ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1992, P. 446-452
Vol. 36, No. 2
0066-4804/92/020446-07$02.00/0
Antimicrobial Activity of MDL 62,873, a Semisynthetic Derivative of Teicoplanin, In Vitro and in Experimental Infections MARISA BERTI, GIAMPAOLO CANDIANI, MONICA BORGONOVI, PAOLO LANDINI, FRANCA RIPAMONTI, ROBERTO SCOTTI, LUIGI CAVENAGHI, MAURIZIO DENARO, AND BETH P. GOLDSTEIN*
Lepetit Research Center, Marion Merrell Dow Research Institute, Via R. Lepetit 34, 21040 Gerenzano (Varese), Italy Received 16 August 1991/Accepted 5 December 1991
MDL 62,873 is an amide derivative of teicoplanin A2-2. Like those of natural glycopeptides, its antibacterial activity is mediated by inhibition of cell wall peptidoglycan synthesis. Against streptococci and enterococci, the in vitro activity of MDL 62,873 was similar to that of teicoplanin and greater than that of vancomycin. Against staphylococci, it has activity similar to that of vancomycin, and it was significantly more active than teicoplanin against coagulase-negative isolates. Like teicoplanin and vancomycin, MDL 62,873 had slow but significant bactericidal activity (99 to 99.9% killing in 24 h) against staphylococci at concentrations near the MIC. In murine septicemia studies with Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae, the 50% effective doses were lower than those of vancomycin. In staphylococcal endocarditis in rats, MDL 62,873 at 20 mg/kg of body weight and vancomycin at 40 mg/kg, both doses given intravenously twice daily, had similar efficacies in reducing the heart bacterial load. These results probably reflect the longer half-life of MDL 62,873, which has a pharmacokinetic profile in rats similar to that of teicoplanin.
Because of the emergence of methicillin-resistant staphylococci, glycopeptide antibiotics have received more attention over the last several years. Among coagulase-negative isolates, whose incidence in serious infection has been increasing (3, 13, 19), teicoplanin-resistant and occasional vancomycin-resistant strains have been reported (2, 9, 11, 12, 22, 23, 27, 31). We have been seeking glycopeptide derivatives with improved activities against these strains. MDL 62,211 (CTA-A-1), the amide obtained by the condensation of teicoplanin complex with 3,3-dimethylamino-1propylamine, had greater in vitro activity than teicoplanin against coagulase-negative staphylococci and was as active as teicoplanin against other bacterial species (15, 21). We have continued our studies with MDL 62,873, the amide of the major peak (A2-2) of the teicoplanin complex. We present data for the antibacterial activity of MDL 62,873 in vitro and in experimental infections in animals as well as data for its pharmacokinetics in rats.
the coagulase-negative staphylococci (CNS) we chose a number of strains for which teicoplanin MICs were relatively high. For mouse septicemia experiments, three laboratory strains, Staphylococcus aureus Smith, Streptococcus pyogenes C203, and Streptococcus pneumoniae Felton UC41 were used. For rat endocarditis experiments, we used clinical isolates for which teicoplanin MICs were 8 to 16 ,ug/ml: S. aureus SA12873, kindly provided by G. W. Kaatz (17), and two methicillin-resistant CNS (Staphylococcus epidermidis L537 and Staphylococcus haemolyticus L1520). We used Bacillus subtilis 566/1 (thyA thyB) for mechanism of action studies. MIC determination. MICs were determined by broth microdilution. Streptococci were grown in Todd-Hewitt broth (Difco), and other organisms were grown in Iso-Sensitest broth (Oxoid). Inocula were approximately 5 x 105 CFU/ml from overnight culture plates (24); incubation was at 37°C for 24 h. Bactericidal activity. Logarithmically growing cultures were diluted and inoculated into Iso-Sensitest broth at a density of 106 to 107 CFU/ml; 10-ml portions were distributed into flasks with and without antibiotics. The time-kill studies were performed with the lowest concentration of each antibiotic which reproducibly inhibited visible growth of the isolate under test conditions and twice this concentration. These concentrations were usually two- to eightfold the MICs determined by broth microdilution (see Results). At intervals after incubation in a stationary 37°C water bath, samples were suitably diluted (at least 10-fold) in saline with 0.1% peptone; the addition of peptone has been reported to increase the recovery of bacteria (28). Duplicate 0.1-ml samples were plated by inclusion in 2.5 ml of Iso-Sensitest soft agar (0.7% agar) on Iso-Sensitest agar plates. This results in an immediate 250-fold dilution of the antibiotic, which, in our experience, is sufficient to prevent carryover effects with the concentrations used. At the inocula used, 99.9% killing can be reliably detected as 10 to 100 colonies per plate at the lowest dilution. Mechanism of action. B. subtilis was grown to an optical
MATERIALS AND METHODS Antibacterial agents. MDL 62,873 and teicoplanin powders were from Lepetit. The MDL 62,873 preparations used contained 75 to 80% MDL 62,873 and 20 to 25% the corresponding amides of other components of the teicoplanin complex. This is the preparation intended for further development. Different components of the teicoplanin complex have similar in vitro and in vivo activities (7), and the MDL 62,873 preparation used here had an in vitro activity similar to that of MDL 62,211 (the amide derivative of the teicoplanin complex) (16). Therefore, we do not expect there to be significant differences in activities among the components of the MDL 62,873 preparation. We also used vancomycin (Vancocin HCI; Eli Lilly), ampicillin (Amplital; Farmitalia-Carlo Erba), and erythromycin (Sigma). Bacterial strains. For in vitro studies, we used clinical isolates of staphylococci, streptococci, and enterococci. Of *
Corresponding author. 446
TABLE 1. Activity of MDL 62,873 against staphylococci Organism (no. of isolates)
S. aureus (50)
S. epidermidis (30)
S. haemolyticus (34)
Other CNS (21)"
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Antibiotic MDL 62,873 Teicoplanin Vancomycin
MIC (Rg/ml)a
Range 0.13-2 0.13-8
MIC50 0.25 0.5
MIC90
0.25-4
1
1 1 2
MDL 62,873 Teicoplanin Vancomycin
0.06-0.5 0.13-16
0.13 4 2
0.25 8 2
MDL 62,873 Teicoplanin Vancomycin
0.13-8 1-64
MDL 62,873 Teicoplanin Vancomycin
0.06-1 0.06-16 0.25-2
1-4
1-4
0.5 16 2 0.13 1 0.5
4 64 4 1 8 1
a MIC50, MIC for 509o of the isolates tested; MIC90, MIC for 90%o of the isolates tested. b S. simulans (7), S. hominis (5), S. saprophyticus (3), S. cohnii (2), S. capitis, S. lugdunensis, S. warneri, and S. xylosus (1 each).
density at 590 nm of 0.2 in Davis-Mingioli medium supplemented with 2% glucose, 0.4% Casamino Acids, 100 ,±g of asparagine per ml, and 1 ,ug of thymine per ml. Macromolecular syntheses (DNA, RNA, protein, and cell wall peptidoglycan) were followed by incorporation of the appropriate radioactive precursors: for DNA synthesis, [methyl-3H]thymidine (2 puCi/ml) diluted with 2 ,ug of cold thymidine per ml; for RNA synthesis, [3H]uridine (1 ,uCi/ml, 10 ,ug/ml); for protein synthesis, [3H]tryptophan (1 ,uCi/mmol, 1 ,ug/ml); for peptidoglycan synthesis, [3H]N-acetylglucosamine (2 ,uCi/ ml, 3.5 ,ug/ml). Samples were precipitated with cold 5% trichloroacetic acid. Samples for protein synthesis were heated for 10 min at 80°C. Trichloroacetic acid-precipitated material was collected on glass fiber filters with a cell harvester (LKB) and counted in a P-plate scintillation counter (Pharmacia). Experimental septicemia in mice. CD-1 mice (Charles River Breeding Laboratories) weighing 18 to 22 g were infected intraperitoneally with 8 x 104 CFU of S. pyogenes, S x 10' CFU of S. pneumoniae (both in 0.5 ml of saline plus 1% peptone), or 2 x 106 CFU of S. aureus (in 0.5 ml of 5% Difco bacteriological mucin). These challenges corresponded to 50 to 150 times the 50% lethal doses for the infecting organisms. Untreated animals died within 48 h after infection. Within 10 min after infection, the animals were given a single subcutaneous administration of the antibiotics. In each experiment, four or five groups of 10 mice each (five males and five females) were treated with different dose levels of each antibiotic. The 50% effective doses and 95% confidence limits were calculated by the Spearman-Kaerber method (8) from the percentages of animals surviving to day 7 at each dose. Experimental endocarditis. Endocarditis was induced in male CD rats (Charles River Breeding Laboratories) weighing about 200 g. A polyethylene catheter (inside diameter = 0.4 mm, outside diameter = 0.8 mm; Portex) was inserted through the aortic valve into the left ventricle via the right carotid artery and secured with a silk ligature. Correct positioning of the catheter was controlled with a recording amplifier (Hewlett-Packard model 7782A) with a pressure transducer. Two days later, rats were injected intravenously
(i.v.) with 0.5 ml of a bacterial suspension. Undiluted overnight cultures in Todd-Hewitt broth were used for S. epidermidis and S. haemolyticus; an overnight culture of S. aureus in Trypticase soy broth (BBL) was diluted to 2 x 104 CFU/ml in saline with 1% peptone. In each experiment, four or five animals were killed just before the first treatment to provide baseline bacterial counts. Rats were treated i.v. with 20 mg of MDL 62,873 per kg of body weight or 40 mg of vancomycin per kg every 12 h for 5 days. The first treatment was given 24 h (CNS) or 16 h (S. aureus) after infection. With these doses, mean peak levels in plasma (1 h after the ninth treatment) were 48 jxg/ml for MDL 62,873 and 46 jig/ml for vancomycin; mean trough levels (12 h after the tenth treatment) were 3 pug/ml for MDL 62,873 and 0.2 jig/ml for vancomycin. Animals that died before the end of the experiment were autopsied and their hearts were removed; surviving animals were killed 12 h after the last treatment. The hearts were weighed and homogenized. To avoid antibiotic carryover, the homogenates were centrifuged, suspended in saline with 1% peptone, and washed three times by centrifugation. Duplicate 0.025-ml aliquots of appropriate dilutions were plated on Todd-Hewitt agar to determine the bacterial heart loads. The presence of blood in the heart homogenates did not influence the results, as bacterial titers in the blood were always at least 1,000-fold lower than the bacterial heart loads. Rats that died within 40 or 48 h after infection (with S. aureus and CNS, respectively) were excluded from statistical analysis because these deaths were likely the result of catheter placement (14) and because the length of treatment would be too short to expect any significant effect on bacterial load. Data were examined by covariance analysis, with time of death as the covariant. Least-square means were used to evaluate differences in bacterial titers between
TABLE 2. Activity of MDL 62,873 against streptococci and enterococci Organism (no. of isolates)
S. pyogenes (15)
Antibiotic MDL 62,873 Ampicillin Erythromycin Teicoplanin Vancomycin
MIC (Jug/ml)a Range M1C50 MIC90 0.06 0.03-0.13 0.03 0.03 0.016 0.004-0.13 0.06 0.03-0.06 0.06 0.13 0.06-0.13 0.06 0.25 0.5 0.13-0.5
0.06-0.13 0.06-4 Ampicillin Erythromycin 0.016->128 0.03-0.25 Teicoplanin 0.06-0.5 Vancomycin
S. pneumoniae (15) MDL 62,873
Other streptococci MDL 62,873 Ampicillin (19)b
Erythromycin Teicoplanin Vancomycin Enterococci (15)C
0.016-0.25 0.016-0.5 0.008->128 0.03-1 0.5-2
MDL 62,873
0.06-0.25
Ampicillin Teicoplanin Vancomycin
1-16 0.06-0.25 0.5-2
MIC0,,
0.13 0.06 0.06 0.06 0.25
0.13 0.13 >128 0.13 0.5
0.13 0.06 0.25 0.06 0.016 >128 0.25 0.13 2 1 0.13 1 0.06 1
0.25 16 0.25 2
a MIC for 90% of the MIC50, MIC for 509o of the isolates tested; isolates tested. b S. agalactiae, S. salivarius, S. sanguis (4 each); S. bovis, S. mutans (3 each); and S. acidominimus (1). c
E. faecalis (12) and E. faecium (3).
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10
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U-E
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JL D
LL
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-Jr
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FIG. 1. Bactericidal activities of MDL 62,873 (0, 0), teicoplanin (O, U), and vancomycin (A, A); untreated control (0). Concentrations chosen were the MIC (0, 0, A) and twice the MIC (0, *, A) as determined under the same conditions used for the experiments. MICs of MDL 62,873, teicoplanin, and vancomycin were, respectively, as follows: (A) for S. aureus L448, 2, 2, and 2 ,ug/ml; (B) for S. aureus L1524, 2, 2, and 4 ,ug/ml; (C) for S. epidermidis L533, 1, 8, and 4 ,ug/ml; (D) for S. epidermidis L1654, 2, 16, and 8 ,g/ml; (E) for S. haemolyticus L1706, 8, 32, and 8 ,Ig/ml; (F) for S. haemolyticus L1707, 4, 32, and 8 ,ug/ml.
control and treatment groups, and P was corrected for multiple comparisons by using the formula P' = 1 - (1 P)n, where n is the number of preplanned comparisons. Mean survival time the standard error was calculated by survival analysis by the SAS Lifetest procedure (26); differences in survival times between control and treatment groups were evaluated by analysis of variance of the reciprocal values of survival time. Pharmacokinetics in rats. Two groups of five male rats each were treated i.v. with a single 20-mg/kg dose of MDL 62,873 administered in 2 ml of 5% glucose per kg. At the time of treatment, the mean weight the standard deviation of rats used for plasma kinetics was 182 5 g; the rats used for urinary excretion analysis weighed 189 5 g. The rats were lightly anesthetized with halothane, and 0.3 to 0.5 ml of blood was collected in heparinized tubes at 0.05, 1, 2, 4, 6, 8, 12, 24, 36, 48, 56, 72, 80, and 96 h after drug administration. The plasma was collected by centrifugation. Urine specimens were collected in metabolic cages at 12, 24, 48, 72, and ±
±
±
±
96 h after drug administration. Concentrations of MDL 62,873 in urine and plasma samples were determined by microbiological assay by using Micrococcus luteus ATCC 9341 as the test organism.
RESULTS In vitro activity. Table 1 shows the MICs of MDL 62,873, teicoplanin, and vancomycin for a series of 134 staphylococci. We included strains for which teicoplanin MICs were known to be :8 ,ug/ml (3 of S. aureus, 8 of S. epidermidis, 27 of S. haemolyticus, and 3 isolates of other coagulase-negative species). MDL 62,873 showed excellent activity, with a MIC of si ,ug/ml for the majority of the isolates. The few isolates for which MDL 62,873 MICs were higher than 1 ,ug/ml included three S. aureus isolates (MIC of 2 ,ug/ml versus 8 ,ug/ml for teicoplanin and 2 to 4 ,ug/ml for vancomycin) and four S. haemolyticus isolates, two of which were inhibited by 4 pug/ml and two of which were inhibited by 8
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FIG. 2. Mechanism of action of MDL 62,873. (A) DNA synthesis; (B) RNA synthesis; (C) protein synthesis; (D) peptidoglycan synthesis. Symbols: 0, control; A, MDL 62,873 at 0.25 ,ug/ml; 4, teicoplanin at 0.25 ,ug/ml. Arrows indicate the times of addition of the antibiotics.
,ug/ml of MDL 62,873; for the four S. haemolyticus isolates, teicoplanin MICs were 16 to 64 p,g/ml and vancomycin MICs were 4 ,ug/ml. With the exception of two of these S. haemolyticus strains, the activity of MDL 62,873 was similar to or greater than that of vancomycin against all isolates. Against streptococci and enterococci, the activity of MDL 62,873 was similar to that of teicoplanin and, for most of the strains, greater than that of vancomycin (Table 2). The MICs of MDL 62,873 were similar to those of ampicillin and erythromycin; as expected, MDL 62,873 was also active against isolates resistant to these antibiotics (Table 2). MDL 62,873 was as active as teicoplanin against a vanB strain of Enterococcusfaecium (30) which is resistant to vancomycin
and susceptible to teicoplanin; it had no activity against vanA strains of E. faecium (20, 29), which are resistant to both teicoplanin and vancomycin (data not shown). Bactericidal activity. The bactericidal activity of MDL 62,873 was compared with those of teicoplanin and vancomycin against two isolates each of S. aureus, S. epidermidis, and S. haemolyticus (Fig. 1). For these experiments, the MIC was defined as the lowest concentration of an antibiotic which reproducibly suppressed visible growth for 24 h under test conditions (10-ml cultures with inocula of 106 to 107 CFU/ml). For all three antibiotics, these MICs were usually two- to eightfold higher than MICs determined by broth microdilution; exceptions were for vancomycin against S.
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TABLE 3. Efficacy of MDL 62,873 in experimental septicemia in micea Organism
S. aureus
Antibiotic
mic ED50 (MIgCm) (pg/m) (mEg/kgD (mgkg)"
MDL 62,873
Teicoplanin
Vancomycin
S. pyogenes
MDL 62,873
Teicoplanin Vancomycin S. pneumoniae
MDL 62,873
Teicoplanin Vancomycin a
95%
confidence
limits of ED50
0.5 1 1
0.19 0.15 0.82
0.16-0.23 0.13-0.18 0.53-1.28
0.06 0.06 0.13
0.09 0.16 0.47
0.08-0.11 0.15-0.19 0.43-0.52
0.13 0.03 0.25
0.71 0.29 1.9
0.61-0.82 0.26-0.32 1.53-2.28
ED,0 50%o effective dose.
bSingle administration.
epidermidis L533 and for teicoplanin against the two S. haemolyticus isolates, for which the inhibitory concentrations under test conditions were the same as the broth microdilution MICs. Against S. aureus L448, 2 or 4 ,g/ml of all three antibiotics killed about 99% of the inoculum within 6 h (vancomycin) or 24 h (MDL 62,873 and teicoplanin), although there was some regrowth at 24 h in the cultures exposed to vancomycin. Against S. aureus L1524, about a 99% kill was obtained with 4 jig of MDL 62,873 per ml and 8 ,ug of vancomycin per ml (twice the inhibitory concentrations) and 2 ,ug of teicoplanin per ml (the inhibitory concentration). MDL 62,873 was particularly bactericidal for both S. epidermidis isolates; a more than 99.9% kill was obtained with twice the inhibitory concentration (2 ,ug/ml for strain L533 and 4 ,g/ml for L1654). Similar results were obtained with teicoplanin, but the concentrations required were 16 and 32 ,g/ml. During the first 6 h, vancomycin killed the S. epidermidis isolates as rapidly as did MDL 62,873 (more than 99%), but the concentrations used were four times higher and no additional killing was observed at 24 h. Against the S. haemolyticus isolates, which were chosen because of their resistance to teicoplanin, MDL 62,873 and vancomycin had similar bactericidal activities. At 16 ,g/ml
(twice the inhibitory concentration), both antibiotics killed 99.9% of the inoculum of strain L1706 in 24 h. For isolate L1707, the bactericidal action of vancomycin was more rapid in the first 6 h, but the inhibitory concentration of either antibiotic (4 ,g of MDL 62,873 per ml or 8 ,ug of vancomycin per ml) killed more than 99% of the inoculum within 24 h. Mechanism of action. In B. subtilis, 0.25 ,ug of MDL 62,873 per ml specifically inhibited cell wall peptidoglycan synthesis to the same extent as the same concentration of teicoplanin, with little effect on other macromolecular syntheses (Fig. 2). Experimental infections. Against S. aureus and S. pyogenes septicemia in mice, the 50% effective doses of MDL 62,873 and teicoplanin were similar and were lower than those of vancomycin (Table 3). Against S. pneumoniae septicemia, teicoplanin was more active than MDL 62,873 while vancomycin was less active. In staphylococcal endocarditis in rats, 20 mg of MDL 62,873 per kg every 12 h and 40 mg of vancomycin per kg every 12 h had similar efficacies (P > 0.5) in lowering the bacterial titers in the hearts of rats infected with S. aureus, S. epidermidis, or S. haemolyticus (Table 4). In all three experiments, survival was similar for both treatment groups (P > 0.2). In the S. aureus experiment, 75% of the untreated animals died within 3 days and treatment with either antibiotic significantly increased the mean survival time (P < 0.01). The survival rates and the mean survival times were higher in untreated animals infected with S. epidermidis or S. haemolyticus; antibiotic treatment did not increase the survival time. Pharmacokinetics of MDL 62,873 in rats. The kinetics of MDL 62,873 in plasma is shown in Fig. 3. The mean concentrations in plasma were 193 ,ug/ml at 3 min after administration, 0.9 ,ug/ml at 24 h, and 0.1 ,ug/ml at 48 h, after which MDL 62,873 was no longer detectable. Mean levels in plasma declined with an apparent three-exponent decay, as previously observed for teicoplanin (5). The half-lives of the initial, intermediate, and terminal disposition phases were 0.06, 3.1, and 12.1 h. The terminal half-life may be underestimated because of the inability to detect concentrations below 0.1 ,ug/ml. The area under the concentration-time curve calculated by the trapezoidal rule (10) was 478 mg * h/ liter. Forty-one percent of the administered dose was excreted in the urine within the first 12 h. The cumulative
TABLE 4. Activity of MDL 62,873 and vancomycin in 5-day therapya for endocarditis Organism
S. aureus
S. epidermidis S. haemolyticus
Mean loglo CFU/g of heart tissue ± SDI
No. of rats,
survivors/total
Mean survival time ± (days SE)
All cases
Fatal cases
2/12 9/15 10/15
3.1 + 0.5 4.6 ± 0.3 5.2 ± 0.3
7.0 ± 2.8 3.9 ± 2.4C 3.2 ± 2.4d
7.9 ± 2.2 4.2 ± 2.5 3.8 ± 2.7
Vancomycin
0.25 2
6/12 9/14 7/14
5.1 ± 0.4 5.4 ± 0.3 5.4 ± 0.3
7.3 ± 0.5 4.7 ± 1.7d 4.8 ± 2.3d
7.3 ± 0.6 5.7 ± 0.9 5.4 ± 2.5
None MDL 62,873 Vancomycin
2 2
6/12 5/12 8/15
5.0 ± 0.4 4.6 ± 0.4 4.6 ± 0.2
6.4 ± 0.3 2.5 ± 1.2e 2.2 ± 1.6e
6.5 ± 0.4 3.2 ± 1.3 3.3 ± 1.9
Antibiotic
None MDL 62,873 Vancomycin None MDL 62,873
MIC (,ug/ml)
2 2
MDL 62,873 (20 mg/kg, i.v.) and vancomycin (40 mg/kg, i.v.) were administered every 12 h for 5 days or until death of the animal. Pretreatment titers determined in separate groups of infected animals were as follows: S. aureus, 5.8 ± 1.0 (four animals); S. epidermidis, 6.4 ± 0.4 (five animals); S. haemolyticus, 6.1 ± 0.2 (five animals). c P < 0.05 compared with untreated animals. d p < 0.01 compared with untreated animals. P p < 0.001 compared with untreated animals. a
b
ANTIMICROBIAL ACTIVITY OF MDL 62,873
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E)
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16 24
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40 48
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FIG. 3. Concentrations of MDL 62,873 in plasma. Each point is the mean of results with five animals treated i.v. with 20 mg/kg.
of antimicrobial activity in the urine 96 h after drug administration was 59%, indicating that, like teicoplanin (5), most of the MDL 62,873 was renally eliminated. The detection method does not distinguish between unaltered drug and microbiologically active metabolites which might be generated.
recovery
DISCUSSION
Glycopeptides are an important part of our armamentarium for combatting serious gram-positive infections and are particularly important against methicillin-resistant staphylococci. Infections due to coagulase-negative species have been increasing (3, 13, 19). Teicoplanin has a broad MIC distribution for coagulase-negative strains, and resistance or intermediate susceptibility has been reported previously (2, 9, 11, 12, 22, 23, 27, 31); vancomycin resistance also occurs (11, 27). Glycopeptide resistance is most often found among methicillin-resistant S. haemolyticus isolates. Many strains with reduced in vitro susceptibility may respond to therapy with existing glycopeptides. In rats and rabbits, endocarditis caused by staphylococcal isolates for which teicoplanin MICs were as high as 16 p.g/ml were successfully treated with 20 or 40 mg of teicoplanin per kg, given i.v. (6, 18). In particular cases, patients have been treated with teicoplanin at up to 30 mg/kg/day, a dosage which was well tolerated and gave trough levels of at least 20 ,ug/ml (25). Nevertheless, we have been searching for teicoplanin derivatives which have better activity against CNS and the pharmacokinetics and safety advantages of teicoplanin. MDL 62,211, the amide complex obtained by condensation of teicoplanin with 3,3-dimethylamino-1-propylamine, has excellent activity against CNS (4, 15, 21). MDL 62,873 is the corresponding amide derivative of teicoplanin A2-2. Its mechanism of antibacterial action is the same as that of teicoplanin, inhibition of cell wall peptidoglycan synthesis. In our studies of the in vitro activity of MDL 62,873, we biased the set of coagulase-negative isolates toward the high end of the teicoplanin MIC range, and we also included three of the rare S. aureus isolates for which teicoplanin MICs are as high as 8 jig/ml. MDL 62,873 was as active as teicoplanin against S. aureus isolates and somewhat more active than vancomycin; against the three S. aureus strains for which teicoplanin MICs were 8 jig/ml, the MIC of MDL 62,873 was 2 jig/ml. MDL 62,873 was more active than either vancomycin or teicoplanin against S. epidermidis and as
451
active as vancomycin against the S. haemolyticus isolates (which included teicoplanin-resistant but no vancomycinresistant strains) and against other coagulase-negative species (which included strains with intermediate susceptibility to teicoplanin). This is in agreement with the recent results of Jones et al. (16), who found MDL 62,873 to be more active than either teicoplanin or vancomycin against a large set of coagulase-negative isolates which were not divided by species. These authors also found the MIC of MDL 62,873 for 90%o of the S. aureus isolates tested to be lower than those of teicoplanin or vancomycin. We observed that MDL 62,873 was more active than vancomycin and similar to teicoplanin in its activity against streptococci and enterococci. Against six strains of staphylococci, MDL 62,873 had slow but significant bactericidal activity (as is typically observed with glycopeptides) at concentrations between 1 and 8 ,ug/ml. Against the S. aureus strains, for which the three glycopeptides had similar MICs, they also had comparable bactericidal activity. Against coagulase-negative isolates, chosen for reduced susceptibility or resistance to teicoplanin, the bactericidal activity of MDL 62,873 was equivalent to (for S. haemolyticus) or better than (for S. epidermidis) that of vancomycin when considered in terms of the concentrations of the antibiotics that produced significant killing. In mouse septicemia studies, in which single doses of antibiotics were administered, the efficacies of MDL 62,873 and teicoplanin were similar and were greater than those of vancomycin, probably because of the longer half-lives of MDL 62,873 and teicoplanin. In staphylococcal endocarditis in rats, MDL 62,873 (20 mg/kg i.v. every 12 h) was as efficacious as twice that dose of vancomycin in lowering heart bacterial titers. Thus, MDL 62,873 has an activity similar to what we previously observed for teicoplanin in this experimental model (1). The better activity of teicoplanin relative to that of vancomycin in our animal models (1) has been attributed to the longer half-life of teicoplanin (5). The pharmacokinetics of MDL 62,873 in rats are similar to those of teicoplanin and may be represented by a three-compartment model. Most of its elimination (87%) occurred during the second phase of disposition, which thus is the main contributor to the duration of antimicrobial activity in plasma; the third phase presumably: represents the slow release of the drug from the tissues to plasma and, as for teicoplanin (5), contributes little to levels in plasma. The microbiological and pharmacokinetic data suggest that MDL 62,873 may have potential as a new glycopeptide for the therapy for severe gram-positive infection. Toxicological evaluation and human pharmacokinetics studies are needed to further assess its therapeutic potential. ACKNOWLEDGMENTS We acknowledge the expert technical assistance of A. Resconi, P. Kaltofen, C. Acquarella, and R. Merati.
REFERENCES 1. Arioli, A., M. Berti, and G. Candiani. 1986. Activity of teicoplanin in localized experimental infections in rats. J. Hosp.
Infect. 7(Suppl. A):91-99.
2. Arioli, V., and R. Pallanza. 1987. Teicoplanin-resistant coagu-
lase-negative Staphylococcus. Lancet i:30. 3. Bailey, E. M., T. D. Constance, L. M. Albrecht, and M. J. Rybak. 1990. Coagulase-negative staphylococci: incidence, pathogenicity, and treatment in the 1990s. DICP Ann. Pharmacother. 24:714-720.
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4. Bartoloni, A., M. G. Colao, A. Orsi, R. Dei, E. Giganti, and F. Parenti. 1990. In vitro activity of vancomycin, daptomycin, ramoplanin, MDL 62,873 and other agents against staphylococci, enterococci and Clostridium difficile. J. Antimicrob. Chemother. 26:627-633. 5. Bernareggi, A., L. Cavenaghi, and A. Assandri. 1986. Pharmacokinetics of ['4C]teicoplanin in male rats after single intravenous dose. Antimicrob. Agents Chemother. 30:733-738. 6. Berti, M., and G. P. Candiani. 1989. Activity of teicoplanin in rat endocarditis caused by staphylococci with reduced in vitro susceptibility to teicoplanin. Program Abstr. 29th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 976. 7. Borghi, A., C. Coroneili, L. Faniuolo, G. Allievi, R. Pallanza, and G. G. Gallo. 1984. Teichomycins, new antibiotics from Actinoplanes teichomyceticus nov. sp. IV. Separation and characterization of the components of teichomycin (teicoplanin). J. Antibiot. 37:615-620. 8. Finney, D. J. 1952. The Spearman-Kaerber method, p. 524-530. In D. J. Finney (ed.), Statistical method in biological assay. Charles Griffin & Company Limited, London. 9. Froggatt, J. W., J. L. Johnston, D. W. Galetto, and G. L. Archer. 1989. Antimicrobial resistance in nosocomial isolates of Staphylococcus haemolyticus. Antimicrob. Agents Chemother.
33:460-466. 10. Gibaldi, M., and D. Perrier. 1982. Pharmacokinetics, 2nd ed., p. 445-449. Marcel Dekker, Inc., New York. 11. Goldstein, F., A. Coutrot, A. Seiffer, and J. F. Acar. 1990. Percentages and distribution of teicoplanin- and vancomycinresistant strains among coagulase-negative staphylococci. Antimicrob. Agents Chemother. 34:899-900. 12. Grant, A. C., R. W. Lacey, A. M. Brownjohn, and J. H. Turney. 1986. Teicoplanin-resistant coagulase-negative Staphylococcus. Lancet ii:1166-1167. 13. Gruer, L. D., R. Bartlett, and G. A. J. Ayliffe. 1984. Species identification and antibiotic sensitivity of coagulase-negative staphylococci from CAPD peritonitis. J. Antimicrob. Chemother. 13:577-583. 14. Heraief, E., M. P. Glauser, and L. R. Freedman. 1982. Natural history of aortic valve endocarditis in rats. Infect. Immun. 37:127-131. 15. Hunter, P. R., R. C. George, and J. W. Griffiths. 1990. Mathematical modeling of antimicrobial susceptibility data of Staphylococcus haemolyticus for 11 antimicrobial agents, including three experimental glycopeptides and an experimental lipoglycopeptide. Antimicrob. Agents Chemother. 34:1769-1772. 16. Jones, R. N., F. W. Goldstein, and X. Y. Zhou. 1991. Activities of two new teicoplanin amide derivatives (MDL 62211 and MDL 62873) compared with activities of teicoplanin and vancomycin against 800 recent staphylococcal isolates from France and the United States. Antimicrob. Agents Chemother. 35:584-586. 17. Kaatz, G. W., S. M. Seo, N. J. Dorman, and S. A. Lerner. 1988. Emergence of teicoplanin resistance during therapy of Staphy-
ANTIMICROB. AGENTS CHEMOTHER. lococcus aureus endocarditis. J. Infect. Dis. 162:103-108. 18. Kaatz, G. W., S. M. Seo, V. N. Reddy, E. M. Bailey, and M. J. Rybak. 1990. Daptomycin compared with teicoplanin and vancomycin for therapy of experimental Staphylococcus aureus endocarditis. Antimicrob. Agents Chemother. 34:2081-2085. 19. Karchmer, A. W., G. L. Archer, and W. E. Dismukes. 1983. Staphylococcus epidermidis causing prosthetic valve endocarditis: microbiologic and clinical observations as guides to therapy. Ann. Intern. Med. 98:447-455. 20. Leclercq, R., E. Derlot, J. Duval, and P. Courvalin. 1988. Plasmid-mediated resistance to vancomycin and teicoplanin in Enterococcus faecium. N. Engl. J. Med. 319:157-161. 21. Malabarba, A., A. Trani, P. Strazzolini, G. Cietto, P. Ferrari, G. Tarzia, R. Pallanza, and M. Berti. 1989. Synthesis and biological properties of N63-carboxamides of teicoplanin antibiotics. Structure-activity relationships. J. Med. Chem. 32:2450-2460. 22. Maugein, J., J. L. PeHlegrin, G. Brossard, J. Fourche, B. Leng, and J. Reiffers. 1990. In vitro activities of vancomycin and teicoplanin against coagulase-negative staphylococci isolated from neutropenic patients. Antimicrob. Agents Chemother. 34:901-903. 23. Moore, E. P., and D. C. E. Speller. 1988. In vitro teicoplaninresistance in coagulase-negative staphylococci from patients with endocarditis and from a cardiac surgery unit. J. Antimicrob. Chemother. 21:417-424. 24. National Committee for Clinical Laboratory Standards. 1990. Approved standard M7-A2. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 2nd ed. National Committee for Clinical Laboratory Standards, Villanova, Pa. 25. Rybak, M. J., S. A. Lerner, D. P. Levine, L. M. Albrecht, P. L. McNeil, G. A. Thompson, M. T. Kenny, and L. Yuh. 1991. Teicoplanin pharmacokinetics in intravenous drug abusers being treated for bacterial endocarditis. Antimicrob. Agents Chemother. 35:696-700. 26. SAS Institute, Inc. 1990. SAS user's guide: statistics. Version 6.06.-1990. SAS Institute, Inc., Cary, N.C. 27. Schwalbe, R. S., J. T. Stapleton, and P. H. Gilligan. 1987. Emergence of vancomycin resistance in coagulase-negative staphylococci. N. Engl. J. Med. 316:927-931. 28. Straka, R. P., and J. L. Stokes. 1957. Rapid destruction of bacteria in commonly used diluents and its elimination. Appl. Microbiol. 5:21-25. 29. Uttley, A. H., C. H. Collins, J. Naidoo, and R. C. George. 1988. Vancomycin-resistant enterococci. Lancet i:57-58. 30. Williamson, R., S. Al-Obeid, J. H. Shlaes, F. W. Goldstein, and D. M. Shlaes. 1989. Inducible resistance to vancomycin in Enterococcus faecium D366. J. Infect. Dis. 159:1095-1104. 31. Wilson, A. P. R., M. D. O'Hare, D. Felmingham, and R. N. Gruneberg. 1986. Teicoplanin-resistant coagulase-negative Staphylococcus. Lancet ii:973.
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Antimicrobial Activity of MDL 62,873, a Semisynthetic Derivative of Teicoplanin, In Vitro and in Experimental Infections MARISA BERTI, GIAMPAOLO CANDIANI, MONICA BORGONOVI, PAOLO LANDINI, FRANCA RIPAMONTI, ROBERTO SCOTTI, LUIGI CAVENAGHI, MAURIZIO DENARO, AND BETH P. GOLDSTEIN*
Lepetit Research Center, Marion Merrell Dow Research Institute, Via R. Lepetit 34, 21040 Gerenzano (Varese), Italy Received 16 August 1991/Accepted 5 December 1991
MDL 62,873 is an amide derivative of teicoplanin A2-2. Like those of natural glycopeptides, its antibacterial activity is mediated by inhibition of cell wall peptidoglycan synthesis. Against streptococci and enterococci, the in vitro activity of MDL 62,873 was similar to that of teicoplanin and greater than that of vancomycin. Against staphylococci, it has activity similar to that of vancomycin, and it was significantly more active than teicoplanin against coagulase-negative isolates. Like teicoplanin and vancomycin, MDL 62,873 had slow but significant bactericidal activity (99 to 99.9% killing in 24 h) against staphylococci at concentrations near the MIC. In murine septicemia studies with Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae, the 50% effective doses were lower than those of vancomycin. In staphylococcal endocarditis in rats, MDL 62,873 at 20 mg/kg of body weight and vancomycin at 40 mg/kg, both doses given intravenously twice daily, had similar efficacies in reducing the heart bacterial load. These results probably reflect the longer half-life of MDL 62,873, which has a pharmacokinetic profile in rats similar to that of teicoplanin.
Because of the emergence of methicillin-resistant staphylococci, glycopeptide antibiotics have received more attention over the last several years. Among coagulase-negative isolates, whose incidence in serious infection has been increasing (3, 13, 19), teicoplanin-resistant and occasional vancomycin-resistant strains have been reported (2, 9, 11, 12, 22, 23, 27, 31). We have been seeking glycopeptide derivatives with improved activities against these strains. MDL 62,211 (CTA-A-1), the amide obtained by the condensation of teicoplanin complex with 3,3-dimethylamino-1propylamine, had greater in vitro activity than teicoplanin against coagulase-negative staphylococci and was as active as teicoplanin against other bacterial species (15, 21). We have continued our studies with MDL 62,873, the amide of the major peak (A2-2) of the teicoplanin complex. We present data for the antibacterial activity of MDL 62,873 in vitro and in experimental infections in animals as well as data for its pharmacokinetics in rats.
the coagulase-negative staphylococci (CNS) we chose a number of strains for which teicoplanin MICs were relatively high. For mouse septicemia experiments, three laboratory strains, Staphylococcus aureus Smith, Streptococcus pyogenes C203, and Streptococcus pneumoniae Felton UC41 were used. For rat endocarditis experiments, we used clinical isolates for which teicoplanin MICs were 8 to 16 ,ug/ml: S. aureus SA12873, kindly provided by G. W. Kaatz (17), and two methicillin-resistant CNS (Staphylococcus epidermidis L537 and Staphylococcus haemolyticus L1520). We used Bacillus subtilis 566/1 (thyA thyB) for mechanism of action studies. MIC determination. MICs were determined by broth microdilution. Streptococci were grown in Todd-Hewitt broth (Difco), and other organisms were grown in Iso-Sensitest broth (Oxoid). Inocula were approximately 5 x 105 CFU/ml from overnight culture plates (24); incubation was at 37°C for 24 h. Bactericidal activity. Logarithmically growing cultures were diluted and inoculated into Iso-Sensitest broth at a density of 106 to 107 CFU/ml; 10-ml portions were distributed into flasks with and without antibiotics. The time-kill studies were performed with the lowest concentration of each antibiotic which reproducibly inhibited visible growth of the isolate under test conditions and twice this concentration. These concentrations were usually two- to eightfold the MICs determined by broth microdilution (see Results). At intervals after incubation in a stationary 37°C water bath, samples were suitably diluted (at least 10-fold) in saline with 0.1% peptone; the addition of peptone has been reported to increase the recovery of bacteria (28). Duplicate 0.1-ml samples were plated by inclusion in 2.5 ml of Iso-Sensitest soft agar (0.7% agar) on Iso-Sensitest agar plates. This results in an immediate 250-fold dilution of the antibiotic, which, in our experience, is sufficient to prevent carryover effects with the concentrations used. At the inocula used, 99.9% killing can be reliably detected as 10 to 100 colonies per plate at the lowest dilution. Mechanism of action. B. subtilis was grown to an optical
MATERIALS AND METHODS Antibacterial agents. MDL 62,873 and teicoplanin powders were from Lepetit. The MDL 62,873 preparations used contained 75 to 80% MDL 62,873 and 20 to 25% the corresponding amides of other components of the teicoplanin complex. This is the preparation intended for further development. Different components of the teicoplanin complex have similar in vitro and in vivo activities (7), and the MDL 62,873 preparation used here had an in vitro activity similar to that of MDL 62,211 (the amide derivative of the teicoplanin complex) (16). Therefore, we do not expect there to be significant differences in activities among the components of the MDL 62,873 preparation. We also used vancomycin (Vancocin HCI; Eli Lilly), ampicillin (Amplital; Farmitalia-Carlo Erba), and erythromycin (Sigma). Bacterial strains. For in vitro studies, we used clinical isolates of staphylococci, streptococci, and enterococci. Of *
Corresponding author. 446
TABLE 1. Activity of MDL 62,873 against staphylococci Organism (no. of isolates)
S. aureus (50)
S. epidermidis (30)
S. haemolyticus (34)
Other CNS (21)"
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Antibiotic MDL 62,873 Teicoplanin Vancomycin
MIC (Rg/ml)a
Range 0.13-2 0.13-8
MIC50 0.25 0.5
MIC90
0.25-4
1
1 1 2
MDL 62,873 Teicoplanin Vancomycin
0.06-0.5 0.13-16
0.13 4 2
0.25 8 2
MDL 62,873 Teicoplanin Vancomycin
0.13-8 1-64
MDL 62,873 Teicoplanin Vancomycin
0.06-1 0.06-16 0.25-2
1-4
1-4
0.5 16 2 0.13 1 0.5
4 64 4 1 8 1
a MIC50, MIC for 509o of the isolates tested; MIC90, MIC for 90%o of the isolates tested. b S. simulans (7), S. hominis (5), S. saprophyticus (3), S. cohnii (2), S. capitis, S. lugdunensis, S. warneri, and S. xylosus (1 each).
density at 590 nm of 0.2 in Davis-Mingioli medium supplemented with 2% glucose, 0.4% Casamino Acids, 100 ,±g of asparagine per ml, and 1 ,ug of thymine per ml. Macromolecular syntheses (DNA, RNA, protein, and cell wall peptidoglycan) were followed by incorporation of the appropriate radioactive precursors: for DNA synthesis, [methyl-3H]thymidine (2 puCi/ml) diluted with 2 ,ug of cold thymidine per ml; for RNA synthesis, [3H]uridine (1 ,uCi/ml, 10 ,ug/ml); for protein synthesis, [3H]tryptophan (1 ,uCi/mmol, 1 ,ug/ml); for peptidoglycan synthesis, [3H]N-acetylglucosamine (2 ,uCi/ ml, 3.5 ,ug/ml). Samples were precipitated with cold 5% trichloroacetic acid. Samples for protein synthesis were heated for 10 min at 80°C. Trichloroacetic acid-precipitated material was collected on glass fiber filters with a cell harvester (LKB) and counted in a P-plate scintillation counter (Pharmacia). Experimental septicemia in mice. CD-1 mice (Charles River Breeding Laboratories) weighing 18 to 22 g were infected intraperitoneally with 8 x 104 CFU of S. pyogenes, S x 10' CFU of S. pneumoniae (both in 0.5 ml of saline plus 1% peptone), or 2 x 106 CFU of S. aureus (in 0.5 ml of 5% Difco bacteriological mucin). These challenges corresponded to 50 to 150 times the 50% lethal doses for the infecting organisms. Untreated animals died within 48 h after infection. Within 10 min after infection, the animals were given a single subcutaneous administration of the antibiotics. In each experiment, four or five groups of 10 mice each (five males and five females) were treated with different dose levels of each antibiotic. The 50% effective doses and 95% confidence limits were calculated by the Spearman-Kaerber method (8) from the percentages of animals surviving to day 7 at each dose. Experimental endocarditis. Endocarditis was induced in male CD rats (Charles River Breeding Laboratories) weighing about 200 g. A polyethylene catheter (inside diameter = 0.4 mm, outside diameter = 0.8 mm; Portex) was inserted through the aortic valve into the left ventricle via the right carotid artery and secured with a silk ligature. Correct positioning of the catheter was controlled with a recording amplifier (Hewlett-Packard model 7782A) with a pressure transducer. Two days later, rats were injected intravenously
(i.v.) with 0.5 ml of a bacterial suspension. Undiluted overnight cultures in Todd-Hewitt broth were used for S. epidermidis and S. haemolyticus; an overnight culture of S. aureus in Trypticase soy broth (BBL) was diluted to 2 x 104 CFU/ml in saline with 1% peptone. In each experiment, four or five animals were killed just before the first treatment to provide baseline bacterial counts. Rats were treated i.v. with 20 mg of MDL 62,873 per kg of body weight or 40 mg of vancomycin per kg every 12 h for 5 days. The first treatment was given 24 h (CNS) or 16 h (S. aureus) after infection. With these doses, mean peak levels in plasma (1 h after the ninth treatment) were 48 jxg/ml for MDL 62,873 and 46 jig/ml for vancomycin; mean trough levels (12 h after the tenth treatment) were 3 pug/ml for MDL 62,873 and 0.2 jig/ml for vancomycin. Animals that died before the end of the experiment were autopsied and their hearts were removed; surviving animals were killed 12 h after the last treatment. The hearts were weighed and homogenized. To avoid antibiotic carryover, the homogenates were centrifuged, suspended in saline with 1% peptone, and washed three times by centrifugation. Duplicate 0.025-ml aliquots of appropriate dilutions were plated on Todd-Hewitt agar to determine the bacterial heart loads. The presence of blood in the heart homogenates did not influence the results, as bacterial titers in the blood were always at least 1,000-fold lower than the bacterial heart loads. Rats that died within 40 or 48 h after infection (with S. aureus and CNS, respectively) were excluded from statistical analysis because these deaths were likely the result of catheter placement (14) and because the length of treatment would be too short to expect any significant effect on bacterial load. Data were examined by covariance analysis, with time of death as the covariant. Least-square means were used to evaluate differences in bacterial titers between
TABLE 2. Activity of MDL 62,873 against streptococci and enterococci Organism (no. of isolates)
S. pyogenes (15)
Antibiotic MDL 62,873 Ampicillin Erythromycin Teicoplanin Vancomycin
MIC (Jug/ml)a Range M1C50 MIC90 0.06 0.03-0.13 0.03 0.03 0.016 0.004-0.13 0.06 0.03-0.06 0.06 0.13 0.06-0.13 0.06 0.25 0.5 0.13-0.5
0.06-0.13 0.06-4 Ampicillin Erythromycin 0.016->128 0.03-0.25 Teicoplanin 0.06-0.5 Vancomycin
S. pneumoniae (15) MDL 62,873
Other streptococci MDL 62,873 Ampicillin (19)b
Erythromycin Teicoplanin Vancomycin Enterococci (15)C
0.016-0.25 0.016-0.5 0.008->128 0.03-1 0.5-2
MDL 62,873
0.06-0.25
Ampicillin Teicoplanin Vancomycin
1-16 0.06-0.25 0.5-2
MIC0,,
0.13 0.06 0.06 0.06 0.25
0.13 0.13 >128 0.13 0.5
0.13 0.06 0.25 0.06 0.016 >128 0.25 0.13 2 1 0.13 1 0.06 1
0.25 16 0.25 2
a MIC for 90% of the MIC50, MIC for 509o of the isolates tested; isolates tested. b S. agalactiae, S. salivarius, S. sanguis (4 each); S. bovis, S. mutans (3 each); and S. acidominimus (1). c
E. faecalis (12) and E. faecium (3).
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FIG. 1. Bactericidal activities of MDL 62,873 (0, 0), teicoplanin (O, U), and vancomycin (A, A); untreated control (0). Concentrations chosen were the MIC (0, 0, A) and twice the MIC (0, *, A) as determined under the same conditions used for the experiments. MICs of MDL 62,873, teicoplanin, and vancomycin were, respectively, as follows: (A) for S. aureus L448, 2, 2, and 2 ,ug/ml; (B) for S. aureus L1524, 2, 2, and 4 ,ug/ml; (C) for S. epidermidis L533, 1, 8, and 4 ,ug/ml; (D) for S. epidermidis L1654, 2, 16, and 8 ,g/ml; (E) for S. haemolyticus L1706, 8, 32, and 8 ,Ig/ml; (F) for S. haemolyticus L1707, 4, 32, and 8 ,ug/ml.
control and treatment groups, and P was corrected for multiple comparisons by using the formula P' = 1 - (1 P)n, where n is the number of preplanned comparisons. Mean survival time the standard error was calculated by survival analysis by the SAS Lifetest procedure (26); differences in survival times between control and treatment groups were evaluated by analysis of variance of the reciprocal values of survival time. Pharmacokinetics in rats. Two groups of five male rats each were treated i.v. with a single 20-mg/kg dose of MDL 62,873 administered in 2 ml of 5% glucose per kg. At the time of treatment, the mean weight the standard deviation of rats used for plasma kinetics was 182 5 g; the rats used for urinary excretion analysis weighed 189 5 g. The rats were lightly anesthetized with halothane, and 0.3 to 0.5 ml of blood was collected in heparinized tubes at 0.05, 1, 2, 4, 6, 8, 12, 24, 36, 48, 56, 72, 80, and 96 h after drug administration. The plasma was collected by centrifugation. Urine specimens were collected in metabolic cages at 12, 24, 48, 72, and ±
±
±
±
96 h after drug administration. Concentrations of MDL 62,873 in urine and plasma samples were determined by microbiological assay by using Micrococcus luteus ATCC 9341 as the test organism.
RESULTS In vitro activity. Table 1 shows the MICs of MDL 62,873, teicoplanin, and vancomycin for a series of 134 staphylococci. We included strains for which teicoplanin MICs were known to be :8 ,ug/ml (3 of S. aureus, 8 of S. epidermidis, 27 of S. haemolyticus, and 3 isolates of other coagulase-negative species). MDL 62,873 showed excellent activity, with a MIC of si ,ug/ml for the majority of the isolates. The few isolates for which MDL 62,873 MICs were higher than 1 ,ug/ml included three S. aureus isolates (MIC of 2 ,ug/ml versus 8 ,ug/ml for teicoplanin and 2 to 4 ,ug/ml for vancomycin) and four S. haemolyticus isolates, two of which were inhibited by 4 pug/ml and two of which were inhibited by 8
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FIG. 2. Mechanism of action of MDL 62,873. (A) DNA synthesis; (B) RNA synthesis; (C) protein synthesis; (D) peptidoglycan synthesis. Symbols: 0, control; A, MDL 62,873 at 0.25 ,ug/ml; 4, teicoplanin at 0.25 ,ug/ml. Arrows indicate the times of addition of the antibiotics.
,ug/ml of MDL 62,873; for the four S. haemolyticus isolates, teicoplanin MICs were 16 to 64 p,g/ml and vancomycin MICs were 4 ,ug/ml. With the exception of two of these S. haemolyticus strains, the activity of MDL 62,873 was similar to or greater than that of vancomycin against all isolates. Against streptococci and enterococci, the activity of MDL 62,873 was similar to that of teicoplanin and, for most of the strains, greater than that of vancomycin (Table 2). The MICs of MDL 62,873 were similar to those of ampicillin and erythromycin; as expected, MDL 62,873 was also active against isolates resistant to these antibiotics (Table 2). MDL 62,873 was as active as teicoplanin against a vanB strain of Enterococcusfaecium (30) which is resistant to vancomycin
and susceptible to teicoplanin; it had no activity against vanA strains of E. faecium (20, 29), which are resistant to both teicoplanin and vancomycin (data not shown). Bactericidal activity. The bactericidal activity of MDL 62,873 was compared with those of teicoplanin and vancomycin against two isolates each of S. aureus, S. epidermidis, and S. haemolyticus (Fig. 1). For these experiments, the MIC was defined as the lowest concentration of an antibiotic which reproducibly suppressed visible growth for 24 h under test conditions (10-ml cultures with inocula of 106 to 107 CFU/ml). For all three antibiotics, these MICs were usually two- to eightfold higher than MICs determined by broth microdilution; exceptions were for vancomycin against S.
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ANTIMICROB. AGENTS CHEMOTHER.
TABLE 3. Efficacy of MDL 62,873 in experimental septicemia in micea Organism
S. aureus
Antibiotic
mic ED50 (MIgCm) (pg/m) (mEg/kgD (mgkg)"
MDL 62,873
Teicoplanin
Vancomycin
S. pyogenes
MDL 62,873
Teicoplanin Vancomycin S. pneumoniae
MDL 62,873
Teicoplanin Vancomycin a
95%
confidence
limits of ED50
0.5 1 1
0.19 0.15 0.82
0.16-0.23 0.13-0.18 0.53-1.28
0.06 0.06 0.13
0.09 0.16 0.47
0.08-0.11 0.15-0.19 0.43-0.52
0.13 0.03 0.25
0.71 0.29 1.9
0.61-0.82 0.26-0.32 1.53-2.28
ED,0 50%o effective dose.
bSingle administration.
epidermidis L533 and for teicoplanin against the two S. haemolyticus isolates, for which the inhibitory concentrations under test conditions were the same as the broth microdilution MICs. Against S. aureus L448, 2 or 4 ,g/ml of all three antibiotics killed about 99% of the inoculum within 6 h (vancomycin) or 24 h (MDL 62,873 and teicoplanin), although there was some regrowth at 24 h in the cultures exposed to vancomycin. Against S. aureus L1524, about a 99% kill was obtained with 4 jig of MDL 62,873 per ml and 8 ,ug of vancomycin per ml (twice the inhibitory concentrations) and 2 ,ug of teicoplanin per ml (the inhibitory concentration). MDL 62,873 was particularly bactericidal for both S. epidermidis isolates; a more than 99.9% kill was obtained with twice the inhibitory concentration (2 ,ug/ml for strain L533 and 4 ,g/ml for L1654). Similar results were obtained with teicoplanin, but the concentrations required were 16 and 32 ,g/ml. During the first 6 h, vancomycin killed the S. epidermidis isolates as rapidly as did MDL 62,873 (more than 99%), but the concentrations used were four times higher and no additional killing was observed at 24 h. Against the S. haemolyticus isolates, which were chosen because of their resistance to teicoplanin, MDL 62,873 and vancomycin had similar bactericidal activities. At 16 ,g/ml
(twice the inhibitory concentration), both antibiotics killed 99.9% of the inoculum of strain L1706 in 24 h. For isolate L1707, the bactericidal action of vancomycin was more rapid in the first 6 h, but the inhibitory concentration of either antibiotic (4 ,g of MDL 62,873 per ml or 8 ,ug of vancomycin per ml) killed more than 99% of the inoculum within 24 h. Mechanism of action. In B. subtilis, 0.25 ,ug of MDL 62,873 per ml specifically inhibited cell wall peptidoglycan synthesis to the same extent as the same concentration of teicoplanin, with little effect on other macromolecular syntheses (Fig. 2). Experimental infections. Against S. aureus and S. pyogenes septicemia in mice, the 50% effective doses of MDL 62,873 and teicoplanin were similar and were lower than those of vancomycin (Table 3). Against S. pneumoniae septicemia, teicoplanin was more active than MDL 62,873 while vancomycin was less active. In staphylococcal endocarditis in rats, 20 mg of MDL 62,873 per kg every 12 h and 40 mg of vancomycin per kg every 12 h had similar efficacies (P > 0.5) in lowering the bacterial titers in the hearts of rats infected with S. aureus, S. epidermidis, or S. haemolyticus (Table 4). In all three experiments, survival was similar for both treatment groups (P > 0.2). In the S. aureus experiment, 75% of the untreated animals died within 3 days and treatment with either antibiotic significantly increased the mean survival time (P < 0.01). The survival rates and the mean survival times were higher in untreated animals infected with S. epidermidis or S. haemolyticus; antibiotic treatment did not increase the survival time. Pharmacokinetics of MDL 62,873 in rats. The kinetics of MDL 62,873 in plasma is shown in Fig. 3. The mean concentrations in plasma were 193 ,ug/ml at 3 min after administration, 0.9 ,ug/ml at 24 h, and 0.1 ,ug/ml at 48 h, after which MDL 62,873 was no longer detectable. Mean levels in plasma declined with an apparent three-exponent decay, as previously observed for teicoplanin (5). The half-lives of the initial, intermediate, and terminal disposition phases were 0.06, 3.1, and 12.1 h. The terminal half-life may be underestimated because of the inability to detect concentrations below 0.1 ,ug/ml. The area under the concentration-time curve calculated by the trapezoidal rule (10) was 478 mg * h/ liter. Forty-one percent of the administered dose was excreted in the urine within the first 12 h. The cumulative
TABLE 4. Activity of MDL 62,873 and vancomycin in 5-day therapya for endocarditis Organism
S. aureus
S. epidermidis S. haemolyticus
Mean loglo CFU/g of heart tissue ± SDI
No. of rats,
survivors/total
Mean survival time ± (days SE)
All cases
Fatal cases
2/12 9/15 10/15
3.1 + 0.5 4.6 ± 0.3 5.2 ± 0.3
7.0 ± 2.8 3.9 ± 2.4C 3.2 ± 2.4d
7.9 ± 2.2 4.2 ± 2.5 3.8 ± 2.7
Vancomycin
0.25 2
6/12 9/14 7/14
5.1 ± 0.4 5.4 ± 0.3 5.4 ± 0.3
7.3 ± 0.5 4.7 ± 1.7d 4.8 ± 2.3d
7.3 ± 0.6 5.7 ± 0.9 5.4 ± 2.5
None MDL 62,873 Vancomycin
2 2
6/12 5/12 8/15
5.0 ± 0.4 4.6 ± 0.4 4.6 ± 0.2
6.4 ± 0.3 2.5 ± 1.2e 2.2 ± 1.6e
6.5 ± 0.4 3.2 ± 1.3 3.3 ± 1.9
Antibiotic
None MDL 62,873 Vancomycin None MDL 62,873
MIC (,ug/ml)
2 2
MDL 62,873 (20 mg/kg, i.v.) and vancomycin (40 mg/kg, i.v.) were administered every 12 h for 5 days or until death of the animal. Pretreatment titers determined in separate groups of infected animals were as follows: S. aureus, 5.8 ± 1.0 (four animals); S. epidermidis, 6.4 ± 0.4 (five animals); S. haemolyticus, 6.1 ± 0.2 (five animals). c P < 0.05 compared with untreated animals. d p < 0.01 compared with untreated animals. P p < 0.001 compared with untreated animals. a
b
ANTIMICROBIAL ACTIVITY OF MDL 62,873
VOL. 36, 1992 1000 100-
E)
10
0.1
0
8
16 24
32
40 48
Hours
FIG. 3. Concentrations of MDL 62,873 in plasma. Each point is the mean of results with five animals treated i.v. with 20 mg/kg.
of antimicrobial activity in the urine 96 h after drug administration was 59%, indicating that, like teicoplanin (5), most of the MDL 62,873 was renally eliminated. The detection method does not distinguish between unaltered drug and microbiologically active metabolites which might be generated.
recovery
DISCUSSION
Glycopeptides are an important part of our armamentarium for combatting serious gram-positive infections and are particularly important against methicillin-resistant staphylococci. Infections due to coagulase-negative species have been increasing (3, 13, 19). Teicoplanin has a broad MIC distribution for coagulase-negative strains, and resistance or intermediate susceptibility has been reported previously (2, 9, 11, 12, 22, 23, 27, 31); vancomycin resistance also occurs (11, 27). Glycopeptide resistance is most often found among methicillin-resistant S. haemolyticus isolates. Many strains with reduced in vitro susceptibility may respond to therapy with existing glycopeptides. In rats and rabbits, endocarditis caused by staphylococcal isolates for which teicoplanin MICs were as high as 16 p.g/ml were successfully treated with 20 or 40 mg of teicoplanin per kg, given i.v. (6, 18). In particular cases, patients have been treated with teicoplanin at up to 30 mg/kg/day, a dosage which was well tolerated and gave trough levels of at least 20 ,ug/ml (25). Nevertheless, we have been searching for teicoplanin derivatives which have better activity against CNS and the pharmacokinetics and safety advantages of teicoplanin. MDL 62,211, the amide complex obtained by condensation of teicoplanin with 3,3-dimethylamino-1-propylamine, has excellent activity against CNS (4, 15, 21). MDL 62,873 is the corresponding amide derivative of teicoplanin A2-2. Its mechanism of antibacterial action is the same as that of teicoplanin, inhibition of cell wall peptidoglycan synthesis. In our studies of the in vitro activity of MDL 62,873, we biased the set of coagulase-negative isolates toward the high end of the teicoplanin MIC range, and we also included three of the rare S. aureus isolates for which teicoplanin MICs are as high as 8 jig/ml. MDL 62,873 was as active as teicoplanin against S. aureus isolates and somewhat more active than vancomycin; against the three S. aureus strains for which teicoplanin MICs were 8 jig/ml, the MIC of MDL 62,873 was 2 jig/ml. MDL 62,873 was more active than either vancomycin or teicoplanin against S. epidermidis and as
451
active as vancomycin against the S. haemolyticus isolates (which included teicoplanin-resistant but no vancomycinresistant strains) and against other coagulase-negative species (which included strains with intermediate susceptibility to teicoplanin). This is in agreement with the recent results of Jones et al. (16), who found MDL 62,873 to be more active than either teicoplanin or vancomycin against a large set of coagulase-negative isolates which were not divided by species. These authors also found the MIC of MDL 62,873 for 90%o of the S. aureus isolates tested to be lower than those of teicoplanin or vancomycin. We observed that MDL 62,873 was more active than vancomycin and similar to teicoplanin in its activity against streptococci and enterococci. Against six strains of staphylococci, MDL 62,873 had slow but significant bactericidal activity (as is typically observed with glycopeptides) at concentrations between 1 and 8 ,ug/ml. Against the S. aureus strains, for which the three glycopeptides had similar MICs, they also had comparable bactericidal activity. Against coagulase-negative isolates, chosen for reduced susceptibility or resistance to teicoplanin, the bactericidal activity of MDL 62,873 was equivalent to (for S. haemolyticus) or better than (for S. epidermidis) that of vancomycin when considered in terms of the concentrations of the antibiotics that produced significant killing. In mouse septicemia studies, in which single doses of antibiotics were administered, the efficacies of MDL 62,873 and teicoplanin were similar and were greater than those of vancomycin, probably because of the longer half-lives of MDL 62,873 and teicoplanin. In staphylococcal endocarditis in rats, MDL 62,873 (20 mg/kg i.v. every 12 h) was as efficacious as twice that dose of vancomycin in lowering heart bacterial titers. Thus, MDL 62,873 has an activity similar to what we previously observed for teicoplanin in this experimental model (1). The better activity of teicoplanin relative to that of vancomycin in our animal models (1) has been attributed to the longer half-life of teicoplanin (5). The pharmacokinetics of MDL 62,873 in rats are similar to those of teicoplanin and may be represented by a three-compartment model. Most of its elimination (87%) occurred during the second phase of disposition, which thus is the main contributor to the duration of antimicrobial activity in plasma; the third phase presumably: represents the slow release of the drug from the tissues to plasma and, as for teicoplanin (5), contributes little to levels in plasma. The microbiological and pharmacokinetic data suggest that MDL 62,873 may have potential as a new glycopeptide for the therapy for severe gram-positive infection. Toxicological evaluation and human pharmacokinetics studies are needed to further assess its therapeutic potential. ACKNOWLEDGMENTS We acknowledge the expert technical assistance of A. Resconi, P. Kaltofen, C. Acquarella, and R. Merati.
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