Comparative activity of ozenoxacin and other quinolones in Staphylococcus aureus strains overexpressing the efflux pump-encoding genes mepA and norA.

BACKGROUND: To evaluate the activity of ozenoxacin (OZN) in Staphylococcus aureus strains overexpressing the efflux pump-encoding genes mepA and norA. METHODS: S. aureus NCTC-8325-1, S. aureus NCTC 8225-2 (overexpressing mepA), S. aureus SA 1199 and S. aureus SA 1199B (overexpressing norA) were used. The minimum inhibitory concentrations (MICs) of OZN, moxifloxacin (MOX), levofloxacin (LVX), ciprofloxacin (CIP) and norfloxacin (NOR) in the presence and absence of reserpine (20 mg/L) were determined using the microdilution method. RESULTS: The MIC of OZN was lower in all evaluated strains compared with the other studied quinolones and was independent of the pump being overexpressed. MIC values of OZN ranged from 0.005 to 0.007 mg/L. Similar results were observed with MOX, with MIC values between 0.021 and 0.031 mg/L, without variations in the presence of reserpine. MIC values for LVX were between 0.167 and 1 mg/L with a slight increase in MIC observed in strains overexpressing the mepA or norA genes (from 0.250 to 0.833 mg/L and 0.167 to 1 mg/L, respectively). Overproduction of the efflux pump MepA did not affect CIP whereas it increased 8-fold the MIC of NOR. Overproduction of NorA increased ~5-fold and ~40-fold the MICs of CIP and NOR, respectively, resulting in a high-level of resistance to these antibiotics compared with OZN (0.007 mg/L). CONCLUSION: OZN does not seem to be a substrate for the efflux pumps MepA and NorA, which are commonly found in Gram-positive bacteria and that affect other quinolones.

Mutant prevention concentration of ozenoxacin for quinolone-susceptible or -resistant Staphylococcus aureus and Staphylococcus epidermidis.

Ozenoxacin (OZN) belongs to a new generation of non-fluorinated quinolones for the topical treatment of skin infections which has shown to be effective in the treatment of susceptible and resistant Gram-positive cocci. The mutant prevention concentration (MPC) of ozenoxacin, levofloxacin and ciprofloxacin was determined in quinolone-susceptible and -resistant strains including methicillin-susceptible S. aureus, methicillin-resistant S. aureus, methicillin-susceptible S. epidermidis and methicillin-resistant S. epidermidis with different profile of mutation in the quinolone resistance determining regions (QRDR). The MPC value of OZN for the methicillin-susceptible S. aureus strain susceptible to quinolones, without mutations in QRDR, was 0.05 mg/L, being 280-fold lower than that observed with ciprofloxacin and levofloxacin. In methicillin-susceptible and-resistant S. aureus strains with mutations in the gyrA or/and grlA genes the MPC of OZN went from 0.1 to 6 mg/L, whereas the MPC of levofloxacin and ciprofloxacin was > 50 mg/L for the same strains. For methicillin-susceptible and-resistant S. epidermidis the results were similar to those abovementioned for S. aureus. According to our results, the MPC of OZN was far below the quantity of ozenoxacin achieved in the epidermal layer, suggesting that the in vivo selection of mutants, if it occurs, will take place at low frequency. Ozenoxacin is an excellent candidate for the treatment of bacterial infections caused by susceptible and quinolone-resistant staphylococci isolated usually from skin infections.

In vitro selection of mutants resistant to ozenoxacin compared with levofloxacin and ciprofloxacin in Gram-positive cocci.

OBJECTIVES: To determine the frequency of selecting mutants resistant to ozenoxacin, a des-fluoro-(6)-quinolone active against pathogens involved in skin and skin structure infections, compared with levofloxacin and ciprofloxacin in quinolone-susceptible and -resistant Gram-positive cocci. METHODS: Forty-nine quinolone-susceptible and -resistant Gram-positive cocci strains with different profiles of mutations in the quinolone resistance-determining region (QRDR) were examined to determine the frequency of selecting mutants resistant to ozenoxacin compared with levofloxacin and ciprofloxacin. MICs and mutations in the QRDR were determined by standard broth microdilution and PCR amplification and sequencing, respectively. RESULTS: The mean resistance rates were 3.8 × 10(-9) (range <9 × 10(-11)-1 × 10(-8)) for ozenoxacin, 9.7 × 10(-9) (range <1.1 × 10(-11)-4.2 × 10(-8)) for levofloxacin and 1.2 × 10(-8) (range <1.6 × 10(-10)-2.6 × 10(-7)) for ciprofloxacin. Spontaneous mutants resistant to ozenoxacin showed lower MICs (≤ 16 mg/L) than mutants resistant to levofloxacin and ciprofloxacin (≤ 512 mg/L). Additional mutations were observed only in ParC at Ser-80 in Staphylococcus spp., Ser-79 in Streptococcus agalactiae and Asp-83 and Ser-89 in Streptococcus pyogenes. CONCLUSIONS: The probability of ozenoxacin selecting spontaneous resistant mutants in quinolone-susceptible and -resistant strains with pre-existing mutations in the QRDR is low, supporting the potential utility of ozenoxacin as a therapeutic alternative in the treatment of skin infections caused by strains highly resistant to quinolones.

In vitro activity of Ozenoxacin against quinolone-susceptible and quinolone-resistant gram-positive bacteria.

In vitro activity of ozenoxacin, a novel nonfluorinated topical (L. D. Saravolatz and J. Leggett, Clin. Infect. Dis. 37:1210-1215, 2003) quinolone, was compared with the activities of other quinolones against well-characterized quinolone-susceptible and quinolone-resistant Gram-positive bacteria. Ozenoxacin was 3-fold to 321-fold more active than other quinolones. Ozenoxacin could represent a first-in-class nonfluorinated quinolone for the topical treatment of a broad range of dermatological infections.

High seroprevalence of Pneumocystis infection in Spanish children.

Pneumocystis infection occurs worldwide, and most individuals test seropositive for Pneumocystis early in childhood. Little is known about the epidemiology of this infection in western Europe. The seroprevalence of Pneumocystis infection in 233 Spanish children was determined in a community study by immunoblot analysis of sera. The overall seroprevalence was 73%, with an age-related increase from 52% at 6 years to 66% at 10 years and 80% at 13 years. The data indicated a high seroprevalence of Pneumocystis infection in healthy Spanish children, thereby demonstrating that this pathogen is widespread in southern Spain.

Antifungal activities of two new azasordarins, GW471552 and GW471558, in experimental models of oral and vulvovaginal candidiasis in immunosuppressed rats.

Sordarins constitute a new class of antifungal agents with a novel mechanism of action involving the selective inhibition of fungal protein synthesis. A further evolution of this class of antifungals has led to a new family of sordarin derivatives called azasordarins. The therapeutic efficacies of two new azasordarins, GW471552 and GW471558, were studied in experimental models of oral and vulvovaginal candidiasis in immunosuppressed rats. In all cases rats were immunosuppressed with dexamethasone in the drinking water. Oral candidiasis was established by inoculating 0.1 ml of a yeast suspension containing 5 x 10(8) cells of Candida albicans 4711E with a cotton swab on three alternate days. Vulvovaginal candidiasis was established in ovariectomized and estrus-induced rats by intravaginal inoculation of 10(7) CFU of C. albicans 4711E in 0.1 ml of saline. GW471552 and GW471558 were administered at 1, 5, and 10 mg/kg of body weight via the subcutaneous route. In oral candidiasis, azasordarins were administered each 8 h for 7 consecutive days, while in vaginal candidiasis the compounds were given each 4 h for 3 consecutive days. Antifungal activity of azasordarins was assessed by colony counts and by histological examination 1 day after treatment. In the oral infection model, GW471552 and GW471558 administered at 5 mg/kg significantly reduced (P < 0.05) the number of CFU of C. albicans compared with untreated controls. In addition, GW471552 and GW471558 given at 10 mg/kg eradicated C. albicans from the oral cavities of 100% of infected animals. Against vulvovaginal infection, both compounds showed significant therapeutic efficacy. GW471552 was able to eradicate the vaginal fungal burden at a dose of 10 mg/kg, and it significantly reduced the number of CFU of C. albicans in vaginas of rats treated with a dose of 5 mg/kg (P < 0.05). GW471558 showed greater efficacy, eradicating the fungal burden of 100% of infected rats at a dose of 5 mg/kg and significantly reducing (P < 0.05) the C. albicans vaginal counts even at a dose of 1 mg/kg. In both therapeutic efficacy studies, the histological findings confirmed the microbiological results. The experimental results presented show that the tested azasordarins are effective against oral and vulvovaginal candidiasis in immunosuppressed rats and could be promising antifungal agents for use in humans.

Antibacterial activities and pharmacokinetics of E-4767 and E-5065, two new 8-chlorofluoroquinolones with a 7-azetidin ring substituent.

E-4767 [(-)-7-[3-(R)-amino-2-(S)-methyl-1-azetidinyl]-8-chloro-1-cyclopropyl-1,4-dihydro-6-fluoro-4-oxo-3-quinolinecarboxylic acid] and E-5065 [(-)-7-(3-amino-1-azetidinyl)-8-chloro-1-cyclopropyl-1,4-dihydro-6-fluoro-4-oxo-3-quinolinecarboxylic acid] are two new chlorofluoroquinolones with an azetidine moiety at position 7. Their in vitro activities were evaluated in comparison with those of ciprofloxacin, ofloxacin, fleroxacin, and tosufloxacin, while ciprofloxacin was used as a reference for in vivo studies. Against gram-positive organisms, E-4767 and E-5065 were, in general, eight- and fourfold more active than tosufloxacin, which is the most potent of the reference compounds. E-4767 and E-5065 were also more potent than the reference compounds against all species of enteric bacteria tested. The MICs of E-4767 and E-5065 at which 90% of the isolates tested were inhibited (MIC(90)s) were 0.007 to 0.5 microg/ml and 0.03 to 2 microg/ml, respectively, for gram-positive organisms and

Antifungal activities and cytotoxicity studies of six new azasordarins.

GW 471552, GW 471558, GW 479821, GW 515716, GW 570009, and GW 587270 are members of a new family of sordarin derivatives called azasordarins. The in vitro activities of these compounds were evaluated against clinical isolates of yeasts, including Candida albicans, Candida non-albicans, and Cryptococcus neoformans strains. Activities against Pneumocystis carinii, Aspergillus spp., less common molds, and dermatophytes were also investigated. Azasordarin derivatives displayed significant activities against the most clinically important Candida species, with the exception of C. krusei. Against C. albicans, including fluconazole-resistant strains, MICs at which 90% of the isolates tested are inhibited (MIC(90)s) were 0.002 microg/ml with GW 479821, 0.015 microg/ml with GW 515716 and GW 587270, and 0.06 microg/ml with GW 471552, GW 471558, and GW 570009. The MIC(90)s of GW 471552, GW 471558, GW 479821, GW 515716, GW 570009, and GW 587270 were 0.12, 0.12, 0.03, 0.06, 0.12, and 0.06 microg/ml, respectively, against C. tropicalis and 4, 0.25, 0.06, 0.25, 0.5, and 0.5 microg/ml, respectively, against C. glabrata. In addition, some azasordarin derivatives (GW 479821, GW 515716, GW 570009, and GW 58720) were active against C. parapsilosis, with MIC(90)s of 2, 4, 4, and 1 microg/ml, respectively. The compounds were extremely potent against P. carinii, showing 50% inhibitory concentrations of 16 microg/ml). These azasordarin derivatives also showed significant activity against emerging fungal pathogens, which affect immunocompromised patients, such as Rhizopus arrhizus, Blastoschizomyces capitatus, and Geotrichum clavatum. Against these organisms, the MICs of GW 587270 ranged from 0.12 to 1 microg/ml, those of GW 479821 and GW 515716 ranged from 0.12 to 2 microg/ml, and those of GW 570009 ranged from 0.12 to 4 microg/ml. Against Fusarium oxysporum, Scedosporium apiospermum, Absidia corymbifera, Cunninghamella bertholletiae, and dermatophytes, GW 587270 was the most active compound, with MICs ranging from 4 to 16 microg/ml. Against Aspergillus spp., the MICs of the compounds tested were higher than 16 microg/ml. The in vitro selectivity of azasordarins was investigated by cytotoxicity studies performed with five cell lines and primary hepatocytes. Concentrations of compound required to achieve 50% inhibition of the parameter considered (Tox(50)s) of GW 570009, GW 587270, GW 479281, and GW 515716 in the cell lines ranged from 60 to 96, 49 to 62, 24 to 36, and 16 to 38 microg/ml, respectively. The cytotoxicity values of GW 471552 and GW 471558 were >100 microg/ml for all cell lines tested. Tox(50)s on hepatocytes were in the following order: GW 471558 > GW 471552 > GW 570009 > GW 587270 > GW 515716 > GW 479821, with values ranging from higher than 100 microg/ml to 23 microg/ml. The cytotoxicity results obtained with fully metabolizing rat hepatocytes were in total agreement with those obtained with cell lines. In summary, the in vitro activities against important pathogenic fungi and the selectivity demonstrated in mammalian cell lines justify additional studies to determine the clinical usefulness of azasordarins.

Correlation between in vitro and in vivo activities of GM 237354, a new sordarin derivative, against Candida albicans in an in vitro pharmacokinetic-pharmacodynamic model and influence of protein binding.

The antifungal effect of GM 237354, a sordarin derivative, was studied in an in vitro pharmacokinetic (PK)-pharmacodynamic dynamic system (bioreactor) which reproduces PK profiles observed in a previously described model of drug efficacy against murine systemic candidiasis. Immunocompetent mice infected intravenously with 10(5) CFU of Candida albicans were treated with GM 237354 at 2.5, 10, and 40 mg/kg of body weight every 8 h subcutaneously for 7 days. Free concentrations in serum were calculated by multiplying total concentrations measured in vivo by 0.05, the free fraction determined in vitro by equilibrium dialysis. In the bioreactor the inoculum was approximately 10(6) CFU/ml; and a one-compartment PK model was used to reproduce the PK profiles of free and total GM 237354 in serum obtained in mice, and clearance of C. albicans was measured over 48 h. A good correlation was observed when the in vivo fungal kidney burden and the area under the survival time curve were compared with the in vitro broth "burden," although only when free in vivo levels in serum were reproduced in vitro. GM 237354 displayed a 3-log decrease effect both in vivo and in vitro. The very few reports available on in vitro-in vivo correlations have been obtained with antibiotics. The good in vitro-in vivo correlation obtained with an antifungal agent shows that the in vitro dynamic system could constitute a powerful investigational tool prior to assessment of the efficacy of an anti-infective agent in animals and humans.

Animal pharmacokinetics and interspecies scaling of sordarin derivatives following intravenous administration.

Sordarin derivatives constitute a new group of synthetic antifungal agents that selectively inhibit fungal protein synthesis. They have demonstrated in vitro activity against the most important fungal pathogens, both yeast and filamentous. This new family of compounds has also shown in vivo activity against murine Candida albicans, Histoplasma capsulatum, and Coccidioides immitis experimental infections, as well as against Pneumocystis carinii pneumonia in rats. After intravenous dosing in animals, both the area under the concentration-time curve and the elimination half-life were highest in Cynomolgus monkeys, followed by those in rats, mice, and rabbits. The volume of distribution at steady state for sordarin derivatives was similar in all species tested. The clearance in rats and mice was higher than for other species. GM 237354, a sordarin derivative, was characterized by high serum protein binding in mouse, rat, and monkey serum (unbound fraction, < or =5%). An indirect evaluation of the effect of liver function upon the metabolism of this class of compounds has been made in animals with impaired liver function such as Gunn rats, as well as in allometric studies that showed better correlations of half-life to liver blood flow than to animal body weight. Linearity of the main pharmacokinetic parameters was demonstrated after intravenous dosing of the representative compound GM 193663 at 10 and 20 mg/kg of body weight in rats. Allometry was used to determine whether human pharmacokinetic parameters can be predicted from animal data by regression analysis against body weight and liver blood flow. All these results have demonstrated that the human pharmacokinetics of sordarin derivatives can be forecast from animal data.

Antifungal efficacy of GM237354, a sordarin derivative, in experimental oral candidiasis in immunosuppressed rats.

GM237354 is a novel sordarin derivative with a broad spectrum of potent activity against a wide range of fungi. The members of this new class of antifungal agents act as potent inhibitors of fungal protein synthesis. In this study, the therapeutic effects of GM237354 were investigated in a novel experimental oral Candida albicans infection model in immunosuppressed rats. The animals were immunosuppressed with dexamethasone in their drinking water and infected on three alternate days. GM237354 was given three times per day for seven consecutive days at 1.25, 2.5, 5, or 10 mg/kg of body weight per dose. In addition, to provide a preliminary idea of the correlation between regimen administration and therapeutic efficacy, GM237354 was administered to two additional groups of rats at 5 mg/kg once or twice a day for 7 days. The drug efficacy was assessed microbiologically, histologically, and by a morphometric study of lesions. Evident agreement was observed among results obtained by the different methods in all of the animals studied. Microbiologically, the efficacy of GM237354 was determined by measuring the number of C. albicans organisms in the oral cavities of rats in the middle (day 4) and at the end (day 7) of the treatment. GM237354 administered at 5, 7.5, 10, 15, or 30 mg/kg/day for 7 days significantly reduced the number of CFU in the oral cavities of treated rats compared with the number of CFU in the oral cavities of the untreated controls. A significant reduction was also observed when GM237354 was administered at 7.5, 10, 15, or 30 mg/kg/day for 4 days. Furthermore, C. albicans was not detected in oral swabs from any infected rats after 1 week of treatment when GM237354 was administered at 15 or 30 mg/kg/day or after 4 days of treatment at 30 mg/kg/day. Histologically, untreated control animals showed extensive colonization of the epithelium of the dorsal tongue by numerous hyphae. Animals treated with GM237354 at 7.5 mg/kg/day showed small areas with superficial hyphal penetration into the epithelium that produced intraepithelial microabscesses. However, animals treated with GM237354 at 15 mg/kg/day showed multiple regenerative areas of the covering epithelium, and only focalized zones of the tongue surface were occupied by hyphae. No hyphal colonization of the epithelium was seen in rats treated with GM237354 at 30 mg/kg/day and which showed extensive areas of epithelial regeneration of the tongue. The histopathology findings were confirmed by morphometry studies, and the percentage of epithelium occupied by C. albicans hyphae decreased from 17.5% in the control group to 4.8 and 0.1% in animals treated with GM237354 at 7.5 and 15 mg/kg/day, respectively. These results demonstrated that the sordarin derivative GM237354 was effective against experimental oral candidiasis in immunosuppressed rats, and further studies are needed to determine the potential of GM237354 for use in the treatment of this infection in humans.

Activities of sordarins in experimental models of candidiasis, aspergillosis, and pneumocystosis.

Sordarin derivatives represent a new class of antifungal agents that act as potent inhibitors of fungal protein synthesis and possess a broad spectrum of activity. The in vivo activity of GM193663 and GM237354 was studied in mouse models of disseminated candidiasis and aspergillosis and in a rat model of pneumocystosis. The pharmacokinetic behavior of both sordarin derivatives was studied in mice and rats. In all studies, compounds were administered by the subcutaneous route. After a subcutaneous dose of 50 mg/kg of body weight to mice, the maximum level in serum, area under the concentration-time curve, half-life, and clearance for GM193663 and GM237354 were 51.8 and 23 microg/ml, 79.5 and 46 microg. h/ml, 0.8 and 0.85 h, and 21 and 25 ml/h, respectively. Systemic candidiasis and aspergillosis were established in CD-1 male mice infected with Candida albicans or Aspergillus fumigatus. For systemic candidiasis, compounds were given three times per day for seven consecutive days at 15, 30, 60, or 120 mg/kg/day. GM193663 and GM237354 showed dose-related efficacy against C. albicans, with 50% effective doses, 1 month after infection, of 25.2 and 10.7 mg/kg/dose, respectively. In experimental infections with A. fumigatus, GM237354 was given three times per day at 30, 60, or 120 mg/kg/day for five consecutive days. Animals treated with GM237354 demonstrated irregular responses. The survival of animals treated with GM237354 was 0, 30, and 0% at 30, 60, and 120 mg/kg/day, respectively. The therapeutic efficacy of GM193663 and GM237354 against Pneumocystis carinii was studied in an experimental P. carinii pneumonia (PCP) rat model. After a subcutaneous dose of 10 mg/kg given to rats, the maximum level in serum, area under the concentration-time curve, half-life, and clearance for GM193663 and GM237354 were 6.6 and 7.2 microg/ml, 8.5 and 11.8 microg. h/ml, 0.7 and 0.8 h, and 230 and 133 ml/h, respectively. To induce spontaneous PCP, rats were chronically immunosuppressed with dexamethasone. Infected animals were treated twice daily for 10 days at 0.2, 2, or 10 mg/kg/day. The therapeutic effect was estimated by the reduction in the number of cysts in the lungs of treated versus untreated animals. GM193663 and GM237354 significantly reduced the mean (+/- standard deviation) log number of cysts from 7.6 +/- 0.2 in the untreated group to 4.7 +/- 0.2 and 4.6 +/- 0.1, respectively, when the drugs were administered at a dose of 2 mg/kg/day. The log number of cysts was also reduced in infected animals given lower doses of the compounds (0.2 mg/kg/day). In summary, GM193663 and GM237354 are new sordarin derivatives that may potentially play a major role in the treatment of candidiasis and PCP. Further testing with Aspergillus in other animal models is warranted.

Transmission of Pneumocystis carinii disease from immunocompetent contacts of infected hosts to susceptible hosts.

Pneumocystis carinii organisms constitute a large group of heterogeneous atypical microscopic fungi that are able to infect immunocompromised mammals by an airborne route and to proliferate in their lungs, inducing Pneumocystis carinii pneumonia. This pneumonia remains a crucial epidemiological challenge, since neither the source of Pneumocystis carinii infection in humans nor the process by which humans become infected has been clearly established. Polymerase chain reaction (PCR) assays have shown that profoundly immunosuppressed patients without pneumocystosis can be subclinically infected with Pneumocystis. Other PCR-based studies have suggested that healthy immunocompetent hosts are not latent carriers of the parasite. However, recent reports have indicated that Pneumocystis carinii can persist for limited periods in the lungs of convalescent rats after recovery from corticosteroid-induced pneumocystosis, and also that immunocompetent mammals can be transiently parasitized by Pneumocystis carinii after close contact with hosts with Pneumocystis carinii pneumonia. Can transiently parasitized hosts be a source of infection for immunosuppressed hosts? In order to investigate this important clinical question, the ability of immunocompetent BALB/c mice, which were carrying subclinical levels of Pneumocystis carinii, to transmit the infection by the airborne route to highly susceptible, uninfected mice with severe combined immunodeficiency was studied. The results indicated that the immunocompetent mice, transiently parasitized by Pneumocystis carinii organisms after close contact with Pneumocystis carinii-infected mice, were able to transmit the infection to Pneumocystis carinii-free mice with severe combined immunodeficiency.

Pharmacokinetics-pharmacodynamics of a sordarin derivative (GM 237354) in a murine model of lethal candidiasis.

Sordarins are a new class of antifungal agents which selectively inhibit fungal protein synthesis (FPS) by impairing the function of elongation factor 2. The present study investigates possible correlations between sordarin pharmacokinetic (PK) properties and therapeutic efficacy, based on a murine model of invasive systemic candidiasis, and provides a rationale for dose selection in the first study of efficacy in humans. A significant correlation between PK parameters and the in vivo activity of GM 237354, taken as a representative FPS inhibitor, was demonstrated in a murine model of lethal systemic candidiasis. Area under the concentration-time curve (AUC) and maximum concentration of drug in serum (C(max)) over 24 h were determined after a single GM 237354 subcutaneous (s.c.) dose (50 mg/kg of body weight) in healthy animals (no significant PK changes with infection were observed for other sordarin derivatives). These results have been used to simulate PK profiles obtained after several doses and/or schedules in animal therapy. A PK-pharmacodynamic (PD) parameter such as the time that serum drug concentrations remain above the MIC (t > MIC) was also determined. Treatment efficacies were evaluated in terms of the area under the survival time curve (AUSTC), using Kaplan-Meier survival analysis and in terms of kidney fungal burden (log CFU/gram) after s.c. doses of 2.5, 5, 10, 20, and 40 mg/kg every 4, 8, or 12 h (corresponding to total daily doses of 5 to 240 mg/kg). The results show all treatments to significantly prolong survival versus that of infected and nontreated controls (P < 0.05). Relationships between simulated PK and PK-PD parameters and efficacy were explored. A good correlation independent of the dosing interval was observed with AUC (but not C(max) or t > MIC) and both AUSTC and kidney burden. Following repeated dosing every 8 h, AUC(50) (AUC at which 50% of the maximum therapeutic efficacy is obtained) was estimated as 21.7 and 37.1 microg. h/ml (total concentrations) for AUSTC and kidney burden using a sigmoid E(max) and an inhibitory sigmoid E(max) PK-PD model, respectively. For an efficacy target of 90% survival, AUC was predicted as 67 microg. h/ml. We conclude that the PK-PD approach is useful for evaluating relationships between PK parameters and efficacy in antifungal research. Moreover, the results obtained with this approach could be successfully applied to clinical studies.

In vitro pharmacodynamic parameters of sordarin derivatives in comparison with those of marketed compounds against Pneumocystis carinii isolated from rats.

Pneumocystis carinii pneumonia remains one of the most serious complications of immunosuppressed patients. In this study, the in vitro pharmacodynamic parameters of four sordarin derivatives (GM 191519, GM 237354, GM 193663, and GM 219771) have been evaluated by a new quantitative approach and compared with the commercially available drugs pentamidine, atovaquone, and trimethoprim-sulfamethoxazole (TMP-SMX). In vitro activities and in vivo therapeutic efficacies of sordarin derivatives against P. carinii were also evaluated. In vitro activity was determined by the broth microdilution technique, comparing the total number of microorganisms in treated and drug-free cultures by using Giemsa staining. The in vitro maximum effect (E(max)), the drug concentrations to reach 50% of E(max) (EC(50)), and the slope of the dose-response curve were then estimated by the Hill equation (E(max) sigmoid model). Sordarin derivatives were the most potent agents against P. carinii, with EC(50)s of 0.00025, 0.0007, 0.0043, and 0. 025 microg/ml for GM 191519, GM 237354, GM 193663, and GM 219771, respectively. The EC(50)s of pentamidine, atovaquone, and TMP-SMX were 0.025, 0.16, and 26.7/133.5 microg/ml, respectively. The results obtained with this approach showed GM 237354 and GM 191519 to be approximately 35- and 100-fold more active in vitro than pentamidine, the most active marketed compound. All sordarin derivatives tested were at least 5,000-fold more active in vitro than TMP-SMX. The three sordarin derivatives tested in vivo-GM 191519, GM 237354, and GM 219771-showed a marked therapeutic efficacy, defined as reduction of cyst forms per gram of lung. GM 191519 was the most potent (daily dose reducing 50% of the P. carinii burden in the lungs [ED(50)], 0.05 mg/kg/day) followed by GM 237354 and GM 219771 (ED(50)s, 0.30 and 0.49 mg/kg/day, respectively). Good agreement between in vitro parameters and in vivo outcome was obtained when P. carinii pneumonia in rats was treated with sordarin derivatives.

Magnetic resonance imaging in the evaluation of inflammatory lesions in muscular and soft tissues: an experimental infection model induced by Candida albicans.

We have developed an experimental model to monitor inflammatory lesions in muscle and soft-tissues during the different stages of the disease by means of Magnetic Resonance Imaging (MRI). MRI of mice legs infected with Candida albicans was performed by standard two-dimensional spin echo and fast spin echo (RARE) using customized coils. The MRI findings were compared with pathologic examinations at the initial acute and established acute inflammatory stages, which provided accurate and detailed information on the evolution of the processes involved. The yeast caused inflammation within the first hours post-inoculation, appearing on T2-weighted images as an inhomogeneous mass with increased signal intensity. The presence of fungal hyphae was observed as hypointense signal areas in both T2 and T1 weighted images, with histologic confirmation. Areas of decreased signal intensity on T2 weighted images were apparent on the last experimental day and were attributed to the granulation tissue located within the capsule surrounding the abscess. The close correlation found between MRI and histopathology suggests that MRI is an ideal radiologic technique for monitoring the clinical and therapeutic follow-up of fungal infections in muscle and soft tissues.

Sordarins: in vitro activities of new antifungal derivatives against pathogenic yeasts, Pneumocystis carinii, and filamentous fungi.

GM 193663, GM 211676, GM 222712, and GM 237354 are new semisynthetic derivatives of the sordarin class. The in vitro antifungal activities of GM 193663, GM 211676, GM 222712, and GM 237354 against 111 clinical yeast isolates of Candida albicans, Candida kefyr, Candida glabrata, Candida parapsilosis, Candida krusei, and Cryptococcus neoformans were compared. The in vitro activities of some of these compounds against Pneumocystis carinii, 20 isolates each of Aspergillus fumigatus and Aspergillus flavus, and 30 isolates of emerging less-common mold pathogens and dermatophytes were also compared. The MICs of GM 193663, GM 211676, GM 222712, and GM 237354 at which 90% of the isolates were inhibited (MIC90s) were 0.03, 0.03, 0.004, and 0.015 microg/ml, respectively, for C. albicans, including strains with decreased susceptibility to fluconazole; 0.5, 0.5, 0.06, and 0.12 microg/ml, respectively, for C. tropicalis; and 0.004, 0.015, 0.008, and 0.03 microg/ml, respectively, for C. kefyr. GM 222712 and GM 237354 were the most active compounds against C. glabrata, C. parapsilosis, and Cryptococcus neoformans. Against C. glabrata and C. parapsilosis, the MIC90s of GM 222712 and GM 237354 were 0.5 and 4 microg/ml and 1 and 16 microg/ml, respectively. The MIC90s of GM 222712 and GM 237354 against Cryptococcus neoformans were 0.5 and 0.25 microg/ml, respectively. GM 193663, GM 211676, GM 222712, and GM 237354 were extremely active against P. carinii. The efficacies of sordarin derivatives against this organism were determined by measuring the inhibition of the uptake and incorporation of radiolabelled methionine into newly synthesized proteins. All compounds tested showed 50% inhibitory concentrations of <0.008 microg/ml. Against A. flavus and A. fumigatus, the MIC90s of GM 222712 and GM 237354 were 1 and 32 microg/ml and 32 and >64 microg/ml, respectively. In addition, GM 237354 was tested against the most important emerging fungal pathogens which affect immunocompromised patients. Cladosporium carrioni, Pseudallescheria boydii, and the yeast-like fungi Blastoschizomyces capitatus and Geotrichum clavatum were the most susceptible of the fungi to GM 237354, with MICs ranging from /=2 microg/ml. In summary, we concluded that some sordarin derivatives, such as GM 222712 and GM 237354, showed excellent in vitro activities against a wide range of pathogenic fungi, including Candida spp., Cryptococcus neoformans, P. carinii, and some filamentous fungi and emerging invasive fungal pathogens.

Antibacterial activity and pharmacokinetics of four new 7-azetidinyl fluoroquinolones.

The activity of E-4535, E-4534, E-4528, and E-4527, four new 7-azetidinyl-6-fluoroquinolones, was studied to evaluate the role played by the C-2' and C-3' substitutions of the azetidinyl group, together with a C-8 fluorine atom. In general, like other 6,8-difluoroquinolones, E-4534 and E-4527 showed higher levels of activity than the corresponding monofluorinated derivatives, E-4535 and E-4528. E-4535 and E-4534, having a 7-(2-methyl-3-aminoazetidinyl) substituent, demonstrated higher levels of activity in vitro than their corresponding structural analogs, E-4528 and E-4527, distinguished by a 3-methyl-3-methylaminoazetidinyl group at position 7 of the quinolone nucleus. In consequence, E-4534 was the most potent of the new agents tested.