ANTIMICROBIAL AGENTS AND CHEMOTERAPY, Aug. 1976, p. 245-248 Copyright © 1976 American Society for Microbiology
Vol. 10, No. 2 Printed in U.S.A.
A New Cephalosporin with a Dual Mode of Action CYNTHIA H. O'CALLAGHAN,* R. B. SYKES, AND SUSAN E. STANIFORTH Glaxo Research Ltd., Greenford, Middlesex, England
Received for publication 26 April 1976
A cephalosporin, (6R,7R)-7-[(2R)-2-hydroxy-2-phenylacetamido]-3-(pyrid-2-ylN-oxide)thiomethylceph-3-em-4-carboxylic acid (MCO), that could lead to a novel approach to the problem of 3-lactamase destruction is described. The compound is slightly more resistant to some (3-lactamases than is cephalothin, but it is still hydrolyzed by many to a varying degree. Hydrolysis ofthe (8-lactam bond of a cephalosporin releases the 3-substituent, which in MCO is itself an antibacterial agent, 2-mercaptopyridine-N-oxide. Thus, MCO has a dual mode of action, and bacteria that do not produce an effective amount of a (& lactamase are inhibited by the intact cephalosporin, whereas those that do hydrolyze it are inhibited by the released antibacterial compound.
,&3Lactamases from a wide range of bacteria can attack most currently available cephalosporins and inactivate them by hydrolyzing the ,B lactam ring. For this reason, these cephalosporins cannot be used to treat infections due to gram-negative organisms that produce large amounts of /8-lactamases. One way of overcoming this problem is to alter the substituents on the cephalosporin nucleus so that the compounds are more resistant to attack by 83-lactamases. Although cephalosporins with resistance to some types of f3-lactamase from gramnegative organisms have been known for many years (8), the early compounds were of no therapeutic value because they had very little antibacterial activity. Recently, cephalosporins with both resistance to f3-lactamases and activity against a wider range of bacteria have been described (10, 12). An alternative solution to the problem has been considered whereby antibacterial activity might be retained, even though the (8-lactam ring of the cephalosporin were to be broken. When the 84-lactam ring is hydrolyzed, the substituent at position 3 is expelled from the cephalosporin molecule (6); if that group possessed antibacterial activity of its own, this would still be available after the destruction of the cephalosporin moiety. A cephalosporin meeting these requirements has been synthesized in our laboratories. The 7acyl group of the compound is n-mandeloyl, a group that also appears in cefamandole (12), and the 3-substituent is 2-mercaptopyridine-Noxide (Fig. 1), which is also known as omadine; as the zinc salt, zinc pyrithione, it is used as an antiseptic in toiletries such as shampoos. The properties of the cephalosporin analogue are described in this paper.
MATERIALS AND METHODS (6R,7R)-7- [(2R)-2-hydroxy-2-phenylacetamido]-3(pyrid-2 yl-N-oxide)thiomethylceph-3-em-4-carboxylic acid (MCO) (Fig. 1) was prepared in our laboratories as a creamy white crystalline solid with a molecular weight of 473; as the sodium salt, it is approximately 20%0o soluble in water. Organisms. Most of the organisms tested were recent clinical isolates. The bacteria were maintained on nutrient agar slopes, and the fungi were maintained on Sabouraud maltose agar. MIC determinations. (i) Bacteria. Minimum inhibitory concentrations (MICs) were determined by an agar dilution technique. Serial twofold dilutions of freshly prepared antibiotic solution were made into Oxoid no. 1 nutrient agar and poured into petri dishes. The plates were inoculated with a replicate inoculating device (Denley Instruments Ltd., Bolney, Sussex, England), with inocula containing approximately 105 colony-forming units of bacteria. The MIC, in micrograms per milliliter, was read after 18 h of incubation at 37°C as the lowest concentration that inhibited growth. All bacterial cultures were tested for 83-lactamase production, using the chromogenic cephalosporin 87/312 described by O'Callaghan et al. (7). (ii) Fungi and yeasts. The MICs of fungi and yeasts were determined in Sabouraud maltose broth, as described by Holt (4). Stability studies with ,B-lactamases. ,3Lactamases from Klebsiella aerogenes Kl, Enterobacter cloacae P99, Escherichia coli TEM+, E. coli RGN238, and Pseudomonas aeruginosa 67/70 were prepared in a partially purified form as described by O'Callaghan et al. The activity of each enzyme preparation against MCO was estimated photometrically, using the decrease in optical density at 263 nm to determine the rate of hydrolysis of the compound (9).
Determination of 2-mercaptopyridine-N-oxide. MCO was incubated at 2 mg/ml in M/20 phosphate buffer at pH 7 with equal volumes of K. aerogenes Kl ,-lactamase containing between 10 and 0.15 en245
246
O'CALLAGHAN, SYKES, AND STANIFORTH
FIG. 1. Structure of MCO.
units for 1 h at 370C. One unit of #-lactamase activity is defined as that amount of enzyme capable of hydrolyzing 1 ,umol of MCO in 1 h at 37°C; 2.5 units will hydrolyze 1,000 ug of MCO in 1 h. The samples were filtered after incubation in an Amicon microultrafiltration system 8MC, using a UM10 membrane, to remove enzyme protein. 2-Mercaptopyridine-N-oxide was then estimated either polarographically or by a colorimetric method. (i) Polarographic method. 2-MercaptopyridineN-oxide contains a free sulfhydryl group that can be easily oxidized to give an anodic wave on the polarograph at -0.16 V at pH 2 and at -0.28 V at pH 4, 6, and 8 with reference to a standard calomel electrode. The anode was a dropping mercury electrode with characteristics t = 2.8 m 1.6 mg/s, and h = 65 zyme
s,
=
cm.
A calibration curve of the concentration of 2-mercaptopyridine-N-oxide in pH 7 M/20 phosphate buffer against the height of the anodic step gave a straight line, the minimum amount detectable being 2 x 10-4 M (25 jAg/ml). (ii) Chemical method. Dalziel and Thompson (1) used the reaction of 2-mercaptopyridine-N-oxide with iron salts to form colored complexes that could be used for the estimation of iron in solution. The black iron complex (C5H4NOS)3Fe is precipitated from weakly acidic solutions. To a known volume of 2-mercaptopyridine-N-oxide solution was added an equal volume of 0.01 M solution of ferric nitrate in 0.1 N sulfuric acid. The violet color or precipitate was extracted with a volume of chloroform equal to twice the volume of the original solution. The optical density of this solution was measured at 550 nm in a 1-cm cell. The calibration curve was a straight line, the least amount of 2-mercaptopyridine-N-oxide detectable being 2.5 x 10-4 M (31.8 jAg/ml). No reaction was obtained with untreated MCO. Values from solutions incubated with f-lactamase were read from the calibration curve.
RESULTS
AimmICROD. AGENTS CHEMOTHICR.
MCO by five partially purified enzymes of classes I, III, and IV from gram-negative organisms (11) was made using the spectrophotometric method. The results are given in Table 2; for each enzyme, the resistance of cephalothin is given the arbitrary value of 1. The increase in resistance to class I enzymes, from E. cloacae and P. aerugino8a, was greater than for the class III and IV enzymes, where the improvement was only marginal. However, MCO was not wholly stable to any of the enzymes tested. Expulsion of the 3-substituent after attack by (8-lactamases. It has previously been shown that attack on the ,B-lactam ring of a cephalosporin by a 3-lactamase is accompanied by expulsion of the substituent at position 3 if this is a leaving group, such as acetoxy (3) pyridyl, or azido (6), obviously able to accept an electron. Solutions of MCO, before and after incubation with K. aerogenes Kl ,B-lactamase, were examined by two methods for the presence of free 2mercaptopyridine-N-oxide. K. aerogenes enzyme was chosen because it was the one to which MCO was most susceptible. The various strengths of enzyme were chosen to give a spread from complete hydrolysis in the 1-h period of incubation at 37°C to little or no decomposition in that time. The liberated 3-substituent was estimated by two methods, and the results are given in Table 3. Treatment with 5 or 2.5 enzyme units/ml TABLE 1. Geometric mean MICs in comparison with
cephalothin No. of
Organism
strains tested
Geometric mean MIC (/Ag/ml) of: MCO
Cephalothin
Staphylococcus au-
25
2.7
1.2
24 8 4 10 13 2 1
1.8 11 3.4 3.7 16 2 4
9.2 50 250 74 >250 >250 >250
reus
Escherichia coli Citrobacter freundii Enterobacter cloacae Enterobacter hafniae Klebsiella aerogenes Proteus rettgeri Proteus morganii
Antibacterial activity. The in vitro activity of MCO against a selection of organisms is summarized in Table 1, in comparison with TABLE 2. Stability to 1&lactamases cephalothin. The results are expressed as geometric mean MICs, and it is evident that the Increase in reSource of ,B-lactamase Class sistance over compound has both greater intrinsic activity cephalothin and a wider antimicrobial spectrum than does cephalothin. Apart from the strains of E. coli, K. aerogenes Kl IV 1.7 I 18.6 most of the organisms are f-lactamase pro- E. cloacae P99 E. coli TEM III 2.7 ducers. coli RGN 238 III 4.3 Stability to f8-lactamase. A comparison of E. I 8.8 the rates of destruction of cephalothin and P. aeruginosa 67/70
VOL. 10, 1976
247
A NEW CEPHALOSPORIN WITH A DUAL MODE OF ACTION
produced complete hydrolysis of MCO; smaller amounts of enzyme produced only partial hydrolysis of the compound, as shown by the decreasing amounts of 2-mercaptopyridine-N-oxide liberated. The two methods of estimating the amount of 2-mercaptopyridine-N-oxide produced during incubation with the 3-lactamase agreed quite well and corresponded to the change in ultraviolet absorption at the Xnax for the 8-lactam ring. The polarographic method depends on the presence of the free -SH group only, whereas the chemical method depends on both the -SH group and the N-oxide group. The agreement between the two methods indicates that 2-mercaptopyridine-N-oxide was expelled intact from the cephalosporin after ,8-lactamase hydrolysis of the ,8-lactam ring of MCO. Incubation of cefazolin and cefamandole with 5 enzyme units of /8-lactamase resulted in their total decomposition, and the polarographic method showed that their respective 3-substituents were expelled intact in stoichiometric proportions. The chemical method could not be applied to 2-mercapto-5-methyl-1,2,4-thiadiazole nor to 1-methyl-5-mercaptotetrazole, the 3-substituents of cefazolin and cefamandole, respectively, since these substances do not chelate iron. Antibacterial activity of 2-mercaptopyridine-N-oxide. Unlike most groups substituted in position 3 of the cephalosporins, 2-mercaptopyridine-N-oxide had antibacterial activity in its own right. Its activity, compared with that of the intact cephalosporin MCO, is shown in Table 4. MCO had activity against all the strains tested, including those that produce a 1lactamase, although it was significantly less active against enzyme-producing organisms than against those with no f8-lactamase. However, it had more activity against these organisms than would be explained by its marginally increased /8-lactamase resistance. In contrast, 2-mercaptopyridine-N-oxide was active at the same high level against all strains, regardless of their ,8-lactamase activity. The possibility must, therefore, be considered that the intrinsic activity of the 2-mercaptopyridine-Noxide substituent made some contribution to the total antibacterial activity of MCO.
TABLE 3. Amount of free 2-mercaptopyridine-Noxide released from 1 mg of MCO by incubation with ,-lactam,ase (KI) Amount of free 2-mercaptopyridine-
Enzyme units/ml (1 h at 37°C)
N-oxide (jAg/ml) measured by: Ferric nitrate Polarographic method method
270 268a 260 268 255 240 1.25 150 0.6 170
Vol. 10, No. 2 Printed in U.S.A.
A New Cephalosporin with a Dual Mode of Action CYNTHIA H. O'CALLAGHAN,* R. B. SYKES, AND SUSAN E. STANIFORTH Glaxo Research Ltd., Greenford, Middlesex, England
Received for publication 26 April 1976
A cephalosporin, (6R,7R)-7-[(2R)-2-hydroxy-2-phenylacetamido]-3-(pyrid-2-ylN-oxide)thiomethylceph-3-em-4-carboxylic acid (MCO), that could lead to a novel approach to the problem of 3-lactamase destruction is described. The compound is slightly more resistant to some (3-lactamases than is cephalothin, but it is still hydrolyzed by many to a varying degree. Hydrolysis ofthe (8-lactam bond of a cephalosporin releases the 3-substituent, which in MCO is itself an antibacterial agent, 2-mercaptopyridine-N-oxide. Thus, MCO has a dual mode of action, and bacteria that do not produce an effective amount of a (& lactamase are inhibited by the intact cephalosporin, whereas those that do hydrolyze it are inhibited by the released antibacterial compound.
,&3Lactamases from a wide range of bacteria can attack most currently available cephalosporins and inactivate them by hydrolyzing the ,B lactam ring. For this reason, these cephalosporins cannot be used to treat infections due to gram-negative organisms that produce large amounts of /8-lactamases. One way of overcoming this problem is to alter the substituents on the cephalosporin nucleus so that the compounds are more resistant to attack by 83-lactamases. Although cephalosporins with resistance to some types of f3-lactamase from gramnegative organisms have been known for many years (8), the early compounds were of no therapeutic value because they had very little antibacterial activity. Recently, cephalosporins with both resistance to f3-lactamases and activity against a wider range of bacteria have been described (10, 12). An alternative solution to the problem has been considered whereby antibacterial activity might be retained, even though the (8-lactam ring of the cephalosporin were to be broken. When the 84-lactam ring is hydrolyzed, the substituent at position 3 is expelled from the cephalosporin molecule (6); if that group possessed antibacterial activity of its own, this would still be available after the destruction of the cephalosporin moiety. A cephalosporin meeting these requirements has been synthesized in our laboratories. The 7acyl group of the compound is n-mandeloyl, a group that also appears in cefamandole (12), and the 3-substituent is 2-mercaptopyridine-Noxide (Fig. 1), which is also known as omadine; as the zinc salt, zinc pyrithione, it is used as an antiseptic in toiletries such as shampoos. The properties of the cephalosporin analogue are described in this paper.
MATERIALS AND METHODS (6R,7R)-7- [(2R)-2-hydroxy-2-phenylacetamido]-3(pyrid-2 yl-N-oxide)thiomethylceph-3-em-4-carboxylic acid (MCO) (Fig. 1) was prepared in our laboratories as a creamy white crystalline solid with a molecular weight of 473; as the sodium salt, it is approximately 20%0o soluble in water. Organisms. Most of the organisms tested were recent clinical isolates. The bacteria were maintained on nutrient agar slopes, and the fungi were maintained on Sabouraud maltose agar. MIC determinations. (i) Bacteria. Minimum inhibitory concentrations (MICs) were determined by an agar dilution technique. Serial twofold dilutions of freshly prepared antibiotic solution were made into Oxoid no. 1 nutrient agar and poured into petri dishes. The plates were inoculated with a replicate inoculating device (Denley Instruments Ltd., Bolney, Sussex, England), with inocula containing approximately 105 colony-forming units of bacteria. The MIC, in micrograms per milliliter, was read after 18 h of incubation at 37°C as the lowest concentration that inhibited growth. All bacterial cultures were tested for 83-lactamase production, using the chromogenic cephalosporin 87/312 described by O'Callaghan et al. (7). (ii) Fungi and yeasts. The MICs of fungi and yeasts were determined in Sabouraud maltose broth, as described by Holt (4). Stability studies with ,B-lactamases. ,3Lactamases from Klebsiella aerogenes Kl, Enterobacter cloacae P99, Escherichia coli TEM+, E. coli RGN238, and Pseudomonas aeruginosa 67/70 were prepared in a partially purified form as described by O'Callaghan et al. The activity of each enzyme preparation against MCO was estimated photometrically, using the decrease in optical density at 263 nm to determine the rate of hydrolysis of the compound (9).
Determination of 2-mercaptopyridine-N-oxide. MCO was incubated at 2 mg/ml in M/20 phosphate buffer at pH 7 with equal volumes of K. aerogenes Kl ,-lactamase containing between 10 and 0.15 en245
246
O'CALLAGHAN, SYKES, AND STANIFORTH
FIG. 1. Structure of MCO.
units for 1 h at 370C. One unit of #-lactamase activity is defined as that amount of enzyme capable of hydrolyzing 1 ,umol of MCO in 1 h at 37°C; 2.5 units will hydrolyze 1,000 ug of MCO in 1 h. The samples were filtered after incubation in an Amicon microultrafiltration system 8MC, using a UM10 membrane, to remove enzyme protein. 2-Mercaptopyridine-N-oxide was then estimated either polarographically or by a colorimetric method. (i) Polarographic method. 2-MercaptopyridineN-oxide contains a free sulfhydryl group that can be easily oxidized to give an anodic wave on the polarograph at -0.16 V at pH 2 and at -0.28 V at pH 4, 6, and 8 with reference to a standard calomel electrode. The anode was a dropping mercury electrode with characteristics t = 2.8 m 1.6 mg/s, and h = 65 zyme
s,
=
cm.
A calibration curve of the concentration of 2-mercaptopyridine-N-oxide in pH 7 M/20 phosphate buffer against the height of the anodic step gave a straight line, the minimum amount detectable being 2 x 10-4 M (25 jAg/ml). (ii) Chemical method. Dalziel and Thompson (1) used the reaction of 2-mercaptopyridine-N-oxide with iron salts to form colored complexes that could be used for the estimation of iron in solution. The black iron complex (C5H4NOS)3Fe is precipitated from weakly acidic solutions. To a known volume of 2-mercaptopyridine-N-oxide solution was added an equal volume of 0.01 M solution of ferric nitrate in 0.1 N sulfuric acid. The violet color or precipitate was extracted with a volume of chloroform equal to twice the volume of the original solution. The optical density of this solution was measured at 550 nm in a 1-cm cell. The calibration curve was a straight line, the least amount of 2-mercaptopyridine-N-oxide detectable being 2.5 x 10-4 M (31.8 jAg/ml). No reaction was obtained with untreated MCO. Values from solutions incubated with f-lactamase were read from the calibration curve.
RESULTS
AimmICROD. AGENTS CHEMOTHICR.
MCO by five partially purified enzymes of classes I, III, and IV from gram-negative organisms (11) was made using the spectrophotometric method. The results are given in Table 2; for each enzyme, the resistance of cephalothin is given the arbitrary value of 1. The increase in resistance to class I enzymes, from E. cloacae and P. aerugino8a, was greater than for the class III and IV enzymes, where the improvement was only marginal. However, MCO was not wholly stable to any of the enzymes tested. Expulsion of the 3-substituent after attack by (8-lactamases. It has previously been shown that attack on the ,B-lactam ring of a cephalosporin by a 3-lactamase is accompanied by expulsion of the substituent at position 3 if this is a leaving group, such as acetoxy (3) pyridyl, or azido (6), obviously able to accept an electron. Solutions of MCO, before and after incubation with K. aerogenes Kl ,B-lactamase, were examined by two methods for the presence of free 2mercaptopyridine-N-oxide. K. aerogenes enzyme was chosen because it was the one to which MCO was most susceptible. The various strengths of enzyme were chosen to give a spread from complete hydrolysis in the 1-h period of incubation at 37°C to little or no decomposition in that time. The liberated 3-substituent was estimated by two methods, and the results are given in Table 3. Treatment with 5 or 2.5 enzyme units/ml TABLE 1. Geometric mean MICs in comparison with
cephalothin No. of
Organism
strains tested
Geometric mean MIC (/Ag/ml) of: MCO
Cephalothin
Staphylococcus au-
25
2.7
1.2
24 8 4 10 13 2 1
1.8 11 3.4 3.7 16 2 4
9.2 50 250 74 >250 >250 >250
reus
Escherichia coli Citrobacter freundii Enterobacter cloacae Enterobacter hafniae Klebsiella aerogenes Proteus rettgeri Proteus morganii
Antibacterial activity. The in vitro activity of MCO against a selection of organisms is summarized in Table 1, in comparison with TABLE 2. Stability to 1&lactamases cephalothin. The results are expressed as geometric mean MICs, and it is evident that the Increase in reSource of ,B-lactamase Class sistance over compound has both greater intrinsic activity cephalothin and a wider antimicrobial spectrum than does cephalothin. Apart from the strains of E. coli, K. aerogenes Kl IV 1.7 I 18.6 most of the organisms are f-lactamase pro- E. cloacae P99 E. coli TEM III 2.7 ducers. coli RGN 238 III 4.3 Stability to f8-lactamase. A comparison of E. I 8.8 the rates of destruction of cephalothin and P. aeruginosa 67/70
VOL. 10, 1976
247
A NEW CEPHALOSPORIN WITH A DUAL MODE OF ACTION
produced complete hydrolysis of MCO; smaller amounts of enzyme produced only partial hydrolysis of the compound, as shown by the decreasing amounts of 2-mercaptopyridine-N-oxide liberated. The two methods of estimating the amount of 2-mercaptopyridine-N-oxide produced during incubation with the 3-lactamase agreed quite well and corresponded to the change in ultraviolet absorption at the Xnax for the 8-lactam ring. The polarographic method depends on the presence of the free -SH group only, whereas the chemical method depends on both the -SH group and the N-oxide group. The agreement between the two methods indicates that 2-mercaptopyridine-N-oxide was expelled intact from the cephalosporin after ,8-lactamase hydrolysis of the ,8-lactam ring of MCO. Incubation of cefazolin and cefamandole with 5 enzyme units of /8-lactamase resulted in their total decomposition, and the polarographic method showed that their respective 3-substituents were expelled intact in stoichiometric proportions. The chemical method could not be applied to 2-mercapto-5-methyl-1,2,4-thiadiazole nor to 1-methyl-5-mercaptotetrazole, the 3-substituents of cefazolin and cefamandole, respectively, since these substances do not chelate iron. Antibacterial activity of 2-mercaptopyridine-N-oxide. Unlike most groups substituted in position 3 of the cephalosporins, 2-mercaptopyridine-N-oxide had antibacterial activity in its own right. Its activity, compared with that of the intact cephalosporin MCO, is shown in Table 4. MCO had activity against all the strains tested, including those that produce a 1lactamase, although it was significantly less active against enzyme-producing organisms than against those with no f8-lactamase. However, it had more activity against these organisms than would be explained by its marginally increased /8-lactamase resistance. In contrast, 2-mercaptopyridine-N-oxide was active at the same high level against all strains, regardless of their ,8-lactamase activity. The possibility must, therefore, be considered that the intrinsic activity of the 2-mercaptopyridine-Noxide substituent made some contribution to the total antibacterial activity of MCO.
TABLE 3. Amount of free 2-mercaptopyridine-Noxide released from 1 mg of MCO by incubation with ,-lactam,ase (KI) Amount of free 2-mercaptopyridine-
Enzyme units/ml (1 h at 37°C)
N-oxide (jAg/ml) measured by: Ferric nitrate Polarographic method method
270 268a 260 268 255 240 1.25 150 0.6 170
