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THE EFFECT OF 1^-METHYL AND tive and Gram-negative bacteria. A number of IMIDOYL SUBSTITUENTS ON THE carbapenems, such as olivanic acids3), carpetimy- ANTIPSEUDOMONAL ACTIVITY cins4), asparenomycins5), and others, have been OF CARBAPENEMS discovered. However, none are available for clinical use except imipenem, an 7V-formimidoyl derivative yoshihiro sumita*, yoko eguchi, of thienamycin. Meropenemand panipenem are MasatomoFukasawa, Takao Okuda, new carbapenems under pre-registration in Japan. Hiroshi Yamaga, Haruki Matsumura Meropenem, which differs chemically from imipe- and Makoto Sunagawa nem and panipenem, has a l/?-methyl group and no imidoyl substituent on the 2-sulfur side chain. In Discovery Research Laboratories III, this study, we investigated the antipseudomonal SumitomoPharmaceuticals Research Center, 3-1-98 Kasugade-naka, Konohana, Osaka 554, Japan activity of four series of carbapenems in terms of the structure-activity relationships, especially the (Received for publication June 4, 1993) l/?-methyl and imidoyl moieties. Meropenem and the other carbapenems, sum- Amongthe manyclinical pathogens Pseudomonas marized in Fig. 1, were prepared in Sumitomo aeruginosa is understood as the bacterium that Pharmaceuticals Research Center, Osaka, Japan. causes opportunistic infections in patients under [14C]Benzylpenicillin potassium salt was purchased immunosuppressed conditions or with cystic fib- from AmershamInternational pic, Bucks., U.K. rosis or diffuse panbronchiolitis. It is difficult to VariousP. aeruginosa strains werereference orga- cure P. aeruginosa infections because of its intrinsic nisms stored in our laboratories, and recent clinical resistance to antibiotics1}. Several new /Mactam isolates were obtained from various hospitals in antibiotics with antipseudomonal activity have Japan. The MICs were measured by agar dilution been introduced into clinical therapeutics. Among on Sensitivity Test Agar (Nissui Pharmaceutical, these, carbapenems are of particular interest as Japan), which is modified Mueller-Hinton agar. they have a powerful effect against P. aeruginosa. An overnight culture of the test organism in Sen- The discovery of thienamycin2) created a scientific sitivity Test Broth (Nissui) was diluted in buffered sensation as it has its potent antibacterial activity saline gelatin, and about 104 cfu/spot were inocul- and a wide spectrum of action against Gram-posi- ated onto a drug-containing agar surface. Plates

Fig. 1. Chemical structure of carbapenems used in this study. Compound R ± R2 R3 Meropenem CH 3 H Desmethyl meropenem H V/\^

Table 1. Antipseudomonal activity of various carbapenem series. MIC (fig/ml) MIC Og/ml) of clinical isolates3 Compound NCTC10490 T IFO3451 PAO2152 TL2666b MIC50 MIC90 Range

Meropenem 0.025 0.39 0.39 0.78 0.78 0.55 2.87 0.05-12.5 Desmethyl meropenem 0. 10 12.5 3.13 3.13 6.25 1.92 14.9 0.20- 50 Compound A 0.20 3.13 3.13 3.13 6.25 4.17 18.7 0.39-25 Desmethyl compound A 1.56 25 12.5 12.5 25 14.4 28.1 1.56-50 l jS-Methyl thienamycin 0.78 3.13 1.56 1.56 3.13 1.99 5.26 0.39-12.5 Thienamycin 1.56 6.25 3.13 3.13 6.25 3.23 6.06 0.78-25 l /?-Methyl imipenem 1.56 6.25 1.56 1.56 3.13 2.21 5.83 0.39-12.5 Imipenem 0.78 1.56 1.56 0.78 1.56 0.96 4.42 0.39-12.5 CompoundB 0.20 1.56 1.56 1.56 1.56 1.19 4.05 0.20- 6.25 Desmethyl compound 1.56 3.13 1.56 1.56 3.13 1.51 4.82 0.39-25 1.56 25 6.25 12.5 12.5 13.5 23.1 0.39-50 Panipeneml /?-Methyl panipenem 1.56 12.5 3.13 3.13 6.25 4.42 ll.9 0.39-25 CompoundC 0.39 12.5 1.56 3.13 6.25 4.58 10.1 0.39- 12.5 Desmethyl compoundC 0.78 12.5 3.13 6.25 6.25 6.01 14.0 0.39-25 CompoundD 0.78 25 3.13 12.5 6.25 13.5 22.1 0.20-25 Desmethyl compoundD 0.78 12.5 3.13 6.25 6.25 9.32 21.0 0.20-50 a /i=25. b Typical clinical isolate. were incubated at 37°C for 18hours. The MICwas tophilia IID127311} by the method of Satno12). defined as the lowest concentration that completely Table 1 shows the antipseudomonal activities of inhibited visible growth. MIC5Os and MIC90s were various carbapenems. Weused 4 standard labora- calculated from the equation describing the linear tory strains and 26 clinical isolates of P. aeruginosa. part of the curve. Of 16 carbapenems used in this study meropenem P. aeruginosa cell membraneswere prepared as was most the active one against P. aeruginosa, previously described6). P. aeruginosa grown to followed by imipenem and compound B. In all four late-exponential phase was harvested, and the cells carbapenemseries, the introduction of a l/?-methyl were ruptured by sonication. The membrane substituent to compounds having an imidoyl group fractions were pelleted by ultracentrifugation. The reduced the antipseudomonal activity, whereas affinities of the carbapenems for penicillin-binding ljft-methyl compounds without an imidoyl moiety proteins (PBPs) were determined by means of a were more active than their corresponding des- competition assay using [14C]benzylpenicillin, methyl derivatives. There may be some congeniality essentially as previously described6'"0. We used among l/?-methyl and imidoyl substituents on P. aeruginosa NCTC1 0490 because this strain always antibacterial activity against P. aeruginosa. How- produces very sharp and clear PBP bands on ever, desmethyl compoundA was much less active sodium dodecyl sulfate-polyacrylamide gel electro- than compoundA. This may be because the bulky phoresis (SDS-PAGE). Meropenem and imipenem side chains in compoundA, acetimidoyl and di- show the same binding profiles to PBPs in three methylcarbamoyl groups on the pyrrolidine moiety, strains of P. aeruginosa.6) Therefore, this result interfere with its ownbinding to the target PBPs. could be generalized for all susceptible P. aerugi- The antibacterial activity of carbapenems greatly nosa. depends on their affinity and access to targets The permeability of carbapenems through the (PBPs) of P. aeruginosa. We selected, therefore, outer membraneof P. aeruginosa was measured meropenemand imipenem series of carbapenems using /Mactamaseenclosed proteoliposome recon- as representatives and examined their affinity for stitution assays8'9). The outer membrane was PBPs and the permeation through the outer mem- purified from P. aeruginosa PAO2152 by the brane using proteoliposomes containing L-l /?- procedure previously described10). The L-l type lactamase. The affinities of four carbapenems are /Mactamase was purified from Xanthomonasmal- summarized in Table 2. Meropenemhad higher VOL. 46 NO. 10 THE JOURNAL OF ANTIBIOTICS 1631

Table 2. Affinities of the series of meropenem and imipenem for PBPs in Pseudomonas aeruginosa NCTC10490a. IC50 Cg/ml)" Antibiotic 3 4 5 Meropenem 0.96 0.84 0.13 0.060 0.022 >10 Desmethyl meropenem 0.49 2.6 0.87 0.38 0.054 >10 1 /MViethyl imipenem 1.7 3.2 0.49 0.94 0.018 >10 Imipenem 0.40 0.81 0.23 0.53 0.014 4.6 Membranefractions were incubated with carbapenems at various concentrations for 10 minutes at 30°C. Subsequently, [14C]benzylpenicillin was added and the incubation was continued for another 10 minutes. Reactions were terminated by adding excess non-radioactive benzylpenicillin and sarkosyl. [ 1 4C]Benzylpenicillin- protein complexes were resolved by SDS-PAGE,followed by fluorography. Values indicate concentrations of antibiotics required to reduce [14C]benzylpenicillin binding by 50%. affinities than desmethyl meropenem, for all PBPs Pharmaceutical University, for valuable suggestions on except PBP1A. Conversely, 1^-methyl imipenem permeability studies in vesicles containing /Mactamase. had less affinity than imipenem. Wealso acknowledge the excellent technical assistance of The relative outer membrane permeability of Yuko Shiono and Yasuko Hirai. meropenem, desmethyl meropenem, 1^-methyl imipenem and imipenem were 100, 110+28, 167+ References 25 and 207+44, respectively, assays performed in 1) Quinn, J. P.: Intrinsic antibiotic resistance in triplicate. The permeation of meropenem and Pseudomonas aeruginosa. In Pseudomonas, Mole- desmethyl meropenemthrough the outer membrane cular Biology and Biotechnology. Ed., E. Calli was lower than that of l/?-methyl imipenem and et aL, pp. 154-160, American Society for Micro- imipenem.However,meropenemwas most active biology, 1992 against P. aeruginosa. These results indicate that 2) Kahan, J. S.; F. M. Kahan, R. Goegelman, S. A. the antipseudomonal activity of these four carba- Currie, M. Jackson, E. O. Stapley, T. W. Miller, penems was directly reflected in their affinity for A. K. Miller, D. Hendlin, S. Mochales, S. PBPs but not related to outer membrane perme- Hernandez, H. B. Woodruff & J. Birnbaum: Thienamycin, a new /Mactam antibiotic. I. Disco- ability. With or without the l/?-methyl moiety, very, taxonomy, isolation and physical properties. meropenemand imipenem series showed almost J. Antibiotics 32: 1-12, 1979 the same permeability rate through the outer mem- 3) Brown, A. G.; D. F. Corbett, A. J. Eglington brane of P. aeruginosa. The permeability of carba- & T. T. Howarth: Structures of olivanic acid penem seemed to depend on the structure of their derivatives MM 22380, MM 22381, MM 22382 and 2-side chain. MM 22383, four new antibiotics isolated from In this report, we revealed that the l/?-methyl Streptomyces olivaceus. J. Antibiotics 32: 961 -963, 1979 moiety on carbapenem directly affects its affinity for PBPs and consequently its antipseudomonal activ- 4) Nakayama, M.; A. Iwasaki, S. Kimura, T. Mizoguchi, S. Tanabe, A. Murakami, I. Watanabe, ity. Wealso showed that there is some congeniality M. Okuchi, H. Ito, Y. Saino, F. Kobayashi & T. between the 1^-methyl and imidoyl groups in Mori: Carpetimycins A and B, new /Mactam producing activity against P. aeruginosa. antibiotics. J. Antibiotics 33: 1388- 1390, 1980 It has been clarified that carbapenems utilize 5) Kimura, Y.; K. Motokawa, H. Nagata, Y. their specific channel D2 outer membrane protein Kameda, S. Matsuura, M. Mayama & T. Yoshida: in P. aeruginosa12U4). In addition, a defective D2 Asparenomycins A, B and C, new carbapenem protein considerably affects antipseudomonal ac- antibiotics, IV. Antibacterial activity. J. Antibiotics tivity of carbapenems. It would be of interest to 35: 32-38, 1982 clarify the affinity of our series of carbapenemsfor 6) Sumita,Y.; M. Fukasawa&T. Okuda: Comparison D2protein from the viewpoint of structure-activity of two carbapenems, SM-7338 and imipenem: Affinities for penicillin-binding proteins and mor- relationships. This is currently under way. phological changes. J. Antibiotics 43: 314-320, 1990 Acknowledgments 7) Spratt, B. G.: Properties of the penicillin-binding proteins of Escherichia coli K12. Eur. J. Biochem. The authors are grateful to Naomasa Gotoh, Kyoto 1632 THE JOURNAL OF ANTIBIOTICS OCT. 1993

72: 341-352, 1977 potent antibacterial activity. Chemotherapy (Basel) Mimms, L. T.; G. Zampighi, Y. Nozaki, C. Tan ford 38: 218-224, 1992 & J. A. Reynolds: Phospholipid vesicle formation Saino, Y.; F. Kobayashi, M. Inoue & S. Mitsuhashi: and transmembrane protein incorporation using Purification and properties of inducible penicillin octyl glucoside. Biochemistry 20: 833-840, 1981 /Mactamase isolated from Pseudomonas maltophilia. Trias, J.; J. Dufresne, R. C. Levesque & H. Antimicrob. Agents Chemother. 22: 564~ 570, 1982 Nikaido: Decreased outer membrane permeability Trias, J. & H. Nikaido: Outer membrane protein in imipenem-resistant mutants of Pseudomonas D2 catalyzes facilitated diffusion of carbapenems aeruginosa. Antimicrob. Agents Chemother. 33: and penems through the outer membrane of 1201-1206, 1989 Pseudomonas aeruginosa. Actimicrob. Agents Chem- Mizuno, T. & M. Kageyama: Separation and other. 34: 52-57, 1990 characterization of the outer membraneof Pseudo- Satake, S.; E. Yoshihara & T. Nakae: Diffusion of monas aeruginosa. J. Biochem. 84: 179~ 191, 1978 /Mactamantibiotics through liposome membranes Nouda, H.; E. T. Harabe, Y. Sumita, T. Okuda & reconstituted from purified porins of the outer M. Fukasawa: Beta-lactamase stability and in- membraneof Pseudomonas aeruginosa. Antimicrob. hibitory activity of meropenem combined with a Agents Chemother. 34: 685-690, 1990