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MICHIGAN STATE UNIVERSITY

Radha Kanta Mishra
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THESIS

 

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ANTAGONISTIC EFFECT OF SOME ANALEPTICS AGAINST

DEEP PENTOBARBITAL ANESTHESIA IN DOGS

By

Radha Kanta Mishra

A THESIS

Submitted to the College of Veterinary Medicine
of Michigan State University in partial
fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Pharmacology

1965

Respectfully Dedicated To:

My Loving Parents
and
My Eldest Brother

Sri. Ramakanta Mishra

ACKNOWLEDGEMENT

I express my deepest gratitude to Dr. Clyde F. Cairy,
Professor of Pharmacology, for selecting the problem and for
his able guidance, invaluable suggestions and persistent
encouragement in making this entirety a successful effort.

I am profoundly grateful to Dr. Bernard V. Alfredson,
officiating Chairman of the Department of Pharmacology and
Department of Physiology, for his affectionate attitude,
sympathetic advice and encouragement.

My sincere appreciation to the other members of the
guidance committee: Dr. William D. Collings, Professor of
Physiology and Dr. Raymond F. Johnston, Associate Professor
of Physiology, for their constructive criticism and sugges-
tions.

I wish to thank Dr. E.I?.Reineke, Professor of
Physiology for his interest and encouragement.

Thanks to Dr. M. A. I. Khan for assisting in differ—
ent aspects of experiment.

I cordially thank all the faculty and other staff
members of the Pharmacology and Physiology Department and
all my colleagues who have directly or indirectly contributed
a greater share towards my wonderful experience in this cam-

pus which will be imprinted in my memory for all time to come.

iii

TABLE OF CONTENTS

Page

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1
Chapter

I. LITERATURE REVIEW . . . . . . . . . . . . . . 5

Picrotoxin . . . . . . . . . . . . . . . . . 5

Methylphenidate (Ritalin, Ciba) . . . . . . 23

II. MATERIALS AND METHODS . . . . . . . . . . . . 34

III. RESULTS . . . . . . . . . . . . . . . . . . . 42
IV. DISCUSSION . . . . . . . . . . . . . . . . . . 61
Picrotoxin . . . . . . . . . . . . . . . . . 61

Methylphenidate Hydrochloride (”Ritalin”)

Hydrochloride . . . . . . . . . . . . . . 63
Methylphenidate and Picrotoxin

Combination . . . . . . . . . . . . . . . 67

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . 71

BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . 74

iv

INTRODUCTION

Acute poisoning by the misuse of barbituric acid
derivatives has become a problematic phenomenon in the
twentieth century; it is growing by degrees, in pace with
the rapid advancement of the pharmaceutical industries. In
Scandinavian countries, 871 patients, in comparison to 152
in 1932, with acute barbiturate poisoning symptoms were
admitted to the hospital in 1947, a sixfold increase of such
occurrence within a 15—year period. The ease of obtaining
drugs led to serious maladies in the society. In the modern
age under the veil of destitution, disease, and deSperation,
the overwhelming preponderance of the use of barbiturates as
the means of self—destruction is far from uncommon. Shulman
and co-workers (1955) have stated that over the past decade
the amount of barbiturates used on both sides of the Atlantic
has more than trebled and the incidence of barbiturate coma
has increased fivefold.

On the other hand, reports are not rare of the seri—
ous complications arising from the clinical use of the bar-
biturates——either as sedatives, hypnotics, and narcotics in
human practice or as general anesthetic in veterinary use,

where the lives of the patients were threatened.

all

 

 

 

 

 

A quest for the prevention of the barbiturate hazard
brought forward the existence of another group of drugs, the
central analeptics, which are capable of greatly minimizing
the mortality from barbiturate intoxication due to their
antagonistic action. Even though the detailed mechanism of
action of most of the central analeptics is not fully under-
stood, they have been credited with life saving properties
against mild and severe types of barbiturate intoxication,
because of their CNS stimulating properties. As regards the
determination of the most efficacious analeptic for the pur—
pose, controversy still exists though some of the old analep-
tics, such as nikethamide and amphetamine, have definitely
been proved as less effective than the modern ones.

At present, objections have been raised to the use
of analeptics against barbiturate poisoning. Some research—
ers feel that analeptics are effective in mild or moderate
barbiturate poisoning but not useful at the time of actual
need, i.e., in severe barbiturate intoxication (Mousel, L. H.
and co—worker 1941).

There are, however, two schools of thought as to the
treatment of acute barbiturate intoxication, though both
agree to a thorough and intelligent medical approach.

The conservative school, led by Nilsson, strongly
opposes analeptic use, and they claim that these drugs may
cause synergistic depression from the production of convul—

sion in subjects already depressed by barbiturates. Nilsson

explains this phenomenon in the following way:

An increased activity in the cells of the tissue

is created by the cerebral stimulation. An in—

creased activity indicates an increased need for

oxygen. An increased need for oxygen in a tissue,

which is hypoxic in advance, paves the way for

deepening the already existing depression.
Very significant results were obtained by Nilsson (1951).
Locket and Angus (1952), Clemmenson (1954), who solely
relied on the planned medical management. There are certain
disadvantages to this line of treatment: (1) One cannot be
sure, in deep barbiturate coma patients, if and when they
will regain consciousness, and in more prolonged coma more
risk is incurred despite scrupulous care. (2) Serious
trouble will arise in hOSpitals to pay constant attention on
prolonged coma cases.

The medical nihilism advocated by Nilsson is con-
demned by Koppanyi and Fazekas (1952) who strongly claim the
definite role of central analeptics in cases of severe bar-
biturate intoxication. In fact the barbiturate—analeptic
antagonism has been evidenced by innumerable clinical and
laboratory experimental reports. Because of the lack of
adequate information concerning the mode of action of the
analeptics, the Council of Pharmacy and Chemistry of the
American Medical Association cautions against the routine

use of such drugs in barbiturate poisoning cases. Still the

following advantages of analeptics can not be denied.

1. Rapid arousal effect due to the cerebral stimula-
tion associated with visible reflex mechanisms.

2. Stimulation of the vital centers, i.e., respira-
tory and cardiovascular centers in the medulla
thereby reinstating adequate respiration and
maintaining normal circulation.

3. The prolonged coma stage is converted to a ”safe
state” and the threat to the patient's life is
prevented.

4. Prolonged endotracheal intubation becomes unnec—
essary.

5. They afford relief from prolonged and strict
nursing, favoring minimum hospital management

procedures.

In this project, picrotoxin, a very old analeptic,
and another fairly recent one, methylphenidate (Ritalin)
have been chosen because of the many favorable reports of
these two drugs. Their ability to antagonize barbiturates
in deeply barbitalised dogs has been studied individually.
They are also tried in various fractional dose combinations
with the hope of better therapeutic value of the analeptics.
Combinations of other drugs were proven to be more effective
in previous study in this laboratory (Cairy, C. F., and

Sisodia, C. S. 1961; Cairy, C. F., and Giri, Naraian 1965).

CHAPTER I

LITERATURE REVIEW

Picrotoxin

 

Introduction

Picrotoxin, a unique, controversial drug but unequiv-
ocally accepted as a versatile antidote against severe barbi—
turate poisoning, especially against short-acting ones, had
its origin in the early nineteenth century. In the course
of time the early premature concepts regarding the chemistry
and therapeutic uses of picrotoxin have either been modulated
or completely abandoned and a rational, though not concrete,
detailed picture of it has been put forth in the modern age.

Boullay, M., discovered picrotoxin in 1812 from
cocculus Indicus (Anamirta cocculus) called ”Fish berries.“
Its composition was believed to be C12H14OS. Barth and
Kretschy, first to investigate the chemistry of picrotoxin,
thought it to be a complex consisting of three constituents:
the active component, picrotoxin (C15H1606); inactive non-

poisonous components, picrotin (C15H3OOb); and Animirtin.

However in 1911 and 1912 picrotoxin was proved to be a mix—
ture of equal parts of picrotoxinin and picrotin (Cervello
1911; Structural formula, Angelico 1912).

In 1814 Tschudi expressed picrotoxin to be a suitable
antagonistic to morphine. J. Chrichton Brown in 1875 sug—
gested it as a possible antagonist against chloral hydrate
poisoning. Koppen in 1892 established this effect.

Little attention was paid to this discovery until
1931 when Maloney, Pitch and Tatum, and Maloney and Tatum
(1932), on a laboratory animal basis, advocated its ability
to combat the central depression resulting from administra—
tion of doses higher than LDSO of various barbiturates.

Koppanyi and co—workers (1936) further explored the
limits of picrotoxin efficacy by determining the component
factors and a rational explanation of their pharmacodynamics,
as a striking barbiturate antidote. He was first to treat
human barbiturate poisoning. He also pointed out (1936) that
picrotoxin, which was once postulated to be helpful in case
of morphine poisoning, gives adverse effects due to syner-
gistic convulsive actions.

Besides this, many investigators have worked on this
drug with diversified results. Because of such inconsistent
action, picrotoxin was deleted from United States Pharma-
copoeia (U.S.P.), though it was official in the U.S.P. in
1890. It is still officially accepted in British Pharma—

copoeia (B.P.).

Chemistry

Picrotoxin is a non—nitrogenous neutral principle of
emperical formula C3OH34013. It is a loose chemical com-
pound, believed to be an equimolecular mixture of picrotox-
inin (C15016O6) and picrotin (C15H1807), which break apart
on heating and reunite upon cooling. Excepting that picrotin
has an additional molecule of water than picrotoxinin, they
both are allied chemically, possess identical pharmacological
actions but the latter acts more strongly (Angelico 1912).
According to another school of thought, picrotin is inert and
the entire activity ascribed to picrotoxin resides in the
picrotoxinin (Chistoniznuiothers). Some believe picrotoxin
has a slight fraction of a third component, animirtin.

Above all, picrotoxadiene, a degradation product of picrotin
has been synthesized (Conroy 1952). Picrotoxinin indicates
no superiority over picrotoxin in barbiturate poisoning

trials (Kohn, 6938).

 

 

 

 

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Pharmacodynamics of Picrotoxin

The action of picrotoxin in the system is confined
only to the central nervous system and nothing is known of
its distribution and fate in the body (Cushny, Textbook of
Pharmacology and Therapeutics, 1937). The autonomic nervous
system stimulation, eSpecially the parasympathomimetic action
of the drug, is purely of central origin (Hatcher, R. A. and

Weiss, s. 1923).

Central Nervous System Action

 

In general stimulation and depression are preceded
by severe stimulation. In rats sometimes depression occurs
with convulsive doses of picrotoxin.

The C.N.S. stimulation may be divided into the con-
vulsive effect and the stimulation of vital medullary

centers.

Picrotoxin as a Convulsant Drug

In normal animals, the C.N.S. stimulation does not
appear until convulsion occurs. But in case of patients
treated with C.N.S. depressant, it has a critical dose level
up to which level beneficial C.N.S. stimulating effects,
like arousal symptoms, result, and beyond that a severe con—
vulsion followed with prolonged depression is observed.
This post convulsive depression sometimes leads to fatal

consequences.

There is a time lag of a few minutes for picrotoxin
before any of its C.N.S. action is exhibited. This latency
which decreases upon increase doses complicates its analeptic
effect as convulsion immediately occurs. This is because
picrotoxin has a narrow therapeutic ratio. In rabbits the
difference between the convulsant and lethal dose is very
small in comparison to pentylenetetrazole.

The typical convulsive effects of picrotoxin is char-
acterized by clonic, asymetrical and coordinated type of
response. In the case of frogs with higher doses the Spinal
stimulation becomes more prominent. Although the convulsions
occur spontaneously in cycles alternating with depression,
they are prevented by removal of the skin in frogs. Convul—
sions require the presence of sensory impressions (IL Shriver
and Perschmann 1935). Optic and acoustic stimuli do not
elicit generalized convulsion in cats treated with picro—
toxin. In dogs a simultaneous increase in reflex irritabil-
ity and a convulsant response in spinal cord is seen (Klein,

J. R. 1943).

Site of Convulsion

Although picrotoxin is commonly known as a medullary
stimulant, regions of the C.N.S. share in stimulation as the
drug dose increases. The actual seat of action moves in an
ascending direction as the higher parts of the C.N.S. become

more developed. In fish picrotoxin action is medullary as

10

the convulsion remains unaltered after decerebration, be—
comes slightly abbrogated with destruction of optic lobes
and the typical picrotoxin symptom is completely lost after
section from medulla. At this stage, the convulsion due to
spinal stimulation looks like a strychnine response. In
mammals the site of action is chiefly mid—brain and cerebrum.
In man the cerebrum seems to be more particularly involved.
The glycogen content of the cortex is increased during
picrotoxin induced convulsion (Chance, M. R. A. 1951), and
the acid phOSphatase in the largest cells of the motor cor-
tex is decreased. However, the brain stem seems to be more

involved in picrotoxin convulsion than pentylenetetrazole.

Medullary Stimulation

The life saving property of picrotoxin against barbi-
turate poisoning is due to the stimulation of the vital cen-
ters--re5piratory and circulatory--located in the medulla.
It increases the rate and depth of respiration, and it in-
creases blood pressure. Besides that, the vomiting, salivary
and other centers are also stimulated with the analeptic dose

of picrotoxin.

Depressing Effect of Picrotoxin

Post convulsive depression is more pronounced than
pentylenetetrazol in rats and rabbits. Loss of placement
reaction indicates that cortex is depressed. Confusion fol—

lowed with unconsciousness proves that the drug first

—
—
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stimulates and then depresses the cerebral cortex (Dille and
Hazleton 1939). Depression duration increases with the in—

creased length of convulsion.

Action on Autonomic System

 

The autonomic nervous system stimulating effect of
picrotoxin is well established. The fact remains debatable
whether the parasympathetic or sympathetic action is more
pronounced. In any case all possible autonomic effects are
of central origin.

The old literature favors the parasympathomimetic
action of picrotoxin. In 1909 Grunwald for the first time
proved this fact by establishing a variety of parasympathetic
nerve action. In his vivid description and rational explana-
tion, he suggests that picrotoxin acts on all autonomic cen-
ters; the centers controlling the cranial and sacral autonom—
ic nerves are especially susceptible to this drug. Salivary
secretion is increased by the stimulation of the nucleus of
the chorda tympani; the heart is slowed by vagus center stim-
ulation. Occulomotor center is stimulated causing constric-
tion of the pupil. In the same way sweating with dilation
of skin vessels, contraction of the bladder and uterus, and
lacrymation occur. All these effects cease on severing the

nerves.

12

The fall in body temperature is due to direct effect
on heat regulating and heat producing center (Harnack). In
the late convulsive stage, the mydriasis and tachycardia are
due to paralysis of parasympathetic nerve endings. Increased
peristalsis of intestine is another feature of late convul-
sions. Rogers, F. T. (1918) has shown tetanic stomach con—
traction in turtles with picrotoxin. It induces ovulation
in rabbits probably by stimulation of the hypophysis (Brooks,
C. M. et al. 1940).

Recent investigators give enough evidence about the
sympathetic effect of picrotoxin: pronounced hyperglycemia
(Rosenthal, F. E. 1941), raised blood pressure, inhibition
of diuresis probably by the constriction of renal arteries
and cessation of rectum and bladder contractility in the
rabbit. The central origin of these effects is proved on
section of spinal cord or appropriate Splanchnic nerves.

The peripheral autonomic effect of picrotoxin, if
any, is supposed to be negligible. Weakening of the excised
frog heart with concentrated picrotoxin solution, stimula-
tion and depression of rabbit intestine (excised) with small
and large drug doses respectively, depression of motor
nerves and skeletal muscle (Ogawa, M. 1928) and finally
hemolysis; these factors contribute to the weak peripheral

effect of this drug.

13

Further advanced study on isolated organs depict
many nonspecific effects. There is no indication of neuro-
muscular junction stimulant action of picrotoxin (Huston,

M. J. and Martin, A. W. 1951). It does not inhibit cholin-
esterase or sensitize the leech muscle to acetylcholine.

Its action on the acetylcholine content of the brain depends
upon the anesthetic used.

In sum, the predominant effect of parasympathomi—
metic and sympathomemetic action of picrotoxin establishes

its central autonomic nervous action.

ReSpiratory Action

 

Picrotoxin is a definite respiratory stimulant in
normal and anesthetically depressed patients; full convul-
sant dose is needed for the former case and subconvulsant
dose for the latter. The respiratory response in normal
cases is least observed till convulsion sets in, though
occasionally respiration is stimulated before convulsions
occur.

The pharmacodynamics of picrotoxin is attributed to
its direct reSpiratory center stimulation at the medullary
region; not carotid chemoceptor stimulation (Krantz, J. C.
and co-workers 1937; Marshall, E. K. and co—workers 1937).
Besides, the sensitivity of the inspiratory center to

electrical stimulus is increased.

14

With convulsive doses the respiration becomes irreg-
ular during muscular twitching, ceases during clonic spasms
and this phenomenon continues alternately until the convul-
sive stage is overcome or fatality results due to the pre-
cipitation of permanent respiratory arrest during spasms.

As a barbiturate antagonist, its success in increas—
ing the reSpiration, both rate and depth has been suggested
by a great number of researchers (Maloney et a1. 1931, 1932;
Das, S. C. 1939; Chakravarti, M. 1939; Thorp, R. H. 1947).
It has been further stated that picrotoxin reinstates ade—
quate reSpiration and greatly lightens the degree of anes-
thesia.

Marshall and co—workers (1937) and others have also
indicated that it is capable of stimulating respiration in
chlorbutanol, trimethylbutanol, paraldehyde and Avertin
anesthesia, but ineffective against ethanol. They suggest
in these cases picrotoxin behaves as a chemical antidote
rather than pharmacological antidote.

Moderate reSpiratory depression by morphine poison—
ing is antagonized by picrotoxin (Hahn, F. 1960). Koppanyi
and co—workers in 1936, 1941 and others disregard this drug
as a morphine antagonist because of its synergestic convul-
sive response with morphine leading to more adverse conse—

quences.

15

Cardiovascular Response

 

Subconvulsant dose of picrotoxin increases blood
pressure. An initial rise followed by a fall in blood pres—
sure 8—10 seconds before the onset of convulsion is observed
with convulsive doses (Pollock and Tredway :UIK3 and Pollock
and Holmes 1915). The possible cause, they explain, is by
one or more of the following mechanisms:

1. Vagus stimulation

2. Stimulation of cardiac inhibiting center; both

leading to cardiac slowing

3. Depression of vasomotor center

4. Some cardiac muscle fatigue.

Return of normal blood pressure after the convulsive
seizure is apparently due to fatigue of the vagus nerve.

The central origin of the vasopressor action of picrotoxin
is evidenced by cumulative experimental findings.

Picrotoxin very strongly antagonizes the barbiturate
induced circulatory failure by its central action (Maloney,
A. H. and Tatum, A. L. 1932) and improvement of the carotid
sinus reflex (Chakravarti, M. 1939). The occasional ineffi-
ciency of picrotoxin to combat the decreased pressure re—
sponse in deep barbitualized animals is due to its lack of
peripheral activity which is possibly depressed to some

extent by barbiturates.

16

Action on Body Temperature

 

Reduction in body temperature in normal subject due
to picrotoxin is inevitable either by heat loss (Hahn, F.
1960) via sweat and dilatation of skin vessels due to auto-
nomic center stimulation or by decreased hypothalamic func-
tion. In the absence of convulsions pronounced fall in body
temperature due to skin vessel dilatation and sweating is
observed (Harnack). Paradoxically the subnormal body temper-
ature caused by barbiturates and paraldehyde is prevented by
picrotoxin (Koppanyi and co—workers 1936; Maloney 1932; and

Rosenthal 1941).

As a Barbiturate Antidote

 

Notwithstanding the few shortcomings of the data on
the merits of picrotoxin—barbiturate antagonism, the pre—
ponderance of reports leave no room for doubt about its
superb life saving effect against severe barbiturate poison-
ing.

Maloney and co—workers (1931) were first to proclaim
its pronounced barbiturate antagonistic effect and its clin—
ical use. Immediately many investigators pinpointed their
attention on this aspect and many research articles came out
in support of this; the outstanding, detailed and more
rational explanatory reports of Koppanyi and co—workers

(1936); Barlow, O. W. (1933); Chakravarti, M. (1939);

17

Jarvinen, P. A. and Vartiainen, A. (1949); Lavenson, G. 8.
Jr., Plum, F. and Swanson, A. G. (1958) are some of them.

Picrotoxin acts as a physiological antidote and not
as a chemical antidote. The blood barbiturate level essen—
tially remains the same in deeply barbitalized control
animals and the animals successfully treated with massive
doses of picrotoxin (Koppanyi and co—workers 1936). In some
rare instances the awakened animals destroy large amounts of
hypnotics due to their improved physiological condition and
thereby reduce the blood barbiturate concentration. Picro—
toxin also does not enhance the elimination rate of the bar—
biturate concentration. Picrotoxin also does not enhance
the elimination rate of the barbiturates (Dille 1938).
Further, that picrotoxin and other analeptics cause a com—
plete arousal from barbiturate narcosis before sufficient
time is elapsed for the chemical destruction of the barbi-
turates in the system indicates that the antagonism is
purely of physiological nature.

Some general aSpects of the review of F. Hahn (1960),
Koppanyi and Pazekas (1952), Koppanyi and co-workers (1936),
Reifenstein and co-workers (1939), and of the council on
pharmacy and of chemistry (1939), on picrotoxin—barbiturate
antagonism are discussed below.

Whereas picrotoxin combats the adversities of some
barbiturates—~sodium barbital, pentobarbital, etc. admin—

istered in multiple lethal doses, it is impotent against

18

certain other barbiturates. Picrotoxin has limited action
against barbiturates in the sense that the latter is capable
of reversing several LD50's of picrotoxin whereas the reverse
is not true. This is perhaps due to the barbiturates‘ effect
on a more extensive area on C.N.S. than the picrotoxin
(Koppanyi and co-workers 1936).

When these two drugs are administered together, a
direct reversal of the depressed state does not occur but
instead a combined form of intoxication of the two drugs
intermingled with depression and stimulation is exhibited
from which the animal eventually recovers (Council on Phar-
macy and Chemistry report 1939).

Picrotoxin is rapidly eliminated from the system
(Dille 1938, Dille and Duff 1939). Hence regular repeated
doses of the drug are given to counteract the long acting
barbiturate intoxication. It gives excellent results against
short acting barbiturates with a single dose of 2 mg/kg in
dogs. Furthermore, because of the typical distribution
nature of the two kinds of barbiturates in the living body,
picrotoxin is injected intravenously (i.v.) for short act-
ing barbiturates and either intramuscularly (i.m.) or sub—
cutaneously (s.c.) for long acting barbiturates.

It antagonizes easily higher doses of short acting
and sub-lethal doses of long acting barbiturates. Against

the lethal dose of the latter barbiturate picrotoxin, within

19

limits, brings about a cure and in doses beyond that, the
efficiency of the analeptic is completely lost. But the life
of the patient may be prolonged for a few hours.

Picrotoxin efficiency in poisoning cases depends on
the depth of anesthesia, deeper the better is the cure and
vice versa, though the upper limit of its perfect usefulness
is restricted to two times the lethal dose (Jarvinen, P. A.
and co-worker 1949). Thus a conspicuous increase in the
Speed of recovery against the lethal dose of short acting
barbiturate is more prevalent than with the long ones.

A dual action of cortical and medullary stimulating
properties of picrotoxin brings about the barbiturate antag—

onism.

Cortical Action

Koppanyi and co-workers (1936) have established the
involvement of higher cortical centers in higher vertebrates
by a couple of ways: (1) the barbiturate denarcotising
property and (2) the preventive measure against barbiturate
narcosis. The participation of cortex is also seen by reac-
tivation of the E.E.G. (Hahn, F. 1960). However this prop—
erty becomes insignificant with too small doses of the drug
(Cass, N. M. 1956).

The cortical arousal effect depends on: (1) the
depth of anesthesia and the dose of anesthetics and (2) the

type of anesthesia—cortical stimulation is exhibited against

20

a minimum anesthetic dose, unassociated with convulsion.
With higher doses the awakening effect is superimposed on
convulsive features. In the laboratory, dogs can be awakened
from barbiturate narcosis (long acting and short acting) by
picrotoxin injection, but the action against the former type
is transient and the animal goes back to deep sleep with the
abolition of the analeptic effect, whereas permanent arousal
is achieved against short acting barbiturates. This pecu-
liar characteristic behavior of picrotoxin against long act-
ing barbiturates should not be confused with the assumption
that it reduces the recovery time in such instances.

Maloney and co—workers (1931 and 1932) have been
able to induce sleep in dogs by barbiturates and awakening
them with picrotoxin. Cushney (1934) states picrotoxin to
be less significant than coriamyrtin in its rapidity of
arousal property.

The action which is responsible for the denarcotis-
ing effect is not responsible for the convulsion as experi-
mental animals (dog) exhibit convulsion under deep comatose
condition (Koppanyi and co—workers 1936). This is further
shown by Dille and Duff (1939) with morphine poisoning in
dogs.

Sporadic reports indicate prophylactic action of
picrotoxin retarding paralysis and accelerating recovery if

given prior to administration of narcotics.

21

Medullary Action

The medullary stimulating property of picrotoxin is
by far the most versatile action against barbiturate poison-
ing as it helps restore the vital body function, i.e., in-
crease in ventilation and blood pressure by direct stimula-
tion of reSpiratory and vasomotor centers located in the
medulla oblongata. Other centers in that region are in-
volved with convulsive doses of the drug. In acute barbi—
turate poisoning, it dramatically terminates the poisoning
effect by reinstating adequate respiration and maintaining
normal circulation. These properties have been discussed in
detail elsewhere in the chapter.

Maintenance of normal body temperature by picrotoxin

in barbiturate poisoning cases are also seen.

Mode of Action as an Analeptic

There is no chemical reaction between picrotoxin and
the barbituates in vitro. In vivo also it acts as a physio-
logical antagonist (Koppanyi and co-workers 1936). It has
no action on chemical distribution or elimination of the drug
from the body (Kahn, J. B. Jr. 1952).

McCulloch, W. S. and Roseman, E. (1943) and Fujita,
N. (1956) explains that picrotoxin increases oxygen consump-
tion (in rats) of brain tissue depressed with barbiturates.
Krantz, Carr and Beck (1937), however, have shown this is

not the direct mechanism to abolish the nerve center

22

depression. Hence increased oxygen consumption may thus be
the consequence, rather than action, of picrotoxin—barbitu-
rate antagonism.

Koppanyi, T. and Fazekas, J. F. (1952) in a general-
ized view say picrotoxin acts: (1) either by causing arousal
or awakening if depression is not too marked; (2) if deep
coma exists and arousal is not effected they may be life sav-
ing by virtue of their medullary stimulating activity.

Very recently experimental evidence has been presented
by VanDerKloot and co—workers (1958, 1959) regarding the
counteracting quality of picrotoxin against the peripheral
depressing activity of barbiturates. They have shown the
antagonistic property of picrotoxin on the inhibitory neuron
of the abductor muscle of the crayfish claw. Gamma-amino-
butyric acid mimics the effects of the inhibitory transmitter

on crayfish muscle. Its action is also blocked by picrotoxin.

Absorption and Elimination

Picrotoxin is effective by oral or parenteral admin-
istration, but absorption from the digestive tract is incom-
plete. Absorption through intact skin is questionable.

Traces of picrotoxin are detected in blood 20 minutes
after intravenous injection; by the end of 2 hours it is
impossible to demonstrate at all in blood, liver, or muscles

(Dille and Duff 1939).

23

Its distribution in the body is unknown. Perhaps it
is readily destroyed inside the system. After massive injec—
tion, traces are recovered from urine which indicates that it
is diSposed of from the body in some unknown way. A convul-
sant dose of picrotoxin in rabbit was destroyed within one
hour and only 10% was excreted in the urine (Dille, J. M.
1938). According to Chistoni, A. (1912) picrotoxin, to some
extent, is eliminated unchanged but usually breaks apart to
two components, picrotoxinin being completely destroyed and

picrotin largely eliminated unchanged in the urine.

Methylphenidate (Ritalin, Ciba)

Introduction

Methylphenidate was for the first time introduced in
Europe in clinical practice by Drassado and Schemidt in 1954.
In the same year Meier, Gross and Tripod indicated the ana~
leptic effect of the drug against thiopental anesthesia
based on laboratory animal experiments.

By now, though not much investigation has been done
about its biochemical and detailed pharmacological action on
the system, it is being commonly used in human medicine for
two purposes—-as a mild psychomotor stimulant and as an ana—
leptic, both are the manifestations of central nervous system
stimulation. Its use in veterinary practice is still of

little significance.

24

The psychoanaleptic action of methylphenidate in
human patients was studied in detail by Ferguson, J. T.
(1953 and 1956). Until December 1956, oral administration
was the only way of combating regressive psychiatric condi—
tions (Ferguson, J. T. 1955 and 1956; Carter, C. H. 1956)
with methylphenidate. Ferguson advocated the superiority
of parenterally (i.v.) used methylphenidate to the oral
route in 1956 also. Because of its non-toxicity to tissues
it may be given by sc., i.m. or i.v. route (Plummer, J. and
Yonkman, F. 1958).

The potentiality of methylphenidate against exces-
sive sedation and lassitude exhibited by barbiturates, anti—
histaminics and tranquilizers has been confirmed by both
clinical observation and laboratory experiments. Christensen
and Fox have reported the success of methylphenidate injec—
‘Cion in hastening the recovery from barbiturate anesthesia
used in oral and other surgery.

Plummer, J. and Yonkman, F. F. (1958) and Gale, A. S.
(1959) have presented a vivid description of various pharma-
cological actions of methylphenidate, its peculiar dose
relationship and eSpecially its antidepressant action. Gale
has stated that the effectiveness of methylphenidate is
independent of the kind of depressant agent but is more
related to the depth of depression and to some extent to the
age of the patient; the very old and the very young seem to

respond best.

a
T

 

 

 

 

25

Carter and Maley (1957) reported favorably on the
effectiveness of methylphenidate in nine cases of barbitu—
rate overdose. This beneficial action was claimed by
Adriani and Smith (1958) to be definite after trying its
efficacy against hundreds of barbiturate intoxication
patients. According to them it is more effective than older
analeptics like picrotoxin and pentylenetetrazole in mild
and severe barbiturate intoxication.

However little effort has been made to prove its
usefulness in severe barbiturate poisoning cases in veteri~
nary practice. We have tried to solve this aspect to some

extent in our laboratory.
Chemically

Phenyl—piperdye—acetic acid methyl ester or methyl—

phenidate
CHCOOCH ° HCl
<3.

3
Trade name: Ritalin

26

Pharmacological Action

Circulatory Effects

 

Methylphenidate, in effective doses, does not affect
the blood pressure level of the patient. As an antidote for
psychic depressants (Reserpine etc.), when administered
along with reserpine or afterwards, it has little effect in
altering the hypotensive effects of the latter drug (Plummer,
A. J. and Maxwell, R. A. 1956); Ferguson, J. T. and Funder-
burk, W. H. 1956). With high doses, capable of causing
central motor stimulation (running movements), the blood
pressure rise was not so much deviated from the normal level
(Maxwell and co-workers 1957). It is useful in optimal
doses to counteract the declined blood pressure caused by
prolonged use of anesthesia (Gale, A. S. 1958). Much higher
doses cause a transient rise in blood pressure of about
20 mm.Hg (Plummer, A. J. and Yonkman, F. F. 1958). Further
reports indicated its usefulness in protecting against the
postoperative decline in blood pressure greater than 10 mm.Hg,
though drop below this limit is not uncommon.

A very rational explanation pertaining to the circu-
latory response of methylphenidate and interesting informa—
tion about its synergistic and antagonistic actions with
other pressor agents has been presented by Maxwell and co—

workers (1957). The drug possesses an antihypertensive

27

effect of central origin and a potentiating action on the
response produced by peripherally acting sympathomimetic
drugs. Blockade of elevated pressure elicited by bilateral
carotid occlusion, prompt reduction of pressure raised by
amphetamine and ephedrine (Gale, A. S. 1958, 1959; Plummer,
A. J. and Yonkman, F. F. 1958) and finally initial moderate
decline in the blood pressure of unanesthetized renal hyper-
tensive dogs characterizes the centrally mediating mechanism
of methylphenidate (Charter, C. H. and Maley, M. C. 1957).
On the other hand, it potentiated the pressure response of
epinephrine, nor—epinephrine and naphthazoline the periph-
eral sympathomimetic drugs. This phenomenon was confirmed
by Ivey, E. P. (1959). Maxwell, R. A. and Plummer, A. J.
have explained the above observations as follows:

Methylphenidate (Ritalin) antagonizes the pres-

sure response to amphetamine via a central

blockade of cardiovascular centers which are

influenced by amphetamine and ephedrine. A

peripheral blocking effect of it can only be

supported if one postulates that amphetamine

and ephedrine have different peripheral cardio-

vascular receptor sites than do epinephrine,

nor-epinephrine and naphthazoline, where

methylphenidate selectively blocks the former

pressure raisers and potentiates the pressure

raising effect of the latter groups.
The synergistic pressure response of Ritalin with epinephrine

plays a significant role in shock treatment by reducing the

epinephrine dose to a great extent (Gale, A. S. 1959).

28

Central Nervous System Action

 

The mild C.N.S. stimulating effect of methylpheni-
date has been reported by many investigators and medical
practitioners. But up-to—date information does not indicate
the exact site of action of this drug on the C.N.S. However,
the accumulating evidences of its psychoanaleptic effect
(Ferguson, J. T. 1955 and 1956; Meier at al. 1954) and the
stimulation of the vital centers, i.e., respiratory, circu-
latory centers (Adriane, J. and Smith, B. 1958; Gale, A. S.
1958) against barbiturate and tranquilizer overdoses defi-
nitely depicts its cortical and medullary stimulating prop-
erties. The cortical arousal activity was further proved
by Plummer, A. J. and co—workers (1958) who recorded the
first encephalogram activity especially in the leads from
mesencephalic reticular formation, following methylphenidate
administration. The prompt C.N.S. stimulation property of
methylphenidate unaccompanied by depression rebound has
been taken advantage of in not infrequent clinical use.

In therapeutic doses its convulsant effect is minimal. Even
in cases of severe barbiturate intoxication and pronounced
tranquilizer depression the drug used in repeated doses

exhibitsits antidotal potency with least untoward effects.

29

Methylphenidate is a direct C.N.S. stimulant. It is
not a biological competive agent nor does it have blocking
agent property (Gale, A. S. 1958). One single optimum dose
of methylphenidate maintains C.N.S. stimulation for 3-4
hours.

A peculiar dose relationship has been recorded with
regard to the C.N.S. stimulation. Methylphenidate in doses
of 0.1—0.2 mg/lb. body weight is most effective. Doses below
0.05 mg/lb. and above 0.4 mg/lb. however, markedly reduce the
stimulating property (Gale, A. S. 1958). There is no explana-
tion for this behavior but to observe that other stimulants

might show such response in higher doses.

Action Against Cortical Depression

In this country methylphenidate, as a matter of fact,
was used clinically as a psychoanaleptic agent against men—
tally retarded patients (Charter, C. H. and co—workers 1955).
Besides the preponderance of evidences in support of this
(Ferguson, J. T. 1956; Ferguson and co-workers 1957; Plummer
and Maxwell 1956), it has further been established as an
antidote against more severe forms of psychophysiologic
depressions, depressions which may be drug induced (Charter
and Maley 1957). Ferguson acknowledges it as the forerunner
of all the drugs tested in controlling, ameliorating or
eliminating the abnormal behavior manifestation seen in

elderly patients. The dramatic response brought about by a

30

single parenteral dose is possibly due to a chemicophysiolog—
ical change within the central nervous system rather than the
action at some specific anatomical sites.

Heart rate, blood pressure, respiration and kidney
function remains unaltered in cases of prolonged massive
methylphenidate injection to mentally ill patients.

Methylphenidate Tranquilizer
Antagonism

 

 

Many observations have been made about the methyl—
phenidate, reserpine, and chlorpromazine antagonism at
various dose levels of the latter groups of drugs and this
has been accepted as a very good antidote against the mood
normalizing, mild corticomotor depression and oversedative
effects of tranquilizers. Plummer and Maxwell (1956) report
that methylphenidate produces complete reversal of the so—
called side reactions of reserpine (in tranquilizing dose)
excepting the hypotensive effects. After the lapse of 3
hours the typical reserpine effects i.e., miosis, nictitating
membrane relaxation, bradicardia, etc. are exhibited. Fail-
ure of ganglionic blockade principle to check the nictitating
membrane relaxation in dogs explains its Specific type of
antagonism at C.N.S. In fact a clear cut antagonism to the
sedative effects and other Side reactions of central origin,
of tranquilizers has been Shown by Plummer and co-workers

(1958) in dogs, rabbits and monkeys.

31

Methylphenidate—Barbiturate
Antagonism

 

 

Although the sedative and anesthetic dose reversal
effect of methylphenidate is agreed upon generally, both in
laboratory experiments and clinical use, controversial re—
ports have been presented concerning its efficacy in combat-
ing barbiturate intoxication. Gale, A. S. (1959) and Plummer
and Maxwell (1958) believe this drug is a moderate antidote
against mild and severe barbiturate intoxication, whereas
Adriani, S. A. and co-workers (1958) claim it as a Superb
barbiturate antagonist in comparison to other analeptics
like pentylenetetrazole, picrotoxin and nikethamides.
Charter and Maley (1957) have also given similar opinions.
However in consideration of its apparently Specific central
stimulation of respiration, ”alerting” of consciousness
effect and the high therapeutic index, it seems to be a use—
ful analeptic. Further, it can be injected parenterally by
s.c., i.m. or i.v. route with similar results. The paren-
teral injection seems to be well tolerated both locally and
parenterally as it does not create any tissue changes at the
site of injection, due to extravasation. There was no
evidence of phlebitis (Ferguson, J. T. 1956).

Methylphenidate increases the respiratory rate and
depth in cases of mild and severe type of barbiturate anes—

thesia. Prompt recovery is seen against light anesthetic

32

doses. A reasonable ventilation increase along with rapid
arousing effect adds to the efficacy of methylphenidate as

a barbiturate antagonist, though the latter effect is not
seen promptly in extreme intoxication cases. Unlike other
analeptics of phenylisopropylamine type it does not possess
cardiovascular stimulating activity. Severe postoperative
barbiturate chill and euphoria after intravenous barbiturate
anesthetics—~these two common complications are greatly
eliminated with methylphenidate injection.

It acts better against short acting barbiturates
(eSpecially pentothal) rather than long acting ones. Because
of transient action of methylphenidate it should be injected
at repeated intervals till a significant long acting barbitu-
rate depression reversal effect is observed.

A synergistic analeptic effect has been recorded
when injected along with levallorphan tartarate (Lorfan)

(Gale, A. s. 1959).

Methylphenidate Against Other
Agents

 

A high percentage of patients depressed with general
anesthetics, barbiturates (pentothal, secobarbital, Neraval,
Surital, Na—pentobarbital), alcohols, aSpirin, tranquilizers
and antihistaminics are reported to improve rapidly and
appreciably after methylphenidate injection. But comatose

patients recover slowly.

33

Gale recorded prompt initiation of respiration in
some patients anesthetized with general anesthetics with
prolonged apnea considered to be due to respiratory center
depression rather than muscular relaxation.

Lastly methylphenidate is believed to counteract the

reSpiratory and circulatory effects of anesthetic adjuvants.

CHAPTER II

MATERIALS AND METHODS

Experimental Animals

 

Dogs, irreSpective of age, sex and breed, were
chosen for the project. These animals were obtained from
the local dog pound and were maintained in individual cages.
Commercial dog food* and water were supplied ad libitum.
Forty—two dogs, including 6 dogs for control, were used in
this experiment. Most of them weighed 10 to 12 kgs, though
dogs varying from 7 to 20 kg in weight were used according
to availability. Thirty—six dogs were divided into 6
groups and each group was treated either with a Single drug
or combination of two drugs in different doses. Usually,
unused dogs were taken for each experiment. Rarely the same
dog was selected a second time. On no occasion were the dogs
used which had been anesthetized for any other experimental
work in the preceding week. The diseased animals were com_

pletely eliminated from the project.

 

7”(Purina Dog Chow.

34

35

Drugs Used

 

Sodium pentobarbital 6 per cent solution with 10 per
cent ethyl alcohol was employed to anesthetize the dogs.

Picrotoxin was 0.3% soln. (Eli Lilly & Company,
Indianapolis and Abbott Laboratories).

Methylphenidate hydrochloride (”Ritalin” — Ciba
Pharmaceutical Company, Summit, N.J.). Crystalline powder

was used to make 1 per cent solution with sterile saline.

Apparatus Setup

 

The apparatus consists of a four channel "Grass
polygraph”* and a wet test gas meter. All four channels in
the polygraph, from top to bottom, were used to record the
reSpiratory rate, ventilation, cuff pressure, and the pulse
wave in order and for convenience this principle was ob-
served in all experiments. The third channel used for cuff
pressure was calibrated before each experiment in Such a way
that one cm. deflection represented 100 mm.Hg pressure. The
other three channels, except for bringing them to a certain
base line, were not calibrated as they were primarily of
qualitative importance.

The time was recorded in one—second interval.

 

*Grass Instrument Company, Quincy, Mass., U.S.A.

36

The pulse wave was recorded by means of an electric
pulse pickup (Grass Model PTTl) fixed to the interdigital
Space of one of the hind legs and to the fourth channel of
the polygraph.

In order to record the cuff pressure a high pressure
transducer was used which was connected to the third channel
of the polygraph at one end and at the other end to a 2” wide
cuff fixed below the hock region of the hind leg also bearing
the pulse pickup. The indirect blood pressure was assessed
from the aforementioned two tracings which will be discussed
further in the section ”Experimental Technique.”

A wet test meter was employed to measure expired air
as a measure of ventilation volume and reSpiratory rate.

Both of these values were finally recorded on the polygraph.
The gas meter was so constructed as to record each 0.25
liters (250 ml) of air by means of an electrically operated
signal pointer. This was finally recorded in channel two of
the polygraph. As regards to respiratory rate, in each
expiration a signal was picked up by a low pressure trans—
ducer touching to a thin rubber flutter valve at the gas

meter outlet and transmitted to the Grass polygraph.

37

Experimental Technique

 

At the outset the dog, clipped at the site of injec-
tion, was weighed and sodium pentobarbital was injected
intravenously via a radial vein. Forty mg/kg body weight
were used to induce deep anesthesia, to decrease the level
of minute reSpiratory volume and eventually to curtail the
respiratory rate markedly. A rubber tracheal tube with an
inflation cuff around its distal end was then inserted into
the trachea through the mouth. The cuff was inflated from
outside with an aim to fix the tracheal tube comfortably
enough to allow air to pass freely into the trachea and out
only through the tracheal tube. The degree of inflation was
indicated by a pilot balloon. The tracheal tube was con—
nected to the gas meter by means of a rubber tubing with
special valves for inhalation from the room and exhalation
through the gas meter.

It was observed in our laboratory that the indirect
blood pressure measured by fixing the cuff below the hock
region gave reading closer to direct blood pressure than fix«
ing the same above the hock region. At intervals, by Squeez—
ing the bulb attached to the cuff, the latter was inflated
and pressure was built up around the vessels which caused
complete disappearance of pulse wave. This was indicated by
the fourth pen drawing a straight line on the graph instead

of the usual pulse wave tracings. As pressure was slowly

 

 

38

released from the cuff the third pen gradually moved downward
and the fourth one just in the opposite direction till the
pulse wave returned. Then the pressure was completely
released from the cuff and the cuff pressure curve came down
to the base line. A straight line drawn upward from the
point of reappearance of the pulse wave to the cuff pressure
curve, indicated the indirect systolic blood pressure read-
ing. The indirect blood pressures were recorded from time

to time throughout each experiment.

Now after all these arrangements, the pulse wave,
indirect blood pressure, ventilation and respiratory rate
were recorded for at least 10 minutes. Some dogs responded
pretty well to a single pentobarbital dose in which cases
minute respiratory volume dropped down to one liter or less.
In other cases where the first dose failed to bring about
significant reduction in the ventilation, repeated doses of
10 mg/kg or 5 mg/kg i.v. of pentobarbital were injected
every ten minutes to lower the ventilation to the desired
level, i.e., 1.25 liters or less. Curiously enough some
dogs, irreSpective of their weights and sizes, failed to
respond sufficiently to a total massive dose of 75 mg/kg
pentobarbital and the ventilation stayed at 1.75 liters,
exhibiting a typical individual drug resistance phenomenon.
However, in general, when the minute respiratory volume was

lowered to around 1.25 liters, polygraph tracings were made

39

for 10 minutes to observe the ventilation level. This is
the right time for trying the efficacy of the analeptics.

In our laboratory it was observed by different trial
that 6 injections of methylphenidate hydrochloride 5 mg/kg
i.v. at 20 minute intervals gave the best result for methyl—
phenidate alone; because of this, 6 doses of 5 mg/kg at 20
minute intervals were regarded as the full dose. A 2 mg/kg
i.v. single injection of picrotoxin was also considered as
the single full dose. Accordingly 6 injections of methyl—
phenidate in 2.5 mg/kg and 1.25 mg/kg i.v. at 20 minute
intervals and one injection of picrotoxin 1 mg or 0.5 mg/kg
i.v. had been referred in the description as half and one—
fourth doses.

A group of 6 dogs figge taken for the study of the
deep pentobarbital antagonistic effect of a particular dose
of an individual drug or drugs in different combinations.

In drug combination injections, both the drugs were taken
into different syringes and one was injected immediately
after the other; every time picrotoxin was run in first.
Immediately, one dose of methylphenidate was given. Then at
20 minute intervals, five more doses of methylphenidate were
given.

A tabulation of experimental condition is in Table 1.

40

 

.mcH o

 

 

 

 

 

 

 

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.>.H mxxmw m.o :onHoHUHw Ao.mq-o.oave.ma o mmw.o o k
. CH
.>.fi mxwms W mpwpficmnma>nyoz
.>.H mx\me H :onHoHUHH Ho.mk-o.oqvk.am o mmm.oH a a
.>.H mx\me m :onHoHUHm Amm-oevqw.mq o 0He.mH o m
mHm>HoHCH .cfiE
om Hm .haH o
.>.H mxxwe oH wHachazmHHEHmz Ho.om-o.oqqu.ae a soo.o o a
mHm>Hopcfi .CME
om Hm .mcH o
.>.H mamme m maawHemganapmz Ho.m©-o.oqvm.kq o mk.o a m
mHm>Hopcfl .GME
om Ha .mcH a
.>.H mx\me m mHmuHcmeaHHEHuz Ao.oo-o.oqvm.ka o mk.o o m
AHWMU PXQC
pofib mop mcov
use: mac: Am©-oqv cm 0 o.oH o H
Amx\wev mmSHQ HmHOUHpc< Amx\wev Eswpom mcpmmo Amxv mmom QSOHU
omoQ HmpHDHmQOHCmQ mo .02 .Hz .m>< mo
mo mmmcmm mmoQ .oz

pcm omoo .m><

 

 

mcoflufiocoo HmpcoEmeaxm .H mHQmH

41

In every case after the end of the last injection, record—
ings were taken for one and one-half hours to observe the
analeptic effect of the drugs.

The radial vein was chosen as the site of injection
of analeptics and anesthetics as well. Many times slight
increase in ventilation and reSpiratory rate observed during
or immediately before the injection of the analeptics was
evidently a reflex mechanism manifestation due to the mechan-
ical stimulus originating at the site of injection.

The reasons for using indirect blood pressure and
pulse wave recording devices were three, i.e., to save time,

for convenience and lastly to avoid sacrificing the dog.

CHAPTER III

RESULTS

The pentobarbital antagonistic responses of these

two analeptics——methylphenidate and picrotoxin, used as

four individual drug doses and in two combinations, with

respect to ventilation percent increase above the pre—drug

level are presented below in the following ascending order

of efficiency. These are also shown in the graphs in sub-

sequent pages. Actual ventilations before the injection of

analeptics are also shown in graphs at ”liters at 0 minute.”

1.

Six doses of methylphenidate 10 mg/kg i.v. at
20 min. intervals.

Deep pentobarbital anesthesia without analep-
tics (control group).

Six doses of methylphenidate 3 mg/kg i.v. at
20 min. intervals.

Six doses of methylphenidate 2.5 mg/kg i.v. at
20 min. intervals and picrotoxin 1 mg/kg i.v.
one injection.

Six doses of methylphenidate 5 mg/kg i.v. at
20 min. intervals.

Six doses of methylphenidate 5 mg/kg i.v. at

20 min. intervals and picrotoxin 0.5 mg/kg i.v.
one injection.

42

43

7. Picrotoxin 2 mg/kg i.v. one dose.

Picrotoxin alone in full dose (2 mg/kg) was found to
be most effective in increasing ventilation. Six injections
of methylphenidate 10 mg/kg i.v. at 20 minute intervals was
least effective. Six injections of methylphenidate 5 mg/kg
i.v. at 20 minute intervals used as drug combination pro—
duced very satisfactory overall analeptic effect.

Quite frequent return of pedal, corneal, palpebral
and occasional cough reflexes coupled with severe paddling
movements, raising the head and objecting to the intubation
were the arousal signs of analeptics. In addition some dogs
coughed for a while soon after the tracheal tube was removed
at the end of the experiments.

The arousal effect was further evidenced in some
dogs by rapid heart rate, increase in respiratory depth and
rate, increased minute respiratory volume, violent running
movements, struggling, extreme stage of excitement, twitch~
ing of muscles of neck, eyebrow, lower jaw region with
occasional general muscular twitching, profuse salivation
and so on~—all these being more pronounced in case of picro—
toxin treatment alone. Details of these effects will be
enumerated under the discussion.

Picrotoxin increased blood pressure in all cases

whereas methylphenidate, both alone and in combination,

44

altered blood pressure only slightly.

Abbreviations in the Graphs

 

M

Methylphenidate
P - Picrotoxin
Pb — Pentobarbital
V — Ventilation
R — Respiratory rate
B.P. — Blood pressure

() — Arousal after analeptics and no more ventilation

record due to struggling and excitement

-&'— Indirect blood pressure recording stopped as the
electronic pulse pickup could not be fixed to

the interdigital Space due to struggling

II - Injection of a single drug.

 

45

 

 

 

 

 

 

 

 

 

3 6 INJECTIONS OF METHYLPHENIDATE 10 MG/KG
3 AT 20 MINUTE INTERVALS
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METHYLPHENIDATE 10 MG/KG--Continued

 

 

 

 

 

 

 

 

 

 

 

 

 

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47

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48

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Minutes

' 12. Bitch 9 kg
Pb - 60 mg/kg

 

 

 

 

200 "

 

I

160%‘

increase

80%

cent

V

0%

V. per

 

Average for 6 dogs

 

 

00‘

A
1

r

 

 

 

49

6 INJECTIONS OF METHYLPHENIDATE 5 MG/KG
AT 20 MINUTE INTERVALS

 
 

 

 

(,3

 

U)

3-:

m

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‘2 m Pb - 40 mg/kg
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14. Dog 12 kg
Pb — 50 mg/kg
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15. Dog 12.5 kg
Pb - 40 mg/kg

 

 

 

 

 

 

 

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Pb — 40 mg/kg

 

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50

METHYLPHENIDATE 5 MG/KG-—Continued

 

 

 

 

 

 

 

 

 

 

3 Average of 6 dogs
I: «- ’
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+.§ <v -20 Pb — 50 mg/kg 6‘? -
6" *5
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Minutes

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Pb — 60 mg/kg

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51

PICROTOXIN 1 MG/KG ONE INJECTION + 6 INJECTIONS OF METHYLPHENIDATE
2.5 MG/KG AT 20 MINUTE INTERVALS
19. Dog 10 kg 21. Bitch 8 kg
Pb - 55 mg/kg Pb - 60 mg/kg

 

 

 

 

 

 

 

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Minutes

20. Bitch 7.75 kg
Pb — 55 mg/kg

fir
I
Q

 

 

22. Dog 7 kg
‘ Pb — 40 mg/kg

 

 

 

 

 

 

52

PICROTOXIN 1 MG/KG + METHYLPHENIDATE 2.5 MG/KG——Continued

Average for 6 dogs

liters

 

23. Dog 12 kg

 

 

 

 

 

 

 

 

 

 

 

o Pb — 60 mg/kg 8‘?
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53

AT 20 MINUTE INTERVALS

 

54* L
0 40 80 120 160 200
. Minutes
28. Dog 9 kg

- Pb — 4o mg/k

 

 

3
I
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26. Bit

Pb

 

 

 

 

 

 

 

PICROTOXIN 0.5 MG/KG ONE INJECTION + 6 INJECTIONS OF METHYLPHENIDATE

9 kg
— 45 mg/kg

 

 

 

 

 

 

 

1' - 27. Dog 13.7 kg
Pb — 40 mg/kg n
V/
JP "
#'_/r””7
I I II I II I
¢.L l 1 1 1 I 1 1 1

 

PICROTOXIN 0.5 MG/K

    
 
 
 
 
   
 
 

 

 

54

+ METHYLPHENIDATE 5 MG/KG-—Continued

  

 

A
T

 

 

'160
‘140 ,
29. B1t h 8 kg
Pb 45 mg/kg
120
‘100
‘80
—60
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55

PICROTOXIN 2 MG/KG ONE INJECTION

31. Dog 12 kg
Pb — 55 mg/kg

  

 

 

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Mgnutes

32. Dog 20 kg
Pb - 70 mg/kg

 

 

 

SI

 

 

33. Dog 17 kg
- Pb — 40 mg/kg

 

34. Dog 18 kg
Pb - 40 mg/kg

 

12'0

10 II

I #100

56

PICROTOXIN 2 MG/KG—-Continued

35. Dog 12.5 kg
Pb - 40 mg/kg

'80 n

I
O
O

 

'

I
4S
0

V. pig min. in liters
R S Rate
I
8
.8

 

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60 100

Minutes

36. Dog 13 kg
Pb - 45 mg/kg

.31
:4 J‘

 

 

 

 

400%?

 

240

ncreage

80%u

V. percent i

0%

 

 

Average for 6 dogs

 

 

 

 

I

«400%‘

320%4

240%1

160%"

8(flh“

0%)"

Ventilation percent increase

 

57

AVERAGE PERCENT IN VENTILATION AT TWO DIFFERENT
DOSE LEVELS OF PICROTOXIN AND METHYLPHENIDATE

 

 

I I. II I' ll
H—o—o Picrotoxin 0.5 mg/kg i.v. + 6 injections of
Methylphenidate 5 mg/kg at 20 min. intervals
Picrotoxin 1 mg/kg i.v. + 6 injections of
""" Methylphenidate 2.5 mg/kg i.v. at 20 min.
intervals
I 1 I l I J l L l l -
4O 80 120 160 200

Minutes

58

AVERAGE PERCENT INCREASE IN VENTILATION AT THREE
DIFFERENT DOSE LEVELS OF METHYLPHENIDATE ALONE

 

 

 

 

 

 

280%L
240%"
J»
160%" g
m
o
u
9 U
c
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. ‘23
80%, w
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a I‘d
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-H
4.:
407“:
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1 I l I ,l .l l n 1 I 4 l
0 4O 80 120 160 200
Minutes

~—«—+—~ 6 injections of Methylphenidate 5 mg/kg i.v.
at 20 minute intervals

vwmmwmm 6 injections of Methylphenidate 3 mg/kg i.vo
at 20 minute intervals

“gynan 6 injections of Methylphenidate 10 mg/kg i.v,
at 20 minute intervals

400% -

320%

340% '

160%

80%

0%

Ventilation percent increase

 

59

THREE BEST ”AVERAGE VENTILATION PERCENT INCREASE”
OF ALL TRIALS USED IN THIS PROJECT

0

n

'3

‘I

B—HE—iP1I Picrotoxin 2mg/kg i.v. one injection
Picrotoxin 0.5 mg/kg i.v. one injection +
a—a—G—O 6 injections of methylphenidate 5 mg/kg i.v.
at 20 minute intervals
___._"__ 6 injections of Methylphenidate 5 mg/kg i.v. at
20 minute intervals

I I I l I I J I l I I

0 4O 80 120 160 200

Minutes

L200

1130

 

60

AVERAGE RESPIRATORY RATE, VENTILATION IN LITERS PER MINUTE
AND BLOOD PRESSURE OF SIX DEEPLY BARBITALIZED DOGS WITH NO
ANALEPTIC TREATMENT

 

 

 

 

 

r “20
3 R.
m‘lO
m 9}
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.5
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94°
“8
ans ‘ l l 1 | 1 lo CIL
0 10 20 40 60 80 90

Minutes

CHAPTER IV

DISCUSSION

Picrotoxin

 

Picrotoxin alone in doses of 2 mg/kg i.v. in deeply
barbitalized dogs, did an excellent job to increase the
ventilation level. Immediately after injection, there was
a 40% increase in minute reSpiratory volume; within 30
minutes ventilation sharply increased by as much as 170%,
stayed there for another half hour, and by the end of one
and one-half hours, it was escalated to a highly commendable
level, approximately a 400% increase. This pehnomenon indi-
cates that picrotoxin is a pharmacological barbiturate antag-
onist. The effect is brought about by the direct central
nervous system stimulation, eSpecially the cortex and the
vital centers in the medulla oblongata, i.e., respiratory
and circulatory centers; this agrees thoroughly with the
vivid pharmacological action of picrotoxin described earlier.
However, in contrast to many investigators' reports, none of
the 6 dogs treated with picrotoxin exhibited the 15-20 minute
time lag which is usually sited as the greatest drawback in

its therapeutic use.

61

62

We further noticed that when picrotoxin was injected
slowly and continuously within a period of 4—5 minutes, its
side effects, like severe convulsion and profuse salivation,
were greatly minimized, though vomition, micturition, defeca—
tion and occasional Sporadic muscular twitches were observed
in one or the other animal used in this experiment. The
reduced convulsant effect is possibly due to the combination
of the following two properties of picrotoxin:

1. It is rapidly destroyed in the system.

2. The concentration of the drug in the cortical
region is not sufficient for the precipitation
of convulsions. Instead this drug level is
capable of producing desired cortical stimula-

tion so as to awaken the deeply depressed dogs.

Two out of the six dogs did not show typical rise in
ventilation or arousal when treated with picrotoxin. This
may be attributed to individual variation to drug reSponse.
Another two dogs which showed muscular twitching, excessive
salivation, vomition and defecation, took a full 20 hours to
stand up, which is believed to be due to picrotoxin post-
treatment exhaustion.

In general picrotoxin seemed to be an excellent bar-
biturate antagonist in 100% of the cases by either awakening
the dogs from severe C.N.S. depression or by keeping the

patient in a ”safety zone” from where it slowly recovers to

63

normal health.

Besides, picrotoxin raises the blood pressure of the
deeply anesthetized animals by virtue of its direct vasomo-
tor center stimulation. In this experiment picrotoxin
raised the blood pressure by 35 mm-Hg above the original
level.

Picrotoxin alone in 2 mg/kg i.v. dose was found to
be most effective barbiturate antagonist of all tried in
this project.

Methylphenidate Hydrochloride
(”Ritalin“5 Hydrochloride

 

 

In search for the best methylphenidate dose against
severe barbiturate depression in dogs, various single and
repeated doses were tried in this laboratory. The author
has taken into consideration only three groups of dogs for
comparative study of methylphenidate efficiency when injected
intravenously in 3, 5 and 10 mg/kg doses; such six injections
being given at 20 minute intervals.

Since the experiment was based primarily on the study
of ventilation increase, various methylphenidate antidotal
dose trials which did not show any substantial elevation in
ventilation were rejected.

A deeply barbitalized dog treated with a single dose
of the analeptic — 30 mg/kg i.v. gave a decrease in ventila-

tion. Another dog injected first with 15 mg/kg i.v. produced

64

slight increase in ventilation which died down to a minus
level soon after the same dose was repeated after twenty
minutes. Identical results were obtained with 10 mg/kg i.v.
doses. But then 5 mg/kg i.v. injection in regular repeti—
tive doses did not decrease the ventilation; instead a clear
cut gradual ascending trend of ventilation was demonstrated.
As a result, the previous doses were abandoned as the possi—
ble barbiturate antidotal doses.

A group of 6 dogs was treated each with 3 doses of
methylphenidate - 5 mg/kg i.v. given at 20 minute intervals
and then watched for one and one—half hours after the last
injection for any change in ventilation level. On an aver-
age, a 100% increase in ventilation was recorded with a
minimum of arousal effect.

With the expectation of further beneficial results,
another series of 6 dogs was treated with 6 doses of 5 mg/kg
i.v. at 20 minute intervals, and as usual the effects were
recorded for one and one—half hours. In general a very
remarkable rise in ventilation, rate and depth of respira—
tory rate and finally typical arousal symptoms were exhibited.
Thus, this was considered to be the standard barbiturate
poisoning antidote in this experiment. Two other groups of
dogs were also studied with 3 and 10 mg/kg i.v. 6 doses at
20 minute intervals and their results were obtained which

will be discussed separately later in this chapter. Six

65

doses of 5 mg/kg methylphenidate was found to be the best of
three individual doses of the drug used in this project.

In the case of a 5 mg/kg analeptic dose, ventilation
increased by 35% at the tenth minute, it then gradually in—
creased to 75% by the end of the 6th injection. There fol—
lowed a somewhat abrupt elevation of ventilation which
increased by as much as 230% at the end of the experiment.
This was accompanied by a very significant change in respira-
tory rate and depth also. But the blood pressure was little
affected in the course of treatment. In certain cases corti—
cal stimulation aroused the animals also.

There was some initial fluctuation in ventilation
level with the 3 mg/kg dose of methylphenidate. After a
transient 20% increase in ventilation it dropped down to
12% then rose to 45% after one hour, followed with a drop
to 30% by the nineteenth minute, and finally a slow and
steady rise up to 70% by the end of the experiment. There
was also some rise in the respiratory rate.

The 10 mg/kg dose produced an entirely different
picture. Ventilation remained reduced from the beginning
of the analeptic therapy till to the end of the experiment.
Soon after the first injection, ventilation decreased by 24%.
Then it went up to -4% by the end of the tenth minute fol—
lowed with a gradual descent to -20% for one hour and forty

minutes. Then a very slow rise in ventilation developed,

66

terminating at last on a par with the original pre—treatment
ventilation level. In fact, the 10 mg/kg dose proved to be
the poorest of all trials in this project. Two animals died
24 hours after the therapy as they failed to recover from
the barbiturate depression.

From the above discussion it is strongly believed
that methylphenidate bears a definite dose relationship with
its therapeutic value. When the standard dose (6 injections
of 5 mg/kg at 20 minute intervals) is doubled or reduced to
3 mg/kg, a great deviation from the usual analeptic effect
is seen. The variation is more amplified as the standard
dose is doubled. This clearly indicates that any other dose
of methylphenidate but the standard one will act less effec-
tively to combat the barbiturate depression. A very similar
type of observation has been made by Gale, A. S. (1959) in
his laboratory. Unfortunately this phenomenon is hardly
explained. Perhaps a certain optimum methylphenidate concen~
tration is needed for desired C.N.S. tissue cell excitation.

Methylphenidate used alone or in combination with
other drugs against barbiturate poisoning seems to be devoid
of any cardiovascular action. Even though it does not raise
the depressed blood pressure in this experiment, it is be-
lieved to protect the animals from the drastic fall in blood

pressure expected from barbiturates.

67

Over and above all, any significant rise in ventila-
tion with methylphenidate is noticed after the end of the
sixth injection only. Although considerable time is lost in
the meanwhile methylphenidate seems to maintain the animals
in a very safe stage by increasing the ventilation to some
respectable height.

Methylphenidate and Picrotoxin
Combination

 

 

Picrotoxin 1 mg/kg i.v. + 6 injections of methyl~
phenidate 2.5 mg/kg i.v. at 20 minute intervals (A) and
picrotoxin 0.5 mg/kg i.v. + 6 injections of methylpehnidate
5 mg/kg i.v. at 20 minute intervals (B) were the two combina-
tions used in this project out of which the latter combina—
tion was found to be more effective than the former. As a
matter of fact, so far as improvement in ventilation is con-
cerned, this combination's efficiency ranks second from the
top, with a maximum 326% increase over the original level.

Picrotoxin at 0.5 mg/kg with 6 doses of methylpheni-
date - 5 mg/kg raised the ventilation by 20% initially.

Then there was a gradual increase in ventilation up to 80%

by the end of one and one-half hours. Following that, it
immediately shot up, reaching a 326% increase by the end of
the experiment. This big peak of ventilation is considerably
more than the increase observed with 6 doses of methylpheni-

date alone, though it was in less than the single dose of

68

picrotoxin 2 mg/kg i.v. It is therefore believed that a

full dose of methylphenidate and one-fourth dose of picro-
toxin synergistically increase the ventilation. Blood pres—
sure remained uniform throughout the experiment in all six
dogs; methylphenidate is not expected to alter blood pressure
and one—fourth a full dose of picrotoxin may be too small to

affect blood pressure. In addition, methylphenidate may

antagonize the pressor effect of picrotoxin. Besides, side
effects are minimum. Recovery time is greatly reduced. All
six dogs were able to walk within three to six hours. The

2—drug-cortical stimulation synergism and the protecting
property of methylphenidate from rebound depression—-these
two behaviors might be attributed as a possible explanation
of the aforesaid phenomenon.

Half and half dose combinations of the two drugs,
however, increased ventilation in deeply barbitalized dogs
very poorly in comparison with the preceding one; even the
reSponse in this respect is less than the full dose effect
of methylphenidate alone. In accordance with the efficiency
of this combination on ventilation percent increase, it
occupied the fourth position in the series. Within the first
four minutes, ventilation went up by 36% (twice as much as
the rise by the other combination at the same time) reduced
to 24% by the tenth minute, then rose to 50% by the end of

thirty minutes (same as the other combination at this point);

69

but unlike the previous combination, it remained flat at
this level for one and one—half hours followed with a some-
what appreciable rise in ventilation which was increased by
as much as 130% at the end.

So, it was observed that there was less ventilation
percent increase (130%) with 1 mg/kg i.v. one injection of
picrotoxin and 6 doses of methylphenidate 2.5 mg/kg i.v°
than with 0.5 mg/kg i.v. one injection of picrotoxin and 6
injections of methylphenidate 5 mg/kg i.v. In the latter
combination ventilation percent increase was 326%. Devia—
tion from the optimum methylphenidate dose (discussed ear—
lier in this chapter) might be a possible reason for this
response. Doubling the picrotoxin from 0.5 mg/kg to 1 mg/kg
could not compensate the ventilation percent drop as this is
still a very ineffective analeptic dose of picrotoxin. When
two dogs were treated with 1 mg/kg i.v. picrotoxin, ventila—
tion percent increase averaged to be a mere 50% which is a
very insignificant result in comparison to 400% increase by
the 2 mg/kg injection of picrotoxin. At any rate the 130%
increase by half and half drug combination is definitely a
consequence of the additive, rather slight synergestic,
effect of methylphenidate and picrotoxin since 3 mg/kg dose
of the former drug and 1 mg/kg dose of the latter drug sep-
arately increased the ventilation from the original level

70% and 50% respectively.

70

The arousal effect was less pronounced and recovery
time slightly increased in comparison with the other combina-
tion.

Blood pressure as usual remained almost constant from
the beginning to the end probably due to the failure of the
1 mg/kg picrotoxin and 2.5 mg/kg methylphenidate doses to

stimulate the vasomotor center centrally.

SUMMARY

Methylphenidate and picrotoxin alone and in two

combinations were tried in this project to study their

ability to improve ventilation in dogs overanesthetized by

barbiturates and were found to be in the following descend-

ing order of efficiency:

1.

2.

Picrotoxin 2 mg/kg i.v. one dose.

Six doses of methylphenidate 5 mg/kg i.v. at
20 minute intervals and one dose of picrotoxin
0-5 mg/kg i.v,

Six injections of methylphenidate 5 mg/kg i.v.
at 20 minute intervals.

Six injections of methylphenidate 2.5 mg/kg
i-v. at 20 minute intervals and picrotoxin
1 mg/kg i.v. one injection.

Six injections of methylphenidate 3 mg/kg i.v,
at 20 minute intervals.

Deep pentobarbital anesthesia without analep—
tics (control group).

Six injections of methylphenidate 10 mg/kg i.v.
at 20 minute intervals.

Picrotoxin alone was found to be excellent to in-

crease the ventilation. Six doses of methylphenidate

72

10 mg/kg were poorest of all. In fact this dose was com—
pletely ineffective. It rather produced regressive effects.
Occasionally picrotoxin prolonged recovery time due to post
treatment exhaustion.

Methylphenidate, in therapeutic doses, also worked
as a very good barbiturate depression antagonist. It was
found to possess less side effects and to be free from
rebound depression. Further, methylphenidate used alone or
in combination did not change the blood pressure.

Picrotoxin and methylphenidate were also observed to
be potent enough to bring about arousal effects in barbital-
ized dogs. The superior awakening property was invariably
noticed with picrotoxin alone, not infrequently with drug
combinations and occasionally with 5 mg dose of methylphen—
idate alone. As evidenced from the data, picrotoxin had
better arousal effect than methylphenidate in deep pentobar-
bital anesthesia. No chemical or pharmacological antagonism
was observed between picrotoxin and methylphenidate when
used in combinations.

So far as early recovery time is concerned (when the
animal is fully able to stand up that time is considered in
this project as recovery time) picrotoxin 0.5 mg/kg i.v. one
injection and 6 doses of methylphenidate 5 mg/kg i.v. at 20

minute intervals combination appeared to be the best of all

73

trials against deep pentobarbital anesthesia. Under such
conditions the side effects were least and ventilation
percent increase was quite significant.

The author strongly believes methylphenidate could
be of value in veterinary practice either alone or with
other analeptics in the treatment of severe barbiturate

poisoning.

U1

10.
11.
12.
l3.
14.
15.
16.

17.

BIBLIOGRAPHY

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Cairy, C. F., Leash, A. and Sisodia, C. S. Journal
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Carter, C. H. Psychiatric Research, 4:44-48, 1956.

 

Carter, C. H. and Maley, M. C. Diseases Ner. System,
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Chakravarthi, M. J. Pharmacol., 67:153-174, 1939.

 

Chance, M. R. A. Brit. J. Pharmacol., 6:1-7, 1951

 

Chistoni, A. Zentr. Bioch. Bioph., 132556, 1912.

 

Clemmensen, C. Acta, Med. Scand., 148:83, 1954.

 

Conroy, H. J. Amer. Chem. Soc., 74:3046—3051, 1952.

 

Cushny, A. Text Book of Pharm. and Therapeutics, 1939.

 

Das, S. C. Quart. J. Exp. Physiol., 29:355—365, 1939.

 

Dille, J. M. J. Pharmacol., 64:319-329, 1938.

 

Dille, J. M. and Hazleton, L. W. J. Pharmacol., 67:276-
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74

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J. Pharmacol and Exp. Therap., 116220, 1956.

 

Ferguson, J. T., Linn, F. V. 2. et al. J. A. M. A.,
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Ferguson, J. T. and Funderburk, W. H. J. A. M. A.,
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Fujita, N. Jap. J. Pharmacol., 5:151—173, 1956.

 

Gale, A. 8. Anesthesiology, 192101, 1958.

 

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Grunwald. Arch. F. EXP. Path. U. Pharm., 602249,
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Hahn, F. Pharmacological Reviews, 122477-481, 1960.

 

Huston, M. J. and Martin, A. W. Arch. int. Pharmacodyn.,

 

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