ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1979, p. 424426 0066-4804/79/09-0424/03$02.00/0
Vol. 16, No. 3
Comparison of Cefamandole Nafate to Cefamandole by Microbiological Assay CARL L. WINELY,* JOSEPH C. SPEARS, AND JACK K. SCOTT Lilly Research Laboratories, Indianapolis, Indiana 46206
Received for publication 29 June 1979
Before microbiological assay, cefamandole nafate should be hydrolyzed for 1 h at 37°C in pH 8 buffer (0.1 M) or for 30 min at room temperature in aqueous solutions containing 1.25 molar equivalents of sodium carbonate. Cefamandole nafate (7-mandelamido-3- {[1methyl- 1H-tetrazol-5-yl) -thiol]) 3 cephem-4-carboxylic acid, formate [ester], sodium salt) is partiallyconverted to cefamandole by hydrolysis of the formyl ester in vitro after dissolution of the dosage form. Because of rapid in vivo conversion of cefamandole nafate to cefamandole (3; J. S. Wold, R. R. Joose, H. R. Black, and K. E. Briscoe, Abstr. Ninth Int. Congr. Chemother. M225, 1975), the latter is the predominant circulating antibiotic after administration of cefamandole nafate. Comparative antibacterial studies to date have led to the assumption that routine microbiological evaluations do not distinguish between the two forms of cefamandole (2; R. B. Kammer, D. A. Preston, and J. R. Turner, Abstr. Ninth Int. Congr. Chemother. M226, 1975). Microbiological agar diffusion assays (cylinder) were performed with Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538P, Escherichia coli ATCC 10536, Escherichia coli 7437, and Escherichia coli X163 using antibiotic medium 1 (Difco) in 10-ml, singlelayer plates. The medium was adjusted to either pH 6 or 8 after sterilization. On each assay day, four samples of cefamandole nafate were prepared at a concentration equivalent to 1 mg of cefamandole activity per ml. Two of the samples were hydrolyzed to cefamandole with 1.25 molar equivalents of sodium carbonate for 30 min. The other two samples were diluted in the appropriate buffer (either pH 6 or 8) without sodium carbonate hydrolysis. Each assay condition was investigated on three separate days. Dose response concentrations were prepared with the lithium salt of cefamandole. Significant potency differences occurred between the two compounds, particularly when both diluent and agar were at pH 6 (Table 1). At pH 6, cefamandole nafate was always less potent than cefamandole, but the potency ratio varied depending on the assay 424
organism. The two compounds were most similar against S. aureus and most dissimilar against E. coli 10536, as cefamandole was 1.61 times as active as cefamandole nafate with S. aureus, but 3.95 times as active with E. coli 10536. When either the agar medium or the diluent was at pH 8, the potency difference between the two compounds decreased. In some instances, the two compounds had equal activity with pH 8 diluent and pH 6 medium. Complete hydrolysis, as judged by equivalent potencies, occurred when both the medium and diluent were at pH 8. Since the cefamandole nafate dosage form contains sodium carbonate, the compound is partially hydrolyzed upon dissolution. Additional hydrolysis occurs during incubation of the assay. To avoid variability and erroneous assay results due to incomplete hydrolysis or unequal hydrolysis of standard material and samples, methods for complete hydrolysis of cefamandole nafate were investigated. Indelicato et al. (1) reported rapid hydrolysis of cefamandole nafate to cefamandole by the addition of sodium carbonate or tromethamine. Various concentrations of sodium carbonate were added to aqueous solutions of cefamandole nafate (1 mg of cefamandole nafate per ml). The reaction mixtures were incubated at room temperature and 370C. Samples were obtained at given intervals by dilution of aliquots with pH 6 buffer. Conditions were sought which caused complete hydrolysis in a reasonable time, but did not result in degradation of cefamandole. Samples were evaluated by agar diffusion assay with E. coli 10536 as the test organism and the agar medium at pH 6. Complete hydrolysis was assumed if a potency of 1,000 ,ug of cefamandole per ml was obtained. The results are shown in Table 2. The zero time values in Table 2 indicate that hydrolysis proceeds rapidly during sample preparation. Based on the data shown in Table 2, the routine procedure adopted was to hydrolyze with 1.25 molar
VOL. 16, 1979
NOTES
425
TABLE 1. Potency of cefamandole nafate compared to cefamandole Organism
Diluent pH
Potency' of cefamafama'ndole
Agar pH
mandole/cefa-
mandole nafate
B. subtilis
6 6 8 8
S. aureus
6 8 6
461 ± 20 787±2
997 ± 17
2.16
1,000± 20
8
982± 13 1,026 ± 8
1,014 ± 7 1,011 ± 15
1.27 1.03 0.99
6 6 8 8
6 8 6 8
625 ± 56 931 ± 24 978± 43 1,003 ± 22
1,009 ± 30 997±9 971 ±34 1,010 ± 24
1.61 1.07 0.99 1.01
E. coli X163
6 6 8 8
6 8 6 8
607 ± 18 804±31 1,010± 39 1,032 ± 38
1,001 ± 30 990±41 965 ± 24 1,034 ± 38
1.65 1.23 0.96 1.00
E. coli 7437
6 6 8 8
6 8 6 8
579 ± 12 1,003±23 1,074 ±20 1,024±23
1,009 ± 33 1,011 ± 13 1,004 ± 16 993± 1.4
2.12 1.01 0.93 0.97
E. coli 10536
6 6 8 8
6 8 6 8 n = 6.
256 ± 50 483 ±43 867 ± 74 978±50
1,011 ± 33 1,045±33 1,027 ± 66 1,003 ±37
3.95 1.79 1.18 1.03
In micrograms per nialliter;
TABLE 2. Hydrolysis of cefamandole nafate with sodium carbonate Concn of Na2CO3 (molar equivalent)
0.0 1.00 1.25 1.50 1.75 Measured by E.
10 min
Potency (pg/ml) as function of hydrolysis time' 30 mi
15 min
283±9 848 ± 81 913 ± 20 909 ± 48 984 ± 7 968 ± 46 986 ± 33 991±96 971±27 coli assay with n = 4.
45 min
924 ± 48 1000 ± 14 981 ± 33 972± 12
equivalents of sodium carbonate (260 mg of sodium carbonate per g of cefamandole nafate) for 30 min, but no longer than 60 min. Hydrolysis was also studied in pH 8 buffer as a possible altemative to sodium carbonate hydrolysis. Four solutions of cefamandole nafate were prepared in pH 8 potassium phosphate buffer (0.1 M) at a concentration of 1 mg of cefamandole nafate per ml. Two of the solutions were incubated at 250C, and two were incubated at 370C. At the time intervals shown in Table 3, aliquots were diluted in pH 6 potassium phosphate buffer (0.1 M). These diluted solutions were assayed by an E. coli agar diffusion method. As shown in Table 3, complete hydrolysis, as evidenced by attaining a potency of 1,000 ,ug of activity per ml, was not attained within 90
902 ± 25 977 ± 14 964 ± 24 968± 11
60 min
90 min
928 ± 12 996 ± 17 979 ± 7 949± 14
312± 11 951 ± 15 971 ± 39 967 ± 10 945±8
TABLE 3. Hydrolysis of cefamandole nafate by pH 8 buffer as measured by agar diffusion assaya Time
Potency (pg/ml) at
25°C incubation (min) 0 418± 10 30 653 ± 22 45 769 ± 33 60 802 ± 28 75 833 ± 30 90 879± 11 a E. coli 10536; n = 6.
Potency (jg/ml) at 37°C incubation
420± 17 892 ± 33 952 ± 26 1,033 ± 46 1,009 ± 50 980±53
min at room temperature, but had occurred within 60 min at 37°C. Previously, the in vitro antibiotic activity of cefamandole nafate was shown to be equivalent
426
NOTES
ANTIMICROB. AGENTS CHEMOTHER.
to cefamandole by conventional agar dilution differences between the two compounds in agar and disc plate susceptibility tests (Kamer et al., medium. Since the two compounds differ in anAbstr. Ninth Int. Congr. Chemother. M226, tibacterial activity and cefamandole is the pre1975; Wold et al., Abstr. Ninth Int. Congr. Chem- dominating antibiotic after administration of other. M225, 1975). The only evidence presented cefamandole nafate (3; Wold et al., Abstr. Ninth for differences in antibacterial activity for the Int. Congr. Chemother. 1975, M225), it seems two compounds was obtained in cell-lysis exper- advisable that in vitro microbiological studies be iments in liquid medium whereby cefamandole made with either the sodium or lithium salts of was approximately 10-fold more active than cef- cefamandole. amandole nafate (2). Our studies with the various agar diffusion LITERATURE CTED assays at different pH values illustrated antibacterial differences for cefamandole nafate as com- 1. Indelicato, J. M., W. L. Wilham, and B. M. Cerimele. 1976. Conversion of cefamandole nafate to cefamandole pared to cefamandole. The necessity for hydrosodium. J. Pharm. Sci. 65:1175-1178. lyzing cefamandole before analytical microbio- 2. Turner, J. R., D. A. Preston, and J. S. Wold. 1977. logical evaluations was well illustrated by activDelineation of the relative antibacterial activity of cefamandole and cefamandole nafate. Antimicrob. Agents ity ratios of 1.61 to 3.95 (cefamandole/cefamanChemother. 12:66-72. dole nafate) when several different microorgan- 3. Wold, J. S., R. R. Joost, H. R. Black, and R. S. isms were used in agar diffusion assays at pH 6. Griffith. 1978. Hydrolysis of cefamandole nafate to These results were the first evidence of activity cefamandole in vivo. J. Infect. Dis. 137:S17-S24.
Vol. 16, No. 3
Comparison of Cefamandole Nafate to Cefamandole by Microbiological Assay CARL L. WINELY,* JOSEPH C. SPEARS, AND JACK K. SCOTT Lilly Research Laboratories, Indianapolis, Indiana 46206
Received for publication 29 June 1979
Before microbiological assay, cefamandole nafate should be hydrolyzed for 1 h at 37°C in pH 8 buffer (0.1 M) or for 30 min at room temperature in aqueous solutions containing 1.25 molar equivalents of sodium carbonate. Cefamandole nafate (7-mandelamido-3- {[1methyl- 1H-tetrazol-5-yl) -thiol]) 3 cephem-4-carboxylic acid, formate [ester], sodium salt) is partiallyconverted to cefamandole by hydrolysis of the formyl ester in vitro after dissolution of the dosage form. Because of rapid in vivo conversion of cefamandole nafate to cefamandole (3; J. S. Wold, R. R. Joose, H. R. Black, and K. E. Briscoe, Abstr. Ninth Int. Congr. Chemother. M225, 1975), the latter is the predominant circulating antibiotic after administration of cefamandole nafate. Comparative antibacterial studies to date have led to the assumption that routine microbiological evaluations do not distinguish between the two forms of cefamandole (2; R. B. Kammer, D. A. Preston, and J. R. Turner, Abstr. Ninth Int. Congr. Chemother. M226, 1975). Microbiological agar diffusion assays (cylinder) were performed with Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538P, Escherichia coli ATCC 10536, Escherichia coli 7437, and Escherichia coli X163 using antibiotic medium 1 (Difco) in 10-ml, singlelayer plates. The medium was adjusted to either pH 6 or 8 after sterilization. On each assay day, four samples of cefamandole nafate were prepared at a concentration equivalent to 1 mg of cefamandole activity per ml. Two of the samples were hydrolyzed to cefamandole with 1.25 molar equivalents of sodium carbonate for 30 min. The other two samples were diluted in the appropriate buffer (either pH 6 or 8) without sodium carbonate hydrolysis. Each assay condition was investigated on three separate days. Dose response concentrations were prepared with the lithium salt of cefamandole. Significant potency differences occurred between the two compounds, particularly when both diluent and agar were at pH 6 (Table 1). At pH 6, cefamandole nafate was always less potent than cefamandole, but the potency ratio varied depending on the assay 424
organism. The two compounds were most similar against S. aureus and most dissimilar against E. coli 10536, as cefamandole was 1.61 times as active as cefamandole nafate with S. aureus, but 3.95 times as active with E. coli 10536. When either the agar medium or the diluent was at pH 8, the potency difference between the two compounds decreased. In some instances, the two compounds had equal activity with pH 8 diluent and pH 6 medium. Complete hydrolysis, as judged by equivalent potencies, occurred when both the medium and diluent were at pH 8. Since the cefamandole nafate dosage form contains sodium carbonate, the compound is partially hydrolyzed upon dissolution. Additional hydrolysis occurs during incubation of the assay. To avoid variability and erroneous assay results due to incomplete hydrolysis or unequal hydrolysis of standard material and samples, methods for complete hydrolysis of cefamandole nafate were investigated. Indelicato et al. (1) reported rapid hydrolysis of cefamandole nafate to cefamandole by the addition of sodium carbonate or tromethamine. Various concentrations of sodium carbonate were added to aqueous solutions of cefamandole nafate (1 mg of cefamandole nafate per ml). The reaction mixtures were incubated at room temperature and 370C. Samples were obtained at given intervals by dilution of aliquots with pH 6 buffer. Conditions were sought which caused complete hydrolysis in a reasonable time, but did not result in degradation of cefamandole. Samples were evaluated by agar diffusion assay with E. coli 10536 as the test organism and the agar medium at pH 6. Complete hydrolysis was assumed if a potency of 1,000 ,ug of cefamandole per ml was obtained. The results are shown in Table 2. The zero time values in Table 2 indicate that hydrolysis proceeds rapidly during sample preparation. Based on the data shown in Table 2, the routine procedure adopted was to hydrolyze with 1.25 molar
VOL. 16, 1979
NOTES
425
TABLE 1. Potency of cefamandole nafate compared to cefamandole Organism
Diluent pH
Potency' of cefamafama'ndole
Agar pH
mandole/cefa-
mandole nafate
B. subtilis
6 6 8 8
S. aureus
6 8 6
461 ± 20 787±2
997 ± 17
2.16
1,000± 20
8
982± 13 1,026 ± 8
1,014 ± 7 1,011 ± 15
1.27 1.03 0.99
6 6 8 8
6 8 6 8
625 ± 56 931 ± 24 978± 43 1,003 ± 22
1,009 ± 30 997±9 971 ±34 1,010 ± 24
1.61 1.07 0.99 1.01
E. coli X163
6 6 8 8
6 8 6 8
607 ± 18 804±31 1,010± 39 1,032 ± 38
1,001 ± 30 990±41 965 ± 24 1,034 ± 38
1.65 1.23 0.96 1.00
E. coli 7437
6 6 8 8
6 8 6 8
579 ± 12 1,003±23 1,074 ±20 1,024±23
1,009 ± 33 1,011 ± 13 1,004 ± 16 993± 1.4
2.12 1.01 0.93 0.97
E. coli 10536
6 6 8 8
6 8 6 8 n = 6.
256 ± 50 483 ±43 867 ± 74 978±50
1,011 ± 33 1,045±33 1,027 ± 66 1,003 ±37
3.95 1.79 1.18 1.03
In micrograms per nialliter;
TABLE 2. Hydrolysis of cefamandole nafate with sodium carbonate Concn of Na2CO3 (molar equivalent)
0.0 1.00 1.25 1.50 1.75 Measured by E.
10 min
Potency (pg/ml) as function of hydrolysis time' 30 mi
15 min
283±9 848 ± 81 913 ± 20 909 ± 48 984 ± 7 968 ± 46 986 ± 33 991±96 971±27 coli assay with n = 4.
45 min
924 ± 48 1000 ± 14 981 ± 33 972± 12
equivalents of sodium carbonate (260 mg of sodium carbonate per g of cefamandole nafate) for 30 min, but no longer than 60 min. Hydrolysis was also studied in pH 8 buffer as a possible altemative to sodium carbonate hydrolysis. Four solutions of cefamandole nafate were prepared in pH 8 potassium phosphate buffer (0.1 M) at a concentration of 1 mg of cefamandole nafate per ml. Two of the solutions were incubated at 250C, and two were incubated at 370C. At the time intervals shown in Table 3, aliquots were diluted in pH 6 potassium phosphate buffer (0.1 M). These diluted solutions were assayed by an E. coli agar diffusion method. As shown in Table 3, complete hydrolysis, as evidenced by attaining a potency of 1,000 ,ug of activity per ml, was not attained within 90
902 ± 25 977 ± 14 964 ± 24 968± 11
60 min
90 min
928 ± 12 996 ± 17 979 ± 7 949± 14
312± 11 951 ± 15 971 ± 39 967 ± 10 945±8
TABLE 3. Hydrolysis of cefamandole nafate by pH 8 buffer as measured by agar diffusion assaya Time
Potency (pg/ml) at
25°C incubation (min) 0 418± 10 30 653 ± 22 45 769 ± 33 60 802 ± 28 75 833 ± 30 90 879± 11 a E. coli 10536; n = 6.
Potency (jg/ml) at 37°C incubation
420± 17 892 ± 33 952 ± 26 1,033 ± 46 1,009 ± 50 980±53
min at room temperature, but had occurred within 60 min at 37°C. Previously, the in vitro antibiotic activity of cefamandole nafate was shown to be equivalent
426
NOTES
ANTIMICROB. AGENTS CHEMOTHER.
to cefamandole by conventional agar dilution differences between the two compounds in agar and disc plate susceptibility tests (Kamer et al., medium. Since the two compounds differ in anAbstr. Ninth Int. Congr. Chemother. M226, tibacterial activity and cefamandole is the pre1975; Wold et al., Abstr. Ninth Int. Congr. Chem- dominating antibiotic after administration of other. M225, 1975). The only evidence presented cefamandole nafate (3; Wold et al., Abstr. Ninth for differences in antibacterial activity for the Int. Congr. Chemother. 1975, M225), it seems two compounds was obtained in cell-lysis exper- advisable that in vitro microbiological studies be iments in liquid medium whereby cefamandole made with either the sodium or lithium salts of was approximately 10-fold more active than cef- cefamandole. amandole nafate (2). Our studies with the various agar diffusion LITERATURE CTED assays at different pH values illustrated antibacterial differences for cefamandole nafate as com- 1. Indelicato, J. M., W. L. Wilham, and B. M. Cerimele. 1976. Conversion of cefamandole nafate to cefamandole pared to cefamandole. The necessity for hydrosodium. J. Pharm. Sci. 65:1175-1178. lyzing cefamandole before analytical microbio- 2. Turner, J. R., D. A. Preston, and J. S. Wold. 1977. logical evaluations was well illustrated by activDelineation of the relative antibacterial activity of cefamandole and cefamandole nafate. Antimicrob. Agents ity ratios of 1.61 to 3.95 (cefamandole/cefamanChemother. 12:66-72. dole nafate) when several different microorgan- 3. Wold, J. S., R. R. Joost, H. R. Black, and R. S. isms were used in agar diffusion assays at pH 6. Griffith. 1978. Hydrolysis of cefamandole nafate to These results were the first evidence of activity cefamandole in vivo. J. Infect. Dis. 137:S17-S24.
