INTRODUCTION

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The emergence of multidrug-resistant microorganisms has caused serious concern about infectious diseases worldwide. Although more than three decades have passed since the first report of methicillin-resistant Staphylococcus aureus (MRSA), MRSA still presents a serious problem worldwide as a cause of nosocomial infections (7, 12). Vancomycin, a cyclic glycopeptide antibiotic, has been extensively used in the clinic to treat MRSA infections. However, it is not an ideal antibiotic because of the slow clinical response (6) and potential adverse effects (3). Furthermore, the emergence of MRSA strains with reduced susceptibility to vancomycin accelerated an urgent need for new chemotherapeutic agents for the treatment of MRSA infections (5).

Previously reported -lactam antibiotics with activity against methicillin-resistant staphylococci (MRS) such as L-695,256 (2), SM-17466 (13), BO-3482 (8), TOC-39 (4), MC-02,479/RWJ-54428 (F. Malouin, C. Chan, S. Bond, S. Chamberland, and V. J. Lee, Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F-177, p. 176, 1997), Ro 63-9141 (P. Hohl, P. Angehrn, R. L. Then, P. Hebeisen, and I. Heinze-Krauss, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F-24, p. 239, 1998), and L-786,392 (J. Huber, K. L. Dorso, J. Koheler, H. Kropp, H. Rosen, and L. L. Silver, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F-30, p. 240, 1998) all had weak activities against gram-negative organisms and/or a lack of antipseudomonal activity.

In the course of our derivatization study of 1-methylcarbapenems, a novel trans-3,5-pyrrolidinylthio-1-methylcarbapenem, J-111,347, was identified as a broad-spectrum agent with activity against MRS, as well as gram-positive and -negative organisms, including Pseudomonas aeruginosa, that are usually covered by the marketed broad-spectrum carbapenems (A. Shimizu, Y. Sugimoto, S. Sakuraba, H. Imamura, H. Sato, N. Ohtake, R. Ushijima, S. Nakagawa, C. Suzuki, T. Hashizume, and S. Morishima, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F-52, p. 246, 1998). Subsequently, we synthesized J-111,225, J-114,870, and J-114,871 (Fig. 1) (10). The stereochemistry of the side chains in these compounds is novel; known pyrrolidinylthio-1-carbapenems like meropenem (14), S-4661 (S. Sasaki, K. Murakami, Y. Nishitani, and S. Kuwahara, Abstr. 34th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F-33, p. 32, 1994), BO-2727 (9), MK-826 (formerly L-749,345) (L. Pelak, S. Gerckens, P. M. Scott, C. Gill, C. Pacholok, L. Lynch, K. Dorso, J. Kohler, D. Shungu, and H. Kropp, Abstr. 36th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F-119, p. 120, 1996), and E1010 (formerly ER-35786) (11) share a cis-counterpart at the side chains.

View larger version (10K): [in this window] [in a new window]   FIG. 1.   Chemical structures of the trans-3,5-disubstituted 1-methylcarbapenems J-111,347, J-111,225, J-114,870, and J-114,871. Me, methyl.

In this paper, we describe the in vitro evaluation of the novel trans-3,5-disubstituted pyrrolidinylthio-1-methylcarbapenems J-111,347 (the prototype), J-111,225, J-114,870, and J-114,871.

(This paper was presented in part at the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, Calif., 24 to 27 September 1998.)

	MATERIALS AND METHODS

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Antibiotics. All of the carbapenems used in this study except imipenem and vancomycin were synthesized at Banyu Tsukuba Research Institute, Tsukuba, Japan; imipenem was a product of Banyu Pharmaceutical Co., Ltd., Tokyo, Japan, and vancomycin was purchased from Sigma Chemical Co., St. Louis, Mo. The antibiotics were dissolved in 10 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (pH 7.0) on the day of use.

Organisms. The clinical isolates used in this study were collected from several hospitals in Japan over the past several years. All isolates were maintained in glycerol broth at 80°C.

Determination of MICs. MICs were determined by the twofold serial broth microdilution method with Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.) unless stated otherwise. Susceptibility testing for streptococci was performed with Todd-Hewitt broth (Difco) supplemented with 5% hemolyzed horse blood. Brain heart infusion broth (Difco), brain heart infusion broth (Difco) supplemented with 5% Fildes enrichment (Difco), and GAM broth (Nissui Seiyaku Co., Ltd., Tokyo, Japan) were used for enterococci, Haemophilus influenzae, and anaerobes, respectively. The inoculum sizes of gram-positive or -negative bacteria and anaerobes were 10⁵ and 10⁶ CFU/ml, respectively. The MIC was defined as the lowest antibiotic concentration that completely prevented visible growth after incubation at 37°C for 20 h.

Killing kinetics. MRSA strain BB6226, a -lactamase-negative homogeneously resistant strain, was used. After preincubation at 37°C for 2 h, an antibiotic was added and the test tubes were incubated in a water bath at 37°C with gentle shaking. The viable cells were counted on a Mueller-Hinton medium (Difco) plate after an aliquot of the culture was taken at the times indicated in Fig. 2. The limit of detection of viable counts was 10 CFU/ml.

Affinity of PBP 2a of MRSA. The affinity of PBP 2a of MRSA was determined by a competition assay with [¹⁴C]benzylpenicillin (15). Briefly, membrane fractions were preincubated at 30°C for 10 min with nonlabeled antibiotics and were postincubated with [¹⁴C]benzylpenicillin for 10 min. Binding affinity was expressed as the concentration of nonlabeled antibiotic that inhibited radiolabeling with [¹⁴C]benzylpenicillin by 50% (IC₅₀) compared with the control in the absence of nonlabeled antibiotics.

	RESULTS

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In vitro activity. The comparative antibacterial activities of J-111,347, J-111,225, J-114,870, J-114,871, and the reference antibiotics against the clinical isolates are shown in Table 1. The new carbapenems showed clearly improved activities against the isolates of MRSA and methicillin-resistant coagulase-negative staphylococci (MRCoNS) compared with those of the older carbapenems imipenem, meropenem, and biapenem. J-111,347, J-111,225, J-114,870, and J-114,871 inhibited 90% of the MRSA isolates at concentrations of 2, 4, 4, and 4 µg/ml (MICs at which 90% of isolates are inhibited [MIC₉₀]), respectively, representing activity 32 to 64 times more potent than that of imipenem, which has an MIC₉₀ of 128 µg/ml. The activity of J-111,347 was about half as potent as or equivalent to that of vancomycin against MRSA and MRCoNS in terms of the MIC₉₀. In general, J-111,225, J-114,870, and J-114,871 were almost as active or were slightly less active than J-111,347 against the MRS isolates. Thus, the new carbapenems are definitely differentiated from conventional carbapenems in terms of their anti-MRS activities.

Bactericidal activity. Bactericidal activity against MRSA was determined by constructing time-kill curves (Fig. 2). As expected, J-111,225, J-114,870, and J-114,871 showed bactericidal kinetics at concentrations above the MICs of 1 to 4 µg/ml, although substantial regrowth was observed after 24 h of incubation in the presence of two times the MICs of J-111,225, J-111,347, and imipenem. Vancomycin (MIC, 1 µg/ml) showed slow killing kinetics even at four times the MIC. Imipenem required high concentrations (over 64 µg/ml) to kill MRSA.

View larger version (34K): [in this window] [in a new window]   FIG. 2.   Killing kinetic curves for trans-3,5-disubstituted 1-methyl carbapenems, imipenem, and vancomycin against homogeneous MRSA.

Activity against -lactamase producers. The new carbapenems were active against various -lactamase-producing bacteria except for Burkholderia cepacia and metallo--lactamase producers (Table 2). With the exceptions of B. cepacia and metallo--lactamase producers, the MICs of the new carbapenems for ampicillin- and/or cefazolin-resistant -lactamase producers were within the susceptible range for other carbapenems, as were those of the reference carbapenems. In E. coli, there seemed to be no appreciable influence of -lactamase producing strain on the activity since basal AmpC -lactamase production had similar susceptibility to the respective new carbapenems (MIC, 0.031 µg/ml). It is noteworthy that the new drugs had greater activities than the marketed carbapenems against the IMP-1 -lactamase-producing organism.

View this table: [in this window] [in a new window]   TABLE 2.   Comparative antibacterial activities against -lactamase producers

Mechanism of action against MRSA. Since PBP 2a, which has a low affinity for -lactams, is the resistance determinant for -lactam antibiotics in MRS, the binding affinities of J-111,347 and J-111,225 for PBP 2a were investigated. These two carbapenems had improved IC₅₀s of 2.6 and 2.5 µg/ml, respectively, compared with the IC₅₀ of 85 µg/ml of imipenem in a competition assay with benzylpenicillin. There was a good correlation between anti-MRSA activity and the binding affinity for PBP 2a of the MRSA strain tested, indicating that the mechanism of anti-MRSA activity could be ascribed to the inhibition of PBP 2a.

	DISCUSSION

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MRS and gram-negative organisms including P. aeruginosa are common pathogens in serious infections, and vancomycin has been the agent of choice for the treatment of infections due to MRS, despite its association with a variety of drug-related side effects. Although a number of antibiotics that target MRSA and other resistant gram-positive organisms have been reported, no antibiotic so far provides potent anti-MRS activity while also providing the excellent broad-spectrum activity offered by carbapenems.

We conducted chemical modification studies with 1-methylcarbapenem on the basis of the concept that a new analogue should provide coverage against MRS as well as other gram-negative organisms including P. aeruginosa. J-111,225, J-114,870, and J-114,871 are crystalline forms, and their structural features include a unique stereochemistry of the side chain, i.e., a phenyl ring directly attached to the pyrrolidine ring with a 3,5-trans configuration at the C-2 position of the carbapenem nucleus.

Since previously reported anti-MRS agents have limited activity against gram-negative bacteria, it was noted that the newly synthesized carbapenems with activity against MRSA showed improved activity against gram-negative organisms including P. aeruginosa and metallo--lactamase producers compared with the activity of imipenem. Metallo--lactamase or carbapenemase is known to confer high-level resistance to penicillins and cephems as well as to carbapenems (16). A recent problem is the spread of the transferable plasmid-mediated IMP-1 enzyme, especially in Japan (1). The mechanism underlying improved activity against IMP-1-producing organisms was explained by resistance to hydrolysis by this enzyme (R. Nagano, Y. Adachi, T. Hashizume, and S. Morishima, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F-55, p. 247, 1998).

Although further evaluation of J-111,347 was suspended due to its epileptogenicity, this undesirable adverse effect was clearly eliminated by N methylation (J-111,225) or the introduction of carbamoylmethyl substituents (J-114,870 and J-114,871) at the  position of the benzylamino group.

The new carbapenems could be suited for use as monotherapy for serious polymicrobial infections associated with MRS. Furthermore, monotherapy with the new carbapenems would avoid unexpected adverse reactions due to combination therapy and the emergence of vancomycin-resistant MRS due to the intensive use of vancomycin. In conclusion, the novel trans-3,5-disubstituted pyrrolidinylthio-1-methylcarbapenems J-111,347 (prototype), J-111,225, J-114,870, and J-114,871 possess broad spectra of activity, with coverage of MRSA and P. aeruginosa, and are worthy of further evaluation.