IN MICE,
`THE SERUM CONCENTRATION OF PENICILLIN
`RABBITS, AND MEN AFTER ITS INTRAMUSCULAR
`INJECTION IN AQUEOUS SOLUTION
`
`HARRY EAGLE, RALPH FLEISCHMAN, AND A. D. MUSSELMAN
`
`Laboratory of Experimental Therapeutics of the U. S. Public Health Service and the Johns
`Hopkins School of Hygiene and Public Health, Baltimore 5, Maryland
`
`Received for publication October 13, 1948
`
`The serum levels of penicillin G in man after its intramuscular injection in
`aqueous solution or peanut-oil-beeswax suspension have been described in a pre-
`ceding paper (Tucker and Eagle, 1948). Similar data are here reported with
`respect to the plasma levels afiorded by the intramuscular injection of aqueous
`penicillin G in mice and rabbits.
`
`METHODS AND MATERIALS
`
`The solutions were injected intramuscularly into the thigh, in a volume of 1 ml
`per kg in rabbits, and in 0.2-ml volume in mice.
`In the latter species the injec-
`tion was partially subcutaneous, because of leakage from the muscle into the sur-
`rounding tissues. The courtesy of the manufacturers in supplying the penicillin
`G used in these studies (Squibb lot numbers V-31, CRA214-20, and 17579, and
`Commercial Solvents lot number 46042605) is gratefully acknowledged.
`The method of assay was a serial dilution technique in which inhibition of
`hemolysis by the C-203 strain of Streptococcus pyogenes was the end point (Ram-
`melkamp, 1942; Kirby and Rantz, 1944). The details of the method have been
`given elsewhere (Eagle and Newman, 1947).
`In the assays of human and rabbit
`serum, the data have been corrected for the inhibitory effect of serum in the assay
`(Eagle, l947a,b; Tompsett, Schultz, and McDermott, 1947a,b), using the cor-
`rective factors as experimentally determined for human serum (Eagle and
`Tucker, 1948) and since found to be valid also for rabbit serum. No corrective
`factors have been applied to the mouse data. The values in which, because of
`the high concentration of serum in the indicator tube, that correction would
`probably have been significant are indicated in the tables.
`It is a pleasure to acknowledge our indebtedness to Mr. Nathan Mantel, of the
`Ofiice of Statistical Coordinator, Division of Public Health Methods, for his
`assistance in the calculation of the probable errors of the median serum levels.
`
`EXPERIMENTAL RESULTS
`
`Figure 1 and the first section of table 1, showing the results obtained in man,
`are here reproduced in modified form‘ from a previous paper (Tucker and Eagle,
`
`1 In the previous communication (Tucker and Eagle, 1948) the standard deviation given
`in the tables was a measure of the variability of the data about the median, as calculated by
`
`the formula (0 ==
`
`Ed’ med) and the standard error given was calculated by the for-
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`120
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`H. EAGLE, R. FLEISCHMAN, AND A. D. MUSSELMAN
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`[von 57
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`1948) in order to facilitate comparison between the results obtained in the 3
`species. Figure 2 and the second section of table 1 show the median serum
`levels in rabbits after the injection of penicillin G at 60, 10, 3, 1, and 0.6 mg per
`kg. Figure 3 and the last section of table 1 give the median serum concentra-
`tions obtained in mice similarly injected.
`In general, the initial blood levels, 15 or 30 minutes after the injection, varied '
`more or less linearly with the amount of penicillin injected.
`In figure 4, the
`.»
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`
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`Penicillin G in Man
`Aqueous solution
`injected inlromusculorly
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`Figure 1. The serum concentrations of penicillin G in man after its intramuscular injec-
`tion in aqueous solution at varying dosage.
`(After Tucker and Eagle, 1948; cf. footnote 1.)
`
`TIME IN HOURS
`
`15-minute concentrations in man are approximations only, obtained by the
`extrapolation of the curves of figure 1. These initial values were of the same
`order of magnitude in rabbits and mice, but in man were 3 to 5 times higher.
`This is indicative of a significant species difference, either in the amount of body
`
`Tl
`mula S.E. = ‘f;_, and is a measure of the reliability of the variability measure c. Of
`greater significance as a measure of the reliability of the median itself is its standard error,
`given by the formula S.E. med. = 1.25 - It is the latter value that is indicated
`n n —
`in all the figures and tables of this paper, including the human data of figure 1 and table 1.
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`1949]
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`SERUM CONCENTRATION or PENICILLIN G
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`CONCENTRATIONOF -—2
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`122
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`H. EAGLE, R. FLEISCEMAN, AND A. D. MUSSELMAN
`
`[voL. 57
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`fluid over which the penicillin is initially distributed, or in the rate of that dis-
`tribution.
`
`In
`After the initial period of rapid absorption, the blood levels fell off rapidly.
`rabbits this rate of decrease averaged 65 per cent each hour, so that the residual
`serum concentration at time t was k X 0.35‘, in which t is the time in hours over
`the period of experimental observation, and k is a constant determined by the
`amount of penicillin injected.
`In man, the rate of fall was less consistent, the
`percentage drop per hour over the first 2 hours varying from 65 at the larger
`
`OIIIIII
`
`‘
`
`60 mg /kg of penicillin G
`Intromusculorly
`
`2
`
`3
`IN
`
`TIME
`
`HOURS
`
`4
`
`6
`
`_Fz'g_ure 2. The serum concentrations of penicillin G in rabbits_after its intramuscular
`inyection in aqueous solution at varying dosage. The dashed portion of the curve at 3 mg
`per kg by-passes the low value at 2 hours.
`
`In mice, however,
`doses to approximately 80 per cent at the smaller (figure 1).
`the blood levels during the first 2 hours fell olfat the extraordinary rate of 91
`to 99 per cent per hour (figure 3). At the largest dosage of 200 mg per kg, a
`slower rate of fall developed after several hours. This probably reflects a re-
`versal in the direction of flow of penicillin out of the tissue reservoir established
`by its initial distribution and back into the blood.
`The serum concentrations provided by a given dose of penicillin were usually
`3 to 5 times higher in man than they were in rabbits similarly injected. Al-
`though the concentrations in rabbits and mice began at the same level, they
`rapidly diverged because of the faster disappearance of penicillin in mice. These
`species diflerences are illustrated for a single dosage of penicillin in figure 5.
`
`
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`1949]
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`snmm CONCENTRATION or PENICILLIN G
`
`123
`
`In the therapeutic use of penicillin, one of the most important single factors is
`the length of time for which the tissue levels of penicillin remain at effectively
`bactericidal levels. When the three species were compared on this basis (figure
`6), a given serum level was sustained approximately 50 per cent longer in man
`than it was in rabbits similarly injected.
`In large part this reflects the initially
`higher concentrations provided in man.
`In turn, the levels were sustained
`
`mg/|io'o1 penicillin G
`inmmuleulavly
`
`PENICILLIN
`
`OF
`
`
`
`SERUMCONCENTRATION
`
`
`
`
`
`(No!correctedforserumErrorinAssay)
`
`TIME
`
`IN
`
`HOURS
`
`Figure 3. The serum concentrations of penicillin G in mice after its intramuscular in
`jection in aqueous solution at varying dosage. Unlike the man and rabbit data of figures
`1 and 2, the data in this figure have not been corrected for the inhibitory efiect of serum in
`the assay. That would robably have been significant only for the two values indicated
`by the symbol :3: in the gum.
`
`approximately 3 times longer in rabbits than they were in mice because of the
`faster rate of fall in the latter species. Thus, as is indicated in figure 6, a dose of
`0.6 mg per kg provided serum levels in excess of, e.g., 0.1 microgram per ml for
`0.5, 1.4, and 2.3 hours in mice, rabbits, and men, respectively; and after a dose
`of 10 mg per kg the corresponding time periods were 1.4, 3.9, and 6.6 hours.
`In
`order to prolong by 1 hour the time for which this level of 0.1 microgram per ml
`would be maintained in the serum, the dosage of penicillin would have to be
`approximately doubled in man, and multiplied approximately 3-fold in rabbits,
`
`
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`124
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`H. EAGLE, R. FLEISCEMAN, mp A. D. MUSSELMAN
`
`[voL. 57
`
`whereas a 5- to 20-fold increase would be necessary in mice, depending on the
`dosage range (figure 6).
`The factors responsible for the relatively rapid disappearance of penicillin
`from mouse blood are as yet unknown. An important consideration may be the
`
`SERUMCONCENTRATIONOFPENICILLINAFTERI5MINUTES,MIOROGRAMS
`
`/‘ml
`
`
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`
`0.6
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`1
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`3
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`no
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`9
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`so
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`zoo
`
`DOSAGE OF PENIOILLIN G, M0 [K9
`Figure 4. The effect of the amount of penicillin injected intramuscularly on the initial
`(15-minute) serum concentration in man, mouse, and rabbit. Human data obtained by
`extrapolation of curves in figure 1.
`
`renal clearance of penicillin, which in this species may be higher relative to body
`weight than it is in either rabbits or man (Eagle and Newman, 1947).
`
`SUMMARY
`
`Data are given for the median serum concentrations of penicillin G after its
`intramuscular injection in aqueous solution in men, rabbits, and mice at doses of
`0.6, 1 (1.5), 3, 10, 60, and 200 mg per kg. For equal doses of penicillin the serum
`levels at a given time were in the order man > rabbit > mouse.
`The initial (15- to 30-minute) level in man was 3 to 5 times greater than it was
`
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`10 mg I kg
`lnmmusculorly
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`SERUMCONCENTRATIONOFPENICILLING,MICROGRAMSPERml
`
`I/4 «/2
`
`I
`
`A
`
`2
`
`4
`
`TIME
`
`IN
`
`HOURS
`
`Figyre 6: 'I_‘he co1_1t1_-asting cnrves of the serum penicillin concentration in men, rabbits,
`and mice similarly injected with penicillin G.
`
`
`
`A
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`
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`ROURSFORWHICHSERUMCONCENTRATIONREMAINED
`
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`
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`GREATERTHAN0.!MICROGRAMSPERml
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`DOSAGE OF PENICILLIN G, MILLIGRAMS PER Kg
`
`Figure 6'. The time for which varying dosages of penicillin G_ p_rovide serum concentr§.-
`tions in excess of 0.1 microgram per ml. The value for man injected at 3 mg per kg is
`an approximation only, obtained by extrapolation of the curve in figure 1.
`125
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`126
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`H. EAGLE, R. FLEISCEMAN, Am) A. n. MUSSELMLAN
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`[voL. 57
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`in mice or rabbits, indicative of a significant species diflerence in the initial dis-
`tribution of the drug in the body fluids.
`Thereafter, penicillin disappeared from the serum of rabbits at the rate of 65
`per cent an hour, and somewhat faster in man.
`In mice, however, it fell off at a
`much faster initial rate, varying between 91 and 99 per cent an hour.
`In consequence of these differences in the initial distribution of penicillin and ‘
`its subsequent rate of disappearance from the serum, the time for which a given
`dosage of penicillin provided a measurable serum concentration was in the order
`1 :3 :4i- in mouse, rabbit, and man, respectively.
`To prolong a given serum level by 1 hour would require approximately a 2-fold
`increase in dosage in man, a 3-fold increase in rabbit, but a 5- to 20-fold increase
`in mice.
`
`REFERENCES
`
`1947:: The inactivation of penicillins F, G, K, and X by human and rabbit
`EAGLE, H.
`serum. J. Exptl. Med., 85, 141-161.
`EAGLE, H.
`1947b The varying blood levels afforded by penicillins F, G, K and X in rabbits
`and man. J. Exptl. Med., 85, 163-173.
`EAGLE, H., AND NEWMAN, E. V.
`1947 The renal clearance of penicillins F, G, K and X in
`rabbits and man. J. Clin. Investigation, 26, 903-918.
`EAGLE, H., AND Tucxmn, H. A.
`1948 The eflect of human serum on the dilution biosssay
`of penicillins G, X, and K.
`J. Bact., 56, 59-62.
`Kmnv, W. M., AND R.AN'rz, L. A.
`1944 Methods of measuring penicillin concentrations
`in body fluids.
`J. Bact., 48, 603.
`RAmmLxAm>, C. H.
`1942 A method for determining the concentration of penicillin in
`body fluids and exudates. Proc. Soc. Exptl. Biol. Med., 51, 95.
`Toupsm-r, R., Scmmrz, 8., AND McDmnuo-rr, W.
`1947a The relation of protein-binding
`to the pharmacology and antibacterial activity of penicillins X, G, dihydro F, and K.
`J. Bact., 53. 581-595.
`Influence of protein-binding
`1947b
`Toursnrr, R., Scnuurz, S., AND McDano*r'r, W.
`on the interpetation of penicillin activity in viva. Proc. Soc. Exptl. Biol. Med., 65.
`163-172.
`1948 The serum concentrations of penicillin G in man
`Tucxnn, H. A., AND EAGLE, H.
`following its intramuscular injection in aqueous solution and in peanut oil-beeswax
`suspension. Am. J. Med., 4, 343-354.
`
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