Biosearch Italia S.p.A., Via R. Lepetit, 34-21040 Gerenzano (Varese), Italy
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Abstract |
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Introduction |
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Here we present data on BI 397, a new amide derivative of the natural glycopeptide antibiotic A40926 (Figure 1). This compound shows promising in-vitro and in-vivo activity as well as favourable pharmacokinetic behaviour. It appears to combine, in a single antibiotic, the favourable properties of vancomycin and teicoplanin.
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Materials and methods |
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BI 397 was produced at Biosearch Italia Spa, teicoplanin was obtained from Hoechst Marion Roussel (Italy) and LY 333328 was a kind gift from Eli Lilly and Co. (Indianapolis, IN, USA). Vancomycin and penicillin G sodium salt were obtained from Sigma Chemical Co. (St Louis, MO, USA). A pharmaceutical preparation of procaine penicillin G (Streuli & Co. AG, Uznach, Switzerland) and vancomycin (Eli Lilly) were used in the experimental infections.
MIC determinations
Bacterial isolates were obtained from various clinical sources. The set of staphylococci used for these experiments was biased to include a high proportion of strains for which teicoplanin MICs were known to be high and a number of methicillin-resistant strains. The set of streptococci included some penicillin-resistant strains, while enterococci were chosen to have a representative number of vancomycin-sensitive and -resistant strains. MICs were performed using the broth microdilution methodology with inocula of approximately 5 x 105 cfu/mL. The media employed included cation-adjusted MuellerHinton broth (Difco Laboratories, Detroit, MI, USA) for staphylococci and enterococci or MuellerHinton broth supplemented with 5% (v/v) fetal calf serum for streptococci. Where appropriate, tests were performed in media supplemented with 50% (v/v) bovine serum. Tests were read after 24 h incubation at 37°C.
Bactericidal activity
After overnight growth on MuellerHinton agar plates (Difco), several colonies were inoculated into cation-adjusted MuellerHinton broth and incubated with moderate agitation at 37°C. After two or more generations of logarithmic growth, cultures were diluted to 105106 cfu/mL with fresh, pre-warmed broth, and 10 mL aliquots were added to pre-warmed flasks with or without the addition of antibiotics. The antibiotic concentrations chosen were four-, eight- and 16-fold the lowest concentration that inhibited visible growth for at least 24 h in preliminary flask experiments. The flasks were agitated as described above and samples were withdrawn at intervals up to 24 h. Duplicate 0.1 mL aliquots of suitable dilutions (at least 10-fold) in 0.1% Difco peptone-0.9% NaCl (PSS) were mixed with 2.5 mL MuellerHinton broth containing 0.7% agar and spread on MuellerHinton agar plates. No antibiotic carryover effects were observed in preliminary experiments when low inocula (102103 cfu/mL) were plated immediately after addition of the highest antibiotic concentration. Colonies were counted after 24 h incubation at 37°C.
Animals
CD1 mice (Charles River Breeding Laboratories, Calco, Italy) of both sexes weighing 2022 g and six- to eight-week-old female NMRI mice (Iffa-Credo, France) were used in the models of acute septicaemia in immunocompetent and neutropenic animals, respectively. Female (pneumonia experiments) and male (endocarditis experiments and pharmacokinetic studies) CD rats (Charles River Breeding Laboratories) were used in the models of localized infection. All studies were approved by the Animal Care Committee at our institution.
Induction of neutropenia
Female NMRI mice were treated ip with 150 mg/kg of cyclophosphamide (ASTA Medica AG, Frankfurt, Germany) on days 5, 3 and 1 (before infection), as previously reported.24 For pneumonia experiments in neutropenic animals, rats were given two ip injections of cyclophosphamide 80 mg/kg on days 4 and 1 (before infection).25 The animals were housed throughout the experiments in a cabinet ventilated with HEPA-filtered air. In each experiment, a group of five animals was reserved for monitoring neutropenia. Blood was collected from the tail vein just before the first cyclophosphamide treatment and 24 h after the last treatment. Total leucocytes were determined using a Bürker counting chamber. Differential count was determined by microscopic examination after staining with May-Grünwald-Giemsa (E. Merck, Darmstadt, Germany). In experiments in which neutropenic mice were used, after cyclophosphamide treatment, total leucocyte counts fell from an initial mean of 11.5 x 109/L (c. 10% granulocytes) on day 5 to a mean of 0.8 x 109/L (c. 1.2% granulocytes) at the time the other groups of animals were infected. In experiments with neutropenic rats, following cyclophosphamide treatment, the leucocyte counts fell from a pretreatment mean of 10.0 x 109/L (c. 11.6% granulocytes) of blood to a mean of 1.0 x 109/L (c. 5.2% granulocytes) at the time that other groups of animals were infected.
Prophylaxis of septicaemia
Acute septicaemia experiments were performed as described previously.26 Immunocompetent mice (six animals per treatment group) were infected ip with 0.5 mL of a bacterial suspension containing 1.8 x 106 cfu of S. aureus Smith or 2.6 x 102 cfu of S. pneumoniae L44 (approximately 20 or 100 times the 50% lethal dose, respectively, for untreated animals). Treatment with antibiotics was by the sc route, within 10 min after infection; in the pneumococcal infection, vancomycin was administered a second time, 5 h after infection.
For the infection of neutropenic animals, mice (five animals per treatment group) were inoculated ip with 0.5 mL of a bacterial suspension containing 1.1 x 105 cfu of Staphylococcus epidermidis L1480 or 3.2 x 104 cfu of Enterococcus faecalis L1139, prepared by dilution of an overnight culture with 5% Difco bacteriological mucin. These challenges corresponded to approximately 2000 and 150 times the 50% lethal doses of the infecting organisms, respectively. With this inoculum, all untreated animals died within 72 h. For each antibiotic tested, four or five groups of five mice each were treated sc with different dosages administered once within 10 min after infection. Vancomycin was administered a second time, 5 h after infection. The 50% effective doses (ED50) and 95% confidence limits were calculated by the SpearmanKärber method27 from the percentages of animals surviving to day 7 at each dose.
Experimental pneumonia in rats
The production of lung infection was performed as described previously.25 Two S. pneumoniae strains were employed: L977 for the
penicillin-susceptible (Pen-S) model in normal rats and L2868 for the penicillin-resistant (Pen-R)
model in both immunocompetent and neutropenic rats. Briefly, rats were anaesthetized by im
injection with 0.1 mL fentanyl droperidol (Farmitalia C. Erba, Milan, Italy) and placed in the
supine position on a small animal surgery board. The trachea was exposed and then animals were
infected by delivering, via a sterile blunt-tipped curved needle, attached to a 1 mL syringe, 40
µL of the appropriate bacterial suspension in brain heart infusion broth (Difco)
supplemented with 0.2% agar for the Pen-S model and Pen-R model in neutropenic rats or with
0.5% agar for the Pen-R model in normal rats. The inocula (cfu per rat) were: 8.4 x 106 for the Pen-S strain; 3 x 107 for the Pen-R strain in normal
rats; and 3 x 106 for the Pen-R strain in neutropenic rats. After infection, rats
were maintained with their heads elevated for at least 20 min to facilitate distal alveolar
migration of the bacteria by gravity. Therapy was initiated 12 h after infection. BI 397 was given
as a single iv dose at 10, 4 or 1.6 mg/kg in the experiments with normal rats and at 10 or 4 mg/kg
in the experiment with neutropenic rats. Procaine penicillin G, included in these studies as a
comparator drug, was administered im at 12 h intervals for 3 days at the following doses: 10,000
IU/kg in the Pen-S model; 80,000 IU/kg in the Pen-R model in immunocompetent rats and
100,000 IU/kg in the neutropenic rats. Surviving rats were killed 84 h after infection, the lungs
were homogenized in 5 mL of PSS and filtered through sterile gauze. Duplicate aliquots (0.025
mL) of suitable dilutions (at least 10-fold) of lung homogenates were spread on Difco Columbia
agar plus 5% sheep blood by the inclusion method. Colonies were counted after incubation at
35°C for 4872 h. Bacterial titres were expressed as log10 cfu in the two
lungs. Lung samples were considered sterile when no colonies were seen in the two undiluted
samples; for the purpose of calculating the mean number of cfu/organ, the samples were
considered to contain 1 cfu/0.05 mL. The lowest detection limit was therefore 2 log10 cfu/organ. The mean titres of bacteria in the two lungs in the treatment groups were
compared by analysis of variance, using the StudentNewmanKeuls test for
multiple comparisons.28 Differences in the numbers of
sterile samples between controls and treated groups were analysed by Fisher's exact
probability test. Statistical significance was defined as a P value of <0.05.
Endocarditis in rats
Experimental endocarditis was established in rats by using a slight modification of the method described previously,26 employing either S. aureus L1524 or S. epidermidis L537. The former strain is methicillin-resistant and teicoplanin-susceptible, while for the S. epidermidis isolate the teicoplanin MIC was 8 mg/L. A polyethylene catheter (PE 10; inside diameter 0.282 mm, outside diameter 0.615 mm; Clay Adams, NJ, USA) was inserted through the aortic valve of rats into the left ventricle via the right carotid artery and left in place throughout the experiment. Two or 3 days later, the animals were infected intravenously. The inocula were 1.7 x 104 and 5.5 x 10 8 cfu/rat for S. aureus and S. epidermidis, respectively. Except for vancomycin, which was given im, all other antibiotics were administered iv. Treatment was bd for 5 days starting 17 or 24 h after S. aureus or S. epidermidis infection, respectively. BI 397 was administered every 24 h at dosages of 10 or 1.25 mg/kg (loading dosage at 20 or 2.5 mg/kg) in the S. aureus infection and at dosages of 2.5 or 1.25 mg/kg (loading dosage 5 or 2.5 mg/kg) in the S. epidermidis infection. In both infections, teicoplanin was administered every 12 h at 20 mg/kg (loading dosage 40 mg/kg) and vancomycin at 100 mg/kg im every 12 h. Animals that died before the end of the experiment were autopsied, their hearts removed and stored at -20°C. Surviving animals were killed on day 7 after infection (24 h after the fifth dose of BI 397, 12 h after the tenth dose of teicoplanin or vancomycin). The hearts were weighed, homogenized in 5 mL of PSS and filtered through sterile gauze. Duplicate aliquots (0.025 mL) of suitable dilutions were plated by the inclusion method on ToddHewitt agar (Difco) as described above. Colonies were counted after incubation at 35°C for 48 h. Bacterial titres were expressed as log10 cfu/g of heart. Heart vegetations were considered sterile when no colonies were seen in the two undiluted samples; for the purpose of calculating the mean number of cfu/g of heart, the samples were considered to contain 1 cfu/0.05 mL, which corresponds to 2.0 to 2.2 log10 cfu/g, depending on heart weight. Data from rats that had the catheter correctly positioned across the aortic valve at necropsy and completed at least 4.5 days of treatment were used for statistical analysis. Untreated animals were all included in statistical analysis irrespective of their time of death. The mean titres of bacteria per gram of heart in the treatment groups were compared by analysis of variance. The StudentNewmanKeuls test was used to adjust for multiple comparisons. Differences in the number of surviving animals and of sterile samples between controls and treated groups were analysed by Fisher's exact probability test. Statistical significance was defined as a P value of <0.05.
Pharmacokinetics in rats
A group of five rats was treated iv with a single 20 mg/kg dose of BI 397 administered at 2 mL/kg. At the time of treatment, the mean weight ± S.D. of rats was 212 ± 4 g. The rats were lightly anaesthetized with halothane (Zeneca Ltd, Macclesfield, UK), and 0.3 to 0.5 mL of blood was collected in heparinized tubes at various time-points after drug administration. Plasma was collected by centrifugation. In experiments of localized infections, groups of two to four rats were used to determine the plasma levels of administered compounds.
Antibiotic concentrations were determined by the agar diffusion method, using Micrococcus luteus ATCC 9341 for BI 397 and procaine penicillin G, and Bacillus subtilis ATCC 6633 for teicoplanin. Limits of quantification were 0.4 mg/L for BI 397, 0.2 mg/L for teicoplanin and 0.05 mg/L for procaine penicillin G; the mean reproducibility was determined to be 9.7 ± 2.0%.
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Results |
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The activity of BI 397 against coagulase-negative and -positive staphylococci is reported in Table I. BI 397 appears to be more active than teicoplanin, LY 333328 and vancomycin against all tested staphylococci. No difference in activity was observed between methicillin-susceptible (Met-S) and -resistant (Met-R) strains except in the case of Staphylococcus haemolyticus, in which the MIC of BI 397 was slightly higher against Met-R isolates. Against streptococci (Table II) BI 397 is as active as teicoplanin and four to eight times more active than vancomycin. In comparison with LY 333328, BI 397 appears to be less active against S. pneumoniae, but equally active against S. pyogenes. All compounds were equally active against Pen-S and Pen-R strains. The results for enterococci show that BI 397 is effective against vancomycin-susceptible (Van-S) and -resistant (Van-B) strains, but poorly active against Van-A strains; the exceptions were two E. faecalis isolates for which the MIC was 0.5 mg/L. While Van-A and Van-B strains are both resistant to vancomycin, they can be distinguished by their different response to teicoplanin; the former strains are resistant, whereas the latter ones are sensitive. In this respect, BI 397 behaves like teicoplanin against Van-B strains, although it is slightly more potent. However, in contrast to LY 333328, BI 397 shows little or no activity against Van-A strains.
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The bactericidal effect of BI 397 was studied using two isolates each of S. aureus and S. epidermidis, including one for each strain (isolates L1524 and L537, respectively) used in the experimental endocarditis studies. In the experiments performed with S. aureus, one with a teicoplanin MIC of 0.5 mg/L (L1524) and the other 8 mg/L (L561), BI 397 was bactericidal against L1524 (Figure 2a) at the highest concentration tested (8 mg/L killed 99.9% of the microorganisms within 24 h), which corresponds to 32 times its MIC value (MIC 0.25 mg/L); while teicoplanin and vancomycin (MIC 1 mg/L), both tested at concentrations eight to 16 times their MIC values were poorly bactericidal. Against S. aureus L561 only vancomycin (MIC 4 mg/L) was bactericidal, at eight and 16 times the MIC (32 and 64 mg/L; Figure 2b). BI 397 (MIC 1 mg/L) and teicoplanin (MIC 8 mg/L) at concentrations of 8 and 16 times the MIC were not bactericidal.
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The values of the 50% protective efficacy of BI 397 against experimental septicaemia in immunocompetent and neutropenic mice are reported in Table IV. In the infection caused by the methicillin-susceptible S. aureus Smith strain, BI 397 and teicoplanin had similar potency, whereas vancomycin was much less efficacious. Against infection caused by S. pneumoniae, where vancomycin was administered twice, BI 397 and teicoplanin were slightly more active than vancomycin.
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Pharmacokinetics in the rat
Following iv administration of a 20 mg/kg dose of BI 397 high and prolonged plasma concentrations were found: plasma concentration averaged 333.1 ± 32.1 mg/L 3 min after administration and 0.43 ± 0.03 mg/L at 120 h, after which the compound was no longer detectable (Figure 4).
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Activity of BI 397 against experimental lobar pneumonia
Table V shows the results from treatments of three pneumococcal infections caused by Pen-S and Pen-R S. pneumoniae strains in immunocompetent or neutropenic rats. In all the pneumonia experiments, BI 397 was administered as a single iv injection and procaine penicillin G was given six times at 12 h intervals. Bacterial counts in lungs of infected rats treated with any antimicrobial regimen were significantly lower than those of untreated controls. In the experiment performed with the Pen-S strain, a single administration of 10 mg/kg BI 397 reduced bacterial counts in lungs to undetectable levels. This dosage was significantly more effective in reducing the bacterial load than the two lower dosages of BI 397 (4 and 1.6 mg/kg), or the six 10,000 IU/kg doses of procaine penicillin G. Nevertheless, all dosages tested were effective in reducing the bacterial load in comparison with untreated controls. The efficacy of the highest dose of BI 397 against this infection was also reflected in a significant number of animals with no detectable pneumococci in the lungs (eight out of eight; P < 0.05 compared with untreated controls).
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In neutropenic rats, both 10 and 4 mg/kg single administrations of BI 397 were highly efficacious in reducing the mean bacterial counts in lungs compared with the pretreatment titre. The counts obtained after the 10 mg/kg treatment were significantly lower than those of rats treated with 4 mg/kg. The efficacy of BI 397 was reflected in the 100% protection from death following infection, in comparison with untreated controls or rats given 100,000 IU/kg of procaine penicillin G, for which no survivors were observed.
As shown in Figure 4, the plasma concentration following a single 10 mg/kg iv dose remained above the MIC for both study organisms throughout the experiment. Taking into account the similar time profiles between the 20 and 10 mg/kg doses (Figure 4), it can be reasonably extrapolated that plasma concentrations are above the MIC for S. pneumoniae (0.01 mg/L) throughout the experiment after a 1.6 mg/dose. As previously reported,25 both treatment schedules for procaine penicillin G produced plasma concentrations in excess of the MICs for the organisms for only a few hours.
Activity of BI 397 against experimental rat endocarditis
The results from the S. aureus and S. epidermidis experimental endocarditis are shown in Table VI. For S. aureus, the od regimen of 10 mg/kg (loading dose 20 mg/kg) of BI 397 was as effective as the bd regimen of 100 mg/kg of vancomycin in reducing the heart bacterial load. Although the od regimen of 1.25 mg/kg (loading dosage 2.5 mg/kg) of BI 397 or the bd schedule of teicoplanin (20 mg/kg with a loading dosage of 40 mg/kg) reduced the heart bacterial load in comparison with untreated controls they were not as efficacious as the higher BI 397 dose or as vancomycin. However, only the group treated with the 10 mg/kg dosage of BI 397 had a significant proportion of animals with no detectable bacteria in their hearts compared with untreated controls. In this experiment, >90% of the untreated animals died within 3 days after infection and treatment with any of the antibiotics significantly increased the survival rate.
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The plasma concentrationtime profile of BI 397 in infected rats up to 24 h was very similar to that observed after the same dose in healthy rats (Figure 4). Therefore, the pharmacokinetic parameters estimated from the healthy rats were used to predict the expected plasma concentrationtime profile in infected animals following the regimen of 20 mg/kg and then 10 mg/kg daily. The predicted profile and the experimental data obtained with this regimen are depicted in Figure 5, which shows excellent correlation between predicted and observed values. As expected from its very long half-life, BI 397 produced plasma concentrations higher than teicoplanin and vancomycin at lower dosages and longer treatment intervals (Table VII).
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Discussion |
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The presence of an in-vitro serum effect was also confirmed with rat plasma (data not shown). Nevertheless the serum binding of BI 397 did not apparently affect its in-vivo activity, as demonstrated by the experimental animal models.
In timekill experiments BI 397 showed a generally better performance against S. epidermidis than S. aureus. Concentrations eight-fold the MIC value were required to obtain 99.9% killing of S. epidermidis isolates, whereas against S. aureus isolates the same level of killing was reached only at 32-fold the MIC. The timekill results observed with two of the staphylococcal strains used in animal models are quite consistent with the results obtained for experimental endocarditis in rats. In the S. aureus L1524 infection, daily treatments with 10 mg/kg of BI 397 performed significantly better than 1.25 mg/kg in terms of bacterial load in the heart. Trough plasma levels attained after a 10 mg/kg iv dose (c. 19 mg/L) exceeded 8 mg/L, the value necessary to obtain 99.9% in-vitro killing of S. aureus L1524. In contrast, the BI 397 concentrations achieved in vivo after daily treatments with 1.25 mg/kg (Table VII), may explain the lower efficacy obtained at this dosage. Against S. epidermidis L537, for which timekill experiments demonstrated a 99.9% killing at 1 or 2 mg/L BI 397, large reductions in the heart bacterial load were easily obtained with a daily treatment of 1.25 mg/kg.
In the endocarditis experiments, single daily doses of BI 397 were equal to or better than the bd higher doses of teicoplanin or vancomycin. In particular, in the S. epidermidis infection, a total dose of 7.5 mg/kg of BI 397, administered over 5 days, was as efficacious in reducing the heart bacterial load as a total dose of 220 or 1000 mg/kg of teicoplanin or vancomycin, respectively, given over the same period. This high in-vivo potency of BI 397 is probably attributable to its high and prolonged blood levels as also revealed in other experimental infections in mice and rats described in this paper.
In immunocompetent mice, BI 397 and teicoplanin were more efficacious than vancomycin against staphylococcal septicaemia. Whereas against streptococcal septicaemia the 50% effective dose values were similar for the glycopeptides tested, except vancomycin. The minor activity of vancomycin compared with BI 397 might be related to either the relatively short half-life in mice29 or the higher MICs for the two tested strains. In S. epidermidis septicaemia, with the cell-mediated immunity of mice severely impaired, BI 397 was at least twenty times as potent as the two control drugs, with vancomycin administered twice.
In the pneumonia models caused by Pen-S or Pen-R pneumococcal strains in either immunocompetent or neutropenic animals, a single iv dose of 10 mg/kg of BI 397 was significantly more efficacious in reducing the bacterial load in the lungs than six administrations of procaine penicillin G. The efficacy of BI 397 appeared to be dose dependent even at plasma concentrations greatly in excess of the MIC of the offending organisms, as has been seen with teicoplanin.25 Of particular relevance is the result obtained with neutropenic rats, where the course of infection was more severe than in normal rats as revealed by the high mortality rate of untreated controls and in rats treated with procaine penicillin G. Previously, we reported that a single dose of 10 mg/kg teicoplanin was effective in reducing the lung bacterial load in immunocompetent but not in neutropenic rats, where a 20 mg/kg regimen was necessary. A delayed initiation of treatment (12 h after inoculation) may explain the failure of the 10 mg/kg teicoplanin regimen.25 In contrast to teicoplanin, we report here that a single 10 mg/kg dose of BI 397, administered 12 h after inoculation, was effective in reducing the lung bacterial load regardless of the immunological status of the animals.
Following iv administration to the rat, the plasma concentration of BI 397 was high and long-lasting. The pharmacokinetics of BI 397 can be described by a two-compartment model, but might be best represented by a three-compartment model, as seen with teicoplanin30 and MDL 63,246.31 However, a possible third phase, not seen in these studies, would have little contribution to plasma levels.
We reported previously the microbiological characterization and pharmacokinetic profile of MDL 63,246, another semisynthetic glycopeptide of the teicoplanin family.24,31 Both MDL 63,246 and BI 397 are semisynthetic amide derivatives of the naturally occurring glycopeptide A40926, possessing a dimethylaminopropyl amide at the peptide-carboxy group. BI 397 is only differentiated from MDL 63,246 by the presence of an acylaminoglucuronic acid on amino acid 4 instead of an acylglucosamine. Although no direct comparison was made between MDL 63,246 and BI 397, the results presented here and those from previous works24,31,32 suggest that both compounds have similar in-vitro and in-vivo activities. While MDL 63,246 showed poor tolerability in preliminary safety pharmacological studies, BI 397 appears to be well tolerated (data not shown).
In conclusion, the microbiological data suggest that BI 397 is a promising new glycopeptide for the treatment of infections caused by multiresistant Gram-positive bacteria, particularly staphylococci and streptococci, with the exception of vancomycin-resistant enterococci expressing Van-A. The preliminary pharmacokinetic data support the possibility that the compound can be therapeutically efficacious at lower dosages and at longer treatment intervals than those currently used for vancomycin and teicoplanin.
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Acknowledgments |
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Notes |
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Present address. Hoechst Marion Roussel, 102, Route de Noisy,
F-93235 Romainville Cedex, France.
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References |
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Received 16 November 1998; returned 24 February 1999; revised 24 March 1999; accepted 23 April 1999