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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, November 2000, p. 3022-3027, Vol. 44, No. 11
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Therapeutic Efficacy of GAR-936, a Novel
Glycylcycline, in a Rat Model of Experimental
Endocarditis
Timothy M.
Murphy,*
Jacqueline M.
Deitz,
Peter J.
Petersen,
Susan M.
Mikels, and
William J.
Weiss
Infectious Disease Research Section,
Antimicrobial Chemotherapy, Wyeth-Ayerst Research, Pearl River, New
York 10965
Received 21 October 1999/Returned for modification 4 January
2000/Accepted 10 August 2000
 |
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.
| |
INTRODUCTION |
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.
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TABLE 1.
MICs and MBCs of GAR-936 and vancomycin against
enterococcal and S. aureus strains in a rat
endocarditis modela
|
|
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).
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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).
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TABLE 2.
Pharmacokinetic parameters for GAR-936 after
single-dose subcutaneous administration in male Wistar rats
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FIG. 2.
Serum GAR-936 levels in male Wistar rats after
subcutaneous administration of 7 ( ), 20 ( ), or 40 ( ) mg/kg.
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|
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.
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TABLE 3.
Comparative efficacies of GAR-936 and vancomycin
against experimental endocarditis caused by vancomycin-susceptible
E. faecalis GC6181
|
|
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.
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TABLE 4.
Comparative efficacies of GAR-936 and vancomycin against
experimental endocarditis caused by
vancomycin-resistant E. faecalis
|
|
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).
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TABLE 5.
Comparative efficacies of GAR-936 and vancomycin against
experimental endocarditis caused by
methicillin-resistant S. aureus
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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).
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|
| |
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.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Wyeth-Ayerst
Research, 401 N. Middletown Rd., Pearl River, NY 10965. Phone: (845)
732-2220. Fax: (845) 732-5671. E-mail:
murphytm{at}war.wyeth.com.
| |
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Antimicrobial Agents and Chemotherapy, November 2000, p. 3022-3027, Vol. 44, No. 11
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
