ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1998, p. 981–983 0066-4804/98/$04.0010 Copyright © 1998, American Society for Microbiology
Vol. 42, No. 4
Efficacy of LY333328 against Experimental Methicillin-Resistant Staphylococcus aureus Endocarditis GLENN W. KAATZ,1,2,3* SUSAN M. SEO,1,2 JEFFREY R. AESCHLIMANN,3,4 HEATHER H. HOULIHAN,3,4 RENEE-CLAUDE MERCIER,3,4 AND MICHAEL J. RYBAK1,3,4 Department of Internal Medicine, Division of Infectious Diseases, School of Medicine,1 Department of Veteran’s Affairs Medical Center,2 and College of Pharmacy and Allied Health Professions,3 Wayne State University, and Anti-Infective Research Laboratory, Department of Pharmacy Services, Detroit Receiving Hospital and University Health Center,4 Detroit, Michigan 48201 Received 6 November 1997/Returned for modification 18 December 1997/Accepted 2 February 1998
The in vivo efficacy of LY333328, a new glycopeptide antibiotic, was compared with that of vancomycin by using the rabbit model of left-sided methicillin-resistant Staphylococcus aureus endocarditis. Animals received LY333328 or vancomycin (25 mg/kg of body weight every 24 or 8 h, respectively) for 4 days. These drugs were equally effective in clearing bacteremia and in reducing bacterial counts in vegetations and tissues. We conclude that in this model, LY333328 was microbiologically effective and may be a therapeutic alternative to vancomycin.
withdrawn from all of the animals, and serial dilution and plating techniques were used to determine numbers of CFU per milliliter of blood. Inclusion in the study required that this blood culture be positive and that the catheter be positioned properly across the aortic valve at the time of autopsy. Rabbits were then randomized to receive 4 days of either LY333328 or vancomycin (25 mg/kg of body weight every 24 or 8 h, respectively) or no treatment (controls). Doses were chosen to simulate concentrations achievable in human serum (for vancomycin) or the least effective dose based on preliminary studies performed in our laboratory (for LY333328; unpublished data). Both drugs were administered by i.v. bolus injection, and the dose administered was adjusted for weight on a daily basis. Controls were sacrificed when therapy was begun for animals receiving antimicrobial agents; this was followed by the determination of bacterial counts in vegetations and tissues (see below). We have shown previously that experimental endocarditis with MRSA-494 is a fatal infection without the occurrence of spontaneous cures (8). Serum samples for measurements of peak (obtained 1 h postdose; alpha phase concluded for both drugs) and trough (obtained just before a scheduled dose) antibiotic contents were collected from all animals at the time of the first dose on day 2. Repeat blood cultures were obtained prior to the first dose on day 3. Following 4 days of therapy, all animals were sacrificed 10 to 12 h (for vancomycin) or 26 to 28 h (for LY333328) following the final dose and were autopsied in an aseptic manner. Blood cultures and serum samples for the measurement of antibiotic contents were obtained, followed by the removal of vegetations and 500-mg (mean weight) sections of left kidney and spleen for culture. These specimens were weighed, suspended in 0.9% NaCl (final volume, 1 ml), and homogenized. Quantitative bacterial counts, determined by serial dilution and plating techniques, were expressed as the log10 of CFU per gram (sensitivity limit, 10 CFU per vegetation or tissue section; culture-negative specimens were considered to contain 10 CFU for numerical and statistical purposes). The potential effect of antibiotic carryover was minimized by the volume of agar used in the culture plates; the dilution effect for cultured material was at least 200-fold.
LY333328 is a semisynthetic glycopeptide derived from LY264826, a naturally occurring compound similar to vancomycin (12). It has potent activity against gram-positive bacteria, including multidrug-resistant strains of Staphylococcus aureus, coagulase-negative staphylococci, and vancomycinsusceptible and -resistant strains of Enterococcus faecalis and Enterococcus faecium (5, 10, 12, 14, 16). Similarly to vancomycin, LY333328 has no clinically relevant activity against gramnegative bacteria. The development of new antimicrobial agents with activity against vancomycin-resistant organisms is of utmost importance, especially in light of the widespread distribution of vancomycin resistance in Enterococcus spp. and the recent emergence of low-level vancomycin resistance in S. aureus (1, 2). The in vivo activity of LY333328 in treating serious S. aureus infections has not been determined. In order to address this issue, we compared the therapeutic activities of LY333328 and vancomycin by using the rabbit model of left-sided S. aureus endocarditis. This model affords a good test of antimicrobial activity in a serious systemic infection. LY333328 and vancomycin were obtained from Eli Lilly and Co., Indianapolis, Ind. The methicillin-resistant strain of S. aureus used (MRSA-494) was a bloodstream isolate from a patient with endocarditis (7). The MICs and MBCs of vancomycin and LY333328 for MRSA-494 were determined in duplicate with Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.), which was cation adjusted with calcium and magnesium according to the guidelines of the National Committee for Clinical Laboratory Standards (11). The resistance of this strain to b-lactams has been established previously (7, 8). All studies were done by using male New Zealand White rabbits (weight, 2 to 3 kg). Left-sided endocarditis was established by placing a catheter across the aortic valve, followed in 3 days by an intravenous (i.v.) bacterial inoculum of 106 CFU (7). The catheter was left in place for the duration of the study. Sixteen hours after bacterial challenge, 1 ml of blood was * Corresponding author. Mailing address: C3690 Detroit VA Medical Center, 4646 John R., Detroit, MI 48201. Phone: (313) 576-4487. Fax: (313) 576-1112. E-mail:
[email protected]. 981
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ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Vegetation and tissue bacterial counts Bacterial count (log10 of CFU/g) ina:
Treatment
No. of rabbits
Vegetation
Kidney
Spleen
Vancomycin LY333328 None (control)
15 16 10
2.01 6 0.30 (14) 2.49 6 1.14 (14) 8.43 6 1.16 (0)
1.30 6 0.09 (15) 1.36 6 0.42 (15) 5.65 6 1.65 (0)
1.31 6 0.08 (14) 1.29 6 0.11 (16) 5.57 6 0.80 (0)
a
Values are means 6 standard deviations. Values in parentheses are numbers of culture-negative (sterile) specimens.
LY333328 and internal standard LY314947 were recovered from rabbit serum by solid-phase extraction. Concentrations of LY333328 were determined by using a validated high-performance liquid chromatography method with fluorescence detection (4). Vancomycin concentrations were determined by the fluorescence polarization immunoassay (TDx; Abbott Diagnostics, Irving, Tex.) (15). Pooled normal rabbit serum was used to prepare standards and to dilute unknowns as needed. The limits of detection for LY333328 and vancomycin by these methods were 0.2 and 0.5 mg/ml, respectively. Comparisons of blood, vegetation, and tissue bacterial counts were made by using Kruskal-Wallis one-way analysis of variance on ranks followed by Dunn’s test for multiple comparisons. Comparisons of the frequencies of sterilization of blood, vegetations, and renal and splenic tissues were made by use of the Fisher exact test. A P value of ,0.05 was considered significant. The MICs and MBCs of methicillin, vancomycin, and LY333328 for MRSA-494 were 25 and 60, 0.4 and 0.4, and 0.8 and 0.8 mg/ml, respectively. No differences were found in the intensities of pretreatment bacteremia (means [6 standard deviations] of log10 of CFU per milliliter) for animals infected with MRSA-494 and receiving vancomycin (3.13 6 0.69) or LY333328 (2.65 6 0.65) sacrificed after 4 days of therapy or controls sacrificed 16 h after bacterial challenge (3.16 6 0.75). The peak and trough concentrations in serum achieved with vancomycin were 37.8 6 6.2 and 2.3 6 1.2 mg/ml, respectively. The corresponding concentrations of LY333328 were 108.6 6 25.3 and 13.9 6 4.3 mg/ml, respectively. Terminal antimicrobial agent concentrations were comparable to trough concentrations. The levels of binding of LY333328 and vancomycin to plasma proteins were estimated to be 77 and 40%, respectively (9). Taking these data into account, the free drug-to-MIC ratios were similar for both test agents at each sampling time. No differences between antimicrobial agents were noted in the frequencies of blood culture sterilization during therapy. For vancomycin-treated animals, 93 and 100% of animals had sterile blood cultures after 2 and 4 days of therapy, respectively. For those treated with LY333328, 81 and 100%, respectively, had sterile cultures at both of these times. Quantitative bacterial counts in vegetations and tissues are given in Table 1. Compared to control animals, there were highly significant reductions in bacterial counts at each of these sites in rabbits treated with either drug (P , 0.001 for all comparisons). There were no significant differences in bacterial counts between LY333328- or vancomycin-treated animals and both drugs sterilized all assayed sites at similar rates. Recently, reports describing the emergence of S. aureus strains with diminished susceptibility to vancomycin have appeared (1, 2). This fact, combined with the successful transfer of the vancomycin-resistant phenotype from enterococci to S. aureus in the laboratory setting, makes the development of new agents with activity against such strains a high priority (13). Additionally, infections caused by vancomycin-resistant strains
of Enterococcus spp. can present significant therapeutic challenges to the clinician. Occasionally, infections caused by such strains essentially are not treatable by currently available antimicrobial agents, which underscores the urgent need for new therapeutic options (3, 6). In vitro studies have suggested that LY333328 is an effective vancomycin alternative (5, 10, 12, 14, 16). In the present study, we have shown that, compared to vancomycin as standard therapy, LY333328 was just as efficacious in clearing bacteremias and reducing vegetation and tissue bacterial counts in animals infected with a clinical isolate of methicillin-resistant S. aureus. Both drugs were also equally effective at sterilizing all assayed sites. These data support the microbiological effectiveness of LY333328 in a serious S. aureus infection and support the contention that the drug may be effective in treating similar infections in humans. Further testing of the efficacy of LY333328 in the therapy of experimental infections caused by other multidrug-resistant strains, such as Enterococcus spp., would be in order. If LY333328 demonstrates good in vivo activity in animal models and has an acceptable toxicity profile in humans, it would be reasonable to proceed to therapeutic trials in humans. This study was supported by a grant from Eli Lilly and Co., Indianapolis, Ind. We thank Anandeep Kumar and Xuan Wu for their technical assistance and Kathleen Emery for LY333328 determinations of concentrations in serum. REFERENCES 1. Centers for Disease Control and Prevention. 1997. Reduced susceptibility of Staphylococcus aureus to vancomycin—Japan, 1996. Morbid. Mortal. Weekly Rep. 46:624–626. 2. Centers for Disease Control and Prevention. 1997. Staphylococcus aureus with reduced susceptibility to vancomycin—United States, 1997. Morbid. Mortal. Weekly Rep. 46:765–766. 3. Handwerger, S., B. Raucher, D. Altarac, J. Monka, S. Marchione, K. Y. Singh, B. E. Murray, J. Wolff, and B. Walters. 1993. Nosocomial outbreak due to Enterococcus faecium highly resistant to vancomycin, penicillin, and gentamicin. Clin. Infect. Dis. 16:750–755. 4. Inman, E. L. 1987. Determination of vancomycin related substances by gradient high-performance liquid chromatography. J. Chromatogr. 410:363– 372. 5. Jones, R. N., M. S. Barrett, and M. E. Erwin. 1996. In vitro activity and spectrum of LY333328, a novel glycopeptide derivative. Antimicrob. Agents Chemother. 41:488–493. 6. Jones, R. N., H. Sader, M. E. Erwin, S. C. Anderson, and The Enterococcus Study Group. 1995. Emerging multiply resistant enterococci among clinical isolates. Diagn. Microbiol. Infect. Dis. 21:85–93. 7. Kaatz, G. W., S. L. Barriere, D. R. Schaberg, and R. Fekety. 1987. Ciprofloxacin versus vancomycin in the therapy of experimental methicillin-resistant Staphylococcus aureus endocarditis. Antimicrob. Agents Chemother. 31:527–530. 8. Kaatz, G. W., S. M. Seo, S. L. Barriere, L. M. Albrecht, and M. J. Rybak. 1990. Efficacy of ofloxacin in experimental Staphylococcus aureus endocarditis. Antimicrob. Agents Chemother. 34:257–260. 9. Lin, Y., R. E. Stratford, L. L. Zornes, W. L. Confer, V. Vasudevan, T. W. Jones, T. I. Nicas, D. A. Preston, C. J. Boylan, D. L. Zeckner, B. J. Boyll, P. A. Raab, N. J. Snyder, M. J. Zweifel, S. C. Wilkie, M. J. Rodriguez, R. C. Thompson, and R. D. G. Cooper. 1995. Nonclinical pharmacokinetics of LY333328, a semisynthetic glycopeptide antibiotic active against vancomycin-resistant enterococci, abstr. F254, p. 157. In Program and Abstracts of
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