Contributors
ALLEN BARNETT
MARIAN MAY
SAM P.
JACK N.
BATTISTA
CLYDE M.
ESAM Z.
E.
BURNETT
DAJANI
R.
GLASSMAN
R.
R.
KADATZ
STEGER
ROBERT L.
V.
PETER HEBBORN
P.
MULLEN
CHARLES J. PAGET
GlLLIARD
JEROME M.
K.
MOSS
C.
SWAMY
ROBERT I. TABER
HEDWALL
ROBERT A.
H.
STONE
J.
WILKENS
TURNER
Screening Methods in
Pharmacology
Edited by
ROBERT A. TURNER
Turner Associates
Greenwich, Connecticut
PETER
HEBBORN
Department of Biochemical Pharmacology
School of Pharmacy
State University of New York at Buffalo
Buffalo, New York
VOLUME II
1971
ACADEMIC PRESS · New York and London
COPYRIGHT © 1971, BY ACADEMIC PRESS, I N C .
ALL RIGHTS RESERVED
NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM,
BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY
OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM
THE PUBLISHERS.
ACADEMIC PRESS, INC.
I l l Fifth Avenue, New York, New York 10003
United Kingdom Edition published by
ACADEMIC PRESS, INC. (LONDON) LTD.
Berkeley Square House, London W1X 6BA
LIBRARY OF CONGRESS CATALOG CARD N U M B E R : 64-24674
PRINTED IN THE UNITED STATES OF AMERICA
List of Contributors
Numbers in parentheses indicate the pages on which the authors' contributions begin.
(209). Department of Pharmacology, Schering Corporation, Bloomfield, New Jersey
ALLEN BARNETT
SAM
P. BATTISTA (167), Life Sciences Division, Arthur D. Little, Incorporated, Cambridge, Massachusetts
M. BURNETT (203), Revlon Research Center, Incorporated, Bronx,
New York
ESAM Z. DAJANI (121), Department of Pharmacology, Rohm and Haas
Research Laboratories, Spring House, Pennsylvania
CLYDE
E.
(249), Biological Laboratories of the Pharmaceutical Department of CIBA, Ltd., Basel, Switzerland
GILLIARD
M. GLASSMAN (227), Director, Clinical Research and Pharmacology, Denver Chemical Manufacturing Company, Stamford,
Connecticut
JEROME
(75, 105), Department of Biochemical Pharmacology,
School of Pharmacy, State University of New York at Buffalo,
Buffalo, New York
PETER HEBBORN
P. R. HEDWALL (249), Biological Laboratories of the Pharmaceutical
Department of CIBA, Ltd., Basel, Switzerland
R.
(41), The Pharmacological Laboratories of the Dr. Karl
Thomae GmbH, Biberach an der Riss, Germany
KADATZ
IX
LIST OF CONTRIBUTORS
X
(85, 101), Center for Theoretical Biology, State University
of New York at Buffalo, Buffalo, New York
MARIAN MAY
JACK
K.
N. MOSS (121), Department of Pharmacology, Rohm and Haas
Research Laboratories, Spring House, Pennsylvania
(249), Biological Laboratories of the Pharmaceutical Department of CIBA, Ltd., Basel, Switzerland
MULLEN
J.
Indiana
CHARLES
R.
PAGET
(145), Lilly Research Laboratories, Indianapolis,
(61), Department of Biochemical Pharmacology, School of
Pharmacy, State University of New York at Buffalo, Buffalo, New
York
STEGER
L.
Indiana
ROBERT
STONE
(145), Lilly Research Laboratories, Indianapolis,
V. C. SWAMY (1), Department of Biochemical Pharmacology, School of
Pharmacy, State University of New York at Buffalo, Buffalo, New
York
I. TABER (209), Department of Pharmacology, Schering Corporation, Bloomfield, New Jersey
ROBERT
ROBERT
A. TURNER ( 21 ), Turner Associates, Greenwich, Connecticut
H. J. WILKENS (61), Department of Biochemical Pharmacology, School
of Pharmacy, State University of New York at Buffalo, Buffalo,
New York
Preface
The second volume of "Screening Methods in Pharmacology" has the
same basic purpose as Volume I, namely, to present sufficient practical
information about techniques so that it would be possible for the reader,
even with little experience, to establish a screening program for a particular pharmacological activity. The contributors to this volume have presented typical results obtained for selected reference compounds, which
are intended to show the responses with a known substance and to guide
the reader during the initial use of a test method so that he may select
suitable doses of the reference drugs and may know the intensity of the
response expected for a certain dose level.
Because the progress in developing methods has been so rapid since the
appearance of the previous volume, it became impossible for one person
to review the pharmacological literature. Thus, unlike Volume I, Volume
II is a multiauthored, coedited work.
ROBERT A. TURNER
PETER HEBBORN
xi
Contents of Volume I
Introduction
A Brief Review of the Biochemistry
of the Nervous System
The Organization of Screening
General Methods
Quantal Responses. Calculation of
the ED 5 0
Depressants of the Central Nervous
System
Ataractic ( Tranquillizing, Neuroleptic) Agents
Analgesics
Oxytocic Agents
Antiserotonin Agents
Parasympatholytic Agents
Sympatholytic Agents
Anti-inflammatory Agents
Anticonvulsants
Sympathomimetic Agents
Central Stimulants
Muscarinic Agents
Ganglion-Blocking Agents
Antifibrillatory Agents
Cardiotonic Agents
Histamine-like Agents
Antihistamine Agents
Antitussive Agents
Antacid Agents
Thyromimetic Agents
Hypoglycémie Agents
Choleretic Agents
Antiparkinson Agents
Anti-inflammatory and Glucocorticoidal Agents
Antiemetic Agents
Bronchodilatant Agents
Curariform Agents
Anabolic, Androgenic, and Antiandrogenic Agents
Potentiators and Antagonists of
Tryptamine
Vasopressive Peptides
Diuretic and Natriuretic Agents
Anticholinesterase Agents
Anticholesterol Agents
Uricosuric Agents
Antishock Agents
Hemostatic Agents
Local and Spinal Anesthetics
Abortifacient Agents
Thymoleptic Agents
Dermal Irritants
Teratogenic Agents
Appendix
References
Author Index-Subject Index
Introduction
Numerous methods often exist for screening a series of compounds
for a given pharmacological activity. Many, but not all, available methods
are described in this volume. They have been selected because they are
the most reliable, the simplest, and, in the opinion of the respective
authors, the preferred of the available methods. The sensitivity of the
assay procedure and the possibility of ranking the compounds that have
proved clinical effectiveness are important factors in the selection of a
screening method.
Those who have been involved with screening drugs for pharmacological activity for even a short time have realized that only a few in a
group of substances have activity. An alternative situation exists if one
has a group of compounds, all of which have varying degrees of activity.
In both cases, the screening process is an attempt to identify, by one or
more tests, those few substances which are gems among a group of
pebbles.
Generally it is better to use a screening method which may give a
few false positives rather than one which will yield some false negatives.
If a substance has no true activity and is shown by a test to have activity,
a false positive results. Sooner or later, as testing with the substance is
continued, its inactivity will be revealed. Some time may be wasted in
studying the compound, but in the end the investigator is not misled.
On the other hand, a false negative may result in the removal of a
substance from further study, so that its activity will remain forever
undetected.
The developer of a new drug is always seeking a relation between
xv
XVI
INTRODUCTION
chemical structure and biological activity, which, if found, is rare
and retrospective, rather than deductive. Sometimes structural changes
in a molecule that appear minor cause unpredictable and extensive
changes in the pharmacological activity, including loss of all activity
and introduction of new side effects. Often the first member of a homologous series of compounds is the most active pharmacologically. Because
the biological consequences of small changes in chemical structure are
not understood, the structural changes cannot be programmed logically.
New drugs of a unique character will probably be derived in the future
from novel structures rather than from modifications of old structures,
study of enzyme systems involved in the disease state, unexpected clinical observations, and an understanding of the metabolism of known,
active drugs.
Experience and scientific intuition play their important roles. Screening
efficiently for certain pharmacological activities is necessary for progress. Since activity is unpredictable, the number of activities covered
by the screening program should be considerable. If several tests have
indicated that a compound has some activity, it is usually advantageous
to study it further rather than to start with a new compound ab initio.
Contemporary investigators of new drugs tend to screen with a broad
program.
No procedure for screening can be perfect. Therefore, anyone performing screening in pharmacology should always be vigilant for borderline results and for results indicating an inactive substance when one
strongly suspects that activity is present. If one has good theoretical
grounds for anticipating activity of a substance, one should continue to
study it, even if one screening procedure indicates that activity of a
certain kind is absent. One should not rigidly accept the results of
screening procedures, if, by doing so, one would relegate to the shelf a
substance which might be valuable clinically.
It is possible for a drug to be metabolized or eliminated very rapidly
by laboratory animals and yet to have a prolonged half-life in man.
Phenylbutazone is an example of a drug having antirheumatic activity in
man, but whose activity as an antiinflammatory agent in rodents is demonstrable only at doses approaching a lethal level. Moreover, in some
disease states, available, clinically effective drugs are only palliative and
not curative. It is reasonable to conclude that pharmacological screening
tests in which such clinically active drugs have a positive effect can be
used to select new drugs which are also palliative and not curative. One
should, therefore, be continually searching for new screening methods
based on animal models of human disease processes.
Elucidation of the etiology of clinical disease states still requires ex-
INTRODUCTION
XV11
tensive effort. When an abnormality in cellular function can be identified
as the consequence of a biochemical lesion, then the primary screening
method for new drugs will involve a biochemical assay procedure. In the
meantime, the pharmacological screening methods of the types described
in this volume will be needed for the discovery of new drugs.
Finally, there are no screening methods that do not require the exercise
of judgment and discretion on the part of the researcher.
ROBERT A. TURNER
PETER HEBBORN
1
a-Adrenergic Blocking Agents
V. C. Swamy
I. General Considerations
A. Adrenergic Receptors
B. Factors Influencing Drug Action
II. Methods
A. Isolated Organ Systems
B. Intact Animal Systems
References
1
1
4
4
4
15
18
I. General Considerations
A. ADRENERGIC RECEPTORS
If receptors may be defined as tissue components with which a drug
interacts to produce its characteristic physiological effects, then the
adrenergic receptors specifically refer to those components of the effector
cells through which the sympathomimetic amines exert their actions.
The adrenergic receptors have been further classified into a- and ß-receptors on the basis of their relative responsiveness to sympathomimetic
amines ( Ahlquist, 1948 ). Although the catecholamines act on both kinds
of receptor, some compounds stimulate or block adrenergic responses
specifically at either a- or ß-receptors; those agents, therefore, can be
1
2
V. C. SWAMY
divided into a- and ß-adrenergic stimulants and a- and ß-adrenergic
blocking agents.
Blockade at the α-adrenergic receptors can be recognized by comparison of a test substance with the actions of two established sympatholytic
agents, now more precisely termed α-adrenergic blocking agents, namely,
phentolamine and phenoxybenzamine. The former compound causes a
parallel and rightward shift of the agonist ( catecholamine ) dose-response curve, and the inhibition of response to a dose of an agonist
may be reversed by larger doses of the agonist. Phentolamine, thus,
is termed a competitive, reversible antagonist. The blocking action of
phenoxybenzamine (POB) and other 2-halogenoethylamines has been
described by a variety of terms: nonequilibrium antagonism (Nickerson,
1957), insurmountable antagonism (Gaddum, 1957), and competitive,
irreversible antagonism (Furchgott, 1955; Kimelberg et al, 1965).
In contrast to phentolamine, phenoxybenzamine does not form a dissociable complex with the receptor. Its binding to the receptor probably
involves covalent bond formation and the blockade is prolonged. Experimentally, an effective adrenergic blockade produced by phenoxybenzamine cannot be overcome even by large doses of the agonist. Consequently, in experiments performed in vitro, increasing the concentration
of phenoxybenzamine results in a progressive depression of response
to the agonist until complete abolition of the response is achieved.
The use of pA# values (Schild, 1947) is a convenient method for
evaluating competitive antagonism. pA* is defined as the negative logarithm of the molar concentration of the antagonist which will reduce
the effect of a multiple dose of an agonist to that of a single dose.
If the interaction of the drugs at the receptor is bimolecular, then
log (x - 1) = log K2 - npA*
(1)
where x is the ratio of equiactive doses of agonist in the presence and
in the absence of antagonist; n and K2 are constants.
Thus, when log ( x — 1 ) is plotted against pA*, a straight line results
with a slope equal to (—n), which intersects the pA* axis at a point
corresponding to pA2 ( Fig. 1 ). When n = 1, pA2 — pA10 = 0.95, and
this difference in pA2 and pA10 values can be used as a test for competitive antagonism, although it is preferable to use a plot of log (x — 1)
over a wide range of antagonist concentrations.
Antagonist activity may be evaluated, also, in terms of the apparent
dissociation constant KB of the receptor-antagonist complex (Furchgott,
1967). The theoretical basis for this procedure is the equation
KB = - ? x —1
(2)
1.
α-ADRENERGIC BLOCKING AGENTS
3
1.6
1.2
2
α>
ω
O
■α
0.8
Q)
_c
"o
c
0)
■σ
o
0.4
0
-0.4
Negative log molar concentration of
thymoxamine
FIG. 1. The antagonistic interaction of thymoxamine with norepinephrine on the
guinea pig vas deferens. Thymoxamine was added to the bath 2 min before contractile responses to norepinephrine were obtained. The pA2 value of 7.57 corresponds to the point of intersection of the regression line with the abscissa. Where
the dose ratio equals 0.95, a perpendicular dropped from the regression line to the
abscissa gives the pAio value of 6.42. ( From Birmingham and Szolcsanyi, 1965. )
where B is the molar concentration of the antagonist and x is the dose
ratio of agonist in the presence and in the absence of the antagonist.
Under true equilibrium conditions —log KB = pA2, as defined by Schild
(1947).
An empirical term, pA/„ may be used as a quantitative index of the
activity of a compound which reduces the attainable maximum of the
dose-response curve for the agonist. ρΑΛ is defined as the negative logarithm of the molar concentration of an antagonist which reduces the
maximum response to an agonist to a value which is 50% of the maximum
4
V. C. SWAMY
obtained previously in the absence of antagonist. This term does not
make any assumptions concerning the mode of action of test compounds
and can be used to quantify all antagonists which reduce the maximum.
To obtain this value, a series of curves are plotted using doses of the
antagonist that cause a flattening of the slope and a progressive decline
of the maximum. The pA/> value is obtained by interpolation or close
extrapolation from two curves whose maxima were reduced to approximately 501
B. FACTORS INFLUENCING DRUG ACTION
A number of factors contribute to the physiological effects of adrenergic drugs. The factors include the processes of uptake and enzymic
modification, which regulate the concentration of sympathomimetic
amines at the receptor sites ( Trendelenburg, 1966, 1968). In addition,
spontaneous changes in tissue sensitivity and interaction of drugs at
sites other than receptors may cause misinterpretations in evaluating
the activity of antagonists (Furchgott, 1968). Finally, given the multiplicity of sites of action in the adrenergic system, the tests may reveal
the action of potential drugs not only at the receptors but also at the
ganglia and sympathetic neurons. This last feature, understandably, is
more likely to occur in intact animal studies than in tissues studied
in vitro.
It may not be possible, therefore, to utilize experimental procedures
that are ideal in all respects. Selection of a particular method necessarily
represents a compromise between its convenience and the qualitative
or quantitative significance of the data obtained. For example, identification of receptor-blocking properties is possible when examining the
cardiovascular activity of the compound. However, a detailed assessment
of the antagonistic properties of test compounds, involving determination
of quantitative indexes (e.g., pA2 values), invariably requires the use
of in vitro studies and a careful appraisal of possible experimental
variables.
II. Methods
A. ISOLATED ORGAN SYSTEMS
The use of isolated organ systems offers obvious advantages over in
vivo studies. Relatively accurate measurement of responses can be made
1.
α-ADRENERGIC BLOCKING AGENTS
5
from several preparations, usually obtained from one animal. The various
complicating factors encountered in vivo such as drug distribution,
humoral activity, and reflex activity are largely minimized or avoided.
Finally, these methods serve to identify and make comparative estimates
of the receptor-blocking properties of test compounds.
The stimulation of «-receptors generally induces contraction of smooth
muscle which may be recorded via isotonic or isometric systems. The
availability of automated recording systems (Vickers Corp.) makes
routine determination of pA2 values convenient. Ideally, the tissues used
in the experiments should contain only «-receptors and should show
only minimal changes in sensitivity over the duration of the experiment. The use of phenylephrine, in place of more commonly used
agonists such as norepinephrine, is more appropriate since it combines
low affinity for presynaptic sites (Burgen and Iversen, 1965) with strong,
preferential action at «-receptors.
The experimental procedure consists of plotting a series of dose-response curves—one curve in the absence of an antagonist and the others
in the presence of varying concentrations of the antagonist (Fig. 1).
The tissue, is made to contract maximally 2 or 3 times at the beginning
of the experiment. Construction of dose-response curves may be carried
out by the method described by van Rossum and van den Brink (1963),
where successive doses of the agonist are added to the bath after the
tissue has acquired steady-state equilibrium to the previous dose. After
the maximum response has been achieved, the agonist is washed out
of the bath and a complete relaxation of the tissue occurs. The antagonist
is then added to the bath and allowed to equilibrate with the tissue
for a given period of time. The tissue is then exposed to the agonist
and a new dose-response curve is obtained, using a range of doses sufficient to duplicate the initial dose-response curve. Similar dose-response
curves are plotted for a wide range of antagonist concentrations. If
the curves are parallel, they may be interpreted as indicating competitive
antagonism (Fig. 2). The dose ratio x is calculated from the parallel
shift of the dose-response curves and is utilized in plotting log (x—1)
against pA*.
A common source of error in this procedure is the failure of the
antagonist to reach equilibrium. The time required to reach equilibrium
varies with experimental preparations and the concentration and nature
of the antagonist (Furchgott, 1967; Schild, 1947). It is possible to determine the time for equilibrium for an antagonist by challenging the tissue
with the agonist at different periods during continuous exposure to the
antagonist. In common practice, the duration of exposure is chosen arbitrarily and reported with the experimentally determined pA2 value.
6
V. C. SWAMY
I0" 7
I0" 6
IO"5
I0" 4
IO"3
Norepinephrine (molar)
FIG. 2. The effect of phentolamine on the contractile responses of the rat vas
deferens to norepinephrine. Responses to norepinephrine were obtained in the
presence of various concentrations of phentolamine. Competitive antagonism is indicated by a parallel shift of the dose-response curves of norepinephrine. (From
van Rossum, 1965.)
Changes in the sensitivity of the preparation to the agonist may result
in erroneous estimates of ρΑ# values. Such a possibility may be accounted for by using a control preparation which is treated in a manner
similar to the experimental preparation, except that the antagonist is
not added. Any shifts in the dose-response curves that occur in the
control preparation are then used to correct the shift caused by the
antagonist in the experimental preparation. Finally, low concentrations
of 2-halogenoethylamines and various nonspecific depressants of smooth
muscle may cause parallel shifts of dose-response curves, thereby leading
to the false conclusion that they are competitive antagonists. However,
employment of a test compound in a wide range of concentrations will
confirm the identity of its antagonistic properties. Increasing the concentrations of a nonspecific depressant results in gradual loss of parallelism,
and a progressive decline in maximal response becomes evident ( Fig. 3 ).
1. Vas Deferens
The vas deferens fulfills many of the optimal conditions for quantitative evaluation of adrenergic antagonists. The response of this organ
1.
7
α-ADRENERGIC BLOCKING AGENTS
9
8
7
6
5
-log Epinephrine concentration
FIG. 3. Effect of Dibenamine-HCl ( D B ) on the response of the rabbit aortic
strip to epinephrine. Increasing concentrations of DB cause a progressive reduction
of the contractile response to epinephrine. Responses to epinephrine were tested
at the end of the exposure period after washing DB from the organ bath. (From
Furchgott, 1955.)
to α-adrenergic agonists consists of a strong rapid contraction followed
by a quick relaxation on washing the agonists out of the tissue. Although
the vasa deferentia of both rat and the guinea pig (Leach, 1956) are
commonly used, the relative preponderance of α-receptors in the vas
deferens of the rat (van Rossum, 1965; Vohra and Reiffenstein, 1967)
makes the latter more suitable for evaluation of α-adrenergic antagonists.
The rat is killed by a sharp blow on the head, the vasa deferentia
are dissected free from the extraneous tissues and are suspended in
organ baths containing Tyrode's solution or a modified form of Krebs'
solution (Hukovic, 1961). The system requires aeration by a mixture
of 02 (95%) and C 0 2 (5%). A simple isotonic lever system (1:15; 0.3 gm)
provides satisfactory recordings of contractile responses which remain
stable for over 3 hr.
Cumulative dose-response curves may be obtained conveniently using
this preparation. The tissue is allowed to equilibrate for 20-30 min before
inducing maximal contractions one or two times. Following this initial
treatment, the second and third dose-response curves for norepinephrine
are usually identical (Patil et al, 1967). This property of the tissue
may be utilized to make accurate estimations of the parallel shift of
the curves caused by reversible antagonists (Fig. 2). The magnitude
8
V. C. SWAMY
of the shifts caused by different concentrations of the antagonist are
then used to estimate the pA2 value by graphical means (Fig. 1). The
pA2 values of some common «-receptor antagonists determined on
the vas deferens of the rat and guinea pig are listed in Table I.
TABLE I
COMPARISON OF pA 2
Tissue
Rat
Agonist
vas deferens
Norepinephrine
Guinea pig
vas deferens
Norepinephrine
Epinephrine
Cat spleen
R a t seminal
vesicle
Norepinephrine
Epinephrine
Norepinephrine
Epinephrine
V A L U E S OF « - A D R E N E R G I C
Antagonist
ANTAGONISTS
Contact
time
(min) pA 2
Reference
Phentolamine
—
6.9
van Rossum (1965)
Piperoxan
Aceperone
Droperidol
Levopromazine
Yohimbine
Dihydroergotamine
Thymoxamine
—
—
—
—
—
30
6.0
8.3
7.9
7.3
5.3
8.25
2
7.57
Piperoxan
Yohimbine
5
5
6.4
4.17
Piperoxan
5
7.05
Yohimbine
5
4.47
Macusine B
Macusine B
Tolazoline
2
10
5
5.57
6.07
4.84
van Rossum (1965)
v a n Rossum (1965)
v a n Rossum (1965)
van Rossum (1965)
van Rossum (1965)
Birmingham and
Szolcsanyi (1965)
Birmingham and
Szolcsanyi (1965)
Calculated from
d a t a of Leach
(1956)
Calculated from
d a t a of Leach
(1956)
Calculated from
d a t a of Leach
(1956)
Leonard (1965)
Leonard (1965)
Bickerton (1963)
Tolazoline
Chlorpromazine
5
15
4.85
14.2
Chlorpromazine
15
14.08
Bickerton (1963)
Gokhale et al.
(1964)
Gokhale et al.
(1964)
A modification of the preparation described here is that in which
the vas deferens of the guinea pig or rat is dissected from the animal
with its accompanying hypogastric nerves (Hukovic, 1961; Graham et
al., 1968). Stimulation of the postganglionic nerve induces a strong rapid
1.
α-ADRENERGIC BLOCKING AGENTS
9
contraction of the vas deferens. The hypogastric nerve-vas deferens
preparation is more difficult to use and possesses no inherent advantage
over the isolated vas deferens preparations described here for assaying
antagonistic activity. It is a useful preparation, however, for detecting
depressant activity of a test compound on sympathetic nerve function.
2. Vascular Smooth Muscle
A commonly used preparation in this category is one utilizing spirally
cut strips of the rabbit aorta (Furchgott and Bhadrakom, 1953;
Furchgott, 1960). This preparation has been extensively used in the
analysis of the action of sympathomimetic amines and their antagonists
at receptor sites (Bevan, 1960; Furchgott, 1954, 1967). It possess many
advantages of an isolated organ system. For example, three or four tissue
preparations are available from each aorta, enabling "paired-control"
studies to be made. It is sensitive to low concentrations of adrenergic
agonists, and the tissue remains stable for long periods of time. Contractile responses may be recorded using an apparatus that permits simultaneous recordings from ten arterial preparations (Nash and Luchka,
1965). However, setting the preparation up requires great care, and
an equilibrium period of approximately 2 hr is needed before drugs
can be administered. In addition, contractile responses to «-adrenergic
agonists are slow, and after washing the preparation relaxation is slow.
Rabbits, preferably weighing 2-3 kg, are killed by a sharp blow on
the head, and the thorax is opened to expose the aorta. An incision
is made on the descending part of the aorta, and a glass rod (3-4 mm
in diameter) is slowly inserted. The aorta is carefully removed, using
the glass rod as a guide, and a continuous spiral is cut to obtain lengths
of tissue 2-3 mm wide and 3 cm long. The aortic strips are allowed
to equilibrate for 2 hr in organ baths containing oxygenated Krebs'
solution at 37°C. Recordings of the aortic contractions may be made
via isotonic levers (1:10; 3.0 gm) or through a force displacement
transducer. Furchgott (1967) recommends evaluation of shifts in the
lower part of the dose-response curve (25-50% of maximum contraction)
when studying the activity of antagonists. Log dose-response curves
are plotted for the agonist alone and in the presence of the antagonist.
The shift of the latter dose-response curve from the control gives an
estimate of x, the dose ratio, which is then used to calculate pA2 or
the apparent KB of the antagonist.
The procedures described here for rabbit aortic strips have been successfully employed to study adrenergic activity in various vascular tissues. For example, Birmingham and Szolcsanyi (1965) used spirally
10
V. C. SWAMY
cut strips of the aorta from rabbits, guinea pigs, and cats and from
the carotid arteries of dogs to assess the adrenergic blocking properties
of thymoxamine. The experimental conditions for the aortic strips from
guinea pigs differ in one respect. While the arterial strips from rabbits,
cats, and dogs are suspended in Krebs' solution at 37°C, the tissues
from the guinea pig are bathed in Krebs' solution maintained at 32°C.
Helically cut coronary arteries have been studied for their responses
to catecholamines (Zuberbuhler and Bohr, 1965). Isolated veins, also,
have been used in the form of spirally cut strips to characterize their
adrenergic receptors (Sutter, 1965; Gulati et al., 1968). The pA2 values
for some common α-adrenergic antagonists, obtained on various vascular
TABLE II
COMPARISON OF « - A D R E N E R G I C BLOCKING ACTIVITY ON VASCULAR
Tissue
Agonist
Antagonist
TISSUE
Contact
time
(min)
pA 2
Reference
Rabbit posterior vena
cava
R a t aorta
Norepinephrine
Phentolamine
20
8.00
Calculated from
Furchgott (1955)
Calculated from
Furchgott (1955)
Calculated from
Furchgott (1955)
Leonard (1965)
Birmingham and
Szolcsanyi (1965)
Birmingham and
Szolcsanyi (1965)
Birmingham and
Szolcsanyi (1965)
Birmingham and
Szolcsanyi (1965)
Gulati et al. (1968)
Norepineph-
Phentolamine
15
8.69
Wohl et al. (1967)
Guinea pig
aortic strip
Dog carotid
strip
Cat aortic
strip
nne
Norepinephrine
Norepinephrine
Norepinephrine
Thymoxamine
5
7.20
Thymoxamine
13
6.99
Thymoxamine
—
6.10
Birmingham
Szolcsanyi
Birmingham
Szolcsanyi
Birmingham
Szolcsanyi
Rabbit aortic
strip
30
7.70
Epinephrine
Dihydroergotamine
Phentolamine
20
7.52
Epinephrine
Yohimbine
30
6.70
Epinephrine
Norepinephrine
Macusine B
Piperoxan
10
10
6.67
6.28
Piperoxan
25
6.39
Thymoxamine
10
6.80
Thymoxamine
25
6.88
Epinephrine
and
(1965)
and
(1965)
and
(1965)
1.
α-ADRENERGIC BLOCKING AGENTS
11
preparations, are given in Table II, and are seen to show good agreement
within the limits of experimental variation.
Interpretation of experimental results obtained from vascular smooth
muscle systems must be accompanied by an awareness of differences
in sensitivities of blood vessels to adrenergic agonists. Bevan (1961)
and Bevan and Osher (1965) have reported on the variability in function
of α-receptors in the thoracic aorta, pulmonary artery, inferior vena cava,
and the anterior mesenteric artery of rabbits. Helical preparations of
these vessels demonstrated that the adrenergic «-receptors in the aorta
and pulmonary artery were identical and that they differed in their
adrenoceptive responses from those of the anterior mesenteric artery
or the inferior vena cava. The experimentally determined pA2 values
for thymoxamine on the cat aorta differed from those obtained on the
arterial preparations of rabbits, guinea pigs, and dogs; the noticeably
greater thickness of the cat aorta is suggested as an explanation for
the discrepancy in the pA2 values (Birmingham and Szolcsanyi, 1965).
The pharmacological analysis of the responses of the isolated veins of
the rabbit also led to the conclusion that they do not form a homogeneous system (Sutter, 1965).
Circular segments of the rat aorta are used in the experiments of
Wohl et al. ( 1967 ). The aortic segments are suspended between stainless
hooks inserted into the lumen so that the contractions of the circular
muscle give rise to increases in isometric tension which are measured
by force displacement transducers (Statham, 0.3-1.0 oz). The tissues
are allowed to equilibrate under tension (2 g) for 1 hr before measuring
responses to drugs. Satisfactory dose-response curves to norepinephrine
can be obtained by cumulative addition of the catecholamine in volumes
of 0.05 ml or less. Responses of the tissue remain stable and reproducible
over long periods of time. Antagonists are added to the bath after two
or three dose-response curves for norepinephrine show close similarities.
An arterial preparation with high sensitivity to norepinephrine has
been described by de la Lande and Harvey ( 1965 ). Lop-eared rabbits
are anesthetized with urethane (1.76 gm/kg, i.p.); the central artery
of the ear is exposed, and a segment of this artery 5-7 cm in length
is suspended in the organ bath. The artery is cannulated at the proximal
end, and the lumen is perfused with oxygenated Krebs' bicarbonate
solution maintained at 37°C and containing 5-hydroxytryptamine
creatinine sulfate (0.4 jug/ml). The outflow from the artery is allowed
to drain by upward displacement; the rate of perfusion is maintained
at approximately 8 ml/min. Drugs are injected into the system through
the rubber tubing attached to the proximal end of the cannula. Changes
in the diameter of the artery caused by norepinephrine result in changes
12
V. C. SWAMY
in perfusion pressure which are measured by mercury manometers or
pressure transducers. The main advantage of this preparation is the
stability of its responses and its high sensitivity to norepinephrine (1-2
ng/ml). Responses to norepinephrine are consistent for 6 hr after commencing infusion, and the tissue may be used after storage at 4°C overnight. Responses to norepinephrine in the presence of antagonists may
be studied by adding the antagonist to the perfusion fluid.
3. Seminal Vesicles
The isolated seminal vesicles of the rat and guinea pig have been
used in the evaluation of adrenergic blockade caused by a wide variety
of compounds (Brugger, 1945; Rothlin and Brugger, 1945; Stone and
Loew, 1952; Lewis and Miller, 1966). The seminal vesicles of the guinea
pig were found to be a particularly suitable tissue for the study of
2-halogenoethylamines since they contract in the presence of catecholamines, histamine, and acetylcholine and exhibit little or no spontaneous
rhythmic activity (Meier, 1950; Stone and Loew, 1952).
Male guinea pigs weighing 300-600 gm are killed, and their seminal
vesicles are removed. The contents of the seminal vesicle can produce
excessive distention and prevent maximal contractions. A small opening
into the lumen, therefore, is made at the proximal end, where the ligature
is tied. The vesicles prepared in this manner are straight tubular structures varying from 4 to 7 cm in length. The tissues are suspended
in organ baths containing oxygenated Locke's solution with 0.1%
dextrose and maintained at 39°C. Contractions induced by sympathomimetic amines may be recorded via an isotonic lever system
(1:15, 1.0 gm).
The seminal vesicles of the rat are removed from animals in essentially
the same manner as described for the guinea pig. An added precaution
to follow is to dissect carefully the coagulation glands from the vicinity
of the seminal vesicles. The removal of the coagulation glands must
be made without injury to the seminal vesicles to avoid abnormal responses or reduced sensitivity. The vesicles are allowed to equilibrate
for 15-30 min before being stimulated by drugs. Recordings of drug-induced contraction may be made with an isotonic lever system (1:10;
0.3 gm) or by using force displacement transducers.
The contractile response of this organ to catecholamines may be utilized for studying antagonistic activity of test compounds according to
the general procedures described in the beginning of this section. The
reproducibility and stability of the responses over 3-4 hr prove advantageous in determining pA2 values (see Table I ) . The property of the
1.
α-ADRENERGIC BLOCKING AGENTS
13
seminal vesicles of the guinea pig to contract to acetylcholine and histamine as well as to adrenergic agonists makes this organ particularly
suitable for examination of a wide spectrum of potential antagonistic
activity. In contrast, the seminal vesicles of the rat are insensitive to
histamine, and although they respond in a linear fashion to epinephrine
and acetylcholine, the tissue shows a greater sensitivity to the former
agonist. Another noteworthy feature of the seminal vesicles from the
rat is that their adrenergic responses are mediated almost exclusively
through the α-receptors (Clark et al., 1961).
4. Spleen
Isolated strips of a cat's spleen are sensitive to catecholamines when
suspended in glucose-deficient Tyrode's solution or McEwen's solution
( 1956 ) and are suitable for analyzing the activity of α-adrenergic antagonists (Bickerton et al, 1962; Bickerton, 1963; Bickerton et al, 1966).
The spleen is removed from the cat through a lateral abdominal incision
and washed in warm Tyrode's solution containing one-half the usual
amounts of sodium bicarbonate and dextrose. The sides and the ends
of the spleen are trimmed, and the central rectangular portion is divided
to give two or more strips of splenic smooth muscle, each measuring
approximately 4.5 cm long and 1.5 cm wide. The tissues are allowed
to equilibrate for 30 min in oxygenated, glucose-deficient Tyrode's solution maintained at 39°C. The contractions of splenic strips may be recorded by an isotonic lever system (1:15; 5.0 gm). The response of
this preparation to given doses of catecholamines increased in magnitude over the first four or five trials and then remained relatively uniform
over several hours (Bickerton et al., 1962).
The response of the splenic smooth muscle to catecholamines is slow.
An initially rapid contraction is followed by a slower phase that reaches
a sustained peak in 3-5 minutes. Significant changes in sensitivity reportedly occur when epinephrine and norepinephrine are given in cumulative
doses (Bickerton, 1963). The largest changes in sensitivity over two
consecutive cumulative dose-response curves were observed at lower
dose levels of the catecholamine, i.e., below one-half maximal responses.
This can be a potential source of error in estimating the activity of
a reversible antagonist because the shift of the curves due to changes
in sensitivity may be construed as an effect of the antagonist. This difficulty may be overcome by using paired strips from each spleen, one
which receives the antagonist and one which serves as a control. The
experiments of Bickerton (1963) have utilized such procedures in determining pA2 values of tolazoline, given in Table I.
14
V. C. SWAMY
5. Intestine
The isolated rabbit ileum with intact mesenteric nerves provides a
useful and simple system in which to observe the action of antagonists
at adrenergic neurons and postsynaptic receptors (Finkleman, 1930;
Bowman and Hall, 1970). Young rabbits starved for 24 hr are killed
and the abdomens are opened. Segments (2-3 cm) of small intestine
(duodenum, jejunum, or ileum) and the mesentery, including the
mesenteric artery and accompanying sympathetic nerve, are mounted
in an organ bath containing oxygenated (95% 0 2 + 5% C 0 2 ) Tyrode's
or Krebs' solution at 37°C. A ligature is tied around the central end
of the mesenteric artery, and the mesentery is threaded through electrodes connected to a stimulator. Stimulation ( 15 Hz ) of the periarterial
adrenergic nerves, preferably, is restricted to periods of less than 30
sec at 3-min intervals to avoid fatigue of the preparation. The pendular
movements of the intestine may be recorded on a kymograph with an
isotonic lever system (1:10; 2.0 gm) or with force displacement transducers. Apparently, no differences are seen between isotonic and isometric recording (Bowman and Hall, 1970).
Stimulation of the sympathetic nerve or the presence of exogenous
catecholamines inhibit the intestinal contractions. This inhibitory action
is mediated by a- and /^-receptors and is blocked by both types of
adrenoreceptor antagonists. Separation of α-adrenergic inhibitory effects
from those mediated by ^-receptors is based on the experimental observations that stimulation of a-adrenoreceptors produced rapid onset
of inhibition, whereas the onset of action at the ß-receptor sites was
slow (van Rossum and Mugic, 1965; Bowman and Hall, 1970). Estimation of α-adrenergic antagonists may best be carried out by using
phenylephrine, an α-adrenoreceptor agonist whose inhibitory effects are
blocked by phentolamine while remaining unaffected by ß-receptor antagonists propranolol and MJ-1999 (Bowman and Hall, 1970). Initially,
responses to phenylephrine are obtained and followed by washout of
the agonist from the system. The antagonist is then added to the bath,
and its blocking activity is determined by stimulating the tissue with
the doses of phenylephrine used initially. Due to the variations in the
sensitivity of the tissue, only a rough estimate of pA2 is possible. This
preparation, however, is a simple and convenient one for rapid qualitative characterization of compounds at adrenoreceptive sites.
6. Uterus
The uterus from nonpregnant rabbits responds to α-receptor stimulation by contracting vigorously, and these contractile responses may be
1.
α-ADRENERGIC BLOCKING AGENTS
15
used to estimate the activity of α-receptor antagonists ( Broom and Clark,
1923). Rabbits weighing at least 2 kg are killed and the abdomens
are opened. The intestine is pulled aside to expose the two horns of
the uterus, which are dissected free from their mesenteric attachments.
Each uterine horn is cut into lengths of 2-3 cm, and each portion is
divided longitudinally to obtain a pair of matched strips. An advantage
of this isolated uterus preparation is that a number ( 6-8 ) of such pairedcontrol preparations can be obtained from each animal. The tissues are
bathed in oxygenated Ringer's solution. If necessary, magnesium chloride
(0.1 gm/liter) is added to inhibit spontaneous motility. An isotonic
lever system (1:5; 1.0 gm) provides satisfactory recordings of uterine
contractions.
B. INTACT ANIMAL SYSTEMS
I. Arterial Blood Pressure Responses
a. Anesthetized Cats and Dogs. The chief advantage of this experimental method is that it serves to reveal the activity of the test compound at various sites in the sympathetic neuroeffector system. (Smith,
1961). Dogs or cats may be anesthetized with barbiturates, although
a-chloralose is preferred for cats because of the stable anesthesia it
induces and its generally weaker inhibitory effects on autonomie functions. The trachea is cannulated routinely, and the vagosympathetic
trunks are cut bilaterally. The blood pressure is recorded from the
femoral or carotid arteries, and the drugs are administered through the
femoral or jugular veins.
Epinephrine is a potent stimulant of a- and ò-reeeptors. The stimulation of ô-receptors causes vasoconstriction with consequent pressor response, while vasodilatation and depressor response result from the activation of vascular ^-receptor system. An adrenergic antagonist such
as phentolamine or phenoxybenzamine converts the normal pressor response of epinephrine to a depressor response by blocking «-receptors
and thereby allowing ^-effects of epinephrine to prevail. An experimental
sequence designed to demonstrate α-adrenergic antagonism thus should
(1) produce a reversal of the pressor response of epinephrine to a depressor response and (2) diminish, but not reverse, the pressor response
of epinephrine in an animal pretreated with a /^-receptor antagonist,
e.g., propranolol ( 1-3 mg/kg, i.V.).
A broader and more sophisticated screening procedure for characterizing adrenergic blockade is the one described by Levy and Ahlquist