Screening Methods in Pharmacology

336 Pages·1965·14.88 MB·English

SCREENING
METHODS IN
PHARMACOLOGY
By ROBERT A. TURNER
BIOLOGICAL SCIENCE LABORATORY
FOSTER D. SNELL, INC.
SUBSIDIARY OF BOOZ-ALLEN
APPLIED RESEARCH, INC.
BRONX, NEW YORK
1965
ACADEMIC PRE S S.New York and London
COPYRIGHT © 1965, BY ACADEMIC PRESS INC.
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PRINTED IN THE UNITED STATES OF AMERICA.
To
PROF. HERIBERT KONZETT,
Pharmacology Institute, Innsbruck
and
DR. WALTER R. SCHALCH,
Pharmacology Division, Sandoz Ltd., Basle
who taught me much about the methods and principles of screening.
PREFACE
"It is important to explore many testing procedures and test conditions;
the continuing examination of a possible new test procedure should be a part
of each major program of drug testing."—Dwight J. Ingle (1962).
This book assembles most of the methods for screening substances
for pharmacological activity and includes discussions of the organi
zation of screening programs. Until now there has been no book
devoted solely to pharmacological screening.
While screening is widely practised in pharmacology laboratories,
a definition of its meaning is perhaps worthy of attention. The term
screening indicates the use of a combination of tests for the purpose
of making a decision. The results of the tests provide information on
the presence of certain pharmacological properties. A decision
whether the tested substance should be studied further may be
founded on that information. Incidentally, the direction of further
study may also be determined by this information.
The operation of retaining from a group of substances those
possessing a certain property is fundamental. Screening is the most
efficient method of detection and of decision-making in any empirical
operation. The finding of substances with a pharmacological prop
erty is empirical because no general theory correlating such a
property with molecular structure exists.
Whereas some of the classic methods of pharmacology are useful
for screening, most of the methods now employed were introduced in
recent years. In writing this book, I have tried to collect the simplest
and most widely used methods. Absolute completeness has not been
attempted, but for the determination of some of the activities (e.g.,
analgesics) several methods have been described where necessary.
It is hoped that most of the common methods are included and are
of such diversity that new methods will occur to the reader for use
in his own studies. Acquaintance with the methods described here
ought to aid in his adaptation of new methods to the problem
at hand. Whenever possible, methods have been chosen which could
become quantitative if small changes were made in the procedure,
such as the use of larger groups of animals. The guiding principles
vii
viii PREFACE
which determined the presentation of the methods are as follows:
(1) A complete and carefully described procedure is given, which
includes particularly modifications of the orginal method, so that
unnecessary manipulations are eliminated while the accuracy is
sustained; (2) the application of the method is described for both
well-known drugs and new substances; an active, new substance is
detected through the use of the method; (3) a discussion is provided
of other methods of screening for the activity and of the reasons for
preferring the method given; (4) the reliability of the method and
its limitations are stated.
The present work is obviously directed to those whose occupation
it is to search for new pharmaceuticals. A variety of investigators
in the fields of pharmacology, physiology, toxicology, and thera
peutics may also find useful methods for their research. Chemists
in the pharmaceutical industry who read this book will be aided in
their understanding of the basis for testing the compounds that
they synthesize and in their understanding of the programs for test
ing that the pharmacologist has devised.
In preparing the sections on blind screening, I have borrowed
considerably from the excellent chapter in "Progress in Medicinal
Chemistry/ ' Volume 1, by Dr. W. G. Smith of Sunderland Technical
College (England). I should like to thank the author, and Butter-
worth, the publisher. The sections on neuropharmacological screen
ing rely on the procedures of Dr. Samuel Irwin of the Oregon
Regional Primate Research Center. Discussions with him have been
most helpful.
A few of the methods in this book were developed in the Biological
and Medical Research Division, Sandoz Ltd. (Basle), and I thank
its director, Dr. Aurelio Cerletti, for permission to publish those
methods. I also wish to thank Dr. B. Berde of the same Division for
help in locating certain references.
I gratefully acknowledge the help of Mrs. Gillian Bateman who
typed the manuscript, Miss Karen Meyer who assisted in the
preparation of the indexes, and Mr. David C. Turner for making
corrections in the manuscript.
My thanks are also due to the publishers and the members of the
editorial staff for their advice and help in the production of this
book.
March, 1965 ROBERT ARNOLD TURNER
CHAPTER 1
INTRODUCTION
The testing of synthetic organic compounds and of compounds
found in nature is being performed on a vast scale. Most of this
testing takes place in the pharmacological laboratories of pharma
ceutical companies, although an increasing volume of it takes place
in the laboratories of universities and'institutes. The chief purpose
of the testing is to find new substances with pharmacological activ
ity. In the companies, the research departments search for new and
more potent pharmaceuticals. The next step, after isolating the
test substance, is a screening procedure. The testing performed in
universities is not often directed to the finding of new pharma
ceuticals, but may be directed to finding substances that exhibit
interesting biological activity, which may aid in understanding
physiological effects. Of course, the screening program undertaken
by the National Cancer Institute in an effort to find anticancer
agents differs in no significant respect from any similar program in
a pharmaceutical company. The same may be said for a search for a
histamine-releaser that is needed in relation to an immunological
investigation in a university laboratory.
All of these testing programs demand a systematic study of sub
stances so that useful ones may be found readily and so that inert
substances may be easily recognized and rejected. Because of the
complex character of biological activity, no system of tests can be
expected to function perfectly, that is, to exhibit all active sub
stances with no falsely active substances included, and to exhibit
all inactive substances without including any active substances
among them. Since a falsely active substance will be revealed
sooner or later as not worthy of further study, a system which allows
1
2 1. INTRODUCTION
a few falsely active substances has not a grave defect. It is more
serious if the system allows active substances to be rejected, for the
reason that, the majority of substances being inactive, it is impor
tant not to miss one of the few active ones. However, any system
will probably have both of these defects, and the persons performing
screening tests must exercise vigilance in order to minimize these
defects.
In evaluating the results of any specific test, the most frequent
purpose will be to decide whether the compound is to be rejected,
as the term screening implies. If a battery of tests is employed,
the defects of an individual test are less important. Ultimately the
efficiency of any screening system cannot be determined. One system
will prove to be more efficient in the hands of one investigator and
less efficient in the hands of another.
The large number of screening tests available permits considerable
ingenuity in the design of a battery of tests that will screen com
pounds for many activities at once. There are three kinds of screen
ing "programs." The simplest employs a single test, or perhaps two
similar tests, to find substances that are active in a single way. An
example is a hypoglycémie test, which measures the ability of a
compound to diminish the concentration of sugar in the blood. The
second kind of program employs several tests in order to determine
what compounds of a group are active and in what ways. The pro
gram will show what is the main activity and what are the subsidiary
activities. A comparison of potency with known active compounds
will permit a decision concerning further study. Also the test will
have multiple purposes rather than a single purpose. The third kind
of program is often called blind testing. The purpose is to find if
there is any biological activity of a new group of compounds, and to
find new areas for research. Since no activity of a definite type is
anticipated, the program will evolve as the experience of the in
vestigator increases.
As soon as an active compound is found in any except the blind-
screening program, further tests to define the compound's activities
and to quantify its effects are undertaken, as a second stage. There
fore tests and methods that are modifiable into quantitative pro
cedures have been favored for inclusion here.
Before further discussion of programs, the biochemistry of the
nervous systems is reviewed, with particular attention to the
mechanisms whereby recently discovered drugs exert their effects.
CHAPTER 2
A BRIEF REVIEW OF THE
BIOCHEMISTRY OF THE
NERVOUS SYSTEM
I. CHEMICAL MEDIATORS
This review is intended to touch on only those parts of the nervous
system which are of constant interest in pharmacology and which
function through known chemical mediators.* To the professional
pharmacologist the review will be quite elementary. However, it
will introduce to some readers a few of the new substances, as well as
the old, which are now commonly used in the investigation of drugs,
and which are standards for comparison with test substances. Even
in a book on methods, some theoretical discussion is needed,
especially in a period when the theoretical side of the science is
beginning to flourish. Moreover, there is a practical aspect to the
mechanistic, or theoretical, side of pharmacology which is related
to screening, namely the deductions about mechanism that may be
* In this book certain synonymous terms are avoided in order to employ a
single word throughout. In the following groups the word in italics is the one
employed:
1. adrenergic, sympathomimetic
2. cholinergic, parasympathomimetic, muscarinic
3. ganglion-stimulating, nicotinic
4. adrenergic-blocking, sympatholytic, antiadrenaline, adrenolytic
5. anticholinergic, parasympatholytic, antimuscarinic, muscarinic-blocking
6. ganglion-blocking, antinicotinic
7. neuromuscular, myoneural
3
4 2. BIOCHEMISTRY OF THE NERVOUS SYSTEM
made from the screening results. If such deductions can be made,
there may be immediate consequences. First, the deductions may-
provide a plan of the next experiments after screening to be per
formed in preparing the "pharmacological profile" of a new sub
stance. Second, a little knowledge of the mechanism may be decisive
in relation to further investigation. For example, if it was found that
a substance caused a delayed fall in blood pressure in the cat, the
substance might not have an intrinsic hypotensive or vasodepressive
activity, but might be a histamine-releasing agent. If it happened
that the screening program was intended to find hypotensive agents,
the next step would be to test for histamine-releasing activity, since
this activity, when demonstrated, might preclude further interest in
the test substance. Thus it is seen that the observation of a delayed
vasodepressive response, pointing to the mechanism of histamine
release rather than to another vasodilatative mechanism, influences
the next steps in the investigation.
Of the two nervous systems, the autonomie (vegetative, involun
tary) and the somatic (voluntary), the autonomie is of greater
interest to pharmacology. It has two parts: the sympathetic system
and the parasympathetic system.
The total nervous apparatus, including all systems, is divided into
two regions: the central nervous system, CNS, consisting of the
brain and spinal cord, and the peripheral nervous system, PNS, con
sisting of those nerves or parts of nerves outside of the brain and
spinal cord. Nervous impulses are conveyed from the central jiervous
system to the periphery along the three routes shown in Fig. 1.
The spinal nerve controls the action of voluntary (striated) muscle.
There is only one synapse, at the neuromuscular junction, where the
chemical transmitter is acetylcholine (I), designated by ACh(n) to
indicate that its action simulates that of a small dose of nicotine (V).
The sympathetic nerve has two synapses: one near the spinal cord,
the ganglionic synapse, in which the transmitter substance is
acetylcholine, and one at the effector end-plate, which may lie in
smooth muscle, cardiac muscle, or in a gland. The chemical trans
mitter in the terminal synapse is epinephrine (III), or norepineph-
rine (II), or a mixture of these designated EpiNor. The second
autonomie nerve in Fig. 1 is a parasympathetic nerve. Both synapses
are near the peripheral end of the nerve. In both the ganglionic
synapse and the terminal synapse, acetylcholine is the chemical
mediator, but its actions are not identical, perhaps because of a
I. CHEMICAL MEDIATORS 5
difference in the acetylcholine receptors at the two synapses. In the
terminal synapse, acetylcholine simulates the action of muscarine
(IV), a drug discovered before acetylcholine, and this kind of action
has been called muscarinic. Drugs that imitate acetylcholine in its
muscarinic actions are therefore said to have muscarinic (cholinergic,
parasympathomimetic) activity.
Substances that imitate the action of acetylcholine in the gangli-
onic synapse are called ganglionic stimulants. A substance inhibiting
CENTRAL NERVOUS PERIPHERAL NERVOUS
SYSTEM SYSTEM
>*\ SSPPIINNAALL NNEERRVVEE /[ Y
\£} (
INVOLUNTARY
SYMPATHETIC NERVE /
— EpiNor MUSCLE OR
ORGAN
GANGLIONIC TERMINAL
SYNAPSE SYNAPSE
<·> PARASYMPATHETIC NERVE jAC*(nWg( ÌACh(m) INVOLUNTARY
MUSCLE
GANGLIONIC TERMINAL
SYNAPSE SYNAPSE
FIG. 1. Routes of nervous impulses proceeding from the central nervous
system to the periphery. ACh(n), Acetylcholine with nicotinic action; ACh(m),
acetylcholine with muscarinic action; EpiNor, a mixture of epinephrine and
norepinephrine. [From Smith (1961).]
the action of acetylcholine in the same locality is called a ganglion-
blocking agent. Dimethylphenylpiperazinium (VIII) is a commonly
used ganglionic stimulant, while hexamethonium (VII) is a ganglion-
blocking agent. Nicotine in small doses acts as a stimulant; in larger
doses, as a blocking agent.
Substances which imitate the activity of the catecholamines
epinephrine (adrenaline) and norepinephrine (noradrenaline), so
that the effect is like stimulation of a sympathetic nerve, are called
sympathomimetic. An example is phenylephrine (VI). Drugs which
antagonize the catecholamines in the terminal sympathetic synapse
are called sympatholytic agents (adrenolytic agents, adrenergic
blocking-agents), of which an example is dibenamine (XLIV).