Vol. 36, No. 7
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 1992, P. 1577-1579
0066-4804/92/071577-03$02.00/0 Copyright © 1992, American Society for Microbiology
Stability of Meropenem and Effect of 1,-Methyl Substitution on Its Stability in the Presence of Renal Dehydropeptidase I MASATOMO FUKASAWA, YOSHIHIRO SUMITA,* EIKO T. HARABE, TOMOHARU TANIO, HIROSHI NOUDA, TSUNEO KOHZUKI, TAKAO OKUDA, HARUKI MATSUMURA, A'ND MAKOTO SUNAGAWA
Research Laboratories, Sumitomo Pharmaceuticals Co., Ltd., 3-1-98, Kasugade-naka, Konohana-ku, Osaka 554, Japan Received 13 February 1992/Accepted 7 May 1992
The stability of meropenem in the presence of renal dehydropeptidase I (DHP-I) varied extremely with the animal source of the enzyme. Meropenem, compared with imipenem, was rather easily hydrolyzed by DHP-Is from mice, rabbits, and monkeys, while it showed a higher resistance to guinea pig and beagle dog DHP-Is. In addition, meropenem was four times more resistant than imipenem to human DHP-I. The 113-methyl substituent on carbapenems, i.e., meropenem and 1-methyl imipenem, made them considerably more resistant to mouse and swine DHP-Is than the 1-unsubstituted derivatives are.
During the last several years, a number of new ,4-lactam antibiotics have been introduced into clinical practice. Some of these antibiotics include carbapenems which are of particular interest. Discovery of thienamycin produced by Streptomyces cattleya (10) marked an epoch for 3-lactam antibiotics which have a wide antibacterial spectrum and high activity against gram-positive and gram-negative bacteria. Subsequently, a number of carbapenems, such as olivanic acids (3), carpetimycins (15), asparenomycins (11), and others, were discovered and some of them were chemically modified. However, none have been made available for clinical use except imipenem, an N-formimidoyl thienamycin. Imipenem and other related carbapenems were considerably inactivated by the renal enzyme dehydropeptidase I (DHP-I) (12, 14). However, imipenem instability was overcome by coadministration of cilastatin, a specific DHP-I inhibitor (16). This combination has been used to treat a variety of infections. Meropenem is a new carbapenem that is undergoing worldwide clinical trials by Sumitomo Pharmaceuticals and the ICI Pharmaceuticals group. One of its most prominent features is that it can be used alone, without any DHP-I inhibitor, since it is highly resistant to hydrolysis by human DHP-I. We investigated the resistance of meropenem to renal DHP-Is from various animal sources, including humans, in comparison with that of imipenem. The effect of 13-methyl substitution on resistance to DHP-I was also examined. (Part of this study was presented previously [22].) Meropenem and imipenem were prepared in the Research Laboratories of Sumitomo Pharmaceuticals Co., Ltd., Osaka, Japan. The other derivatives of meropenem and imipenem (Fig. 1) and glycyldehydrophenylalanine (Gdp) were also synthesized in our laboratories. Renal DHP-Is from various animal sources and humans were used in this study. Human tissue was supplied by the Kobe University School of Medicine. The enzymes were either purified or partially purified by the methods of Campbell et al. (4, 5) and Sugiura et al. (20), modified as described below. Kidney tissues from various animals were minced and *
homogenized by a Polytron (Kinematica GmbH, Lucerne, Switzerland) with n-butanol to solubilize DHP-I, and then the homogenates were centrifuged at 10,000 x g for 10 min. The supernatants were mixed with acetone for 60 min at 4°C. It was centrifuged again at 30,000 x g for 30 min, and the precipitates were solubilized in H20. The enzyme, which was in the supernatant from 50 to 75% saturation of ammonium sulfate, was dialyzed against 10 mM Tris-HCl buffer (pH 7.0) containing 0.01 mM MnCl2 and further purified by DEAE-Toyopearl 650M (Tosoh Corp., Tokyo, Japan) ionexchange column chromatography. In these procedures, DHP-Is from various animal sources were purified about 10to 100-fold, with a recovery of 15 to 75% against the n-butanol-solubilized enzyme. Mouse and swine DHP-Is were further purified to homogeneity by using cilastatinlinked Sepharose affinity chromatography. The activity of DHP-I was spectrophotometrically determined by measuring the hydrolysis of Gdp as a substrate by using a 320 spectrophotometer (Hitachi Ltd., Tokyo, Japan) at 37°C. One unit of enzyme activity was defined as the amount of enzyme which hydrolyzed 1 ,umol of a substrate per min per mg of protein at 37°C. The protein concentration was estimated by the method of Lowry et al. (13) with bovine serum albumin as the standard. The rate of enzyme-catalyzed hydrolysis of carbapenems was measured by spectrophotometry. The enzyme concentration employed was adjusted to 0.02 U/ml, and its activity was confirmed in every experiment by using Gdp. Velocity was expressed in micromoles per minute per unit. The Km and Vm. values of respective enzymes were determined
OH
OH
N
2S/\NH w,N COOH
COOH
X:
CH,
Des-methyl meropenem
CHCH,(P)
Meropem
S
Penem analogue of meropenem
Y:
CH2 CHCH,U)
Imipenem 1p-Methyl IMipenem
FIG. 1. Chemical structures of meropenem and imipenem logs.
Corresponding author. 1577
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ANTIMICROB. AGENTS CHEMOTHER.
NOTES
TABLE 1. Kinetic parameters of meropenem hydrolysis by renal DHP-Is from various animals Meropenema DHP-I source
Mouse Rat Guinea pig Rabbit Swine Beagle dog Rhesus monkey Human
Km
Vma.
5.0 5.0 3.2 2.8 8.3 0.91 1.9 5.7 8.3 1.0 14 2.0 10 56 2.0 0.090
VmaxIK ratio
Km
1.0 0.88 0.11 3.0 0.12 0.14 1.7 0.045
3.0 8.3 13 2.8 11 17 4.8 6.3
Imipenema Vmax/Km
Vmax
ratio
1.6 8.3 14 2.5 11 14 2.9 1.0
0.53 1.0 1.1 0.89 1.0 0.82 0.43 0.16
a Km and Vm,a are expressed as millimolar and micromoles per minute per enzyme unit, respectively.
from a Lineweaver-Burk plot. These determinations were performed in duplicate. Table 1 shows the kinetic parameters of hydrolysis of meropenem and imipenem by renal DHP-Is. The stability of these two carbapenems was determined from their VmaIKKm ratios. The resistance of meropenem to renal DHP-Is varied extremely with the animal source of the enzyme. Compared with imipenem, meropenem was easily hydrolyzed by mouse, rabbit, and rhesus monkey DHP-Is, while it showed greater resistance to guinea pig and beagle dog DHP-Is. In addition, meropenem was four times more resistant than imipenem to human DHP-I. Meropenem and imipenem had almost the same affinities for various DHP-Is. In general, meropenem showed slightly higher affinity for these DHP-Is than did imipenem, although its affinities for mouse and rhesus monkey DHP-Is were about half of those of imipenem. On the other hand, the Vm. values of these carbapenems differed markedly with the source of the enzyme. The Vm. of meropenem for guinea pig DHP-I was about 1/10 of that of imipenem, but it was 10 times higher for rhesus monkey DHP-I. The Vm. of meropenem for human DHP-I was 1 order of magnitude lower than that of imipenem, although it showed three times higher affinity for this DHP-I. To assess the effect of 1B-methyl substitution on resistance to DHP-I, meropenem and imipenem were compared with desmethyl meropenem and 1l-methyl imipenem, respectively (Table 2). In this study, we used mouse and swine DHP-Is. Since these enzymes had different substrate specificities and biochemical properties, mouse DHP-I more readily hydrolyzed meropenem and imipenem than Gdp. By contrast, swine DHP-I more readily hydrolyzed Gdp than
and imipenem. In addition, these enzymes were clearly distinguished by their biochemical properties. Swine DHP-I was inactivated by EDTA and 1,10-phenanthroline, but the mouse enzyme was not (unpublished data). Introduction of a 1f-methyl substituent to a carbapenem skeleton drastically reduced the Vm. values of both mouse and swine enzymes on meropenem and imipenem series. However, it did not affect the Km values. Judging from the VmeiKm ratio, meropenem and 13-methyl imipenem had fivefold higher resistance to mouse DHP-I than did the corresponding 1-unsubstituted analogs. On the other hand, meropenem was about 50 times and 1,B-methyl imipenem was about 10 times more resistant to hydrolysis by swine DHP-I than were desmethyl meropenem and imipenem, respectively. The resistance of the penem analog of meropenem to these enzymes was intermediate between those of meropenem and its desmethyl derivative. Shih et al. (19) have already reported that the 13-methyl carbapenem antibiotics are highly resistant to renal DHP-I. Our results support his suggestion. In our previous study, the percentages of the dose of meropenem excreted in the urine after intravenous administration of 20 mg/kg were 34% in mice, 25% in rats, 43% in guinea pigs, 35% in rabbits, 43% in beagle dogs, and 26% in rhesus monkeys (22). Meropenem had relatively high resistance to DHP-I from guinea pigs and beagle dogs, which is responsible for the comparatively high urinary recovery in these animals. On the other hand, the urinary recoveries of meropenem and imipenem in mice and rats were more than 20% (10), despite their low resistance to DHP-Is from these animals. This indicates that the carbapenems are excreted in the urine to some extent, even though they are well hydrolyzed by DHP-Is. This may be explained by the fact that the turnover rate of the carbapenem-hydrolyzing activity of DHP-I is relatively low compared with the renal clearance rate in those animals. It is also possible that urinary recovery of carbapenems is affected by the amount and localization of the enzyme in the kidneys (8) and/or the activity of extrarenal DHP-I (9, 21). On the whole, however, the more DHP-I-resistant carbapenems tend to have higher urinary meropenem
recoveries.
As a carbapenem-hydrolyzing enzyme, DHP-I resembles the metalloenzyme 1-lactamases produced byXanthomonas maltophilia (17), Flavobactenium odoratum (18), Bacteroides fragilis (6, 23), and Aeromonas spp. (1). At almost the same rate, these ,B-lactamases can hydrolyze any carbapenem, with or without a 1p-methyl group, as a substrate (data not shown). However, the activities of mouse and swine DHP-Is were affected by the 13-methyl substituent.
TABLE 2. Hydrolysis of meropenem analogs and Gdp by mouse and swine DHP-Is Compound
Mouse DHP-Ia
Km (mM)
Gdp
0.28
Desmethyl meropenem Meropenem Penem analog of meropenem
8.4 5.7 1.8
Vm. 100 1,800 (100) 250 (14) 190 (11)
Swine DHP-Ia
Vm,a,/Km ratio 100 58 (100) 12 (21) 30 (51)
Km (mM) 0.92 5.8 11 53
Vm. 100 65 (100) 2.3 (3.5) 44 (68)
Vm,,,aKm ratio 100 10 (100) 0.18 (1.8) 0.76 (0.78)
Imipenem 3.6 74 (100) 5.7 (100) 7.0 59 (100) 7.7 (100) 113-methyl imipenem 2.7 16 (21) 1.5 (26) 4.9 5.2 (8.9) 0.98 (13) a Vna,. and the Vmax/Km ratio are expressed as percentages of Gdp hydrolysis. The values in parentheses are relative rates of hydrolysis expressed as hydrolysis
of desmethyl-type carbapenems.
NOTES
VOL. 36, 1992
These findings indicate that DHP-I recognizes the l-methyl moiety on the carbapenem more strictly than do metalloenzyme 3-lactamases. DHP-Is from various animals showed different activities on the carbapenems. Substrate specificities and biochemical properties of mouse and swine DHP-Is were extremely different. More detailed examination of the biological and biochemical properties of DHP-Is from other animals may reveal the interactions between DHP-I enzymes and carbapenems.
We also studied the resistance of various analogs of and imipenem to these DHP-Is. However, the substrate specificities of none of the DHP-Is correlated well with those of human DHP-I for all of these analogous compounds. Swine DHP-I had a profile similar to that of human DHP-I, but it was a far-from-perfect match. The present findings show that the 13-methyl substituent on meropenem confers higher resistance to renal DHP-I. In clinical trials of meropenem with healthy human volunteers, about 60 to 80% of the dose was excreted in the urine (2, 7). This high urinary recovery is due to the high resistance of meropenem to human renal DHP-I. meropenem
9. Hirota, T., Y. Nishikawa, M. Tanaka, T. Igarashi, and H. Kitagawa. 1986. Characterization of dehydropeptidase I in the rat lung. Eur. J. Biochem. 160:521-525. 10. Kahan, J. S., F. M. Kahan, R. Goegelman, S. A. Currie, M. Jackson, E. 0. Stapley, T. W. Miller, A. K. Miller, D. Hendlin, S. Mochales, S. Hernandez, H. B. Woodruff, and J. Birnbaum. 1979. Thienamycin, a new t-lactam antibiotic. Discovery, taxonomy, isolation and physical properties. J. Antibiot. 32:1-12. 11. Kimura, Y., K. Motokawa, H. Nagata, Y. Kameda, S. Matsuura, M. Mayama, and T. Yoshida. 1982. Asparenomycins A, B and C, new carbapenem antibiotics. VI. Antibacterial activity. J.
Antibiot. 35:32-38. 12. Kropp, H., J. G. Sundelof, R. Hajdu, and F. M. Kahan. 1982. Metabolism of thienamycin and related carbapenem antibiotics by the renal dipeptidase, dehydropeptidase-I. Antimicrob. Agents Chemother. 22:62-70.
13. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 14.
15.
We are grateful to S. Kamidono, Kobe University School of Medicine, for supplying human kidney tissue.
16. 1.
2.
3.
4.
REFERENCES Bakken, J. S., C. C. Sanders, R. B. Clark, and M. Hori. 1988. P-Lactam resistance in Aeromonas spp. caused by inducible ,B-lactamases active against penicillins, cephalosporins, and carbapenems. Antimicrob. Agents Chemother. 32:1314-1319. Bax, R. P., W. Bastain, A. Featherstone, D. M. Wilkinson, M. Hutchison, and S. J. Haworth. 1989. The pharmacokinetics of meropenem in volunteers. J. Antimicrob. Chemother. 24(Suppl. A):311-320. Brown, A. G., D. F. Corbett, A. J. Eglington, and T. T. Howath. 1979. Structures of olivanic acid derivatives MM22380, MM22381, MM22382 and MM22383, four new antibiotics isolated from Streptomyces olivaceus. J. Antibiot. 32:961-963. Campbell, B. J. 1970. Renal dipeptidase. Methods Enzymol. 19:722729.
5. Campbell, B. J., Y. C. Lyn, R. V. Davis, and E. Balew. 1966. The purification and properties of a particulate renal dipeptidase. Biochim. Biophys. Acta 118:371-386. 6. Cuchural, G. J., Jr., M. H. Malamy, and F. P. Tally. 1986. 1-Lactamase-mediated imipenem resistance in Bacteroides fragilis. Antimicrob. Agents Chemother. 30:645-648. 7. Harrison, M. P., S. R. Moss, A. Featherstone, A. G. Fowkes, A. M. Sanders, and D. E. Case. 1989. The disposition and metabolism of meropenem in laboratory animals and man. J. Antimicrob. Chemother. 24(Suppl. A):265-277. 8. Hirota, T., Y. Nishikawa, M. Tanaka, K. Fukuda, T. Igarashi, and H. Kitagawa. 1987. Localization of dehydropeptidase-I, an enzyme processing glutathione, in the rat kidney. J. Biochem. 102:547-550.
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20. 21. 22. 23.
1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Mikami, H., M. Ogashiwa, Y. Saino, M. Inoue, and S. Mitsuhashi. 1982. Comparative stability of newly introduced 3-lactam antibiotics to renal dipeptidase. Antimicrob. Agents Chemother. 22:693-695. Nakayama, M., A. Iwasaki, S. Kimura, T. Mizoguchi, S. Tanabe, A. Murakami, I. Watanabe, M. Okuchi, H. Ito, Y. Saino, F. Kobayashi, and T. Mori. 1980. Carpetimycins A and B, new P-lactam antibiotics. J. Antibiot. 33:1388-1390. Norrby, S. R., K. Alestig, B. Bj6rnegard, L. A. Burman, F. Ferber, J. L. Huber, K. H. Jones, F. M. Kahan, J. S. Kahan, H. Kropp, M. A. P. Meisinger, and J. G. Sundelof. 1983. Urinary recovery of N-formimidoyl thienamycin (MK0787) as affected by coadministration of N-formimidoyl thienamycin dehydropeptidase inhibitors. Antimicrob. Agents Chemother. 23: 300-307. Saino, Y., F. Kobayashi, M. Inoue, and S. Mitsuhashi. 1982. Purification and properties of inducible penicillin 1-lactamase isolated from Pseudomonas maltophilia. Antimicrob. Agents Chemother. 22:564-570. Sato, K., T. Fujil, R. Okamoto, M. Inoue, and S. Mitsuhashi. 1985. Biochemical properties of 1-lactamase produced by Flavobacterium odoratum. Antimicrob. Agents Chemother. 27: 612-614. Shih, D. H., J. A. Fayter, F. Baker, L. Cama, and B. G. Christensen. 1983. Program Abstr. 23rd Intersci. Conf. Antimicrob. Agents Chemother., abstr. 333. Sugiura, M., Y. Ito, K. Hirana, and S. Sawaki. 1978. Purification and properties of human kidney dipeptidases. Biochim. Biophys. Acta 522:541-550. Takahagi, H., T. Hirota, Y. Matsushita, M. Tanaka, S. Muramatsu, H. Naganuma, and Y. Kawahara. 1990. Program Abstr. 30th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 896. Tanio, T., H. Nouda, E. Tada, T. Kohzuki, M. Kato, M. Fukasawa, T. Okuda, and S. Kamidono. 1987. Program Abstr. 27th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 758. Yotsuji, A., S. Minami, M. Inoue, and S. Mitsuhashi. 1983. Properties of novel P-lactamase produced by Bacteroides fragilis. Antimicrob. Agents Chemother. 24:925-929.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 1992, P. 1577-1579
0066-4804/92/071577-03$02.00/0 Copyright © 1992, American Society for Microbiology
Stability of Meropenem and Effect of 1,-Methyl Substitution on Its Stability in the Presence of Renal Dehydropeptidase I MASATOMO FUKASAWA, YOSHIHIRO SUMITA,* EIKO T. HARABE, TOMOHARU TANIO, HIROSHI NOUDA, TSUNEO KOHZUKI, TAKAO OKUDA, HARUKI MATSUMURA, A'ND MAKOTO SUNAGAWA
Research Laboratories, Sumitomo Pharmaceuticals Co., Ltd., 3-1-98, Kasugade-naka, Konohana-ku, Osaka 554, Japan Received 13 February 1992/Accepted 7 May 1992
The stability of meropenem in the presence of renal dehydropeptidase I (DHP-I) varied extremely with the animal source of the enzyme. Meropenem, compared with imipenem, was rather easily hydrolyzed by DHP-Is from mice, rabbits, and monkeys, while it showed a higher resistance to guinea pig and beagle dog DHP-Is. In addition, meropenem was four times more resistant than imipenem to human DHP-I. The 113-methyl substituent on carbapenems, i.e., meropenem and 1-methyl imipenem, made them considerably more resistant to mouse and swine DHP-Is than the 1-unsubstituted derivatives are.
During the last several years, a number of new ,4-lactam antibiotics have been introduced into clinical practice. Some of these antibiotics include carbapenems which are of particular interest. Discovery of thienamycin produced by Streptomyces cattleya (10) marked an epoch for 3-lactam antibiotics which have a wide antibacterial spectrum and high activity against gram-positive and gram-negative bacteria. Subsequently, a number of carbapenems, such as olivanic acids (3), carpetimycins (15), asparenomycins (11), and others, were discovered and some of them were chemically modified. However, none have been made available for clinical use except imipenem, an N-formimidoyl thienamycin. Imipenem and other related carbapenems were considerably inactivated by the renal enzyme dehydropeptidase I (DHP-I) (12, 14). However, imipenem instability was overcome by coadministration of cilastatin, a specific DHP-I inhibitor (16). This combination has been used to treat a variety of infections. Meropenem is a new carbapenem that is undergoing worldwide clinical trials by Sumitomo Pharmaceuticals and the ICI Pharmaceuticals group. One of its most prominent features is that it can be used alone, without any DHP-I inhibitor, since it is highly resistant to hydrolysis by human DHP-I. We investigated the resistance of meropenem to renal DHP-Is from various animal sources, including humans, in comparison with that of imipenem. The effect of 13-methyl substitution on resistance to DHP-I was also examined. (Part of this study was presented previously [22].) Meropenem and imipenem were prepared in the Research Laboratories of Sumitomo Pharmaceuticals Co., Ltd., Osaka, Japan. The other derivatives of meropenem and imipenem (Fig. 1) and glycyldehydrophenylalanine (Gdp) were also synthesized in our laboratories. Renal DHP-Is from various animal sources and humans were used in this study. Human tissue was supplied by the Kobe University School of Medicine. The enzymes were either purified or partially purified by the methods of Campbell et al. (4, 5) and Sugiura et al. (20), modified as described below. Kidney tissues from various animals were minced and *
homogenized by a Polytron (Kinematica GmbH, Lucerne, Switzerland) with n-butanol to solubilize DHP-I, and then the homogenates were centrifuged at 10,000 x g for 10 min. The supernatants were mixed with acetone for 60 min at 4°C. It was centrifuged again at 30,000 x g for 30 min, and the precipitates were solubilized in H20. The enzyme, which was in the supernatant from 50 to 75% saturation of ammonium sulfate, was dialyzed against 10 mM Tris-HCl buffer (pH 7.0) containing 0.01 mM MnCl2 and further purified by DEAE-Toyopearl 650M (Tosoh Corp., Tokyo, Japan) ionexchange column chromatography. In these procedures, DHP-Is from various animal sources were purified about 10to 100-fold, with a recovery of 15 to 75% against the n-butanol-solubilized enzyme. Mouse and swine DHP-Is were further purified to homogeneity by using cilastatinlinked Sepharose affinity chromatography. The activity of DHP-I was spectrophotometrically determined by measuring the hydrolysis of Gdp as a substrate by using a 320 spectrophotometer (Hitachi Ltd., Tokyo, Japan) at 37°C. One unit of enzyme activity was defined as the amount of enzyme which hydrolyzed 1 ,umol of a substrate per min per mg of protein at 37°C. The protein concentration was estimated by the method of Lowry et al. (13) with bovine serum albumin as the standard. The rate of enzyme-catalyzed hydrolysis of carbapenems was measured by spectrophotometry. The enzyme concentration employed was adjusted to 0.02 U/ml, and its activity was confirmed in every experiment by using Gdp. Velocity was expressed in micromoles per minute per unit. The Km and Vm. values of respective enzymes were determined
OH
OH
N
2S/\NH w,N COOH
COOH
X:
CH,
Des-methyl meropenem
CHCH,(P)
Meropem
S
Penem analogue of meropenem
Y:
CH2 CHCH,U)
Imipenem 1p-Methyl IMipenem
FIG. 1. Chemical structures of meropenem and imipenem logs.
Corresponding author. 1577
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ANTIMICROB. AGENTS CHEMOTHER.
NOTES
TABLE 1. Kinetic parameters of meropenem hydrolysis by renal DHP-Is from various animals Meropenema DHP-I source
Mouse Rat Guinea pig Rabbit Swine Beagle dog Rhesus monkey Human
Km
Vma.
5.0 5.0 3.2 2.8 8.3 0.91 1.9 5.7 8.3 1.0 14 2.0 10 56 2.0 0.090
VmaxIK ratio
Km
1.0 0.88 0.11 3.0 0.12 0.14 1.7 0.045
3.0 8.3 13 2.8 11 17 4.8 6.3
Imipenema Vmax/Km
Vmax
ratio
1.6 8.3 14 2.5 11 14 2.9 1.0
0.53 1.0 1.1 0.89 1.0 0.82 0.43 0.16
a Km and Vm,a are expressed as millimolar and micromoles per minute per enzyme unit, respectively.
from a Lineweaver-Burk plot. These determinations were performed in duplicate. Table 1 shows the kinetic parameters of hydrolysis of meropenem and imipenem by renal DHP-Is. The stability of these two carbapenems was determined from their VmaIKKm ratios. The resistance of meropenem to renal DHP-Is varied extremely with the animal source of the enzyme. Compared with imipenem, meropenem was easily hydrolyzed by mouse, rabbit, and rhesus monkey DHP-Is, while it showed greater resistance to guinea pig and beagle dog DHP-Is. In addition, meropenem was four times more resistant than imipenem to human DHP-I. Meropenem and imipenem had almost the same affinities for various DHP-Is. In general, meropenem showed slightly higher affinity for these DHP-Is than did imipenem, although its affinities for mouse and rhesus monkey DHP-Is were about half of those of imipenem. On the other hand, the Vm. values of these carbapenems differed markedly with the source of the enzyme. The Vm. of meropenem for guinea pig DHP-I was about 1/10 of that of imipenem, but it was 10 times higher for rhesus monkey DHP-I. The Vm. of meropenem for human DHP-I was 1 order of magnitude lower than that of imipenem, although it showed three times higher affinity for this DHP-I. To assess the effect of 1B-methyl substitution on resistance to DHP-I, meropenem and imipenem were compared with desmethyl meropenem and 1l-methyl imipenem, respectively (Table 2). In this study, we used mouse and swine DHP-Is. Since these enzymes had different substrate specificities and biochemical properties, mouse DHP-I more readily hydrolyzed meropenem and imipenem than Gdp. By contrast, swine DHP-I more readily hydrolyzed Gdp than
and imipenem. In addition, these enzymes were clearly distinguished by their biochemical properties. Swine DHP-I was inactivated by EDTA and 1,10-phenanthroline, but the mouse enzyme was not (unpublished data). Introduction of a 1f-methyl substituent to a carbapenem skeleton drastically reduced the Vm. values of both mouse and swine enzymes on meropenem and imipenem series. However, it did not affect the Km values. Judging from the VmeiKm ratio, meropenem and 13-methyl imipenem had fivefold higher resistance to mouse DHP-I than did the corresponding 1-unsubstituted analogs. On the other hand, meropenem was about 50 times and 1,B-methyl imipenem was about 10 times more resistant to hydrolysis by swine DHP-I than were desmethyl meropenem and imipenem, respectively. The resistance of the penem analog of meropenem to these enzymes was intermediate between those of meropenem and its desmethyl derivative. Shih et al. (19) have already reported that the 13-methyl carbapenem antibiotics are highly resistant to renal DHP-I. Our results support his suggestion. In our previous study, the percentages of the dose of meropenem excreted in the urine after intravenous administration of 20 mg/kg were 34% in mice, 25% in rats, 43% in guinea pigs, 35% in rabbits, 43% in beagle dogs, and 26% in rhesus monkeys (22). Meropenem had relatively high resistance to DHP-I from guinea pigs and beagle dogs, which is responsible for the comparatively high urinary recovery in these animals. On the other hand, the urinary recoveries of meropenem and imipenem in mice and rats were more than 20% (10), despite their low resistance to DHP-Is from these animals. This indicates that the carbapenems are excreted in the urine to some extent, even though they are well hydrolyzed by DHP-Is. This may be explained by the fact that the turnover rate of the carbapenem-hydrolyzing activity of DHP-I is relatively low compared with the renal clearance rate in those animals. It is also possible that urinary recovery of carbapenems is affected by the amount and localization of the enzyme in the kidneys (8) and/or the activity of extrarenal DHP-I (9, 21). On the whole, however, the more DHP-I-resistant carbapenems tend to have higher urinary meropenem
recoveries.
As a carbapenem-hydrolyzing enzyme, DHP-I resembles the metalloenzyme 1-lactamases produced byXanthomonas maltophilia (17), Flavobactenium odoratum (18), Bacteroides fragilis (6, 23), and Aeromonas spp. (1). At almost the same rate, these ,B-lactamases can hydrolyze any carbapenem, with or without a 1p-methyl group, as a substrate (data not shown). However, the activities of mouse and swine DHP-Is were affected by the 13-methyl substituent.
TABLE 2. Hydrolysis of meropenem analogs and Gdp by mouse and swine DHP-Is Compound
Mouse DHP-Ia
Km (mM)
Gdp
0.28
Desmethyl meropenem Meropenem Penem analog of meropenem
8.4 5.7 1.8
Vm. 100 1,800 (100) 250 (14) 190 (11)
Swine DHP-Ia
Vm,a,/Km ratio 100 58 (100) 12 (21) 30 (51)
Km (mM) 0.92 5.8 11 53
Vm. 100 65 (100) 2.3 (3.5) 44 (68)
Vm,,,aKm ratio 100 10 (100) 0.18 (1.8) 0.76 (0.78)
Imipenem 3.6 74 (100) 5.7 (100) 7.0 59 (100) 7.7 (100) 113-methyl imipenem 2.7 16 (21) 1.5 (26) 4.9 5.2 (8.9) 0.98 (13) a Vna,. and the Vmax/Km ratio are expressed as percentages of Gdp hydrolysis. The values in parentheses are relative rates of hydrolysis expressed as hydrolysis
of desmethyl-type carbapenems.
NOTES
VOL. 36, 1992
These findings indicate that DHP-I recognizes the l-methyl moiety on the carbapenem more strictly than do metalloenzyme 3-lactamases. DHP-Is from various animals showed different activities on the carbapenems. Substrate specificities and biochemical properties of mouse and swine DHP-Is were extremely different. More detailed examination of the biological and biochemical properties of DHP-Is from other animals may reveal the interactions between DHP-I enzymes and carbapenems.
We also studied the resistance of various analogs of and imipenem to these DHP-Is. However, the substrate specificities of none of the DHP-Is correlated well with those of human DHP-I for all of these analogous compounds. Swine DHP-I had a profile similar to that of human DHP-I, but it was a far-from-perfect match. The present findings show that the 13-methyl substituent on meropenem confers higher resistance to renal DHP-I. In clinical trials of meropenem with healthy human volunteers, about 60 to 80% of the dose was excreted in the urine (2, 7). This high urinary recovery is due to the high resistance of meropenem to human renal DHP-I. meropenem
9. Hirota, T., Y. Nishikawa, M. Tanaka, T. Igarashi, and H. Kitagawa. 1986. Characterization of dehydropeptidase I in the rat lung. Eur. J. Biochem. 160:521-525. 10. Kahan, J. S., F. M. Kahan, R. Goegelman, S. A. Currie, M. Jackson, E. 0. Stapley, T. W. Miller, A. K. Miller, D. Hendlin, S. Mochales, S. Hernandez, H. B. Woodruff, and J. Birnbaum. 1979. Thienamycin, a new t-lactam antibiotic. Discovery, taxonomy, isolation and physical properties. J. Antibiot. 32:1-12. 11. Kimura, Y., K. Motokawa, H. Nagata, Y. Kameda, S. Matsuura, M. Mayama, and T. Yoshida. 1982. Asparenomycins A, B and C, new carbapenem antibiotics. VI. Antibacterial activity. J.
Antibiot. 35:32-38. 12. Kropp, H., J. G. Sundelof, R. Hajdu, and F. M. Kahan. 1982. Metabolism of thienamycin and related carbapenem antibiotics by the renal dipeptidase, dehydropeptidase-I. Antimicrob. Agents Chemother. 22:62-70.
13. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 14.
15.
We are grateful to S. Kamidono, Kobe University School of Medicine, for supplying human kidney tissue.
16. 1.
2.
3.
4.
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