ANTmcRoBIAL AGzNTS AND CHSMoHzRwY, Oct. 1977, p. 455-460 Copyright 0 1977 American Society for Microbiology

Vol. 12, No. 4 Printed in U.S.A.

In Vitro and In Vivo Antibacterial Activity of T-1220, a New Semisynthetic Penicillin K. UEO,1 Y. FUKUOKA,1 T. HAYASHI,1, T. YASUDA,2 H. TAKI,2 M. TAI,2 Y. WATANABE,2 I. SAIKAWA,2 AND S. MITSUHASHIl* Department of Microbiology, School of Medicine, Gunma University, Maebashi, Gunma,' and Research and Development Center, Toyama Chemical Co., Ltd., Toyama,2 Japan

Received for publication 14 March 1977

T-1220, sodium 6-[D-(-)-a-(4-ethyl-2,3-dioxo-1-piperazinylcarbonyl-amino)-aphenylacetamido] penicillanate, is a new semisynthetic penicillin derivative that possesses a broad spectrum of in vitro antibacterial activity against grampositive and gram-negative bacteria. T-1220 is more effective than carbenicillin (CB-PC) against Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus species, and Serratia marcescens. Addition ofhuman serum to culture media did not significantly alter the antibacterial activity of T-1220. Greater bactericidal activity toward various strains of gram-negative bacteria was demonstrated with T-1220 than with CB-PC. T-1220, like penicillin G, was hydrolyzed by penicillinase, but was sable to a type IV penicillinase produced by P. aeruginosa strains. In vivo antibacterial activities of T-1220, ampicillin (AB-PC), and CB-PC were compared, using systemic infections of mice with P. aeruginosa, K. pneumoniae, and Escherichia coli. The 50% effective doses (milligrams per kilogram) of T-1220 were consistently lower than those of AB-PC and CB-PC. The incidence of Pseudomonas aeruginosa infection has progressively increased, along with infections due to other gram-negative bacteria (2, 5, 10, 14). Carbenicillin (CB-PC) was the first penicillin effective against P. aeruginosa infection (1, 4). In our laboratory, 4-ethyl-2,3-dioxo-1-piperazinylcarboxylic acid substituted on the a-amino group of ampicillin (AB-PC) showed more potent antipseudomonal activity, in addition to high activity against other gram-negative bacteria. This paper presents the in vitro and in vivo microbiological evaluation of this compound, designated T-1220. MATERIALS AND METHODS Antibiotics. T-1220, shown in Fig. 1, was synthesized in the Research Laboratory of Toyama Chemical Co., Ltd. The crystalline form used in these studies was prepared by freeze-drying an aqueous solution of T-1220 and was more than 98% pure. Other antibiotics used for comparison were commercial products. Test strains. Strains stocked in this laboratory were used as standards. Stock cultures of the Reference Laboratory of Drug-Resistant Bacteria, Gunma University, were originally isolated from clinical materials. Media. Media used in this experiment were as follows. Heart infusion (HI) agar, brain heart infusion agar, HI broth, and brain heart infusion broth were products of Eiken Chemical Co., Ltd. Other media used were peptone water and medium-B. Pep-

tone water consisted of 10 g of polypeptone, 5 g of NaCl, and 1,000 ml of distilled water. Medium-B consisted of 2 g of yeast extract, 10 g of polypeptone, 7 g of Na2HPO4 12H20, 2 g of KH2PO4, 1.2 g of glucose, and 0.4 g of MgSO4 7H20 in 1,000 ml of distilled water. In vitro antibacterial activity. Minimal inhibitory concentrations (MICs) of antibiotics were determined by a standard twofold serial dilution method, using HI agar. Overnight cultures in peptone broth were used for precultures of tested strains. Brain heart infusion agar was used for determination of the MIC against Streptococcus pyogenes. KNO3 was added to peptone broth in a final concentration of 0.4% to yield a homogeneous culture of the P. aeruginosa strain. MICs were determined after overnight incubation at 37°C, with an inoculum equivalent to a 10-2 dilution of an 18-h culture in peptone broth (about 108 cells/ml). Each inoculum was seeded onto the agar plates by use of an inoculum-replicating apparatus and transferred by a 0.005-ml calibrated loop. Effects of human serum on the antibacterial activity were examined by using a twofold dilution method in HI broth. Bacterial inocula consisted of approximately 104 to 105 organisms/ml. Bactericidal activity. An overnight culture of each strain in HI broth was diluted to a final concentration of about 104 cells/ml with HI broth containing serial twofold dilutions of antibiotics. MICs were recorded after incubation at 37°C for 18 h. One loopful of each culture tube in the MIC test series was spotted onto antibiotic-free HI agar plates, and, after incubation at 37°C for 18 h, the minimum bactericidal concentrations of antibiotic were re455

456

ANTMICROB. AGENTS CHEMOTHER.

UEO ET AL. 0

0 S

C2H5N

CH3

NCONC oNHH3

HCHCONHJj§'HCOONa FIG. 1. Structure of T-1220.

corded as the lowest concentration of drug that had prevented visible growth on HI agar plates. Stability to 13-lactamase. The enzyme samples were prepared as follows. A 20-ml brain heart infusion culture of each strain was diluted 10-fold with medium-B and incubated at 37°C. Penicillin G was added to the culture of P. aeruginosa GN918 in a final concentration of 5 mg/ml for the induction of Plactamase formation. After incubation for 6 h, the cells were harvested by centrifugation, washed with 0.1 M phosphate buffer (pH 7.0), and resuspended in 5 ml of the same buffer. The cells were disrupted in an ultrasonicator for about 5 min at 00C. (3-Lactamase activity was determined by a modification of Perret's method (11). Penicillinase (PCase) activity was expressed as units per milligram of protein when penicillin G was used as a substrate. Substrate specificity was expressed as a relative rate of the five substrates, taking the absolute rate of penicillin G hydrolysis as 100. Cephalosporinase activity was expressed as units per milligram of protein when cephaloridine was used as a substrate. Substrate specificity was expressed as a relative rate, taking the absolute rate of cephaloridine hydrolysis as 100. Escherichia coli W3630 Rms212+ (3, 18), W3630 Rms213+ (3, 18), and W3630 Rte16+ (15) and P. aeruginosa ML4259 Rms139+, ML4259 Rms149+, and ML4259 Rms242+ (13) were used as the standard strains capable of producing the known types of PCase. P. aeruginosa GN918 and E. coli GN5482 were used as the standard strains capable of producing cephalosporinase (16). In vivo antibacterial activity. Fifteen ICR strain male mice, weighing 18 to 20 g each, were used for each dose level. Mice were challenged intraperitoneally with sufficient microorganisms to kill all nontreated mice within 72 h. Microorganisms grown on an HI agar plate were suspended in physiological saline solution. Klebsiella pneumoniae GN6445 and P. aeruginosa NC-5 were suspended in physiological saline containing 1 and 5% mucin (Wako Chemical Co., Tokyo; hog gastric mucin, LP-K type), respectively. Mice infected with E. coli ML4707 were treated 1 h after infection. Mice infected with K. pneumoniae GN6445 and P. aeruginosa NC-5 were treated, respectively, at 1 and 4 h and 1, 4, and 8 h after infection. The total number of surviving mice was recorded, usually 1 week after infection, and the amount of a single dose (milligrams per kilogram) that gave protection to 50% of the infected mice was estimated by means of a log-probit plot (6).

RESULTS AND DISCUSSION Antibacterial spectrum. The spectrum of antibacterial activity of T-1220 against gram-posi-

tive and gram-negative bacteria is shown in Table 1. T-1220 was active against gram-positive organisms susceptible to CB-PC and ABPC. T-1220 was slightly more active than CBPC but less active than AB-PC against grampositive organisms that do not produce PCase. T-1220 was also active against gram-negative organisms, and the MICs of T-1220 were less than those of CB-PC and AB-PC against gramnegative organisms susceptible to both drugs. It was characteristic that T-1220 was highly active against gram-negative organisms, i.e., Serratia marcescens, K. pneumoniae, Proteus species, Enterobacter cloacae, and P. aeruginosa, which were resistant to either or both drugs. TABLE 1. Antibacterial activity of T-1220 against standard strains of bacteria MIC (6i&gm) Test organism

CB-PC

AB-PC

Staphylococcus aureus FDA 209P S. aureus Terashima S. aureus Smith S. epidermidis IID866 Bacillus subtilis ATCC 6633

0.78

0.39

200

0.78 6.25

1.56 100

T-1220

H1D620

10490 P. aeruginosa IFO 3445

3.13 0.2

3.13

50 1.56

50

0.39 1.56 0.39 200

>200 200 >200

457

VOL. 12, 1977

ANTIBACTERIAL ACTIVITY OF T-1220

Antibacterial activity. The antibacterial activity of T-1220 against gram-positive and gram-negative organisms was compared with that of AB-PC and CB-PC by cumulative percentages of strains inhibited by various concentrations of the drugs. The representative results at 3.13 and 12.5 ,g/ml are shown in Table 2. The percentage of isolates of gram-negative bacteria inhibited by T-1220 was somewhat larger than that of CB-PC and AB-PC. Cumulative percentages of 200 E. coli strains inhibited by T-1220, AB-PC, and CB-PC are illustrated in Fig. 2. The concentration of T1220 required to inhibit the growth of 50% ofthe total number of tested E. coli strains (MIC.)

was 1.56 to 3.13 ug/ml; MIC50 values of AB-PC and CB-PC were 6.25 ,tg/ml, respectively. The MIC50 of T-1220 against K. pneumoniae strains was 3.13 to 6.25 ug/ml; MIC50 values of AB-PC and CB-PC were 50 to 100 ,ug/ml, respectively. Therefore, T-1220 was approximately 10 times more active than AB-PC and CB-PC (Fig. 3). Large differences were seen in the MIC distributions of T-1220 and CB-PC against 300 P. aeruginosa strains; the peaks of T-1220 and CBPC were located at 6.25 and 50.0 ,ug/ml, respectively. The M1C. values of T-1220 and CB-PC against P. aeruginosa strains were 3.13 and 50.0 ug/ml, respectively; T-1220 was approxi-

TABLE 2. Comparison of the antibacterial activites of T-1220, CB-PC, and AB-PC against clinical isolatesa Inhibition (%) by drug at:b No. of

Organism

3.13

ug/ml

CB-PC

T-1220

12.5

AB-PC

ug/ml

CB-PC

T-1220

AB-PC

S. aureus 89 100 99 70 70 93 100 S. epidermidis 100 100 100 100 100 100 100 S. pyogenes 100 100 100 100 100 100 100 64 57 56 55 25 31 E. coli 100 62 3 9 33 3 4 K.pneumoniae 200 96 96 89 93 82 68 P. mirabilis 100 87 83 6 79 75 4 P. morganii 100 P. vulagris 80 73 44 70 61 38 100 P. rettgeri 88 80 36 84 68 8 75 S. marcescens 50 43 6 31 11 0 100 60 50 3 49 8 0 E. cloacae 136 85 6 NT 35 0.5 NTC P. aeruginosa 300 a Clinical isolates are the stock cultures of the Reference Laboratory of Drug-Resistant Bacteria, School of Medicine, Gunma University. b Percentage of inhibition was calculated from the cumulative percentages of strains inhibited by various concentrations of each drug. c NT, Not tested. %

100 -S

C

C

a)cr C

(a)_50

Z.4

cL

LL

a-

ELL-)

0.2

0.78 3 13 12.5

50

0.2 0.78 3.13 >100 Concentration of Drug (mcgfml)

12.5

FIG. 2. Susceptibility of 200 E. coli strains to T-1220, AB-PC,

and

50

'100

CB-PC.

458

ANTiMICROB. AGzNTS CHEMOTHER.

UEO ET AL.

U

a

CB-PC

0 or ._ h-

T-1220

0.2

0.78 3.13 12.5

50 '100 0.2 0.78 3.13 12.5 50 >100 Concentration of Drug (mc9mI) FIG. 3. Susceptibility of 200 K. pneumoniae strains to T-1220, AB-PC, and CB-PC. s

%

100r C c

C GE C

,0

T 1220

L-

0

C-

0.78 3.13 12.5 50 -100 0.2 0.78 3.13 12.5 50 >100 Concentration of Drug (mcgfml) FIG. 4. Susceptibility of 300 P. aeruginosa strains to T-1220 and CB-PC. 0.2

TABLE 3. Effect of human serum on antibacterial activity of T-1220 Human serum

('M 0 10 40

E. coli NIHJ

aureu FDA 209P

0.39 0.39 0.2

1.56 1.56

S.

P. aeruginosa NCTC 10490

1.56

0.78 0.78 0.78

mately 10 times more active than CBPC (Fig. 4). Effects of different culture media and pH values on the MICs of T-1220 were not great. The addition of human serum at 10 and 40% did not alter the inhibitory effect of T-1220 against E. coli NIHJ, Staphylococcus aureus FDA 209P, or P. aeruginosa NCTC 10490 (Table 3). Bactericidal activity. Correlation of MICs

and minimum bactericidal concentrations of T1220 is shown in Table 4. The minimum bactericidal concentrations of T-1220 against most of the tested strains seemed to be the same or only twofold higher than the MICs. However, the minimum bactericidal concentrations of T-1220 against PCase-producing strains, i.e., K. pneumoniae GN5559, Proteus vulgaris GN1543, P. vulgaris IID874, and P. aeruginosa GN6727, were four- or eightfold higher than the MICs. Susceptibility to PCase. The R-mediated PCases were classified into four types (3, 13, 17, 18), and a type IV PCase capable of specifically hydrolyzing CB-PC was demonstrated from P. aeruginosa strains carrying R plasmids (9, 13). The relative rate of hydrolysis of four penicillins and cephaloridine by R-mediated PCases and cephalosporinase is shown in Table 5. T-1220, CB-PC, and AB-PC were

ANTIBACTERIAL ACTIVITY OF T-1220

VOL. 12, 1977

459

quite stable to cephalosporinases produced by produced by P. aeruginosa strains carrying R E. coli GN5482 and P. aeruginosa GN918 (16). plasmids. By contrast, T-1220 was susceptible to type I, II, In vivo antibacterial activity. Chemotheraand III PCases. It is characteristic, however, peutic effects of T-1220 on experimental infecthat T-1220 is quite stable to a type IV PCase tions of mice with E. coli ML4707, K. pneumoTABLE 4. Correlation between MICs and minimum bactericidal concentrations (MBCs) (micrograms per milliliter) of T-1220 T-1220

CB-PC

AB-PC

Organism

E. coli NIH JC-2 E. coli GN6281 K. pneumoniae IID875 K. pneumoniae GN5559 P. morganii IID602 P. morganii GN1893 P. vulgaris IID874 P. vulgaris GN1543 P. mirabilis GN1537 P. rettgeri GN1725 S. marcescens IID620 S. marcescens GN6467 P. aeruginosa NCTC 10490 P. aeruginosa GN6727 a NT, Not tested.

Enzyme sourceb

W3630 Rms212+ W3630 Rms213+ W3630 Rte16+ ML4259 Rmsl39+ ML4259 Rmsl49+ ML4259 Rms242+

MIC

MBC

MIC

MBC

MIC

MBC

3.13 0.78 6.25 6.25 1.56 1.56 25 3.13 0.78 0.78 0.78 0.78 0.39 6.25

3.13 0.78 6.25 25 1.56 1.56 50 50 0.78 0.78 3.13 1.56 0.78 50

12.5 6.25 100 200 3.13 0.78 50 3.13 0.39 0.78 6.25 6.25 0.78 200

25 6.25 200 200 6.25 1.56 100 50 0.78 0.78 12.5 50 1.56 >200

6.25 6.25 12.5 50 200 200 800 >200 1.56 >200 25 3.13 NTa NT

6.25 6.25 25 200 400 200 1600 >200 3.13 >200 50 100 NT NT

TABLE 5. Substrate profiles of various f-lactamasesa Relative rate of hydrolysis Type of 6-lacta- Sp act (U/

Type m PCase I PCase II PCase III PCase IV PCase IV PCase IV

mg of pro- PC-G tein)

74 3 4 285 160 128

100 100 100 100 100 100

T-1220

CB-PC

AB-PC

CER

89 118 90 5 17 8

8 60 57 96 253 100

92 545 125 105 101 110

137 95 23 18 10 15

P. aeruginosa GN918 CSase 253 1 20 0.2 E. coli GN5482 22 CSase 46 0.1 0.8 a PC-G, Penicillin G; CER, cephaloridine; CSase, cephalosporinase. b W3630, substrain of E. coli K-12; ML4259, substrain of P. aeruginosa 1008.

0.8 0.7

100 100

TABLE 6. In vivo antibacterial activity of T-1220 against systemic infectiona Challenge orgaChallenge dose Drug MIC (.tg/ ED. (mg/ 95% confidence limit nism ml) kg) 1.56 47.0 E. coli ML4707 2.5 x 106 cells (5 x LD5O) in CB-PC 36.5-60.0 P > 0.05 saline T-1220 1.56 48.0 37.5-61.5 3.13 20.5 AB-PC 16.0-25.0 P < 0.05 K. pneumoniae GN6445

106 cells (25 x LD50) in saline containing 1% mucin

CB-PC T-1220 AB-PC

800 6.25 100

550 19.0 37.5

350-870 9.5-37.0 26.0-54.0

P < 0.05 P > 0.05

3.5 x 104 cells (100 x LD50) in CB-PC 200 1,250 880-1,775 t p < 0.05 12.5 350 saline containing 5% mu- T-1220 195-630 ) cin a Subcutaneous injection of each drug. Challenge, Intraperitoneal injection with a saline suspension of each organism. ED., 50% effective dose; LD50, 50% lethal dose.

P. aeruginosa NC-5

460

UEO ET AL.

niae GN6445, and P. aeruginosa NC-5 are shown in Table 6. Against infection of mice with E. coli MIA707, T-1220 was as active as CB-PC, but less active than AB-PC. Against infections with K. pneumoniae, in vivo effectiveness of T-1220 was much higher than that of CB-PC and AB-PC. T-1220 also showed much higher in vivo effectiveness against P. aeruginosa than did CB-PC. LITERATURE CITED 1. Acred, P., D. M. Brown, E. T. Knudsen, G. N. Rolinson, and R. Sutherland. 1967. New semi-synthetic penicillin active against Pseudomonas pyocyanea. Nature (London) New Biol. 215:25-30. 2. Altermeier, W. A., R. P. Hummel, E. 0. Hill, and S. Lewis. 1973. Changing patterns in surgical infections. Ann. Surg. 178:436-445. 3. Egawa, R., T. Sawai, and S. Mitauhashi. 1967. Drug resistance of enteric bacteria. XII. Unique substrate specificity of penicillinase produced by R factor. Jpn. J. Microbiol. 11:173-178. 4. English, A. R. 1969. Laboratory studies with carbenicillin, p. 482-488. Antimicrob. Agents Chemother. 1968. 5. Finland, M. 1970. Changing ecology of bacterial infections as related to antibacterial therapy. J. Infect.

Dis. 122:419-431. 6. Litchfield, J. T., and F. Wilcoxon. 1948. A simplified method of evaluating dose-effect experiments. J. Pharmacol. 92:99-113. 7. Morimoto, S., H. Nomura, T. Fugono, I. Minami, M. Hori, and T. Masuda. 1972. Semisynthetic p-lactam antibiotics. 2. Synthesis and properties of D- and L-asulfobenzylpenicillins. J. Med. Chem. 15:1108-1111. 8. Naide, Y., K. Suzuki, I. Nagakubo, and M. Ohkoshi. 1971. Basic and clinical evaluation of sulfobenzylpenicillin. Chemotherapy 19:1023-1025.

ANTIMICROB. AGENTS CHEMOTHER. 9. Newson, S. W. B., R. B. Sykes, and M. H. Richmond. 1970. Detection of a ,-lactamase markedly active against carbenicillin in a strain of Pseudomonas aerugino8a. J. Bacteriol. 101:1079-1080. 10. Ohkoshi, M. 1970. Drug resistance patterns of bacteria isolated from clinical sources in Japan, p. 717-722. In International Society of Chemotherapy (ed.), Progress in antimicrobial and anticancer chemotherapy, vol. 2. University of Tokyo Press, Tokyo. 11. Perret, C. J. 1954. lodometric assay of penicillinase. Nature (London) 174:1012-1013. 12. Rodrignez, V., J. Inagaki, and G. P. Bodey. 1973. Clinical pharmacology of ticarcillin (a-carboxy-3-thienylmethyl penicillin BRL 2288). Antimicrob. Agents

Chemother. 4:31-36. 13. Sawada, Y., S. Yaginuma, M. Tai, S. Iyobe, and S. Mitauhashi. 1976. Plasmid-mediated penicillin betalactamase in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 9:55-0. 14. Schimpff, S. C., W. H. Greene, V. M. Yonung, and P. H. Wiernic. 1974. Significance of Pseudomonas aeruginosa in the patient with leukemia or lymphoma. J. Infect. Dis. 130(Suppl):24-31. 15. Sutherland, R., J. Burnett, and G. N. Rolinson. 1971. a-Carboxy-3-thienylmethyl penicillin (BRL 2288), a new semisynthetic penicillin: in vitro evaluation, p. 390-395. Antimicrob. Agents Chemother. 1970. 16. Yaginuma, S., T. Sawai, H. Ono, S. Yamagishi, and S. Mitauhashi. 1973. Biochemical properties of a cephalosporin 9-lactamase from Pseudomonas aeruginosa. Jpn. J. Microbiol. 17:141-149. 17. Yaginuma, S., N. Terakado, and S. Mitsuhashi. 1975. Biochemical properties of a penicillin beta-lactamase mediated by R factor from Bordetella bronchiseptica. Antimicrob. Agents Chemother. 8:238-242. 18. Yamagishi, S., K. O'Hara, T. Sawai, and S. Mitsuhashi. 1969. The purification and properties of penicillins #-lactamases mediated by transmissible R factors in Escherichia coli. J. Biochem. 66:11-20.

In vitro and in vivo antibacterial activity of T-1220, a new semisynthetic penicillin.

ANTmcRoBIAL AGzNTS AND CHSMoHzRwY, Oct. 1977, p. 455-460 Copyright 0 1977 American Society for Microbiology Vol. 12, No. 4 Printed in U.S.A. In Vitr...
718KB Sizes 0 Downloads 0 Views