undesirable events. In certain cases, successive

and increasingly bigger samples may be introduced one after another in

an ever expanding clinical study, thus allowing for continually improving

detection abilities of, at first, high, and, later, lower frequency events.

This general approach allows us to resolutely move ahead while

minimizing in the extreme unavoidable iatrogenic risks.

PRINCIPLES GOVERNING THE CLINICAL STUDIES OF NEW DRUGS

General rules

The FDA should approve a new drug only after at least two

well-controlled clinical trials have shown it to be safe and effective in

the treatment of a defined clinical condition. All human studies in the

United States and abroad using drugs manufactured in the United States

must be performed under the scrutiny of the FDA. Clinical studies are

permitted after adequate chemical definition of the drug and suitable

animal studies, and these studies generally pass through four distinct

phases.

Phase I studies are almost always conducted with normal volunteers and

are meant to define the general safety of the drug and its significant

pharmacological properties. The clinical effectiveness of the drug for a

given condition is tested during phase II studies, albeit with relatively

small groups of patients. Phase III enlarges such studies into Iarge-scaIe,

well-controlled clinical trials, at the end of which a new drug may be

approved if it is found to be, as mandated by law, safe and affective.

Phase IV, or post approval, studies are sometimes needed to answer some

secondary, yet still important, questions or to resolve some long-term

safety considerations.

412

U.S. GOVERNMENT PRINTING OFFICE:1987-181-332:60328
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AUTHORS

John L. Gueriguian. Ph.D.

Yuan-Yuan H. Chiu, Ph.D.

Division of Metabolism and Endocrine Drug Products

Center for Drugs and Biologics

Food and Drug Administration

5600 Fishers Lane, Room 14B-04

Rockville, Maryland 20857

414
DHHS Publication No. (ADM)87-1455

Alcohol, Drug Abuse, and Mental Health Administration

Printed 1986 Reprinted 1987