BLOOD STUDIES IN DOGS
FOLLOWING THE INJECTION
OF PENICILLIN

Thesis for the Degree of M. S-
MICHIGAN STATE COLLEGE

W. O. Brinker
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BLOOD STUDIES IN DOGS

FOLLOWING THE INJECTION OF PENICILLIN

 

_ ___

BLOOD STUDIES IN DOGS
FOLLOWING THE INJECTION OF PENICILLIN

w. o. gamma

A THESIS

Submitted to the Graduate School of Michigan
State College of Agriculture and Applied
Science in partial fulfilment of the
requirements for the degree of

MASTER OF SCIENCE
Department of Animal Pathology

1947

'01:,

ACKNOWLEDGEMENT

The writer wishes to express his sincere apprecia-
tion to Dr. E. T. Hallman for making available the facili-
ties for this investigation and for his helpful advice.

Gratitude is also expressed to Dr. C. S. Bryan for
his able guidance in solving many technical problems and
for supplying various materials, to Dr. L. B. Sholl and
Dr. Bernard V. Alfredson for their helpful and encourag-
ing suggestions during the course of this study and their
contributions on the final arrangement of this manuscript,
and to Dr. Frank Thorp Jr. for many valuable suggestions
concerning the final arrangement of this manuscript.

Thanks are due many others who have aided in

various ways.

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TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . 1
REVIEW OF LITERATURE . . . . . .'. . . . . . . 2
METHODS . . . . . . . . . . . . . . . . . . . . 25
RESULTS AND DISCUSSION . . . . . . . . . . . . 29
SUMMARY AND CONCLUSIONS . . . . . . . . . . . . 124

BIBLIOGRAPH O O O O O O O O O O O O O O O O O 127

 

INTRODUCTION

One of the major problems confronting the veteri-.
nary practitioner today is the paucity of basic knowledge
underlying the use of drugs. With the advent of newer
antibiotics, which are more specific in action, and more
specific in methods of administration this defect becomes
increasingly apparent. If these agents are to be used to
the best advantage in the treatment of infections caused
by susceptible organisms, basic studies are essential.

The following study was limited to certain obser-
vations on penicillin in dogs. The two main objectives
upon which this investigation was based were: (1) The
determination of penicillin blood plasma levels in dogs
following the administration of this agent in Various ve-
hicles and by the different routes of injection. (2) A
consideration of the immediate effect of penicillin on the

blood picture following d.ngle injections.

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REVIEW 9}: LITERATURE

The results obtained by the use of vehicles to re-
tard absorption of penicillin compared with those obtained
by dissolving it in physiological saline have been report-
ed by various authors.

Armstrong, Halpern, and Cutting (1945) after ex-
perimental work on dogs using various vehicles concluded
that none were more effective than 5 per cent dextrose a-
lone in sustaining penicillin levels in the blood sera.
The various vehicles which they used included methyl
cellulose, human plasma, 12 per cent sucrose, 12 per cent
lactose, sesame or peanut oil, 4.5 to 25 per cent Sorbitol,
8.5 per cent gelatin and urea, congo red and dextrose,
epinephrine and gelatin, and pitressin and dextrose. In us-
ing 5 per cent dextrose as a vehicle in human patients,
they found that with smaller GIBBS of penicillin the in-
creased level could hardly be called important, but with
larger doses it was great enough to be theoretically val-
uable. With doses of 40,000 units and 80,000 units, the
measurable serum level was maintained for one hour longer
than a similar dose given in physiological saline.

Fish, Foord and Allis (1945) compared the serum
levels obtained in rabbits after injections of penicillin
in physiological saline with those obtained from the in-

jection of penicillin in physiological saline and adrena-

 

line. Using saline as a vehicle, measurable levels were
maintained for approximately 1.5 hours, whereas, saline plus
adrenaline maintained the measurable level for 5 to 4 hours.
The addition of adrenaline was observed to level off the
peak and increase the duration of the blood concentration
curve. Similar results were obtained in human patients.
Saline used as a vehicle maintained a measurable level for
approximately 2 hours while saline plus adrenaline maintain-
ed it for approximately 2.5 to 5 hours.

Parkins, Wiley, Chendy and Zintel (1945) report that
the use of colloidal gelatin as a vehicle with the addi-
tion of privine or neosynephrine had a definite action on
sustaining blood levels in the dog and in man. They com-
ment that the colloidal gelatin delayed absorption and exv
tended the effect of the vasoconstrictor as well as that
of the penicillin, and the vasoconstrictor, in turn, delay-
ed the absorption of the gleatin acting as a vehicle for
penicillin. In comparison with physiological saline as a
vehicle, colloidal gelatin sustained the level twice as
long, and colloidal gelatin plus privine or neosynephrine
sustained the penicillin level about three times as long.

The efficacy of various agents for delaying absorp-
tion of penicillin in the horse was reported by Doll and
Dhmock (1946). They point out that the absorption of peni-
cillin was somewhat erratic when vasoconstrictors were used
in conjunction with.physiologica1 saline, saline and dextrose

or gelatin solutions. Since the vasoconstrictors produced

only a short prolongation of the effective blood level
period, the authors consider them as offering very little
advantage in the therapeutic use of penicillin. Their re-
sults also indicate that the use of dextrose or gelatin in
combination with vasoconstrictor drugs, offers no signifi-
cant advantage over saline preparations.

A simple rapid technique for preparing water-in-
oil emulsions of penicillin was reported by Freund and
Thompson (1945). They prepared the emulsion by first in-
jecting 1.4 cc. of sterile physiological saline into a
sterile vial containing 100,000 units of penicillin. To
this solution was added 3.1 cc. of a lanolin-peanut oil
mixture. This addition was made with a sterile syringe a-
dapted with a seventeen gauge needle. Emulsification was
accomplished by repeated with-drawals and injections of the
penicillin—oil mixture from the syringe into the vial con-
taining the original penicillin.

Cohn and others (1945) using the emulsion technique
reported measurable serum levels in man at four hours in
comparison to measurable levels at three hours following a
single injection of 150,000 units in aqueous solution. They
also reported that giving part of the total unitage in aqueous
solution and part in water-in-oil emulsion sustained the
level somewhat longer. This appears to be substantiated in
practical clinical tests in the treatment of gonorrhea as
thn, Kornblith, Crumstein, Freund and Thompson.(1946) found
that one injection of 150,000 units of sodium penicillin.in a

water-in—oil emulsion effected a cure of 101 patients in a
series of 105; also that the simultaneous injection of
100,000 units in water-in-oil and 50,000 units in aqueous
solution cured all 49 patients treated.

The results reported by Ory, Iilcox and Finland (1946)
on comparative serum levels following single intramuscular
injections of penicillin in saline and penicillin in water-
in-oil emulsion did not substantiate those of Cohn and others
in that the latter did not sustain the serum level longer.
When tested in the same subject, they found the serum con-
centrations obtained after a single injection of penicillin
in a water-in-oil emulsion were not superior to those obtain-
ed with the Same dose given in the same volume of saline,
and the levels were not better sustained. Their findings
were based on single intramuscular injections of 100,000,
200,000 and 500,000 units.

Horus, Wilcox and Finland (1946) reported on a second
series of observations on comparative penicillin serum
levels. This time "emulgan" which is composed of sesame
oil and cholestrin base was compared with saline as a
vehicle. When tested on the same patient, saline com-
pared very favorably with.the water-inpoil emulsion as a
vehicle in sustaining the penicillin serum level.

Doll and Hull (1947) found that an emulsion of
water-in-oil as a vehicle for the intramuscular administra-
tion of penicillin produced no delay in absorption as comp

pared with.physiological saline as a vehicle in'horses and

sheep. On the basis of 500 units per pound of body weight
the saline vehicle maintained a measurable level for 2.5 to
5 hours, and in the case of water-in-oil emulsion the level
was maintained for 5 hours in horses. In sheep, on the basis
of 500 units per pound of body weight, saline maintained the
level 4 hours and water-in-oil emulsion maintained the levdl
4 to 5 hours.

Jones, Donaldson and Allen (1946) found that the use
of magnesium sulfate monohydrate with sodium penicillin and
peanut oil delayed the absorption of penicillin. They re-
ported finding 0.05 units per cc. of serum in eight of elev-
en patients twenty-four hours after a single subcutaneous tn-
jection of 250,000 units in this vehicle. Clinical results
showed 108 of 115 cases of gonorrhea (95.6 per cent) "cured"
using this method.

Penicillin was suspended in a dry sterile vegetable
oil (peanut oil) by Raiziss (1944). The blood of rabbits
was found to be bacteriostatic against Staphylococcus 325225
for a longer period of time following an intramuscular in—
jection of penicillin in the oil suspendion when compared
with a similar injection in aqueous solution. He also founi
that penicillin in oil.was therapeutically more effective
in.the treatment of experimental rabbit syphilis than were
aqueous solutions of penicillin.

Favorable therapeutic serum concentrations (0.007
and 0.019 units per cc.) were maintained for eight hours in

25 patients after a single intramuscular injection of 150,030

 

units of penicillin in a beeswax (1 to 2 per cent) and se-
same oil mixture by Zinnomon and Seeberg (1945).

Trumper and Butter (1944) and later Trumper and
Thompson (1945) reported that the absorption of penicillin
could be delayed by chilling the site of injection before
and after the administration of penicillin, thus giving a
prolonged therapeutic value using a minimum amount of peni-
cillin. In checking forearm blood flow they found that it
ranged from 0.5 cc. (per hundred cubic cc. of arm volume
per minute) at a temperature of 55.40 F to a speed of 5.9 cc.
at 98.60 F. By using a thermocouple, the temperature 0.5
of an inch.below the skin after an ice bag was placed on the
deltoid muscle was found to be reduced to 650 F. In the
clinical use of penicillin, ice bags were applied to the
site of injection two hours previous and twelve hours fol-
lowing intramuscular injection. This procedure maintained
effective bacteriostatic levels for a period of 12 hours
in five out of six cases.

Chow and McKee (1945) in working with crystalline
penicillin found that it combines with human serum albumin
to make a large penicillin albumin complex. In mouse ex»
cretion studies, the penicillin-albumin complex was found
to be excreted more slowly than the sodium salt of peni-
cillin. The authors suggested this as another means of sus-
taining penicillin serum levels.

In 1941 Code and Lewis and in 1942 Code and Varco

found that the action of desoxycorticosterone acetate and

 

histamine could be markedly prolonged by administering them
parenterally in beeswax mixtures. Bryson and Code (1944)
reported prolonging the action of heparin as an anticoagu-
lant when administered in beeswax.

Romansky and Rittman (1944) observing these results
reported by Code and associates in the delaying action of
beeswax added it to peanut Oil in preparing a vehicle to de-
lay absorption of penicillin. They prepared their peni-
cillin beeswaxroil suspension as follows: All equipment
used was sterilized at 17 pounds steam pressure for 20 min-
utes. The beeswax was heated and filtered through six lay-
ers of gauze, while the peanut oil was sterilized by filt-
ering through a Seitz filter. Various amounts of beeswax
were added to the peanut oil, ranging from 0.75 to 6 per

cent. The vial containing the penicillin was shaken to

break it into a powdery state. To the penicillin vial were

next added 5 to 5 beads and the beeswax-peanut oil mixture.

It was then shaken for 15 minutes to make a uniform sus-

pension. Calcium penicillin was used as it was less hydro-

scopic than sodium penicillin.
Stability tests on Romansky's mixture showed no de-

terioration when kept under refrigeration, at room and
570 C. temperatures for 50 to 62 days.
Rabbits given single intramuscular injections of
5,000 to 10,000 units in the suspension, sistained a measur-
able penicillin blood level for 6 to lZ‘hours, while similar

amounts of penicillin injected in saline resulted in a de-

\

tectable level for only 2 hours.

When single intramuscular injections of 41,500 to
66,400 units in the suspension was given to humans the peni-
cillin blood level was maintained for 6 to 7 hours. None of
the patients complained of local pain or irritation. In the
initial clinical trials twelve patients with gonorrhea were
given from 51,250 to 100,000 units in a single intramuscu-
lar injection; eleven of the twelve were pronounced cured.

In studying tissue reactions to intramuscular in-
jections of 5 per cent beeswax—peanut oil mixture, ten ham-
sters were given 0.05 to 0.10 cc. (500 to 1,000 units peni-
cillin) twice a day for five days. They were then sacri-
ficed at the rate of one per week and tissue sections were
prepared. At twenty-four hours the muscle fibers were sep-
arated by oil cysts surrounded by polymorphonuclear leuco-
cytes: no necrosis was present. At the end of ten days,
particles of beeswax were surrounded by giant cells and
scattered disintegrating leukocytes. At thirty days the
beeswax had completely disappeared and the cyst walls, whidi
were composed of minute amounts of fibrous tissue and giant
cells, were partially collapsed.

A number of interesting points were bought out in
a study of absorption and excretion of penicillin by Kirby,
Leifer, Martin, Rammelkamp and Kinsman (1945). Three-
hundred and fifty-nine patients were given intramuscular
and subcutaneous injections of 500,000 units in beeswax and

peanut 011. These workers noted quite a wide variation in

absorption and excretion. The sojourn of assayable levels

in the blood stream of different patients ranged from 4 to
28 hours. Part of the patients in whom levels were present
for 16 to 28 hours showed irregularity of absorption during
the second twelve hours; i.e., there were one or more occa-
sions on which assayable levels were not present. Excre-
tion in the urine after the first 12 hours was found to de-
cline rapidly, however, small amounts could be detected

for 72 hours in some patients. No significant cumulative
evidence was found in patients given intramuscular injec-
tions of 500,000 units of the mixture daily for eight days.
Only 7 per cent had assayable levels at 24 hours following
the injection. They also found that results with subcutane-
ous administration were superior to results with intramuscu-
lar injection both in uniformity of absorption and in pro-
longation of blood levels. No induration, nodule formation
or sterile abscesses were observed when the mixture was
administered subcutaneously.

Doll and Dimock (1946) in working on horses found
that single intramuscular injections of 500 to 500 units
ofpenicillin per pound of bOdy weight given in a suspen-
sion of beeswax and peanut oil maintained effective blood
concentration for four to six hours. This interval was
approximately double that when saline solutions were used
as a vehicle.

Bdhls and Cook (1945) thought it of interest to

study the effect of aluminum-potassium sulfate and peni-

 

- 11 -

cillin mixtures in delaying absorption following intramus-
cular injection since this aluminum material was so suc-
cessful in delaying the absorption of diphtheria toxoid.
Local and systemic (temperature) reactions of aluminum po-
tassium sulfate-penicillin mixtures containing 16,000 units
of penicillin and 20 mg. of aluminum were studied in nor-
mal rabbits. Control animals were injected with penicillin-
beeswax-peanut oil mixtures of Romansky and Rittman (1944).
No significant reactions were noted except that a small
nodule could be felt at the site of injection in some of
the animals for several days. Blood concentrations of

the animals receiving the aluminum-penicillin mixtures com-
pared favorably with those receiving Romansky's Formula.
Both mixtures maintained assayable levels for 8 hours. Hu-
man patients injected with 50,000 units of penicillin in
aluminum potassium sulfate maintained a therapeutic level
for from 6 to 9 hours. No local or systemic reactions were
noted.

A method of estimating penicillin levels in body fluids
was reported by Randall, Price and Welch (1945). This was
a serial dilution method employing a strain of Bacillus
subtilis as the test organism. The strain selected was
equally sensitive to penicillin when compared with.most
strains of hemolytic streptococci. They reported that
thousands of determinations had been made with.satisfac-

tory results.

- 12 -

Elias, Merrion and Speicher (1945) observed that
some human blood sera contained a substance inhibitory to
B. subtilis and streptococcus strain C 205. They pointed
out that the effects of these substances may be very easily
misinterpreted as penicillin activity, especially in con-
centrations of the order of 0.02 to 0.05 units per cc. of
serum. The sera from both children and adults contained
this inhibitory substance and its activity was mofe pro-
nounced against B. subtilis than towards the streptococci.
Sera from ailing adults showed pronounced bacteriostatic
and bactericidal activity against both microorganisms.

Chandler, Price and Randall (1945) made the same ob-
servations and also found that the inhibitory substance
was both variable and transitory in a given individual.
Forty per cent of the human sera tested contained an anti-
subtilis factor and they also pointed out that these inhi-
bitory substances may materially affect the interpretation
of the panicillin concentration of the blood samples in a-
mounts of 0.125 units or less.

In searching for organisms to use in determining the
concentration of streptomycin in body fluids Buggs et al
(1946) found that 50 out of 55 sera tested from normal per-
sons, who had received no previous medication, contained
inhibitory substances against B. subtilis, and none against
Staph. aureus. This inhibitory substance was present in di-
lutions of serum up to 1:52.

In a recent report Hoffman (1946) commented on this

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- 13 -

inhibitory factor. In a total of 120 plasma specimens he
found only one that showed inhibition in a level equiValent
to 0.05 units per cc. (1:4 dilution): the remaining speci-
mens gave negative levels.

The Committee on Medical Research and Medical Research
Council (1945) jointly made known the fact that penicillin,
as it is distributed commercially, is a mixture of several
substances. At least four different penicillin types are
known to exist. These are disignated in the United States
as F, G, K and X. Chemically they differ from one another
in side groups attached to a common nuclear structure.

Prior to the above report information had begun to
appear indicating that several types of penicillin were
present in commercial penicillin, also that these types
Varied significantly in their in vitro and in vivo activi—
ty against a variety of bacteria. Libby and Holmberg (1945)
found that penicillins G and X appeared equally effective
on a unit basis in inhibiting growth of Staph. aureus and

 

B. subtilis. On the basis of weight penicillin G was more
effective than X. 0n the other organisms tested penicillin
X was found to be more effective than G in inhibiting growth
in vitro.

Penicillins X and G were compared with respect to
sensitivity of pathogenic organisms and serum levels by
Ory, Wilcox and Finland (1945). An alpha hemolytic strep-
tococcus, a gonococcus, pneumococcus, streptococcus viri-
dans and meningococcus were found to be two to eight times

more sensitive to X than to G. Staphylococci were found to

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be equally sensitive to both types. Comparative penicillin
serum levels on ten convalescent patients following single
intramuscular injections of 20,000 units on two successive
days were significantly higher and were sustained longer
using penicillin X than regular penicillin. a comparison
of clinical results using the two penicillins left the
authors with the impression that gonococcal infection re-
sponded somewhat more rapidly and completely when penicil-
lin X was used, while on other infections the two showed up
equally effective.
Eagle (1946) using lots of crystalline penicillins F,
G, K and X compared their activity in vitro against a
streptococcus on a milligram basis. Their order of effic-

iency was X, K, G and F. Against §pirocheta pallida the or-

 

der of efficiency was G, K, F and X, while with §tgph. aureus
the efficiency was K, G, F and X.
The pharmalogical basis for the low therapeutic activ-
ity of penicillin K compared with that of penicillins F,
G and.X was determined and reported by Eagle and Musselman
(1946). They found that penicillin K disappeared from the
blood far more_rapidly than did the other penicillins, also
that a relatively smaller amount was excreted in the urine.
In rabbits injected with penicillin K'a measurable blood
level was maintained for 45 minutes; using F, G or X the
level was maintained for two hours. Urinary excretion of
K'averaged 55 per cent; the other three averaged 74 per

cent. Approximately the same results were obtained in man.

 

In experimental infection in white mice the ratio of activ-
ity was found to be K-9, F-50, G-lOO and X-260. These ther»
apeutic results compared with their in vitro activity sug-
gested that it would seem desirable to standardize impure
mixtures of penicillin (since these contain various amounts
of the different types) for therapeutic use by some method
other than their bactericidal activity in vitro.

The Committee on Medical Research, The United States
Health Service, and The Food and Drug Administration (1946)
jointly reported on the changing character of penicillin.
They brought out the fact that commercial penicillin might
vary from time to time in the relative content of the various
penicillins and that in recent months some commercial peni-
cillins have contained a substantial proportion of penicil-
lin K. This report also indicates thvt penicillin X was
relatively inefficient as compared to F, G and X against
several infections studied. Commercial penicillin has
also undergone a change in the direction of increased puri-
ty with a consequent decrease in certain impurities which
may have possessed some therapeutic activity.

The results with penicillin in the treatment of
early syphilis reflects this change in character as treat-
ment of this disease has been less satisfactory since May
1944 than prior to this date. They point out that factors
responsible for the apparent decrease in efficiency are un-
der intensive study and that practical steps to meet the

difficulty are in progress by industrial producers.

 

The administration of penicillin by various routes
was studied by Rammelkamp and Keefer (1945) to determine
its absorption, excretion, and distribution. Intravenous
injections of penicillin were followed by an immediate
rise and very rapid fall in blood serum levels in contrast
to intramuscular and subcutaneous injections in which the
rise was someWhat delayed, serum levels reached were not
so high, but remained for a longer period of time. Intra-
venous injection of 10,000 units showed an immediate value
of 0.625 to 1.25 units per cc. of serum. In 50 minutes
the level ranged between 0.059 and 0.156 units, 0.19 units
in 60 minutes and 0.019 to 0.007 units in 90 minutes. The
occurrence of the last readable levels in the various pati-
ents tested was at intervals ranging from 90 to 210 minutes.

After an intramuscular injection of 10,000 units
the serum level was 0.58 units at 5 minutes, 0.078 units
at 50 minutes, 0.015 to 0.039 at 90 minutes. In two sub-
jects given 10,000 units subcutaneously a high of 0.007
units was reached, disappearing at 205 to 500 minutes.

Fleming, Suchet, Young and Rowe (1944) also studied
the concentration of penicillin in the blood serum of pati-
ents who had been receining the drug in Various doses in-
travenously, intramuscularly, or subcutaneously as single
injections. Following an injection of 15,000 units in-
travenously they found a maximum.concentration of about

4 units per cc. almost immediately, followed by a very

 

- 17 -

rapid fall so that after ten minutes it might be only 0.5
units. Thereafter the fall was slower reaching about 0.03
units in one hour. Following a similar intramuscular in-
jection the blood concentration at two minutes was 0.12
units per cc., reaching a maximum at 6 minutes, after which
the content dropped rapidly, but not as quickly as with

the intravenous injection, so that after 5 hours little or
no penicillin was detectable. Subcutaneous administration
was found to give almost the same curve as the intramuscul-
ar route, except that the forst appearance in the blood was
delayed for a minute or two. In fifteen minutes it had
reached the same maximum level as with intramuscular in-
jection, and the fall followed the same curve.

Various doses were given intramuscularly and the
time of disappearance determined. Fifteen-thousand units
disappeared in 2 to 5 hours, 20,000 units in 3 hours,
55,000 units in 4 hours, 50,000 units in 4 to 5 hours,
100,000 units in 5 to 6 hours.

When purified preparations of penicillins became
available, Hoffman (1946) studied the blood serum levels
obtained following the subcutaneous and intramuscular
routes of injection. Results of a series of 206 assays
revealed that absorption and elimination from the blood
stream.were almost identical when given.byeither route
of injection. The author also reported that the patients

preferred the subcutaneous injection to the intramuscular

 

- 18 -

injection since less pain was produced.

Waltz and Zintel (1945) reported their findings on
the transmission of penicillin to the amniotic fluid and
fetal blood in the human. Twenty-five thousand units in
physiological saline were injected intramuscularly to each
member of the first three groups: In group I (those de-
livering within 60 minutes after injection of penicillin)
comparatively high maternal blood levels were reached while
very little of the drug was found in the cord blood or am-
niotic fluid. In group II (those delivering between 60
and 90 minutes) maternal levels 0.156, cord blood 0.039
to 0.078, amniotic fluid 0.039 to 0.156. In group III
(those delivering between 90 and 180 minutes) maternal
levels 0.178 or less. Penicillin was not found consis-
tantly in cord blood or amniotic fluid. Group IV received
penicillin by continuous intravenous drip at the rate of
10,000 units per hour. After 2 hours the maternal blood
was 0.078 to 0.312 units, cord blood 0.039 to 0.078 units
and amniotic fluid 0.039 to 0.078 units. After 20 hours
the maternal blood, cord blood, and amniotic fluid all con-
tained 0.156 units. No evidence of toxicity was observed
either to the mother or child in any of the groups.

Some observations on the toxicity of penicillin to
animals were included in Flemings (1929) first article re-
porting the discovery of the new antibiotic. In working
with mold broth filtrates containing penicillin he found
that its toxicity to animals was very low. He reported

that intravenous and intraperitoneal injections into rab-

bits and mice, respectively, resulted in no more toxic
reaction than the same quantity of broth. Irrigation of
human conjunctiva and infected surfaces had no irritating
effect. He also reported that in vitro, penicillin which
completely inhibited the growth of staphylococcus in a
dilution of l to 600 did not interfere with leukocytic
function to a greater extent than did ordinary broth.

Abrahan et a1 (1941) studied the effect of peni-
cillin on leukocytes at various dilutions. They found
that penicillin at a dilution of l to 100 inactivated the
leukocytes almost immediately; at l to 250 more than 50 per
cent of the leukocytes were active for 4 hours; at l to
500 the preparation was indistinguishable from the control.
Injections of 20 mg. of penicillin (40 to 50 units per mg.
strength) caused serious embarassment but not death in mice.

Later Flory and Jennings (1942), working with a more
purified product, injected mice intravenously with 20 mg.
of penicillin (250 and 350 units per mg. strength) with
no observable reaction.

A toxicity investigation comparing crude prepara-
tions of penicillin (60 units per mg.) with a more puri-
fied product (300 and 400 units per mg.) was made by Rob-
inson (1943). He found that on the basis of weight a puri-
fied penicillin preparation was somewhat less toxic than
crude penicillin in spite of the marked increase in anti-

bacterial activity.

Homre, Rake, McKee and MacPhillamy (1943) found
that guinea pigs tolerated less penicillin units per kilo-
gram of body weight when fatal doses were administered
than rabbits or mice. 0n autopsy the guinea pigs exhibited
no gross or microscopic lesions in the central nervous
system, lungs, spleen, lymph nodes, intestines, pancreas,
kidney, adrenal, gonads or voluntary muscles. In 20 per
cent of the cases the heart showed necrosis of several
muscle fibers in the left ventricle and a surrounding in-
filtration with monocytes and a few polymorphonuclear cells.
On gross examination the site of injection was edematous
and yellowish red in color. MicroscOpically the picture was
one of subcutaneous edema and infiltration of monocytes
and leukocytes. Focal areas of necrosis were present in
many of the livers.

The authors also conducted experiments with mice
comparing the toxicity of crude and purified preparations
of penicillin and further verified the work of Abraham,
Flory and Robinson in that purification lowers toxicity.

In a compiled report of 500 cases by the Committee
0f Chemotherapeutic and Other Agents (1943) it was con-
cluded that the remarkable feature of penicillin was its
relatively low toxicity and the extremely low incidence of
reactions of a.systemic nature. Material available at the
time of this report was 10 to 15 per cent pure penicillin.
Toxic reactions were as follows: fever - 5 cases, chills

and fever - 12 cases, thrombophlebitis at site of injection

- 19 cases, urticaria - 14 cases, gluteal tenderness at
site of injection - 5 cases, headaches and flushing cf the
face - 10 cases, tingling of testes - 2 cases, and pains
in muscles -2 cases. They commented that urticaria might
occur during the course of treatment or after treatment
had been discontinued.

Lyons (1943) reviewed the results observed in 209
cases. He divided the reactions into two groups. (1) Re-
actions associated with particular batches and thought to
be due to impurities were chills with or without fever
after intravenous injections, eosinophilia (20 to 30 per
cent), burning pains at site of intramuscular injection,
headaches, faintness and flushing of face, unpleasant
taste after parenteral injections, tingling of the testes,
muscular cramps, and femoral phleothrombosis. (2) Re-
actions which were not limited to particular batches of
penicillin were - urticaria 5.7 per cent, fever without
urticaria, transient azotemia, and thrombophlebitis.

A summary of 100 clinical cases receiving penicillin
therapy was reported by Dawson and Hobby (1944). Three
patients develOped mild urticaria. Chills and fever had not
been observed by the authors since the early cases when the
material Was known to contain pyrogenic substances. Throm-
bophlebitis was observed in only one case. Complaints of
discomfort at site of injection seemed to be connected
with particular lots of material. In a majority of cases

no symptoms of any nature were observed, nor had prolonged

administration led to the deveIOpment of any intolerance
or sensitivity.

Kolodny and Denhoff (1946) reported their observa-
tions on 124 patients. Immediate reactions following par-
enteral administration were shown by 16 per cent while 7
per cent showed delayed reactions. No significant rela-
tionship was found between the incidence of reactions,
previous penicillin therapy, recurrence of symptoms follow-
ing additional therapy, time interval between courses of
penicillin or past history of personal or familial allergies.
Relief by epinephrine or ephedrine was definite.

Hypersensitivity of the tuberculin type was report-
ed by Welch and Rostenberg (1944) following intradermal
injections of penicillin. The authors later (1945) test-
ed 144 persons and found that 5.5 per cent exhibited this
type of sensitivity. It did not occur following intramus-
cular or subcutaneous injections as the penicillin was
absorbed and excreted too rapidly.

An investigation was carried out by Herwick, Welch,
Putman and Gamboa (1945) to determine why certain lots of
commercial penicillin produced severe pain on intramuscular
injection. They found no correlation between the irritation
produced by intradermal injections of penicillin in man or
rabbits and that produced by intramuscular injections of
this material in 100 patients. They also found, by using
the blindfold test on 100 patients, that intramuscular in-
jections of penicillin produced a greater incidence and in-

tensity of pain than did the injection of isotonic salt sol-

- 25 -

ution. A significant correlation between purity (units
per mg.) and irritation following intramuscular injection
was found. An increase in potency was associated with a
corresponding decrease in pain produced.

Using purified preparations of penicillin, Hoffman
(1946) reported that there were no recognized cases of
urticaria or other allergic manifestations. He suggested
that these findings might be related to the purity of the
preparation used.

Five patients who developed urticaria associated
with penicillin therapy were studied for hypersensitivity
and for circulating antibodies by Colloway and Barefoot
(1946). They found all five to be negative when subjected
to intracutaneous or passive transfer tests. Precipitation
tests were inconclusive as all.patients, including controls,
showed a fine precipitate. The authors assumed that an
excessive amount of histamine was present as in all instances
the urticaria was controlled by Benadryl.

The effect of prolonged administration of penicillin
upon the blood picture of canines was studied by Bailey
(1946). Examinations made included coagulation time,
erythrocyte counts, leukocyte counts, differential leukocyte
counts, hemoglobin determinations and erythrocyte sedimenta-
tion rates, bleeding time and percentage of packed blood
cells. The only two changes observed were an increase in
eosinophiles and a decrease in the erythrocyte sedimenta-

tion rate.

- 24 -

Malkamus and Oppermann (1944) reported the normal
ranges of blood constituents in the dog as follows:
erythrocytes 5.5 to 8 million per cu. mm., leukocytes 9 to
10 thousand per cu. mm., and the leukocyte differential
count was 2 to 4 per cent eosinophiles, 0 to 0.5 per cent
basophiles, 13 to 32 per cent lymphocytes, 3 to 5 per cent
monocytes.

The normal ranges of the canine blood constituents
reported by Coffin (1945) were as follows: erythrocytes
5.5 to 8.8 millions per cu. mm., leukocytes 5 to 20 thou-
sand per cu. mm., hemoglobin 12.5 to 17.3 g. per 100 cc.,
neutrophiles 3.6 to 15 thousand per cu. mm., eosinOphiles
0.1 to 2 thousand per cu. mm., basOphiles 0 to 0.4 thou-
sand per cu. mm., lymphocytes 0.6 to 6 thousand per cu.
mm., monocytes 0.1 to 2.4 thousand per cu. mm.

Boddie (1946) reported the following as normal
ranges in dogs: erythrocytes 5.1 to 7.6 million per cu.
.mm., leukocytes 8 to 14.6 thousand per cu. mm., hemoglobin
11.2 to 14.8 g. per 100 cc., and the leukocyte differential
count was 0 to 4 per cent eosinOphiles, 67 to 81 per cent
neutrOphiles, 14.6 to 25.4 per cent lymphocytes, 1 to 7

per cent monocytes.

- 25 -

METHODS

The procedures used in these experiments were as
follows:

Estimation 2£_Penicillin in Body Fluids. The offi-

 

 

 

cial test of the Food and Drug Administration, develop-

ed by Randall, Price and Telch (1945) was used. The tech-
nique was as follows: One-half cc. amounts of sterile
yeast beef broth*‘were placed in regular size sterile test

tubes and serial dilutionsby halves made by adding one-

A _ gﬂ—ﬂ”

half cc. of the fluid being tested to one of the tubes and
carrying one-half cc. in serial dilution for as many tubes
as necessary (usually 5 to 15 tubes). The first tube in
the series contained one-half cc. of the material under
test only. A standard was prepared for comparison by dilut-
ing a known potency penicillin** to one-unit per co. in
sterile water. This one-unit standard was diluted exactly
as above in serial dilution by halves. One and one-half
cc. of a 1:100 dilution of the test organism*** in yeast
beef broth was added to all tubes; then the tubes were in-
cubated at 37° C over night (18 hours). The last tube in
which no growhh.occurs is taken as the end-point. This
was usually sharp, inasmuch as one tube was clear while

* Difco Yeast Beef Broth Dehydrated, Difco Lab., Detroit.
** Reference Standard of known potency was obtained from

Food and Dr Administration.
***0rigina1 culﬁure of test organism (B. subtilis) was ob-

tained from.Food and Drug Administration.

the next one in the series would have the typical pellicle
of B, subtilis on the surface of the medium.

The concentration of penicillin in the unknown was
then determined by comparing the end-point of the unknown .
with that of the standard. Ordinarily the test as describ-
ed here is sufficiently sensitive to determine potencies as
low as 0.03 units of penicillin per cc. of blood plasma.

Determination 2£_Hemoglobin. The Sheard - Sanford

 

 

Photelometer was used in determining the grams of hemoglobin
per 100 cc. of blood (Sanford et a1, 1933). Preparation
of the sample of blood was as follows: Twenty cc. of the
diluting fluid (0.1 per cent solution of s>dium carbonate
in water) was measured accurately into a suitable contain-
er such as a 50 cc. Erlenmeyer flask. To this was added 0.1
cc. of blood. The mixture was thoroughly shaken and then
transfered to the photelometer for reading using a green
filter. This reading was then obtained in grams of hemo-
globin per 100 cc. of blood from a calculated table.
Leukocyt§_and Egythrocyte Enumeration;_ Leukocyte
and erythrocyte counts were made within 24 hours after
bleeding using Thoma diluting pipettes and Bright-Line
Improved Neubauer Counting Chambers (Coffin, 1945). Two
per cent oxalic acid (Jones, 1927) was used as the diluting
fluid for counting leukocytes, while Leak and Guy diluting
fluid (Todd, 1943) was used for making erythrocyte counts.

Differential Leukocyte Counts. Blood smears were pre-

- 27 -

pared within one hour after bleeding by the two-slide
method and stained with Vright's stain (Coffin, 1945)
immediately following drying.

The dogs used in this study were obtained from a
city dog pound. On arrival from the pound each individual
was given 40 cc. of an homologous anti-canine distemper
serum subcutaneously. In ten days an additional 40 cc. of
serum was given to each dog. None of the dogs developed
distemper and all remained free of the disease throughout
the course of study. They were checked routinely for inter-
nal and external parasites and then treated when necessary.
Their ages ranged from approximately four months to ten
years. The animals were fed once each day; the ration con-
sisted of one part of cooked meat scraps and two parts of
commercial cereal dog food. This was fed at approximately
six p.m. Water was kept before the dogs at all times.
Penicillin injections and drawing of blood samples were al-
ways begun in the morning or at noon, depending on the num-
ber of samples collected. All the animals gained weight
steadily during the entire course of study, the individual
gains ranged from 10 to 25 pounds. At the termination, the
dogs were autopsied and examined for lesions, particularly
at the sites of injection.

The anticoagulant used in collecting blood samples was
7.5 per cent potassium oxalate; two drops“ of this solution
*A commercial eye dropper was used. The delivery end had

a diameter equal to the No. 16 Opening on U. S. Standard
Gauge for Sheet and Plate Iron and Steel N. 283.

- 28 -

was used to each 5 cc. of blood.

RESULTS AND DISCUSSION

The problem of immediate importance was to deter-
mine if the amount of anticoagulant used (potassium
oxalate) had any effect on inhibiting the growth of B.
subtilis, the test organism. It was also thought to be
of interest to determine the amount necessary to stop the
growth of the organism. This was accomplished in the follow-
ing manner: Five series of ten tubes each were set up. To
each was added 0.5 cc. of B. subtilis inoculated yeast
(beef broth. Serial dilutions were then prepared using
0.5 cc. amounts of aqueous solutions containing various
quantities of 7.5 per cent potassium oxalate. The results

of this experiment are presented in Table I.

Table I. BACTERIOSTATIC EFFECT OF POTASSIUM OXALATE ON

B. SUBTILIS.

Kmount oTI
Potassium Oxalate Tubes
Added

 

l drOp
2 drOps

"UFU’UH

5 drops

'1! '1') "U "U (0
"U "U "U ‘11 ()0
'17 "d "d "U +#
"U "U "U "U

"U "U "U "U 03
"U 1’11 "U ’1‘) <1
'11 "U *U "U (I)
"U "U "U '1'} <0
’1! "U "U "U

10 drops -

- no growth
P growth with pellicle formation

It was found that approximately an equal amount of
anticoagulant, 7.5 per cent potassium oxalate, could be add-
ed before inhibiting the growth of B, subtilis. Since
only two drops were added per 5 cc. of blood in drawing
blood samples, its inhibitory action was considered insig-
nificant.

Another problem of immediate importance was to de-
termine if the blood of any of the dogs had an inhibitory

factor against B. Subtilig as this would influence the in-

 

terpretation of results. An inhibitory factor in human
blood sera was found to be quite variable. Chandler, Price
and Randall (1945) reported that 40 per cent of the patients
examined carried this factor, Buggs et a1 (1946) reported
85.7 per cent and Hoffman (1946) reported less than 1 per
cent. The possible significance of this factor in the pre-
sent study was determined in the following manner: A
series of 4 tubes was set up for each dog. One-half cc.

of yeast beef broth was added to the third and fourth

tube of each series. One cc. of serum.was added to the
first tube, 0.5 cc. to the second tube, 0.5 cc. to the
third tube and serially diluted through the fourth tube.
One and one-half cc. of a 1:100 dilution of the test organ-
ism in yeast beef broth was added to all tubes; then in-
cubated at 37° C for 18 hours.- The blood plasma of all
dogs used in this experiment was checked at different in-
tervals throughout the course of study using the above pro-
cedure. The results of the initial, midway and fi-

nal check are shown in Table II. Tube 1 represents a serum

dilution of 1:2.5, tube 2 a dilution of 1:4, tube 3 a
dilution of 1:8 and tube 4 a dilution of 1:16.

Table II. EVIDENCE OF INHIBITORY FACTOR IN DOG SERA

AGAINsm B. SUBTILIS.

 

 

Feb./46 Test June/46 Test Jan./47 Test

 

 

Dog No. Tubes Tubes Tubes

1 2 s 4 1 2 s 4 1 2 s 4
100 G P P P - P P P P P P P
200 P P P P - P P P P P P P
500 P P P P I G P P P P P P P
400 G P P P - G P P G G P P
500 G P P P - P P P P P P P
600 G P P P G G P P G P P P
700 G G P P G G P P
800 G P P P G G P P P P P P
900 G P P P - P P P P P P P
000 G G P P - P P P G G P P
L00 G P P P G G P P G P P P
B00 G P P P - G P P G G P P

 

P growth with typical pellicle formation
G flocculent growth without pellicle formation
- no growth

- 52 -

These data agree with that reported by Chandler,

Price and Randall (1945) in that the inhibitory substance
was both variable and transitory in a given individual. In
all the dogs the 1:4 dilution of serum showed growth,
either typical pellicle formation or flocculent type. In
the actual testing for the blood serum concentration of
penicillin a sufficient number of hourly blood samples

were drawn so that in every case the study was carried for-
ward far enough to show growth in the 1:4 dilution tube.
Thus each series of samples on an individual acted as a
check on the presence or absence of the inhibitory factor
even though a sample was not drawn Specifically for this
purpose prior to injecting the penicillin. The number of
animals used in this experiment was too small to draw

any conclusions, however, this group of dogs at no time
carried the inhibitory factor in sufficient concentration
to interfere with the method used for estimating penicillin
levels in body fluids.

The first objective in this study was to compare
penicillin blood plasma levels following a single injection
of a fixed unitage of penicillin given by the different
routes of administration, namely: intravenously, intra-
peritoneally, intramuscularly, and subcutaneously. One-
thousand units per pound of body weight was decided upon
as the trial dosage.

Five per cent dextrose was used as the vehicle since

Armstrong, Halpern and Cutting (1945) concluded from their

experiments that it was equally efficient or more so than
other vehicles tested in sustaining penicillin levels in the
blood sera. The test solution was made up so that each 00.
contained 10,000 units of penicillin.

In the series of trials using the intravenous route
the penicillin was injected into the left radial vein. In
5 minutes the first blood srmple (5 to 7 cc.) was drawn
from the right radial vein. Blood samples were then taken
at half-hour.intervals for the next three hours. Beginning
with the first sample drawn, blood was taken at hourly in-
tervals and checked for red and white cell counts, hemoglobin
content, and leukocyte differential counts, to determine if
penicillin had any effect on altering these in the normal
healthy animal.

In the series of trials using the intraperitoneal,
intramuscular, and subcutaneous routes blood samples were
drawn one-half hour following the injection, and then at
hourly intervals. In these three series of studies samples
were taken at the beginning and end of each individual tri-
al to determine the effects of penicillin on the blood
picture.

Intraperitoneal injections were made in the lateral
abdominal region. The subcutaneous injections were made
in the nuchal region approximately on the midline. This
location was chosen as it is readily accessible and caused

least discomfort to the patient, particularly When a 24

- 54 -

gauge needle was used. Intramuscular injections were made
in the posterior portion of the external femoral region
about midway between the stifle and hip joint. The needle
was inserted deep into the muscle, usually about 5/4 of

an inch.

Four dogswwere used in the series injected intra—
venously - 601, 201, 101 and 701; four in the intraperi-
toneal - 103, 203, 501 and 405; eight in the subcutaneous -

901, 001, 502, B02, 801, 002, 902 and BOB; and four in the
intramuscular - L01, 801, 102 and 202. Tables III to XXII
inclusive.

As the penicillin was introduced directly into the
circulatory system when given intravenously it was found
almost immediately at r concentration of 4 to 8 units per
cc. of blood plasma. The concentration was found to fall
quite rapidly so that after 50 minutes it ranged between
0.5 and 1 unit in the four dogs tested. From this point
on the drOp was more gradual, disappearing below measurable
levels in 2 to 2.5 hours. -

The intraperitoneal, subcutaneous and intramuscular
penicillin blood plasma levels compared very closely with
each other both in regard to the concentration reached at
30 minutes, and to the gradual drOp in concentration from
this point until the penicillin was no longer measurable,
which in each case was between 3.5 and 4.5 hours.

The highest concentration at 30 minutes was shown

by the intraperitoneal route; this ranged between 0.5 and

- 35 -

2 units. The subcutaneous was lowest, ranging between 0.125
and 1 unit, and the intramuscular ranged between 0.5 and 1
unit. At 1.5 hours the intraperitoneal and intramuscular
levels ranged between 0.06 and 0.5 units, while the subcu-
taneous ranged between 0.125 and 0.5 units. at 2.5 hours
the intraperitoneal route levels ranged at 0.03 and 0.25
units, while the subcutaneous and intramuscular held at
0.05 to 0.125 units. The measurable levels of penicillin
on all three routes disappeared between 5.5 and 4.5 hours,
however, seven out of eight dogs still had a level of 0.015
units or higher at 5.5 hours when penicillin was adminis-
tered by the subcutaneous route while two out of four sus-
tained this level by the intraperitoneal route and three
out of four by the intramuscular route.

Graph 1 shows the average penicillin blood plasma
level curves for each of the four routes of injection.
When these curves on canines were compared with curves on
human penicillin blood plasma levels (Fleming et a1, 1944,
and Hoffman, 1946) following single injections of penicillhl
made by different routes of injection, they were found to
be quite similar.

From a clinical standpoint the primary interest was
(1) to determine the route which was most convenient for
administration and (2) to determine the route or routes
which were equally effective or advantageous in maintain-

ing effective penicillin plasma levels.

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- 44 -

 

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- 50 -

 

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- 52 -

 

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- 53 -

 

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-54..

 

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- 55 -

 

 

 

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HHNN Odnma

After analyzing the results of the four routes of
injection, the subcutaneous and intramuscular routes of
administration seemed to more nearly meet the clinical
qualifications required. The intravenous route was tem-
porarily discarded because of occasional difficulty in
administration and primarily because of poorly sustained
vlood plasma levels. The intraperitoneal route, although
equally effective in sustaining blood plasma levels, was
considered less practical because of necessary additional
restraint of the dog on administration.

The next series of experiments were to determine
the penicillin blood plasma levels using single injections
of vaeious dosages per pound of body weight (250, 500
and 1,000 units) and giving the penicillin either intramus-
cularly or subcutaneously. The vehicle used in this series
was 5 per cent dextrose in physiological saline. Studies
on the blood picture were continued.

In the intramuscular series the following dogs
were used:

1000 units per 1b. body weight - L04, 604, 505 and 107.
500 units per lb. body weight - B04, 802, 805 and 004.
250 units per lb. body weight - 505, 602, L02 and 605.

Tables XXIII to XXXIV inclusive.

In the subcutaneous series the following d0gs were
used:

1000 units per 1b. body weight - 605, 905, 904 and 504.
500 units per lb. body weight - 405, 205, 104, 204, 502

- 57 -

and 404.
250 units per lb. body weight - 905, 005, 505 and 105.
Tables XXV to XLVIII inclusive.

Graph II shows the average penicillin blood plasma
level curves for different dosages administered by the in-
tramuscular route of injection. Graph III shows the same
for the subcutaneous route of injection.

The penicillin blood plasma level curves were found
to be quite regular. The height and maintenance of the
penicillin concentrations were also found to be directly
related to dosage administered. When penicillin was ad-
ministered by the intramuscular route of injection the 50
minute plasma level for 1000 units per pound of body weight
was 2 to 4 units, 500 units resulted in a level of 2
units and 250 units in a level of 0.25 to 0.5 units. In
all cases the drop in blood concentration was quite re-
gular and gradual. With 1000 units per pound of body weight,
values drOpped below the measurable level at 5.5 to 4.5
hours, with 500 units at 5.5 hours add with 250 units at
2.5 to 5.5 hours.

Various dosages given by the subcuataneous route re-
sulted in blood plasma levels which were found to corre-
late very closely with those obtained by the intramuscular
route. At 50 minutes, 1000 units per pound of body weight
resulted in a blood level of 4 units, 500 units per pound
resulted in a level of 2 units and 250 units per pound re-

sulted in a level of 0.5 to 1 unit. From this point the

 

 

 

 

 

 

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- 55 -

 

 

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- 73 -

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mo. .ns m.»

m. .n: m.m

.H .pn m.a
a ma mm m m.oa ooo.oo>.o ooo~¢a .¢ .nn 0.

.5 .q .z .m .m 400 ooa\w .o.m.m .o.m.; memwam1.ou\w Hwbnman
Hwﬁuqopommﬁm .nm nHHHHOHnom maﬁa

 

 

moﬂuaam cooam

.oSmmap omomHUG oanwpoUHmcoo wnﬁhamwo .hsuawon hapnonwmmd I knopnﬁm

.oGHme .uhnm nH omomuxov mm I oHoH£o> on; hoop .DH\5 Good I Oman

.uhh n .mQH m¢ onEmm .msoonwpsopdm I oudom

®¢\o\HH $00 .02 on no unoapwqﬁanmpom HH>NNN oanma

 

NH om mm

- 74 -

 

m w.¢a ooo~ooo.m ooo.¢a mo.v .nn m.m
00. v .95 muss.
wo.v :5 90
mo. v :3 m.m
mo. .un m.¢
mma. .an m.m
mm. .gn m.m
.H .nn mra
m om be m H.¢H ooo.om>.o oom.na .w .an m.
.s .q .z .m .m .00 ooa\w‘ .o.m.m .o.m.a mamaam .oo\p aapnman
Hmﬁpcoao%gﬂn .nm nHHHHOHgom maﬁa

 

 

.onﬁamm .mhﬂm GH omonpxou Rm I mHOH£o>

.mhh o

m§\m\aa

.mnH mm

mama

won .02 won no macapwnaanopmm

moacdpm vooam

.hﬂpamoﬂ hapnopwmm< I hhomnam
.p; hcon .na\s oooa I omen
msomnwpsondm I opsom

HHH>NNN OHDNB

- 75 -

 

m wa Hb m m.mH

ooo.ooo.m

 

 

 

om>.na mo.v .9: m.¢
oo.v .95 m.n
mma. .hn m.m
.H .nn m.H
m ma or w ¢.¢H ooo.ooa.o omm.ma .m .an m.
.2 .q .z .m .m .00 ooa\m, .o.m.m .o.m.a wamwag .oo\m‘ pracunH
HprQonoMQﬁn _ .nm aaaaaoaQom made
In. moauspm cooam
.hnpawon hapcmpwmm4 I hhopnam
.oﬁaamm .mhzg ca mmonpxmw mm I oaoa£o> .pa hoop .QH\¢ com I omen
.mhh w .mna wm oaws mdoonmpSoQSm I opsom
©w\m\aa mow .02 won 20 mnoapwsﬁanopom

MHNNN magma

 

a ma mm m ¢.ma ooo.omo.> omo.ma mo.v .nn m.¢

. mo. .nn m.m
5
7 mma. .an m.m
.
m. .nn m.H
ma am we m .mH ooo.omm.m oom.na .m .ng m.

 

 

 

.2 .q .z .m .m .00 ooa\w. .o.m.m .o.m.§ wsmwam.omwb Hapnoan
Hwﬁpnomow%am .pm GHHHHoHQOm maﬁa

 

uoﬁusum cooam

.hﬂpa¢o£ hapnogwnm4 I hAOpmHm

.onﬁamm .mhﬂg ma omoapxoc mm I maoﬁno> .ps hoop .DH\5 com I omen

.unh m .mnH mm cams msommprOnbw I ousom

ow\m\aa mom .02 won no mqoapmnaanopmm ax oapag

 

 

 

 

 

ooo.OO¢.m oo¢.sa no.v .nn m.¢

00. .ns m.»

mm. .nn m.m

m .H .2 3
. 0 pH mp n «.ma ooo.oom.o ooa.ma .m .m: m.

.2 .H .z .m .m .00 ooa\w .o.m.m .o.m.a wamwam .oo\b Hwbkoan
Hwﬁpnomo%wﬁa .nm GHHHHoanom maﬁa

moauspm wooam

.hﬂpamon hapmonwmm¢ I hhopmam

.oaaawm .mham QH omOApxou mm I oaoa£®> .9; hﬁon .pH\s oom.I omen

.mah m .mna mm mam: msomsmpSODsm I ouuom

ow\m\m

woa .oz mom no mﬂOﬁuwQHEnopmm

qu oanda

oa ow mm H w H.ma ooo.oom.o 00¢.ma no.v .nn m.¢

mo. .hn 0.a
.
8 mm. .hn m.m
7
. m. .An 0.a
NH ma no 0 ©.mH ooo.oom.o Iom¢.ma .m .An m.

 

.2 .q .z .m .m .00 ooa\w .o.m.m .o.m.a «Emwam .oo\b Ha>hoqu
Hwapcohm%hﬁm .Qm nHHHHOHGom maﬁa

moﬁcapm UOOHm

.hﬁpawon hapmohwmm¢ I bHOpmHm

.onHme .mhnm GH omoapxmu mm I oaoﬁno> .9: hwon .na\s com I anon

.mnh m .mna mm cams adoonwpsopsm I ounom

ow\m\m «om .02 won no acoﬁpmqﬂanmpom Hqu manna

 

ma ma mm n .ma ooo.om¢.m omn.HH oo.v. .9: m.¢

. 00. .pg 0.a

w mma. .nn m.m

. m. .nn_m.a
oa Hm $0 m m.ma ooo.omn.m oo¢.oa .m .nn m.

 

.2 .q .z .m .m .00 ooa\w, .o.m.m .o.m.3 wamwaa .oﬂ\D Hwbﬁoan
Huﬁaqopogmam .nm aaaadoasom maﬁa .

moadﬂpm Gooam

.hﬂpawoa hapaonwmmd I knapmam

.oqaamo .mhnm ca emogpxmw Rm I maoﬁ£o> .pa hcon .pa\d com I mmom

.&h H .mpa an mam: msoonpronbm I opSOm

ow\>a\> mom .02 woe :0 maoﬁpauﬁEAopom HHqu oapms

a ma mo ¢ m.ma ooo.oao.© 00¢.ma mo.v .an m.¢

mo.v .9: m.n
. mad. .pn m.m
0
.u .H .ng m.a
.
m mm mm H m.©a ooo.ooo.> omo.ma .m .pn m.

 

.2 .q .z .m .m .00 ooa\w. .o.m.m .o.m.a wsmwam.oo\b Hwbnoan
HwapnmnoMMﬂm .nm nﬁaaaoaamm cede
moaospw wooam

.hnpammn hangomwmm¢ I knapuam
.oQanm .mhnm ma omoapxmv mm I maowﬂo>

.mmh ¢

.ps hcop .pH\s com I cuon
.mpa rm mam: msooqusonam I opsom

ow\>a\> wow .02 mom :0 unoHmeHsnmpom >qu oanaa

 

a ma mm m ¢.na ooo.omo.m omm.o mo.v .92 m.¢

mo.v .nn m.»
mo. .an m.m
.
1. mma. .9: m.a
8
. ma am no ¢ m.ma ooo.omo.m oom.m m. .m: n.

 

.2 .q .z .m .m .ooooa\m, .o.m.m .o.m.3 wamwam .oo\D HmpnopaH
Haapaonommam .nm aaaaﬁoazom maﬁa

moﬂospm cooam

.Qﬁnp Haapm was p59 psmﬁo; swam on meaczﬁmop mm; mow 029 .hnpammn hapaopwmm4 I hpopnam
.onﬁawm .thQ :2 mmonpxmu Rm I maoano> .p; hoop .QH\5 0mm I omen
.mah n

.mpa mm mawamm maoonwpdonsm I opsom

ow\oa\m now .02 won no mcoauquEAmpom >AN magma

 

oa om be m m.¢a ooo.oom.w oom.ma mo.v .nn m.¢

mo.v .9; m.»
.
% oo.v. .nn 0.a
. mma. .92 m.H
Ha ma mm o m.ma ooo.omo.> ooa.na m. .92 m.

 

.2 .q .2 .m .m .ooOOHNw .o.m.m .o.m.a mammam.oo\p HdbhOpﬁH
Hwﬁpgonmwmﬁm .92 naaaaoﬁaom maﬁa
moawdpw wooam

.hnpawoﬂ hauaohwamw I hAOpmHm

.oQanm .mhﬂg ca omogpxmv Rm I OHOﬁ£o> .93 hoop .DH\§ 0mm I anon

.08 OH .mQH H2 oamﬁmm msomﬁwpﬁonsm I opdom

ow\oa\m moo .02 man no anoﬁpmqﬁagmpom H>Ax canaa

 

ma ma mm m m.¢a ooo.oa>.o omm.aa oo.v. .92 m.¢

 

mo. v :5 m6
. mo. v :5 m.m
3
8 mma. 93 min
. .
a ma mp w H.¢H ooo.oom.m omH.HH .H .m: m.
.2 .q .2 .m .2 .oo ooa\m .o.m.m .o.m.a mammam.opVD HwbpopaH
Hmﬁpnogoguﬁm ..pm I I qHHHHoanom maﬁa

 

 

moﬁcspm cooam

.hnpawms hapcwmama¢ I knapuam

.onﬁamm .mham aw omopuxou mm I oaoa2m> .p3 2609 .QH\5 0mm I omen
.m» H .mna mm oaw2 msoonmpSOQsm I opsom

wv\m\aa now .02 mom :0 uncameHShmpom HH>QN oanue

 

m ma pm 0 m.mH ooo.oma.> omo.ma oo.v .55 m.¢

oo.v .55 m.»
. ®Oov as mom
mo. 39. :5 m4
. 0 mm ¢m m m.oH ooo.oom.o oom.¢a m. .55 m.

 

.2 .9 .z .m .2 .oo ooa\m .o.m.m .o.m.a. Idemmam .oo\D HwbnounH
Hmapcmhommam .92 aaaﬂaoanmm 0829
moavspm cooam

.299Hmo9 hapﬂonwmgd I h909mam
.wnaamm .mh9m 9H mmoauxow Rm I maaa90>

.mhh m

.5; 2505 .59\5 0mm I omen
.d9H mm oaw2 msoocwpdo95m I mpdom

o¢\m\aa moa .02 man so m5055wnﬁsmopon HHH>qx oﬂnwa

-85-

 

 

 

 

-87-

decline in penicillin blood plasma level was gradual, drOp-
ping below a measurable level at 4.5 to 5.5 hours when the
dosage was 1000 units per pound, 5.5 to 4.5 hours when the
dosage was 500 units per pound, and 2.5 to 3.5 hours when
the dosage was 250 units per pound.

This study of subcutaneous vs. intramuscular peni-
cillin injections at various dosages corresponds to those
reported on human patients by Fleming et a1 (1944) and
Hoffman (1946).

The same brand of penicillin was used throughout
the course of this study, also different lots of the pro-
duct were used as they arrived. The first study, compar-
ing the various routes of injection, was begun February
21, 1946 and completed June 15, 1946. The study comparing
various dosages given either intramuscularly or subcutan-
eously was begun July 6, 1946 and completed November 26,
1946. Comparison of the results obtained from these two
studies on the penicillin blood plasma concentrations for
the subcutaneous and intramuscular routes revealed two
distinct points. One point was that much higher penicillin
blood plasma levels were obtained from penicillin injec-
tions studied after July 6, 1946, the other point was
that the measurable levels persisted for a longer period
of time.

These observations fell in line with.a joint state-
ment reported by the Committee on Medical Research, The

United States Health Service, and The Food and Drug Admin-

-88...

istration (1946) on the changing character of penicillin.
They brought out the fact that commercial penicillin may
vary from time to time in the relative content of the
various penicillins 81d that in recent months some com-
mercial penicillins have contained a substantial propor-
tion of penicillin K. Eagle and musselman (1946) in ex-
perimenting with rabbits found that penicillin K disappears
from the blood far more rapidly than do other penicillins,
thus resulting in a lower initial penicillin blood plasma
level and also a shorter measurable level. The joint
statement on the changing character of penicillin also
pointed out that factors responsible for the apparent de-
crease in efficacy of commercial penicillin are under in-
tensive study and that practical steps to meet the diffi-
culty are in progress by industrial producers.

The two vehicles for delaying absorption of penicil-
lin which.were receiving most publicity at the time of
this study were water-inuoil emulsions and oil and wax
suspensions. Since very few data have been published on
the penicillin levels in dogs following the injection of
penicillin in these two vehicles it was decided to com-
pare their delaying effect with that of 5 per cent dex-
trose in physiological saline as a vehicle.

Two emulsion vehicles were used; one was hydrogenat-

ed vegetable oil (Spry*); the other was a product contain-

“Manufactured by Lever Brothers 00., Cambridge, Mass.

I'll

 

mo.v .95 m.¢

 

oa om so n m.ea ooo.oom.w oom.ma
oo.v .95 m.n
—
. . .99 m.m
mw mo v
. mma. .55 m.a
99 me me o m.ma ooo.omo.s ooH.nH m. .mn m.
.2 .5 .z .m .m .ooooa\m .o.m.m .o.m.s «swede .oe\b amppopeH
HwnpﬂoHQMMHQ .92 GHHHHOHnom QEHBII.

 

 

mosespm eoomm

.2999wc9 hapdcpmmmw I knopmam

OOHHHHNW oﬂhgnm HHH meaHpNOU Mum I QHOHSONV OP; hUOD. OQIH\.D 0mm I. OMOAH
.08 OH omQI—u Hﬁ ®Hmaom “doogwﬂdbnﬂgﬂ I. Opﬁom

o¢\oa\m moo .02 meg do anewpmqﬁasopcn H>AN 0H9ma

lull

oo.v .55 m.¢

 

 

 

m9 m5 mm m m.ea ooo.oas.o omm.aa
8. v :5 m...”
. _ mo. v :5 ad
new mNH. .99 m...”
- .
a ma me e 5.e5 ooo.oom.w om5.95 .5 .55 m.
r[.2 .5 .z .m .5 .co ooa\m .o.m.m .o.m.a «smeam .oe\9 Hw>509mw
959950909999 ..9m _ nﬂaaaOHnom maﬁa
Ir, I modespm eoomm
.2999509 2995095992 I 2909m92
.onﬂnmm .mhnm a.“ cmoﬁtnop Rm I 0.39989 .93 9309 .9H\5 0mm I anon
.5» H .m9H mm 0952 msoosm9so9sm I 09502

mw\m\aa won .02 909 do anoa9msasno9cm HH>AN 09955

 

0 09 >0 0 0.09 000.009.s 000.09 00.v .55 0.9

00.v .55 0.0
. 00.v .55 0.0
m. 009. :5 0.9
. 0 mm 90 m 0.09 000.000.0 000.09 0. .55 0.

 

.2 .9 .z .m .m .00 009\m .o.m.m .o.m.3 4080099.00\D 90>50959
909950509999 .92 599990950m 0899
0090550 00090

.2999009 2995090994 I h909m9m
.059900 .mh9m 59 00099500 mm I 090990>

.mhh m

.5; 9005 .59\5 000 I omen
.d99 mm 0902 05005095095m I 0950m
09\0\99 009 .oz 000 no m5090w55a50500 HHH>59 095m5

IIIIFII «ILIFIfl. I I II |

 

 

~85-

 

 

 

 

-86..

 

decline in penicillin blood plasma level was gradual, drOp-
ping below a measurable level at 4.5 to 5.5 hours when the
dosage was 1000 units per pound, 5.5 to 4.5 hours when the
dosage was 500 units per pound, and 2.5 to 5.5 hours when

the dosage was 250 units per pound.

This study of subcutaneous vs. intramuscular peni-
cillin injections at various dosages corresponds to those
reported on human patients by Fleming et al (1944) and
Hoffman (1946).

The same brand of penicillin was used throughout
the course of this study, also different lots of the pro-
duct were used as they arrived. The first study, compar-
ing the various routes of injection, was begun February
21, 1946 and completed June 15, 1946. The study comparing
various dosages given either intramuscularly or subcutan-
eously was begun July 6, 1946 and completed November 26,
1946. Comparison of the results obtained from these two
studies on the penicillin blood plasma concentrations for
the subcutaneous and intramuscular routes revealed two
distinct points. One point was that much higher penicillin
blood plasma levels were obtained from penicillin.injec-
tions studied after July 6, 1946, the other point was
that the measurable levels persisted for a longer period
of time.

These observations fell in line with a joint state-
ment reported by the Committee on medical Research, The
United States Health Service, and The Food and Drug Admin-

- 88 -

istration (1946) on the changing character of penicillin.
They brought out the fact that commercial penicillin may
vary from time to time in the relative content of the
various penicillins 81d that in recent months some com-
mercial penicillins have contained a substantial propor-
tion of penicillin K. Eagle and musselman (1946) in ex-
perimenting with rabbits found that penicillin K disappears
from the blood far more rapidly than do other penicillins,
thus resulting in a lower initial penicillin blood plasma
level and also a shorter measurable level. The joint
statement on the changing character of penicillin also
pointed out that factors responsible for the apparent de-
crease in efficacy of commercial penicillin are under in-
tensive study and that practical steps to meet the diffi-
culty are in prOgress by industrial producers.

The two vehicles for delaying absorption of penicil-
lin which were receiving most publicity at the time of
this study were water-in-oil emulsions and oil and wax
suspensions. Since very few data have been published on
the penicillin levels in dogs following the injection of
penicillin in these two vehicles it was decided to com-
pare their delaying effect with that of 5 per cent dex-
trose in physiological saline as a vehicle.

Two emulsion vehicles were used; one was hydrogenat-

ed vegetable oil (Spry*); the other was a product contain-

“Manufactured by Lever Brothers 00., Cambridge, Mass.

- 89 -

ing wax and lanolin in peanut oil“. The emulsions were
prepared as reported by Freund and Thompson (1945). The
oil and wax suspension used was prepared according to
Romansky's Formula containing potassium penicillin.

Single injections of 1,000 units of penicillin per
pound of body weight were used as the trial dosage. A
limited number of trials were also made using 2,000 units
per pound of body weight to see if a more favorable level
was sustained at a higher dosage. The subcutaneous route
of injection was used as it was the easiest route of ad-
ministration, least objected to by any of the dags, and
since it was found to be equally effective (when compared
with the other routes of administration using 5 per cent
dextrose in physiological saline as a vehicle - Tables III
to XXII inclusive) in sustaining penicillin blood plasma
levels.

The following dogs were used in studying the delaying
action of a product containing wax and lanolin in peanut
oil as an emulsion vehicle: .

1000 units per lb. body weight - L05, 605, 408, 007, BO6
and 304.

2000 units per lb. body weight - 406 and 005.

Tables XLIX to LVI inclusive.

The following dogs were used in studying the delay-

*Exact composition unknown.

-90...

ing action of Romansky's Formula containing potassium

penicillin the following dogs were used:

1000 units per lb. body weight - 805, 206, 407, 006, 506,
208, 108 and 409.

Tables LXV to LXXII inclusive.

Graph.IV shows the average penicillin blood plasma
level curves for 1000 and 2000 units per pound of body
weight when administered in an emulsion using hydrogenated
vegetable oil as the vehicle. Graph V shows the same
using a product containing wax and lanolin in peanut oil
as the vehicle. The average penicillin blood plasma level
curve obtained when a single injection of 1000 units per
pound of body weight in Romansky's Formula is shown in
Graph VI. Graph VI also shows plasma penicillin concen:
trations after single subcuataneous injections of 1000

units per pound of body weight in the 4 vehicles studied.

-91..

WOOV O-Hg

0.0
00.v .55 0.0
00.v .55 0.5
no.v .55 0.0
00.v .55 0.0
00. .55 0.9
00. .55 0.0
00. .55 0.0
0. .55 0.9

0 09 00 0 0.09 000.000.0 000.09 .0 .55 0.

 

02 05H 02 cm I11 cm 0040 OOHE oComom

909950909999 .9m

'.' ‘l‘l

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955009 050 5990509 «N09 I 090990>

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00\09\m9 009 .02 009 50 05099059590909

.o.mm§ 050099 .09\D 90950959

5999909509 0598

0090m00 00090

.9999005 9995090994 I 9909099

N999 0990B

0 09 90 0 0.09 000.090.0 000.09 00.v .55 0.09

 

00.v .55 0.0
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as .9 .z .0 .0 .00 009\0 .0.0.0 .0.0.5 050090 .00\5 90550559
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0090000 00090

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.050 0 .059 00 0905
00\09\09

050050550950 I 09500

moo .oz 009 50 05095059850909 9 09909

 

m om $ A» 0.3 80.086 08.3 no. v :3 m6

 

no. v :2 m6
no. .nn m.¢
no. .nn m.»
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MW .H .ns m.a
. a ma mo ¢ m.ma ooo.o>m.m oom.ma .¢ .an m.
.2 .q .z .m .m .00 ooa\m .o.m.m .o.m.§ mammam .oo\w prmman
.pm nHHHHoasom oSHB

 

 

modudpw cooam
.hﬂpawmn hapamnwmmd u hAOpmHm

.GOﬁmHsam HHo pdcwmm Una naaoawa .NmB u oaoagob .93 hoop .na\s OOOH u mmon
.mph ¢ .mna mo mama msooqudondm a cpdom
o¢\>m\aa mo¢ .02 won so maoﬁpw:HEpmpom Ha sands

 

 

 

 

ouﬁ.a goom m.ma ooo.oaa.o oom.ma mo.v .9: m.o
mo.v .gn m.m
mo. .an m.¢

. no. .mn m.n

04“ mo. :3 m.m

. m. .nﬂ m.H

m om ‘ mm m o.¢H ooo.omm.m oom.ma .m .ng m.
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Hmdpnopommﬂm .pm qﬁaaﬁoanom oaﬁa
1 11 1‘ moduspm oooam
.hnpawoﬁ hapnopmmmd u hhopaam
.Goamadao HHo panama and cﬁaoqwa .Nda I oaoa£o> .ps hues .QH\5 oooa a omen
.9» H .mna mw mawEmm msooupronbm u ousom
m¢\bm\aa poo .oz mom no mcoﬁpmaﬁapopom HHA capaa

-95-

 

m mm oo o p.ma ooo.omm.o oma.ma mo.v .an m.o

mo.v .nn m.>
mo.v. .nn m.o

no. v :3 win

no. v :5 ma...

no. .9: m.n

mod. .nn m.m

m. .9: m.a

oH om mo m ¢.na ooo.om>.m ooo.aa .m .nn m.

 

.2 .Q .2 .m .m .00 ooa\w .o.m.m .o.m.3 wamwdm‘.oo\b Habaoan
Hmﬁpcommmmaa .nm QHHHHOHQom cede

noacdpm cooam

.Aoww mo apnea onov mmammdm m muﬁmpdz .hﬂuawmn hapnopwamd I hhopoam

.aoamasao HHo panama cam oGHHoan .xog u oHoH£o> .pa hoop .nH\s oooa n omen
.mnh ¢ .mpa m¢ oamﬁom odoonwpdonﬁm a opsom
ow\ma\aa oom .02 won no mnoﬂpwcﬁaaopoo

HHHA magma

 

oH oH om w H.¢H ooo.omo.o ooo.ma no.v .pn

no.v .nn

mo.v .an

no.v .nn

mo.v I .nﬂ

mo. .9;

MW oo. .an
. m. .9;
Ha mm mo m m.«a ooo.omo.o ooo.aa .m .nn

m.m
m.b
m.©
m.m
m.¢
m.w
m.m
m.H
m.

 

.2 2H .2 om cm 000 OOH\w gnu-mom oUomog “EmeQM‘ oOO\D. HdPHOPd—H

HwavcohmHhHQ .Qm

gHHHHOHnom oaaa

uodospm oooam

.hﬂpawos hapnonamq4 I knopmam

.noamHsEo HHo panama cam :«Homoa .Kma I 0H0H30>
.nh H .mna mm cams

ow\ma\aa oon .02 meg no muoapanﬁanopoo

.oa hoop .pa\s oooH I omen
msoonwpsonsm I opsom

>HA manda

a.ﬂ «a oo o o.ma ooo.ooo.o ooo.na mo.v .hn NH

 

no.v .9: Ha

no.v .an 0H

no.v .nn o

oo.v .9: m

mo.v .nn o

no. .n: o

oo. .an m

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- 118 -

The penicillin blood plasma levels obtained from a
single injection of 1000 units per pound of body weight
of penicillin in either hydrogenated vegetable oil emulsion
or a wax, lanolin and oil emulsion compared very closely.
Both resulted in a plasma concentration with a range of 2
to 4 units per cc. of blood at 30 minutes, then declined
gradually, dropping below a measurable level at 4.5 to
5.5 hours. When a dosage of 2000 units per pound was used
the penicillin concentrations obtained from using the two
emulsion vehicles also compared favorably, however, only
two trials were run on each vehicle at this dosage.

In the eight trials on Romansky's Fonmula given sub-
cutaneously at 1000 units per pound the penicillin plasma
concentration ranged between 2 and 4 units at 50 minutes.
From this point on the decline was gradual, values drOp-
ping below a measurable level at 7.5 hours for 5 of the 8
dOgs. Of the three remaining one drOpped below the measur-
able level at 9.5 hours, another dropped below at 7.5 hours,
showed a level of 0.05 units at 8.5 hours then drOpped be-
low at 9.5 hours, the third drOpped below at 8.5 hours,
showed a level of 0.03 units at 9.5 hours, then dropped
below at 10.5 hours. This irregularity of absorption was
noted by Kirby et al (1945) in human patients who maintain-
ed assayable levels for a longer period of time.

In delaying the absorption of penicillin the oil
and wax suspension of penicillin was found to be superior

to the water-in-oil emulsion. The suspension given at the

- 119 -

rate of 1000 units per pound maintained a level of 0.05
units or better for 6.5 hours while the emulsion was
found to maintain this level for only 5.5 hours. When
2000 units per pound was administered in either of the
emulsions used a level of 0.03 units per cc. was sustain-
ed 5.5 to 7.5 hours. When compared to 5 per cent dextrose
in physiological saline as a vehicle, neither of the emul-
sions was found to possess any advantage over the former
vehicle in delaying absorption of penicillin.

During the course of these experiments one Opportun-
ity (Deg No. 805 - Table LXXIII) was presented to study
the transmission of penicillin to the amniotic fluid and
fetal blood following an injection of penicillin into the
pregnant bitch. Two-thousand units per pound of body
weight was given in water-in-oil emulsion and administered
by the subcutaneous route. Hourly blood samples were
drawn from the bitch. Four hours after the initial in-
jection of penicillin, the bitch was given ether anesthesia
and the young were removed by caesarian section. Samples
of the amniotic fluid and blood were taken from each of
the first five puppies as they were removed from the uter-
us. The last two puppies were kept 81d not sacrificed for
blood samples, however, samples were taken of the amniotic
fluid.

No evidence of toxicity was observed either in the

bitch or in the two puppies that were saved as all three

 

 

 

 

 

 

 

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- 121 -

made an uneventful recovery and the two puppies grew
normally.

The mother's penicillin blood plasma concentration
at one hour following injection of penicillin was 2 units
percc. At four hours, when the caesarian section Operation
was begun, her level was 0.5 units, dropping below a meas-
urable level at 8 hours. The amniotic fluid from 6 out
of the 7 amnions had a penicillin concentration of 0.06
units. Blood plasma from 3 of the 5 puppies checked had
a penicillin blood plasma concentration of 0.06 units.

Seventy-one penicillin trials were run during the
course of this experiment. In 70 of these, blood samples
were taken at the beginning and end of each individual
trial and checked for red and white cell counts and hemoglo-
bin content. Differential leukocyte counts were made on
67 of the dogs, in three cases the slides were too poorly
stained for accurate counting.

The gre test difference between the initial and final
count in any one trial was 890,000 red blood cells per cu.
mm., 2,350 white blood cells per cu. mm. and 1.0 gram of
hemoglobin. 0n the differential counts, in any one given
trial, the largest difference between initial and final
counts on eosinophiles was 6 cells, neutrophiles 12 cells,
lymphocytes 7 cells and monocytes 8 cells.

Todd and Sanford (1943) state that “Since twice the
Standard Deviation is the usually accepted limits of signi-

ficance, the error of a single estimate of the erythrocyte

- 122 -

count was given by them as Z 16 per cent". The difference
between the initial and final erythrocyte counts in each
case was found to be less than the ”normal" error allowable.
Each trial was further tabulated to find out if there
was a trend towards lowering or raising of the cellular
constituents or hemoglobin content following a single

injection of penicillin. Results of this tabulation are
shown in Table LXXIV.

Table LXXIV. BLOOD PICTURE FOLLOWING A SINGLE INJECTION
OF PENICILLIN COMPARED WITH THE INITIAL
BLOOD PICTURE.

 

 

No. of Showing Showing Trials Showing

 

Test Trials Decrease Increase Identical Results
W. B. C. 70 31 36 5

R. B. C. 70 39 29 l
Hemoglobin 70 35 50 5

% Eosinophiles 67 28 35 4

% Basophiles 67 5 5 57

% Neutriphiles 67 55 27 ‘ 5

3; Lymphocytes 67 27 54 e

% Monocytes 67 26 31 10

 

The results revealed no observable trends as the
number of trials showing an increase approximately balanc-

ed with those showing a decrease. Since the difference bet

- 125 -

tween the initial and final results in each trial was

found to be more or less within the normal error and since.
there was no trend toward raising or lowering, a single
injection of penicillin was thought to have no immediate
effect (within 5 to 12 hours) on altering red and white

cell counts, differential counts or hemoglobin content in
the normal healthy dog. The range of blood constituents

in all trials was found to be within the normal variations
reported by Coffin (1945), Malkamus (1944) and Boddie (1946).

The subcutaneous route of injection was found to be
the one of choice for ease of administration. None of the
dogs objected to this route when a 24 gauge needle was used.
Slight objection was registered by a few of the dogs when
the 17 gauge needle was used for injection of the oil and
wax emulsions and suspensions. Objection here was to the
insertion of the needle and not to the deposition of the
penicillin and vehicle. All dogs objected to the intra-
muscular and intraperitoneal injections. Intravenous in-
jections were well tolerated.

None of the degs eXhibited any local or systemic
reactions following the injection of penicillin, regard-
less of the vehicle used.

All dogs were sacrificed for autOpsy at the term—
ination of the experiment. Examinations of the sites of
injection revealed no macroscOpic lesions in any of the

animals.

- 124 -
SUMMARY AND CONCLUSIONS

Published experimental data obtained by the adminis-
tration of penicillin in dogs using the various injectable
routes, different dosages and vehicles were found to be
quite limited. Data on blood cell counts and hemoglobin
content following administration of penicillin were also
limited.

The vehicles used in this experiment were: 5 per
cent dextrose in physiological saline, two water-in-oil
emulsions (hydrogenated vegetable oil, wax and lanolin
in peanut oil) and Romansky's Formula.

The d0gs exhibited the least objection to injections
by the subcutaneous and intravenous routes and most ob-
jection to those by the intramuscular and intraperitoneal
routes when penicillin was administered. Following a
single injection of 1000 units per pound of body weight
in a vehicle consisting of 5 per cent dextrose in physio-
logicalsaline the penicillin blood plasma concentration
was found to be approximately the same for all four routes
of injection at 50 minutes, however,the penicillin level
of 0.03 units or better was maintained approximately
twice as long when the sucutaneous, intramuscular, and
intraperitoneal routs were used as compared to the in-
travenous route.

Penicillin blood plasma levels following the admin-

istration of various doses (250, 500 and 1000 units per lb.)

- 125 -

in 5 per cent dextrose and physiological saline given
intramuscularly and subcutaneously were studied. In this
series of trials the resulting levels following subcutaneous
administration were found to be slightly superior to those
following intramuscular administration both in concentra-
tions and prolongation of the plasma levels. A concentrat-
ion of 0.03 units or better was found to be maintained an
average of 4 hours following a single injection of 1000
units per pound, 5.5 hours following 500 units per pound
and 2.25 hours following 250 units per pound.

Five per cent dextrose in physiological saline was
found to be equal or slightly superior to the water-in-oil
vehicles in maintaining penicillin plasma levels. Romansky‘s
Formula was found to excell the above vehicles, as a level
of 0.03 units or better were maintained in all cases at
least 6.5 hours following a subcutaneous injection of 1000
units per pound.

A change in the character of penicillin was noted,
as that used after the middle of June 1946 gave rise to a
higher level and maintained a measurable level somewhat
longer.

No local or systemic reactions were noted following
the injection of the various penicillin preparations used
in this experiment. Studies on the blood picture indicate
that single injections of penicillin in dosages from 250
to 2000 units per pound of body weight have no immediate

- 126 -

effect on altering the red and white cell counts, differen-
tial count or hemOglobin content in the normal healthy dog.
No macroscOpic lesions were found at the sites of injection

on autopsy of the dogs at the termination of the experiment.

- 127 -
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- 129 -

Eagle, H. and A. D. Musselman. 1946. Low Therapeutic
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Elias, W. F., H. J. lerrion and T. Speicher. 1945.
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223-225.

Fisk, R. T., A. G. Foord and G. Alles. 1945. Prolongy
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Fleming, A. 1929. On the Antibacterial Action of
Cultures of a Penicillin with Special Ref-
erence to Their Use in the Isolation of
B. Influenzae. Brit. J. Egper. Path.
I0:226-236. "—

 

 

Fleming, A., J. Suchet, M. Y. Young and A. J. E. Rowe.
1944. Penicillin Content of Blood Serum
After Various Doses of Penicillin By
Various Routes. Lancet. 2:621-624.

Flory, H. W. and M. A. Jennings. 1942. Some BiolOgical
Properties of Highly Purified Penicillin.
Brit. J. Exper. Path. 23:120-123.

 

Freund, J. and K. J. Thompson. 1945. A Simple Rapid
Technic of Preparing Water-in-Oil Emulsions
of Penicillin, Drugs and BiolOgies. Science
101:468-469.

Hamre, D., G. Rake, C. M. McKee and H. B. McPhillamy.
1943. The Toxicity of Penicillin as Pre-
pared For Commercial Use. Am. _J_. 1‘_I_. _S__c_.
206:642-652.

Herwick, R. P., H. Welch, L. E. Putman and A. M. Gamboa.
1945. Correlation of the Purity of Peni-
cillin Sodium. _J_. A. M. A. 127:74-76.

Hoffman, W. S. 1946. Subcutaneous vs. Intramuscular
Administration of Penicillin. _J. Lab. §_c_
Clin. Mad. 31:1165-11690

 

Horus, H., C. Wilcox and M. Finland. 1946. Plasma
Levels After Repository Injections of
Penicillin in Water-in-Oil Emulsions.
Proc. Soc. Exp. Biol. §.E§§f 63:199-200.

Jones, R. 1927. Oxolic Acid a Good White Cell Diluting
Fluid. 'J. Lab. Clin. Med. 12:614.

- 150 -

Jones, T. R. L., E. M. Donaldson and S. J. Allen. 1946.
One-Shot Treatment of Acute Gonorrhea with
Penicillin. Lancet. 1:526-529.

Kirby, M. M., M. Leifer, S. P. Martin, C. H. Rammelkamp
and J. M. Kinsman. 1945. Intramuscular
and Subcutaneous Administration of Peni-
cillin in Beeswax-Peanut Oil. J. A. M. A.
129:940-944.

 

Klodney, M. H. and E. Denhoff. 1946. Reactions in Peni-
cillin Therapy. J. A. M. A. 130:1058-1061.

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of Penicillins G and X in Vitro. Science.
102 : 503-504. . ...__....__.

Malkamus, B. and Opperman, Th. 1944 Clinical Diagnosis,
Alex Eger, Inc., Chicago. Normal Aver-
ages of Red Blood Cells 288; Lenkocytes 294.

Ory, E. M., M. Meade and M. Finland. 1945. Penicillin
X Compared with Penicillin G with Reapect
to Sensitivity of Pathogenic Organisms and
Serum Levels. J. A. M. A. 129:257-261.

 

Ory, E. M., C. Wilcox, and M. Finland. 1946. Serum Lev-
els After Repository Injections of Penicil-
lin. Proc. Soc. Exp. Biol. §_Med. 62:86-88.

Parkins, W. M., M. Wiley, J. Chendy and H. A. Zintel.
1945. Maintenance of the Blood Level of
Penicillin After Intramuscular Injection.
Science. 101:203-205.

Raiziss, G. 1944. Penicillin in Oil Suspension. Bacter-
iostatic and Spirocheticidal Agent. Science.

100:412-415.

Rammelkamp, C. H. and C. S. Keefer. 1943. The Absorp-
tion, Distribution, and Excretion of Peni-
cillin. J. Clin. Investigation. 22:425-437.

Randall, W. A., C. W. Price and H. Welch. 1945. The
Estimation of Penicillin in Body Fluids.

30161106. 101:365'3660

Robinson, H. J. 1943. Toxicity and Efficacy of Peni-
cillin. J. Pharmacol. & Exp. Therap.

77:70-79.

- 151 -

Romansky, N. J. and G. E. Rittman. 1944. Penicillin:

Prolonged Action in Beeswax-Peanut Oil

Mixture: Single Injection Treatment of
Gonorrhea. Bull. U. S. Army_Med. Dept.
18:43-49. " "‘

 

 

Romansky, M. J. and E. G. Rittman. 1944. A Method of

Prolonging the Action of Penicillin.
Science. 100:196-198.

Rostenberg, A. and H. Welsh. 1945. A Study of the

Sanford,

Todd, Jo

Trumper,

Trumper,

Waltz,

Types of Hypersensitivity Induced by
Penicillin. Am. J. Med. S2. 210:158-167.

A. H., C. Sheard and A. E. Osterberg. 1933.
The Photolometer and Its Use in the Clin-
ical Laboratory. Am.J. Clin. Path.
3:412. — '-

C. and A. H. Sanford. 1943. Clinical Diagnos-
is by Laboratory Methods. W. A. Saunders
Co., Philadelphia. The "Normal" Error of
the Count 227. Diluting Fluid of Leaks
and Guy 246 o

M. and A. M. Hutter. 1944. Prolonging Effective
Penicillin Action. Science. 100:432-434.

M. and G. Thompson. 1946. Prolonging the Ef-
fects of Penicillin by Chilling. J. A. M._A.

and Zintel. 1945. The Transmission of Peni-
cillin to Amniotic Fluid and Fetal Blood
in the Human. Am. J. Obst. §_Gyne. 50:
338—340. '

Welch, H. and A. Rostenberg. 1944. Hypersensitivity of

the Tuberculine Type to Crystalline Peni-
cillin Sodium. J. A. M. A. 126:10-12.

Zinnamon, B. L. and V. P. Seeberg. 1945. Penicillin

Serum Concentrations in the Treatment of
Gonorfhea by Delayed Intramuscular Absorp-
tion. :1. Van. D18. Inform. 26:31-34.

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HICHIGRN STRTE UNIV. LIBRARIES

31293009898465

 

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m

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Coffin, D. L.

Cohn, A.,

Cohn, A.,

Colloway,

Committee

Committee

Committee

B.

J.

of

on

on

- 128 -

1945. Manual of Veterinary Clinical
Pathology. Comstock Publishing Co. Inc.,
Ithaca, N. Y. Erythrocyte Enumeration 109-
110; Leukocyte Enumeration 106-109;
Leukocyte Differential 92-95; Normal Ranges
of Blood Cellular Elements 118.

A. Kornblith, I. Crumstein, J. Freund and
K. J. Thompson. 1946. Treatment of Gon-
orrhea with Penicillin. Results of Therapy
with Penicillin in Water-in-Oil Emulsion as
Compared with Those Obtained by Single or
Multiple Injections of Aqueous Penicillin
Solutions. J. Van. Dis. Inform. 27:154-159.

 

A. Kornblith, I. Crumstein, K. J. Thompson
and J. Freund. 1945. Repository Injection
of Penicillin in Water-in-Oil Emulsion.

I Effect on Gonorrhea. Proc. Soc. __2.
Biol. §_M§d. 59:145-147.

 

L. and S. W. Barefoot. 1946. Immunological
Studies on Patients Developing Urticaria
Associated with Penicillin Therapy. ‘J.
Investigative Dermatology. 7:285-290.

Chemotherapeutic and Other Agents. 1943.
Penicillin in the Treatment of Infections.

Medical Research and Medical Research Coun-
cil. 1945. Chemistry of Penicillin.
Science. 102:627-629.

Medical Research, The United States Service
and The Food and Drug Administration. 1946.
The Changing Character of Penicillin.

J. _A_. M. A. 131:271-275.

Dawson, M. H. and G. L. Hobby. 1944. The Clinical Use

of Penicillin. J. A_._ M. A. 124:611-622.

Doll, E. R. and W. W. Dimock. 1946. Efficacy of Various

Agents for Delaying Absorption of Pen-
iCillino 9;. E0 lo 2!. £0 108:510'5130

Doll, E. R. and F. E. Hull. 1947. Serum Levels of

Eagle, H.

Penicillin in Horses and Sheep Following
Injection in a Water-in-Oil Vehicle. Vet.

Med. 42:25-26.

1946. The Relative Activity of Penicillin F,

g, E, and X Against Spirochetes and Strept-
ococci in Vitro. 'J. Bacty. 52:81-87.

- 129 -

Eagle, H. and A. D. Musselman. 1946. Low Therapeutic
Activity of Penicillin K Relative to That
of Penicillins F, G and X, and Its Pharm-
acological Basis. Science. 103:2681.

Elias, W. F., H. J. lerrion and T. Speicher. 1945.
Inhibitory Factors in the Determination of
Penicillin in Human Sera. Science. 102:
223-225.

Fisk, R. T., A. G. Foord and G. Alles. 1945. Prolong-
ation of Penicillin Activity By Means of
Adrenaline. Science. 101:124-126.

Fleming, A. 1929. On the Antibacterial Action of
Cultures of a Penicillin with Special Ref-
erence to Their Use in the Isolation of
B. Influenzae. Brit. J. Exper. Path.
1:0ng —'_'

Fleming, A., J. Suchet, M. Y. Young and A. J. E. Rowe.
1944. Penicillin Content of Blood Serum
After Various Doses of Penicillin By
Various Routes. Lancet. 2:621-624.

Flory, H. W. and M. A. Jennings. 1942. Some Biological
PrOperties of Highly Purified Penicillin.
Brit. J. Exper. Path. 23:120-123.

Freund, J. and K. J. Thompson. 1945. A Simple Rapid
Technic of Preparing Water-in-Oil Emulsions
of Penicillin, Drugs and BiolOgies. Science
101:468-469.

Hamre, D., G. Rake, C. M. McKee and H. B. MCPhillamy.
1943. The Toxicity of Penicillin as Pre-
pared For Commercial Use. _A_m. J. M. _S__<_:_.
206:642-652.

Herwick, R. P., H. Welch, L. E. Putman and A. M. Gamboa.
1945. Correlation of the Purity of Peni-

cillin Sodium. J. g. g. 4- 127:74-76.

Hoffman, W. S. 1946. Subcutaneous vs. Intramuscular
Administration of Penicillin. J. Lab. J
Clin. Med. 51:1165-11690

 

Horus, H., C. Wilcox and M. Finland. 1946. Plasma
Levels After Repository Injections of
Penicillin in Water-in-Oil Emulsions.
Proc. Soc. Exp. Biol. §_Mgg. 63:199-200.

Jones R. 1927. Oxolic Acid a Good White Cell Diluting
’ Fluid. J. Lab. Clin. Med. 12:614.

- 130 -

Jones, T. R. L., E. M. Donaldson and S. J. Allen. 1946.
One-Shot Treatment of Acute Gonorrhea with
Penicillin. Lancet. 1:526—529.

Kirby, M. M., W. Leifer, S. P. Martin, C. H. Rammelkamp
and J. M. Kinsman. 1945. Intramuscular
and Subcutaneous Administration of Peni-
cillin in Beeswax-Peanut Oil. J. A. M. A.
129:940-944.

 

Klodney, M. H. and E. Denhoff. 1946. Reactions in Peni-
cillin Therapy. J. A. M. A. 130:1058-1061.

Libby, R. L., and N. L. Holmberg. 1945. The Activity
of Penicillins G and X in Vitro. Science.

102:505-504.

Malkamus, B. and Opperman, Th. 1944 Clinical Diagnosis,
Alex Eger, Inc., Chicago. Normal Aver-
ages of Red Blood Cells 288; Lenkocytes 294.

Ory, E. M., M. Meade and M. Finland. 1945. Penicillin
X Compared with Penicillin G with Respect
to Sensitivity of Pathogenic Organisms and
Serum Levels. J. A. M. A. 129:257-261.

 

Ory, E. M., C. Wilcox, and M. Finland. 1946. Serum Lev-
els After Repository Injections of Penicil-
lin. Proc. Soc. Exp. Biol. §_Med. 62:86-88.

 

Parkins, “’0 M0, M0 VJiley, Jo Chendy and Ho A. Zintel.
1945. Maintenance of the Blood Level of
Penicillin After Intramuscular Injection.
Science. 101:203-205.

Raiziss, G. 1944. Penicillin in Oil Suspension. Bacter-
iostatic and Spirocheticidal Agent. Science.

100:412-413.

Rammelkamp, C. H. and C. S. Keefer. 1943. The Absorp-
tion, Distribution, and Excretion of Peni-
cillin. J. Clin. Investigation. 22:425-437.

Randall, W. A., C. W. Price and H. Welch. 1945. The
Estimation of Penicillin in Body Fluids.

Science. 101:365-366.

Robinson, H. J. 1943. Toxicity and Efficacy of Peni-
cillin. J. Pharmacol. & Exp. Therap.

77:70—79.

- 151 -

Romansky, M. J. and G. E. Rittman. 1944. Penicillin:

Prolonged Action in Beeswax-Peanut Oil

Mixture: Single Injection Treatment of
Gonorrhea. Bull. U. S. Army_Med. Dept.
18:43-49. "' “

 

 

Romansky, M. J. and E. G. Rittman. 1944. A Method of

Rostenberg,

Sanford,

Todd, J.

Trumper,

Trumper,

Waltz,

A.

C.

M.

and

Prolonging the Action of Penicillin.
Science. 100:196-198.

A. and H. Welsh. 1945. A Study of the
Types of Hypersensitivity Induced by
Penicillin. 5m. J. Med. S3. 210:158-167.

H., C. Sheard and A. E. Osterberg. 1933.
The Photolometer and Its Use in the Clin-
ical Laboratory. Am.J. Clin. Path.
3:412. — '-

and A. H. Sanford. 1943. Clinical Diagnos-
is by Laboratory Methods. W. A. Saunders
Co., Philadelphia. The "Normal" Error of
the Count 227. Diluting Fluid of Leaks
and Guy 246.

and A. M. Hutter. 1944. Prolonging Effective
Penicillin Action. Science. 100:432-434.

and G. Thompson. 1946. Prolonging the Ef-
fects of Penicillin by Chilling. J. A. M._A.
130:627-630.

Zintel. 1945. The Transmission of Peni-
cillin to Amniotic Fluid and Fetal Blood
in the Human. Am. J. Obst. g Gyne. 50:
338-340. ‘

Welch, H. and A. Rostenberg. 1944. Hypersensitivity of

the Tuberculine Type to Crystalline Peni-
cillin Sodium. J. A. M. A. 126:10-12.

Zinnamon, B. L. and V. P. Seeberg. 1945. Penicillin

Serum.Concentrations in the Treatment of
Gonorfhea by Delayed Intramuscular Absorp-
tion. 'J. Ven. Dig. Enfogm. 26:31-34.

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