Banyu Tsukuba Research Institute, Okubo 3, Tsukuba 300-2611, Japan
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Abstract |
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Introduction |
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J-111,225 is a novel trans-3,5-disubstituted pyrrolidinylthio carbapenem with an ultra-broad spectrum covering Gram-positive and Gram-negative bacteria including methicillin-resistant staphylococci and P. aeruginosa.10 In the course of evaluation, J-111,225 was found to have better antibacterial activity than marketed carbapenems against IMP-1 metallo-ß-lactamase-producing organisms. In this paper, we describe the interaction of J-111,225 with ß-lactamases.
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Materials and methods |
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J-111,225 and its side chain, as well as meropenem and panipenem, were synthesized at the Tsukuba Research Institute, Banyu Pharmaceutical Co. Ltd, Tsukuba, Japan. J-111,225 has two chiral centres, C3 and C5, bearing a sulphide bond and benzene ring, respectively, in its pyrrolidine ring (Figure 1). Imipenem and amikacin were the products of Banyu Pharmaceutical Co. Ltd. The following drugs were obtained commercially: ceftazidime (Tanabe Pharmaceutical Co. Ltd, Osaka, Japan), piperacillin (Toyama Chemical Co. Ltd, Tokyo, Japan), aztreonam (Eisai Co. Ltd, Tokyo, Japan), ofloxacin (Sigma Chemical Co., St Louis, MO, USA), cephaloridine (Shionogi Pharmaceutical Co., Osaka, Japan) and nitrocefin (Oxoid, Basingstoke, UK).
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Clinical isolates that produced IMP-1 metallo-ß-lactamase were collected from various districts in Japan since 1994, and the blaIMP gene was detected by PCR.5 P. aeruginosa GN17203, which harbours the blaIMP-carrying plasmid, pMS350, and Bacteroides fragilis GAI30079 were generously given by M. Inoue, Kitasato University, School of Medicine, Kanagawa, Japan, and K. Watanabe, Institute of Anaerobic Bacteriology, Gifu University, School of Medicine, Gifu, Japan, respectively.
Determination of MICs
MICs were determined by the two-fold serial broth microdilution method using MuellerHinton broth (Difco Laboratories, Detroit, MI, USA). A culture of test strains grown at 37°C for 6 h in MuellerHinton broth was diluted to 107 cfu/mL, and these dilutions were inoculated into the drug-containing broth with an inoculation apparatus (MIC-2000: Dinatech Laboratories Inc., Chantilly, VA, USA) at the final inoculum size of 105 cfu/mL. The MIC was defined as the lowest antibiotic concentration that completely prevented visible growth after incubation at 37°C for 20 h. Synergy of drug combinations was determined by the chequerboard method using S. marcescens BB5886 and P. aeruginosa GN17203, which showed resistance to imipenem with an MIC of 128 mg/L in MuellerHinton broth. The fractional inhibitory concentration (FIC) index was determined according to the method of Elion et al.11
ß-Lactamase preparation
The IMP-1 metallo-ß-lactamase was purified from P. aeruginosa GN17203 harbouring the blaIMP-carrying plasmid pMS350. Cells were incubated at 4 °C for 1 h in 10 mM 3-(N-morpholino)propanesulphonic acid (MOPS) buffer (pH 7.0) containing 27% sucrose and 2 mg/mL lysozyme (Sigma Chemical Co.), and were then disrupted by sonication. The cellular debris was removed by centrifugation (16,000g, 15 min, 4°C), and the supernatant was precipitated in an 80% saturated concentration of ammonium sulphate. This fraction was dialysed against 10 mM MOPS buffer (pH 7.0) and applied to a CM-Sephadex C-50 column (Pharmacia Biotech AB, Uppsala, Sweden) equilibrated with 10 mM MOPS buffer (pH 7.0). The enzyme was eluted with a linear NaCl gradient. The active fractions were pooled, dialysed against 10 mM MOPS buffer (pH 7.0) containing 1 µM ZnCl2 and concentrated by ultrafiltration with a UK-10 ultra filter (Advantec Toyo Co., Tokyo, Japan). The purity of the preparation was checked by sodium dodecylsulphatepolyacrylamide gel electrophoresis (SDSPAGE) and the purified enzyme solution was stored at 80°C.
The CcrA metallo-ß-lactamase was prepared from B. fragilis GAI30079. Cells were suspended in 50 mM sodium phosphate buffer (pH 7.0) and disrupted by sonication. Cellular debris was removed by centrifugation (13,500g, 15 min, 4°C) and the supernatant was dialysed against 50 mM phosphate buffer (pH 7.8). This fraction was applied to a DEAEToyopearl 650M column (Toso Co., Tokyo, Japan) equilibrated with 50 mM phosphate buffer (pH 7.8) and the enzyme was eluted with a linear NaCl gradient. The pooled active fractions were dialysed against 50 mM phosphate buffer (pH 7.0), concentrated by ultrafiltration with a UK-10 ultrafilter and rechromatographed with a Sephadex G-100 column (Pharmacia Biotech AB) equilibrated with 50 mM phosphate buffer (pH 7.0). The concentrated crude enzyme preparation was dialysed against 50 mM phosphate buffer (pH 7.0) and stored at 80°C.
The L1 metallo-ß-lactamase was prepared from Stenotrophomonas maltophilia GN12873 as described previously.12 The Bacillus cereus type II metallo-ß-lactamase, Escherichia coli TEM-1 penicillinase and Enterobacter cloacae cephalosporinase were purchased from Sigma Chemical Co.
Determination of ß -lactamase activity
The activity at each step of metallo-ß-lactamase preparation was determined by monitoring the hydrolysis of 100 µM imipenem ( = 9.04 mM1 cm1 at 299 nm) at 30°C in 10 mM MOPS buffer (pH 7.0) containing 100 µM of ZnCl2. One unit (U) of ß-lactamase activity was defined as the amount of enzyme that hydrolysed 1 µmol of imipenem in 1 min at 30°C.
Determination of IC 50
The 50% inhibitory concentration (IC50) for the IMP-1 metallo-ß-lactamase was determined by measuring the enzymatic hydrolysis of a chromogenic cephalosporin, nitrocefin, in the presence of inhibitors. This automated assay system was a modification of a previously reported method.13 To avoid identifying the metal chelators, 10 mM MOPS buffer (pH 7.0) containing 100 µM of ZnCl2 was used in this microassay. One microlitre of inhibitor and 25 µL of IMP-1 metallo-ß-lactamase (36 mU/mL) were mixed in a 98-well microplate, and the assay was initiated within 1 min by the rapid addition of 75 µL of nitrocefin solution to create a final nitrocefin concentration of 72.7 µM. Inhibitors were dissolved in 10 mM MOPS buffer (pH 7.0) or dimethyl sulphoxide (DMSO) and prepared at final concentrations of 0.1, 1 and 10 µM. If needed, a concentration of >100 µM was prepared. Control wells contained reaction mixture without inhibitor, enzyme or substrate. Assay plates were incubated with slow shaking in an M-36 microincubator (Taitec Co., Tokyo, Japan) at 30°C, and the hydrolysis of nitrocefin was determined 15 min after incubation by monitoring the increase in absorbance at 492 nm in an MTP-120 plate reader (Corona Electric Co., Ibaraki, Japan). Three wells were prepared at each inhibitor concentration; the mean values were used for determining IC50s. The initial rates of hydrolysis at each inhibitor concentration were calculated, and the IC50s (µM) were determined by plotting percentage inhibition against inhibitor concentration.
ß -Lactamase assay
Kinetic studies were performed at 30°C in 10 mM MOPS buffer (pH 7.0) and the hydrolysis of the substrate, imipenem and cephaloridine ( = 10.2 mM1 cm1 at 260 nm), by metallo- and serine-ß-lactamases, respectively, was monitored in a temperature-controlled spectrophotometer, UV-2200 (Shimadzu, Tokyo, Japan). The initial hydrolysis velocity of J-111,225 (
= 9.82 mM1 cm1 at 298 nm) at a concentration of 100 µM was determined, and was calculated relative to that of imipenem and cephaloridine for metallo- and serine-ß-lactamases, respectively. Kinetic parameters were derived from at least two independent experiments from a HanesWoolf plot of the initial velocity of substrate hydrolysis by ß-lactamases. To calculate the Ki values of the inhibitors from a Dixon plot, rates of substrate hydrolysis at concentrations ranging from 10 to 100 µM were determined in the presence of various concentrations of inhibitors. The total reaction volume was 1 mL in all cases.
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Results |
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Table I presents the antimicrobial activities of J-111,225 and reference antibiotics against metallo-ß-lactamase producers. The IMP-1 metallo-ß-lactamase producers, S. marcescens and P. aeruginosa, were highly resistant to the marketed carbapenems, such as imipenem (MICs from 16 to >128 mg/L) and meropenem (MICs from 32 to >128 mg/L), and to ceftazidime (MICs from 128 to >128 mg/L). The MICs of J-111,225 were four- to eight-fold lower (MICs 432 mg/L) than imipenem and meropenem for the IMP-1 metallo-ß-lactamase producers, S. marcescens and P. aeruginosa. Piperacillin showed lower MICs for some of the IMP-1 metallo-ß-lactamase producers and MICs of aztreonam were lowest, but high-level resistance was observed in some isolates. The metallo-ß-lactamase producers tested were often resistant to other classes of antibiotics such as amikacin and ofloxacin, indicating other coexistent resistance mechanisms.
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J-111,225 showed inhibitory potential with an IC50 value of 0.7 µM. The IC50s of the side chain of J-111,225, meropenem and panipenem were >10, 25 and >100 µM, respectively. Metal chelators such as dipicolinic acids14 and EDTA showed IC50s ranging from 80 to 200 µM. Aztreonam15 had a low affinity for the IMP-1-enzyme, with an IC50 of >100 µM. The comparative hydrolysis rates of aztreonam and J-111,225 were <0.01 and 4, respectively, taking the hydrolysis rate of imipenem as 100.
As shown in Figure 2, the Ki of J-111,225 against the IMP-1 metallo-ß-lactamase was determined to be 0.18 µM from a Dixon plot. The Ki of the cis-counterpart at the C5 position of J-111,225 was 0.12 µM, while the inhibitory activity was markedly reduced by inversion of the C3 chiral centre (IC50 > 10 µM) regardless of C5 stereochemistry.
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Table III presents the combination effect of J-111,225 and imipenem against IMP-1 metallo-ß-lactamase producers of S. marcescens BB5886 and P. aeruginosa GN17203. As expected, synergy (FIC
0.5) was observed between the antibacterial activities of J-111,225 and imipenem.
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Discussion |
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Recent studies on X-ray crystal structures of metallo-ß-lactamases such as B. cereus type II,18,19 Ccr2022 and L123 enzymes and an enzymatic study on IMP-124 have revealed that there are two zinc atoms at the active site. The occupancy of the zinc atoms and the amino acid alignment vary between these metallo-ß-lactamases and seem to affect the catalytic activities of each enzyme. The fact that J-111,225 is an inhibitor of IMP-1 but a substrate for chromosomal metallo-ß-lactamases such as CcrA, L1 and type II might be ascribed to diversity among the metallo-ß-lactamases.
Thus, J-111,225 is a novel carbapenem antibiotic exhibiting inhibitory activity against the transferable IMP-1 metallo-ß-lactamase as compared with the marketed carbapenems such as imipenem, meropenem and panipenem (available only in Japan). Recent reports on the appearance of the blaIMP-carrying organisms in South Korea7 and Europe8 in addition to Japan have indicated that precautions are needed to prevent further spread of the metallo-ß-lactamases, which are a threat to antimicrobial chemotherapy with ß-lactam antibiotics including carbapenems. Previous reports on metallo-ß-lactamase inhibitors mainly described their inhibitory properties against chromosomal metallo-ß-lactamases.22,2528 It is important to take steps to cope with the organisms carrying the blaIMP gene which codes for a resistance mechanism that can be transferred among the enteric bacteria and glucosenon-fermentative Gram-negative rods including pseudomonads.
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Notes |
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References |
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Received 19 July 1999; returned 1 October 1999; revised 4 November 1999; accepted 22 November 1999