Journal of Antimicrobial Chemotherapy (1979) 5, 423-429
Antibiotic levels in experimentally infected mice in relation to therapeutic effect and antibacterial activity in vitro
Beecham Pharmaceuticals Research Division, Chemotherapeutic Research Centre, Brockham Park, Betchworth, Surrey RH3 7AJ, England
Antibiotic levels in blood were determined in experimentally infected mice following administration of a number of different penicillins at the median protective dose (PD i 0 ) and these levels were correlated with in vitro MIC determinations. When administered by repeated dosage the P D i 0 values were found to be lower than when the antibiotics were administered as a single dose and for the majority of the antibiotics tested the resulting peak levels in blood were below the in vitro MIC value. The results are discussed in relation to the nature of the antibiotic, the type of infection and the dosage schedule used. Introduction
In published studies on the evaluation of antibiotics two aspects continue to receive particular attention: one is the antibacterial activity in vitro, commonly expressed as the minimum inhibitory concentration, and the other is the level of antibiotic reached in the blood following a given dose. However, it is not certain what level of antibiotic is indeed required in the blood in order to achieve a therapeutic effect or what the relationship is between this level and the MIC as determined in vitro. It is widely assumed that the level reached in the blood should at least equal the MIC and preferably exceed this value by several fold. Eagle, Fleischman & Musselman (1950a) reported that the minimum effective serum concentrations of benzylpenicillin in a number of experimental infections in mice and rabbits were 2 to 5 times higher than those necessary to kill the same organism in vitro. However, using a schedule of repeated doses of benzylpenicillin in rats experimentally infected with Streptococcus pneumoniae Schmidt and Walley (1951) found that at the ED B0 the plasma levels 15 min after dosing were only equal to the concentration required for inhibition in vitro and 30 min after dosing the levels had fallen to approximately half the MIC. In order to obtain further data on the question of blood levels in relation to therapeutic effect, studies have been carried out in which antibiotic concentrations were determined in blood in experimentally infected mice at dosages corresponding to the median protective dose (PD 60 ) and these levels have been correlated with the in vitro MIC. These results are reported here. *To whom correspondence and reprint requests should be sent. 423 O3O5-7453/79/O4O423+07 801.00/0
© 1979 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at University of California, Santa Barbara on June 24, 2015
D. Merrikin and G. N. Rolinson*
424
D. MerrOdn and G. N. Rolinson Materials and methods
MIC
Protein binding The extent of binding in mouse serum was determined using Centriflo membrane cones type 50A (Amicon Ltd.). After centrifugation the antibiotic concentration in the ultrafiltrate was determined by cup-plate assay using Sarcina lutea (NCTC 8340) as test organism with antibiotic standards prepared in phosphate buffered saline. Experimental infections in mice Male or female albino mice, 18 to 22 g, CS1 strain (Charles River U.K. Ltd.) were used. The bacteria used for infection were Staphylococcus aureus Smith and Staph. aureus strain T43 (non-f}-lactamase producing) and Escherichia coli strain 8 (ampicillin sensitive) and the cultures were grown in double strength veal infusion broth (Difco) at 37°C overnight. The antibiotics were administered in solution in phosphate buffered saline, subcutaneously, either as a single dose immediately after infection or by repeated dosage as indicated in the results. Intraperitoneal infections. Overnight cultures of Staph. aureus Smith or E. coli strain 8 were diluted 1/1000 in 3% hog gastric mucin (1701-W, Wilson Laboratories, Chicago) and 0-5 ml injected intraperitoneally to mice giving an infective inoculum of 100 to 1000 median lethal doses. Groups of ten mice were used and the numbers surviving 4 days after infection were recorded and the dose required to protect 50% of the animals calculated (median protective dose, PD60) using log X probability paper. Intramuscular infection. Groups often mice were infected intramuscularly in the right hind leg with 0-2 ml of an undiluted overnight culture of Staph. aureus strain T43 or a 1/8 dilution of an overnight culture of E. coli strain 8. The inoculation was made into the muscles of the back of the thigh as described by Selbie & O'Grady (1954). The diameter of the infected thigh of each of the mice was measured 24 h after inoculation in the case of E. coli, and 4 days after inoculation in the experiments with Staph. aureus, and the antibiotic dose required to minimize the enlargement of the thigh to 50 % of the infected controls was calculated (median protective dose, PD60) using a log x probability plot of the results. Antibiotic levels in blood After determination of the respective PD 60 dosages for the different antibiotics, using the different infections, each antibiotic was administered at these doses to infected mice and the resulting levels in blood determined by sacrificing groups of mice at intervals of time and collecting from each a sample of blood in heparinized tubes. Another group of mice
Downloaded from http://jac.oxfordjournals.org/ at University of California, Santa Barbara on June 24, 2015
Minimum inhibitory concentrations (MIC) were determined in double strength veal infusion broth (Difco) using the Cooke microtitre system (Dynatech Laboratories Ltd.). Overnight broth cultures of the test organism were diluted 1/1000 and 005 ml amounts were added to an equal volume of medium in each well of the microtitre plate to give a final inoculum of approximately 106 cfu/ml. MIC tests were also carried out by tube dilution using 5 ml amounts of nutrient broth (Oxoid no. 2) inoculated with one drop (approx. 0-03 ml) of undiluted overnight broth culture giving afinalinoculum of approx. 10" cfu/ml. The minimum inhibitory concentrations were determined afteT 18 h at 37°C.
Antibiotic levels in experimentally infected mice
425
Results and discussion Tables I and II show the PD 6 0 values for the antibiotics used in the different experimental infections together with the peak blood levels which resulted at these dosages. The tables also show the extent of binding of the antibiotics in mouse serum and the MIC values against the infecting organisms. In the case of the infections with Staph. aureus, when benzylpenicillin, penicillin V, phenethicillin, ampicillin or amoxycillin were administered by repeated injection at the PD 6 0 dosage the levels in blood failed to reach the MIC. With ampicillin and amoxycillin the levels were even less than one tenth of the MIC value. In the case of the infections with E. coli the levels of benzylpenicillin, ampicillin, amoxycillin and carbenicillin in blood were also found to be below the respective MIC values when these antibiotics were administered by repeated injection at the PD 6 0 dose. However, these findings regarding the levels of antibiotic in blood relative to MIC are not necessarily paradoxical. From the results in Table III it can be seen that over a period of 6 h a marked inhibitory effect on the growth of Staph. aureus occurred with benzylpenicillin and ampicillin at concentrations one tenth the MIC and similar results were obtained with E. coli and ampicillin. Subinhibitory concentrations of antibiotics are already known to exert some effect on bacterial growth in vitro (Lorian, 1975) and the terms minimum antibacterial concentration (MAC) and minimum active concentration (MAC) have been used (Lorian & Popoola, 1972; Shah, Heetderks & Stille, 1976) to denote the minimum concentration causing a significant diminution in either the rate or the final amount of growth compared with antibiotic-free controls. These effects of subMIC levels of antibiotic may well be adequate to result in a therapeutic effect in experimental infections provided the normal body defences are not impaired and provided such levels of antibiotic are maintained for an adequate period of time. It is also known that certain antibiotics at subinhibitory concentrations markedly reduce the ability of Staph. aureus to produce a variety of toxins and enzymes (Gemmell, 1977) and this may contribute to the therapeutic effects observed at such concentrations. In contrast to the results with benzylpenicillin, penicillin V, phenethicillin, ampicillin and amoxycillin, for which the levels in blood were relatively low compared with the MIC, the levels of methicillin and cloxacillin relative to MIC were found to be considerably higher. For example, in the intramuscular infection with Staph. aureus, despite administration by repeated dosage, the levels of cloxacillin in blood were as high as 13 X MIC compared with values of less than 008 X MIC for ampicillin or amoxycillin. It may be significant that of the penicillins tested cloxacillin was the most highly bound in mouse serum and it will be seen from Table I that the levels of free cloxacillin in blood,
Downloaded from http://jac.oxfordjournals.org/ at University of California, Santa Barbara on June 24, 2015
was set aside to confirm that the dosage used did result in approximately 50 % protection. In the experiments involving a single dose of antibiotic, blood samples were taken 10, 20, 30, 60 and 120 min after dosage. In the experiments involving repeated dosage, blood samples were taken at the same time intervals, following both the first and the last dose. No accumulation of antibiotic occurred and the results shown in the tables are the means of the peak levels in the blood following the first and the last dose. Blood samples from each of four mice were assayed individually and the mean values for the group are reported. The antibiotic concentrations in the blood samples were determined by cup-plate assay using standards prepared in heparinized mouse blood. Sarcina lutae (NCTC 8340) was used as assay organism except in the case of carbenicillin assay for whichPseudomonas aeruginosaHCTC 10701 was used.
MIC*
0-03 003 0-06 012 012 10 012 0-03 003 006 012 012 2-0 012
44 73 77 22 19 34 80 44 73 77 22 19 34 80
(mg/1)
*By microtitre. tPeak level of total antibiotic at PDI0 dose. {Calculated from values for extent of binding.
Intraperitoneal infection with Staph. aweus Smith benzylpenicillin penicillin V phenethicillin ampicillin amoxycillin methicillin cloxacillin Intramuscular infection with Staph. aweus T43 benzylpenicillin penicillin V phenethicillin ampicillin amoxycillin methicillin cloxacillin
bound mouse serum riJio
30 3-4 3-0 1-9 0-9 100-0 39-0
0-25 012 018 0-14 013 4-2 12-5
(mg/kg)
20-7 17-0 111 6-5 3-3 29-2 16-3
36-7 63-3 48-3 8-3 4-2 44-2 81-7 11 2-9 10 0-5 88-5 9-8
19
1-6
Antibiotic levels in experimentally infected mice in relation to therapeutic effect and antibacterial activity in vitro
Beecham Pharmaceuticals Research Division, Chemotherapeutic Research Centre, Brockham Park, Betchworth, Surrey RH3 7AJ, England
Antibiotic levels in blood were determined in experimentally infected mice following administration of a number of different penicillins at the median protective dose (PD i 0 ) and these levels were correlated with in vitro MIC determinations. When administered by repeated dosage the P D i 0 values were found to be lower than when the antibiotics were administered as a single dose and for the majority of the antibiotics tested the resulting peak levels in blood were below the in vitro MIC value. The results are discussed in relation to the nature of the antibiotic, the type of infection and the dosage schedule used. Introduction
In published studies on the evaluation of antibiotics two aspects continue to receive particular attention: one is the antibacterial activity in vitro, commonly expressed as the minimum inhibitory concentration, and the other is the level of antibiotic reached in the blood following a given dose. However, it is not certain what level of antibiotic is indeed required in the blood in order to achieve a therapeutic effect or what the relationship is between this level and the MIC as determined in vitro. It is widely assumed that the level reached in the blood should at least equal the MIC and preferably exceed this value by several fold. Eagle, Fleischman & Musselman (1950a) reported that the minimum effective serum concentrations of benzylpenicillin in a number of experimental infections in mice and rabbits were 2 to 5 times higher than those necessary to kill the same organism in vitro. However, using a schedule of repeated doses of benzylpenicillin in rats experimentally infected with Streptococcus pneumoniae Schmidt and Walley (1951) found that at the ED B0 the plasma levels 15 min after dosing were only equal to the concentration required for inhibition in vitro and 30 min after dosing the levels had fallen to approximately half the MIC. In order to obtain further data on the question of blood levels in relation to therapeutic effect, studies have been carried out in which antibiotic concentrations were determined in blood in experimentally infected mice at dosages corresponding to the median protective dose (PD 60 ) and these levels have been correlated with the in vitro MIC. These results are reported here. *To whom correspondence and reprint requests should be sent. 423 O3O5-7453/79/O4O423+07 801.00/0
© 1979 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at University of California, Santa Barbara on June 24, 2015
D. Merrikin and G. N. Rolinson*
424
D. MerrOdn and G. N. Rolinson Materials and methods
MIC
Protein binding The extent of binding in mouse serum was determined using Centriflo membrane cones type 50A (Amicon Ltd.). After centrifugation the antibiotic concentration in the ultrafiltrate was determined by cup-plate assay using Sarcina lutea (NCTC 8340) as test organism with antibiotic standards prepared in phosphate buffered saline. Experimental infections in mice Male or female albino mice, 18 to 22 g, CS1 strain (Charles River U.K. Ltd.) were used. The bacteria used for infection were Staphylococcus aureus Smith and Staph. aureus strain T43 (non-f}-lactamase producing) and Escherichia coli strain 8 (ampicillin sensitive) and the cultures were grown in double strength veal infusion broth (Difco) at 37°C overnight. The antibiotics were administered in solution in phosphate buffered saline, subcutaneously, either as a single dose immediately after infection or by repeated dosage as indicated in the results. Intraperitoneal infections. Overnight cultures of Staph. aureus Smith or E. coli strain 8 were diluted 1/1000 in 3% hog gastric mucin (1701-W, Wilson Laboratories, Chicago) and 0-5 ml injected intraperitoneally to mice giving an infective inoculum of 100 to 1000 median lethal doses. Groups of ten mice were used and the numbers surviving 4 days after infection were recorded and the dose required to protect 50% of the animals calculated (median protective dose, PD60) using log X probability paper. Intramuscular infection. Groups often mice were infected intramuscularly in the right hind leg with 0-2 ml of an undiluted overnight culture of Staph. aureus strain T43 or a 1/8 dilution of an overnight culture of E. coli strain 8. The inoculation was made into the muscles of the back of the thigh as described by Selbie & O'Grady (1954). The diameter of the infected thigh of each of the mice was measured 24 h after inoculation in the case of E. coli, and 4 days after inoculation in the experiments with Staph. aureus, and the antibiotic dose required to minimize the enlargement of the thigh to 50 % of the infected controls was calculated (median protective dose, PD60) using a log x probability plot of the results. Antibiotic levels in blood After determination of the respective PD 60 dosages for the different antibiotics, using the different infections, each antibiotic was administered at these doses to infected mice and the resulting levels in blood determined by sacrificing groups of mice at intervals of time and collecting from each a sample of blood in heparinized tubes. Another group of mice
Downloaded from http://jac.oxfordjournals.org/ at University of California, Santa Barbara on June 24, 2015
Minimum inhibitory concentrations (MIC) were determined in double strength veal infusion broth (Difco) using the Cooke microtitre system (Dynatech Laboratories Ltd.). Overnight broth cultures of the test organism were diluted 1/1000 and 005 ml amounts were added to an equal volume of medium in each well of the microtitre plate to give a final inoculum of approximately 106 cfu/ml. MIC tests were also carried out by tube dilution using 5 ml amounts of nutrient broth (Oxoid no. 2) inoculated with one drop (approx. 0-03 ml) of undiluted overnight broth culture giving afinalinoculum of approx. 10" cfu/ml. The minimum inhibitory concentrations were determined afteT 18 h at 37°C.
Antibiotic levels in experimentally infected mice
425
Results and discussion Tables I and II show the PD 6 0 values for the antibiotics used in the different experimental infections together with the peak blood levels which resulted at these dosages. The tables also show the extent of binding of the antibiotics in mouse serum and the MIC values against the infecting organisms. In the case of the infections with Staph. aureus, when benzylpenicillin, penicillin V, phenethicillin, ampicillin or amoxycillin were administered by repeated injection at the PD 6 0 dosage the levels in blood failed to reach the MIC. With ampicillin and amoxycillin the levels were even less than one tenth of the MIC value. In the case of the infections with E. coli the levels of benzylpenicillin, ampicillin, amoxycillin and carbenicillin in blood were also found to be below the respective MIC values when these antibiotics were administered by repeated injection at the PD 6 0 dose. However, these findings regarding the levels of antibiotic in blood relative to MIC are not necessarily paradoxical. From the results in Table III it can be seen that over a period of 6 h a marked inhibitory effect on the growth of Staph. aureus occurred with benzylpenicillin and ampicillin at concentrations one tenth the MIC and similar results were obtained with E. coli and ampicillin. Subinhibitory concentrations of antibiotics are already known to exert some effect on bacterial growth in vitro (Lorian, 1975) and the terms minimum antibacterial concentration (MAC) and minimum active concentration (MAC) have been used (Lorian & Popoola, 1972; Shah, Heetderks & Stille, 1976) to denote the minimum concentration causing a significant diminution in either the rate or the final amount of growth compared with antibiotic-free controls. These effects of subMIC levels of antibiotic may well be adequate to result in a therapeutic effect in experimental infections provided the normal body defences are not impaired and provided such levels of antibiotic are maintained for an adequate period of time. It is also known that certain antibiotics at subinhibitory concentrations markedly reduce the ability of Staph. aureus to produce a variety of toxins and enzymes (Gemmell, 1977) and this may contribute to the therapeutic effects observed at such concentrations. In contrast to the results with benzylpenicillin, penicillin V, phenethicillin, ampicillin and amoxycillin, for which the levels in blood were relatively low compared with the MIC, the levels of methicillin and cloxacillin relative to MIC were found to be considerably higher. For example, in the intramuscular infection with Staph. aureus, despite administration by repeated dosage, the levels of cloxacillin in blood were as high as 13 X MIC compared with values of less than 008 X MIC for ampicillin or amoxycillin. It may be significant that of the penicillins tested cloxacillin was the most highly bound in mouse serum and it will be seen from Table I that the levels of free cloxacillin in blood,
Downloaded from http://jac.oxfordjournals.org/ at University of California, Santa Barbara on June 24, 2015
was set aside to confirm that the dosage used did result in approximately 50 % protection. In the experiments involving a single dose of antibiotic, blood samples were taken 10, 20, 30, 60 and 120 min after dosage. In the experiments involving repeated dosage, blood samples were taken at the same time intervals, following both the first and the last dose. No accumulation of antibiotic occurred and the results shown in the tables are the means of the peak levels in the blood following the first and the last dose. Blood samples from each of four mice were assayed individually and the mean values for the group are reported. The antibiotic concentrations in the blood samples were determined by cup-plate assay using standards prepared in heparinized mouse blood. Sarcina lutae (NCTC 8340) was used as assay organism except in the case of carbenicillin assay for whichPseudomonas aeruginosaHCTC 10701 was used.
MIC*
0-03 003 0-06 012 012 10 012 0-03 003 006 012 012 2-0 012
44 73 77 22 19 34 80 44 73 77 22 19 34 80
(mg/1)
*By microtitre. tPeak level of total antibiotic at PDI0 dose. {Calculated from values for extent of binding.
Intraperitoneal infection with Staph. aweus Smith benzylpenicillin penicillin V phenethicillin ampicillin amoxycillin methicillin cloxacillin Intramuscular infection with Staph. aweus T43 benzylpenicillin penicillin V phenethicillin ampicillin amoxycillin methicillin cloxacillin
bound mouse serum riJio
30 3-4 3-0 1-9 0-9 100-0 39-0
0-25 012 018 0-14 013 4-2 12-5
(mg/kg)
20-7 17-0 111 6-5 3-3 29-2 16-3
36-7 63-3 48-3 8-3 4-2 44-2 81-7 11 2-9 10 0-5 88-5 9-8
19
1-6
