Abstract
GAR-936, a novel glycylcycline, was investigated with a rat model of experimental endocarditis. It was compared with vancomycin against both vancomycin-susceptible and -resistant Enterococcus faecalis and methicillin-resistant Staphylococcus aureus. GAR-936 exhibited the lowest MICs (≤0.12 μg/ml) in vitro against each of the isolates tested. Endocarditis was established by placement of a catheter across the aortic valve, followed by intravenous injection of 106 CFU of bacteria 48 h later. Treatment with GAR-936 or vancomycin was initiated 24 to 36 h after bacterial infection and administered subcutaneously twice a day for 3 days at ascending doses. GAR-936 reduced bacterial vegetation titers by >2 log10 CFU, compared to those in untreated controls, for both vancomycin-susceptible and -resistant (VanA and VanB) E. faecalis strains and >4 log10 CFU for a methicillin-resistant S. aureus isolate. The glycylcycline was more efficacious at a lower administered dose in the rat model of endocarditis than was vancomycin. The efficacy of GAR-936 in this model was apparently not enhanced by a factor in rat serum, as was observed for vancomycin with a time-kill curve. The results of this study demonstrate the therapeutic potential of GAR-936 for the treatment of enterococcal and staphylococcal infections and warrant further investigation.
Endocarditis has many underlying causes, including complications from intravenous drug use, prosthetic valves, and nosocomial bacteremia, leading to extended hospital stays and high mortality rates (19). Streptococci, staphylococci, and enterococci are considered the three leading causes of infective endocarditis (3, 6). It is recognized as a difficult infection to treat and presents a therapeutic challenge, especially when caused by methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus spp. (3).
Treatment of resistant strains is limited, involving removal of the vegetation or associated device, constant infusion of effective antibiotics, and synergistic combinations of two or more antibacterial agents (8). Several investigational antibiotics may prove efficacious in the treatment of infective endocarditis. The fluoroquinolones, everninomicin, modified glycopeptides (LY 333328), linezolid, and quinupristin-dalfopristin have demonstrated good in vitro activities, and some are under investigation in animal models of infection (4, 7, 9, 14; M. C. Birmingham, G. S. Zimmer, B. Hafkin, W. M. Todd, D. Batts, S. Flavin, C. Rayner, K. E. Welch, P. F. Smith, J. D. Root, N. E. Wilks, and J. J. Schentag. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1098, p. 724, 1999; M. Rupp and J. Ulphani, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F111, p. 260, 1998; Y. Sato, A. Kuga, R. Okamoto, and M. Inoue, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. E112, p. 202, 1998). Animal models of infective endocarditis have been shown to be useful for the study of the human disease by examination of dosing regimens, bactericidal effect, relapse, and antibiotic penetration into the vegetation (1, 2, 11, 17).
GAR-936, a new glycylcycline antibiotic, has demonstrated excellent in vitro activity and in vivo efficacy against a broad spectrum of aerobic and anaerobic bacteria, including methicillin-resistant S. aureus and vancomycin-resistant Enterococcus faecalis (16). This study was performed to evaluate the efficacy of GAR-936 in a rat model of experimental endocarditis involving both methicillin-resistant S. aureus and vancomycin-susceptible and -resistant E. faecalis isolates.
MATERIALS AND METHODS
Organisms.
E. faecalis GC6181 (vancomycin susceptible) was kindly provided by Michael Gilmore of the University of Oklahoma, Oklahoma City; E. faecalis GC6207 (VanB) was obtained from Louis Rice, Cleveland VA Hospital, Cleveland, Ohio; and E. faecalis GC6191 was constructed by mating GC6181 and a vancomycin-resistant E. faecalis clinical isolate by a modified filter method with colony selection on vancomycin agar (18) and verification with Riboprint. Methicillin resistant S. aureus 89-4 is a hospital clinical isolate.
Antibiotics.
A standard powder of GAR-936 was obtained from Wyeth-Ayerst Research, Pearl River, N.Y. Vancomycin was obtained from Sigma Chemical Co., St. Louis, Mo.
Susceptibility testing.
MICs were determined by the microtiter method with Mueller-Hinton II broth as recommended by the National Committee for Clinical Laboratory Standards (12). Microtiter plates containing 50 μl of twofold serial dilutions of the antimicrobial agents per well were inoculated with 50 μl of inoculum to yield a final density of 1 × 105 to 5 × 105 CFU/ml.
MICs were determined after 18 to 22 h of incubation at 35°C. The MIC was defined as the lowest concentration of the antimicrobial agent that completely inhibited the growth of the organism, as detected by the unaided eye. Minimum bactericidal concentrations (MBCs) were measured by removing 10 μl from all wells containing no visible growth and plating the samples on Trypticase soy agar plates. The plates were incubated overnight at 35°C. The MBC was defined as the lowest concentration (the first dilution) that resulted in a ≥3-log reduction from the original inoculum concentration.
Time-kill curve.
Bactericidal activity was determined by use of time-kill curves as recommended by the National Committee for Clinical Laboratory Standards (13). The contents of flasks containing 25 ml of the test organism grown to logarithmic phase in Mueller-Hinton II broth (adjusted to a final density of approximately 106 CFU/ml) were added to flasks containing 25 ml of either rat serum or Mueller-Hinton II broth containing the antimicrobial agent to yield a drug concentration equivalent to four times the MIC (12). The flasks were incubated at 35°C in a shaking water bath. Aliquots were removed at 0, 3, 6, and 24 h and diluted, and 0.1 ml was plated in duplicate on agar plates. Total bacterial CFU per milliliter were determined after 18 h of incubation at 35°C. Bactericidal activity was defined as a 99.9% (>3-log10) reduction in the total count from the original inoculum (13).
Pharmacokinetics.
Male Wistar rats (Charles River Laboratories) weighing 200 to 250 g were administered a single dose of 7, 20, or 40 mg of GAR-936 in phosphate-buffered saline per kg of body weight by subcutaneous injection. Three rats were used for each dose tested. Blood samples were obtained via a surgically implanted jugular cannula at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h.
Plasma GAR-936 levels were determined using a microbiological agar diffusion assay with Bacillus cereus ATCC 11778 as the indicator organism; this organism was grown in nutrient broth with 1.1% agarose and preincubated at 4°C for 2 h before being placed in a humidified 30°C incubator overnight. The limit of detection of the assay was 0.25 μg/ml, with a range of 0.25 to 8.0 μg/ml and a correlation coefficient of ≥0.98 for any given assay. Pharmacokinetic analysis was accomplished through noncompartmental modeling using Winnonln.
Bacterial endocarditis.
Endocarditis was produced in male Wistar rats by insertion of a sealed polyethylene cannula (PE10) through the right carotid artery into the left ventricle; the cannula was sutured in place as a point of adherence for bacterial infection (5, 6, 15).
At 48 h after implantation of the cannula, a 5-h bacterial culture was diluted to 105 to 106 CFU/ml in sterile saline, and 1 ml was injected intravenously (5). Inoculum infection concentration was verified by plate counts.
Antibacterial treatment was initiated 24 or 36 h after bacterial challenge, depending on the bacteria being tested. Treatments were delivered by subcutaneous administration every 12 h for 3 days. The dose ranges were 0.5 to 80 mg/kg/day for GAR-936 and 40 to 240 mg/kg/day for vancomycin. Untreated control rats received injections of phosphate-buffered saline. Both treated and control rats were euthanatized by CO2 inhalation 24 h after the last treatment. Hearts were aseptically removed, weighed, homogenized, and serially diluted in saline for determination of bacterial titers, expressed as log10 CFU per heart.
Statistics.
Data were analyzed using mixed-model analysis of variance (ANOVA) methods. The mean response for a specific compound and dose was compared to the mean response for the control using a two-tailed t test, with the t statistic being based on the error term from the ANOVA.
RESULTS
In vitro studies.
The in vitro activities of GAR-936 and vancomycin against the methicillin-resistant S. aureus and E. faecalis strains used in the rat endocarditis model are summarized in Table 1.
TABLE 1.
MICs and MBCs of GAR-936 and vancomycin against enterococcal and S. aureus strains in a rat endocarditis modela
| Organism | Strain | Type | GAR-936
|
Vancomycin
|
||
|---|---|---|---|---|---|---|
| MIC | MBC | MIC | MBC | |||
| E. faecalis | GC6181 | Susceptible | 0.12 | >4 | 1 | 16 |
| E. faecalis | GC6191 | Resistant (VanA) | ≤0.008 | >0.25 | >256 | >256 |
| E. faecalis | GC6207 | Resistant (VanB) | ≤0.008 | >0.25 | 256 | >256 |
| S. aureus | 89-4 | Methicillin resistant | 0.12 | 0.5 | 1 | 2 |
MICs and MBCs are given in micrograms per milliliter.
GAR-936 was active against all bacteria, with MICs ranging from ≤0.008 to 0.12 μg/ml. GAR-936 was eightfold more active than vancomycin against the vancomycin-susceptible E. faecalis and methicillin-resistant S. aureus strains.
The MBCs of GAR-936 and vancomycin were high relative to their respective MICs for the E. faecalis strains (>16-fold increase) but were only 4-fold higher for the methicillin-resistant S. aureus strain.
GAR-936 and vancomycin exhibited bacteriostatic effects in Mueller-Hinton II broth alone against E. faecalis GC6181 in time-kill curve studies. Vancomycin but not GAR-936 grown in Mueller-Hinton II broth with 50% rat serum added demonstrated an enhanced antibacterial effect, with a 2.6-log10 decrease in viable bacterial counts (Fig. 1).
FIG. 1.
Antibacterial activities of GAR-936 and vancomycin (VAN) against E. faecalis GC6181 in the presence of Mueller-Hinton II broth (MHB) and 50% rat serum (RS).
Pharmacokinetics.
Subcutaneous administration of GAR-936 at single doses of 7, 20, and 40 mg per kg resulted in a dose-proportional relationship for maximum concentration of drug in serum and area under the concentration-time curve (AUC) (Table 2). Times above the MIC for the bacteria included in this study for drug doses of 7, 20, and 40 mg/kg were approximately 9, 12, and 25 h, corresponding to 75, 100, and >100% of the dosing interval, respectively (Fig. 2). Time above the MIC for vancomycin has been calculated from data obtained from Marre et al. to be approximately 3 h for a 10-mg/kg single intravenous dose (10).
TABLE 2.
Pharmacokinetic parameters for GAR-936 after single-dose subcutaneous administration in male Wistar rats
| Dose (mg/kg) | Cmaxa (μg/ml) | AUC (μg · h/ml) | Half-life (h) |
|---|---|---|---|
| 7.0 | 1.3 ± 0.16 | 5.6 ± 1.07 | 2.5 ± 0.71 |
| 20.0 | 3.9 ± 0.50 | 17.7 ± 0.79 | 2.9 ± 0.33 |
| 40.0 | 5.6 ± 1.10 | 43.1 ± 0.57 | 4.6 ± 0.02 |
Maximum concentration of drug in serum.
FIG. 2.
Serum GAR-936 levels in male Wistar rats after subcutaneous administration of 7 (▴), 20 (■), or 40 (●) mg/kg.
Experimental endocarditis.
For experimental endocarditis caused by E. faecalis GC6181 (vancomycin susceptible), GAR-936 doses of ≥14 mg/kg/day resulted in an average log10 decrease of >2.0 CFU from the controls (Table 3). Vancomycin at doses of 40 to 240 mg/kg/day resulted in an average log10 decrease of <1.5 CFU from the controls.
TABLE 3.
Comparative efficacies of GAR-936 and vancomycin against experimental endocarditis caused by vancomycin-susceptible E. faecalis GC6181
| Compound | No. of animals | Dose (mg/kg/day) | Bacterial titer (log10 CFU/heart)
|
Reduction from control (log10 CFU) | |
|---|---|---|---|---|---|
| Mean | SEM | ||||
| None (control) | 30 | 7.01 | 0.22 | ||
| GAR-936 | 8 | 0.5 | 7.60 | 0.40 | +0.59 |
| 4 | 2.0 | 6.27 | 0.54 | −0.73a | |
| 7 | 7.0 | 5.69 | 0.41 | −1.32a | |
| 10 | 14.0 | 4.58 | 0.35 | −2.42a | |
| 11 | 40.0 | 4.70 | 0.33 | −2.26a | |
| 9 | 80.0 | 3.69 | 0.36 | −3.32a | |
| Vancomycin | 10 | 40.0 | 5.52 | 0.35 | −1.49a |
| 3 | 120.0 | 5.54 | 0.60 | −1.46a | |
| 3 | 240.0 | 5.75 | 0.60 | −1.25a | |
Statistically significant difference (P < 0.05), as determined by ANOVA.
The protective effects of GAR-936 and vancomycin against infection with vancomycin-resistant E. faecalis GC6191 (VanA) are summarized in Table 4. At 14 mg/kg/day, GAR-936 exhibited a log10 reduction from the controls of 2.68 CFU, a result comparable to the results obtained with vancomycin-susceptible E. faecalis GC6181. Vancomycin at 240 mg/kg/day had no significant effect on bacterial titers in the cardiac vegetations. GAR-936 also demonstrated efficacy against E. faecalis GC6207 (VanB)-induced endocarditis, with 2.8-, 3.7-, and 4.7-log10 CFU reductions in bacterial titers after subcutaneous administration of 2, 7, and 14 mg/kg/day, respectively.
TABLE 4.
Comparative efficacies of GAR-936 and vancomycin against experimental endocarditis caused by vancomycin-resistant E. faecalis
| E. faecalis | Compound | No. of animals | Dose (mg/kg/day) | Bacterial titer (log10 CFU/heart)
|
Reduction from control (log10 CFU) | |
|---|---|---|---|---|---|---|
| Mean | SEM | |||||
| GC6191 (VanA) | None (control) | 19 | 7.80 | 0.33 | ||
| GAR-936 | 7 | 0.5 | 8.59 | 0.49 | +0.79 | |
| 8 | 7.0 | 5.79 | 0.47 | −2.01a | ||
| 4 | 14.0 | 5.12 | 0.63 | −2.68a | ||
| 7 | 40.0 | 4.94 | 0.49 | −2.86a | ||
| 2 | 80.0 | 4.61 | 0.81 | −3.19a | ||
| Vancomycin | 4 | 40.0 | 7.66 | 0.64 | −0.14 | |
| 2 | 80.0 | 7.44 | 0.81 | −0.36 | ||
| 4 | 240.0 | 7.24 | 0.64 | −0.56 | ||
| GC6207 (VanB) | None (control) | 11 | 7.72 | 0.29 | ||
| GAR-936 | 11 | 0.5 | 7.22 | 0.29 | −0.50 | |
| 7 | 2.0 | 4.89 | 0.36 | −2.83a | ||
| 4 | 7.0 | 3.99 | 0.48 | −3.73a | ||
| 4 | 14.0 | 3.01 | 0.48 | −4.71a | ||
Statistically significant difference (P < 0.05), as determined by ANOVA.
Against a methicillin-resistant S. aureus infection (Table 5), GAR-936 (14 mg/kg/day) was more efficacious than vancomycin (40 mg/kg/day), with observed reductions in bacterial titers of 4.5 and 0.69 log10 CFU, respectively. To determine the effect of GAR-936 on bacterial counts in S. aureus-infected cardiac vegetations at initiation of treatment, counts were determined prior to the first administered dose and 24 h after the last dose (Fig. 3). The results indicate a dose-effect relationship, with doses above 1.0 mg/kg/day yielding reductions in bacterial titers of 0.9 to 2.8 log10 CFU (with 40 mg/kg/day).
TABLE 5.
Comparative efficacies of GAR-936 and vancomycin against experimental endocarditis caused by methicillin-resistant S. aureus
| Compound | No. of animals | Dose (mg/kg/day) | Bacterial titer (log10 CFU/heart)
|
Reduction from control (log10 CFU) | |
|---|---|---|---|---|---|
| Mean | SEM | ||||
| None (control) | 17 | 11.40 | 0.78 | ||
| GAR-936 | 6 | 1.0 | 10.98 | 0.88 | −0.42 |
| 4 | 2.0 | 9.89 | 0.97 | −1.51 | |
| 5 | 10.0 | 7.93 | 0.90 | −3.47a | |
| 5 | 14.0 | 6.85 | 0.95 | −4.54a | |
| 7 | 40.0 | 6.23 | 0.87 | −5.17a | |
| Vancomycin | 3 | 40.0 | 10.70 | 1.06 | −0.69 |
Statistically significant difference (P < 0.05), as determined by ANOVA.
FIG. 3.
Efficacy of GAR-936 for methicillin-resistant S. aureus titers in an endocarditis infection model (log reduction from titers at initiation of treatment).
DISCUSSION
GAR-936, a derivative of minocycline, has been shown to exhibit potent in vitro and in vivo efficacies against a broad spectrum of gram-positive and gram-negative bacteria, including tetracycline-resistant strains carrying both efflux and ribosomal resistance determinants (P. Petersen, W. Weiss, P. Labthavikul, and P. Bradford, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F132, p. 266, 1998). GAR-936 has good activity and is more active than vancomycin against the methicillin-resistant S. aureus and vancomycin-susceptible and -resistant E. faecalis (VanA and VanB) isolates used in this study. The inhibitory activity of GAR-936 was equivalent for vancomycin-susceptible E. faecalis as well as either VanA- or VanB-mediated vancomycin-resistant E. faecalis. This antibacterial activity, like that of other tetracyclines, is bacteriostatic, with MBCs greater than 16-fold the MIC against the E. faecalis strains but only 4-fold higher against the methicillin-resistant S. aureus strain used in this study.
The efficacy of GAR-936 in a rat model of experimental endocarditis with both E. faecalis and methicillin-resistant S. aureus strains has been demonstrated in this set of experiments. When administered subcutaneously twice a day for 3 days, GAR-936 doses of ≥14 mg/kg/day resulted in statistically significant (P = 0.0001) reductions in cardiac titers of a vancomycin-susceptible E. faecalis strain—≥2.42 log10 CFU compared to the results for untreated controls. Vancomycin administered at doses of up to 17 times the minimal efficacious dose of GAR-936 resulted in <1.5-log10 reductions in bacterial titers. GAR-936 demonstrated comparable efficacy against the vancomycin-resistant (VanA) isogenic strain of E. faecalis. The reductions in titers at the site of infection were equivalent per dose level for both of these strains, as was observed for the in vitro activity of GAR-936. The AUC for GAR-936 at an efficacious dose correlates with the exposures from previous therapeutic models, including murine thigh infection and murine pulmonary infection (S. Mikels, E. Lenoy, W. Allen, S. Comptom, and W. J. Weiss, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F135, p. 266, 1998). Vancomycin at doses of up to 240 mg/kg/day exhibited no significant effect against the resistant strain. The efficacy of GAR-936 was also observed against resistant E. faecalis expressing the VanB phenotype, with >4-log10 reductions in CFU/heart observed after administration of 14 mg/kg/day. Against methicillin-resistant S. aureus, GAR-936 at increasing doses resulted in a reduction in bacterial load and a greater efficacy at a lower administered dose than vancomycin.
Previous studies (8) indicated that a factor in rat serum enhances the antibacterial activity of vancomycin against enterococci, suggesting that the efficacy of vancomycin in the rat model of experimental endocarditis may be overstated. As indicated by time-kill curves, the antibacterial activity of GAR-936 was not affected by the same serum factor.
GAR-936, in fact, demonstrated bactericidal activity against methicillin-resistant S. aureus infection when cardiac bacterial titers at the initiation and end of treatment were compared. Administration of GAR-936 at greater than 10 mg/kg/day for the course of treatment resulted in >2-log10 reductions in CFU from titers at the start of treatment. This apparent bactericidal activity warrants further investigation.
In order to be efficacious in endocarditis, an antibiotic must penetrate the fibrin and platelet vegetation and elicit a response (2). The degree of response would therefore depend on the activity of the antimicrobial agent, diffusion into the site, pharmacodynamic parameters required for efficacy, and the in vivo postantibiotic effect. Studies have confirmed the activity of GAR-936 against both enterococci and staphylococci, two of the more common causative agents of endocarditis (5, 15). Petersen et al. (38th ICAAC) observed a long in vitro postantibiotic effect for GAR-936 against both S. aureus (>3 h) and Escherichia coli (1.8 to 2.9 h) isolates. Pharmacokinetic data for several species indicate that GAR-936 exhibits a high volume of distribution (unpublished data). The results of this investigation of GAR-936 in experimental endocarditis and supporting data from other investigators (1, 2, 5) indicate the need for further studies on the therapeutic potential of GAR-936 in endocarditis and other deep-seated infections.
ACKNOWLEDGMENT
We acknowledge the assistance of Fred Immermann for statistical analysis of the endocarditis data.
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