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1.  Dicyclic and Tricyclic Diaminopyrimidine Derivatives as Potent Inhibitors of Cryptosporidium parvum Dihydrofolate Reductase: Structure-Activity and Structure-Selectivity Correlations 
Antimicrobial Agents and Chemotherapy  2001;45(12):3293-3303.
A structurally diverse library of 93 lipophilic di- and tricyclic diaminopyrimidine derivatives was tested for the ability to inhibit recombinant dihydrofolate reductase (DHFR) cloned from human and bovine isolates of Cryptosporidium parvum (J. R. Vásquez et al., Mol. Biochem. Parasitol. 79:153–165, 1996). In parallel, the library was also tested against human DHFR and, for comparison, the enzyme from Escherichia coli. Fifty percent inhibitory concentrations (IC50s) were determined by means of a standard spectrophotometric assay of DHFR activity with dihydrofolate and NADPH as the cosubstrates. Of the compounds tested, 25 had IC50s in the 1 to 10 μM range against one or both C. parvum enzymes and thus were not substantially different from trimethoprim (IC50s, ca. 4 μM). Another 25 compounds had IC50s of <1.0 μM, and 9 of these had IC50s of <0.1 μM and thus were at least 40 times more potent than trimethoprim. The remaining 42 compounds were weak inhibitors (IC50s, >10 μM) and thus were not considered to be of interest as drugs useful against this organism. A good correlation was generally obtained between the results of the spectrophotometric enzyme inhibition assays and those obtained recently in a yeast complementation assay (V. H. Brophy et al., Antimicrob. Agents Chemother. 44:1019–1028, 2000; H. Lau et al., Antimicrob. Agents Chemother. 45:187–195, 2001). Although many of the compounds in the library were more potent than trimethoprim, none had the degree of selectivity of trimethoprim for C. parvum versus human DHFR. Collectively, the results of these assays comprise the largest available database of lipophilic antifolates as potential anticryptosporidial agents. The compounds in the library were also tested as inhibitors of the proliferation of intracellular C. parvum oocysts in canine kidney epithelial cells cultured in folate-free medium containing thymidine (10 μM) and hypoxanthine (100 μM). After 72 h of drug exposure, the number of parasites inside the cells was quantitated by indirect immunofluorescence microscopy. Sixteen compounds had IC50s of <3 μM, and five of these had IC50s of <0.3 μM and thus were comparable in potency to trimetrexate. The finding that submicromolar concentrations of several of the compounds in the library could inhibit in vitro growth of C. parvum in host cells in the presence of thymidine (dThd) and hypoxanthine (Hx) suggests that lipophilic DHFR inhibitors, in combination with leucovorin, may find use in the treatment of intractable C. parvum infections.
PMCID: PMC90829  PMID: 11709300
2.  Macrolide Resistance Gene mreA of Streptococcus agalactiae Encodes a Flavokinase 
The mreA gene from Streptococcus agalactiae COH31 γ/δ, resistant to macrolides and clindamycin by active efflux, has recently been cloned in Escherichia coli, where it was reported to confer macrolide resistance (J. Clancy, F. Dib-Hajj, J. W. Petitpas, and W. Yuan, Antimicrob. Agents Chemother. 41:2719–2723, 1997). Cumulative data suggested that the mreA gene was located on the chromosome of S. agalactiae COH31 γ/δ. Analysis of the deduced amino acid sequence of mreA revealed significant homology with several bifunctional flavokinases/(flavin adenine dinucleotide (FAD) synthetases, which convert riboflavin to flavin mononucleotide (FMN) and FMN to FAD, respectively. High-performance liquid chromatography experiments showed that the mreA gene product had a monofunctional flavokinase activity, similar to that of RibR from Bacillus subtilis. Sequences identical to those of the mreA gene and of a 121-bp upstream region containing a putative promoter were detected in strains of S. agalactiae UCN4, UCN5, and UCN6 susceptible to macrolides. mreA and its allele from S. agalactiae UCN4 were cloned on the shuttle vector pAT28. Both constructs were introduced into E. coli, where they conferred a similar two- to fourfold increase in the MICs of erythromycin, spiramycin, and clindamycin. The MICs of a variety of other molecules, including crystal violet, acriflavin, sodium dodecyl sulfate, and antibiotics, such as certain cephalosporins, chloramphenicol, doxycycline, nalidixic acid, novobiocin, and rifampin, were also increased. In contrast, resistance to these compounds was not detected when the constructs were introduced into E. faecalis JH2–2. In conclusion, the mreA gene was probably resident in S. agalactiae and may encode a metabolic function. We could not provide any evidence that it was responsible for macrolide resistance in S. agalactiae COH31 γ/δ; broad-spectrum resistance conferred by the gene in E. coli could involve multidrug efflux pumps by a mechanism that remains to be elucidated.
PMCID: PMC90643  PMID: 11451686
3.  Role of ATP-Binding-Cassette Transporter Genes in High-Frequency Acquisition of Resistance to Azole Antifungals in Candida glabrata 
Candida glabrata has been often isolated from AIDS patients with oropharyngeal candidiasis treated with azole antifungal agents, especially fluconazole. We recently showed that the ATP-binding-cassette (ABC) transporter gene CgCDR1 was upregulated in C. glabrata clinical isolates resistant to azole antifungal agents (D. Sanglard, F. Ischer, D. Calabrese, P. A. Majcherczyk, and J. Bille, Antimicrob. Agents Chemother. 43:2753–2765, 1999). Deletion of CgCDR1 in C. glabrata rendered the null mutant hypersusceptible to azole derivatives and showed the importance of this gene in mediating azole resistance. We observed that wild-type C. glabrata exposed to fluconazole in a medium containing the drug at 50 μg/ml developed resistance to this agent and other azoles at a surprisingly high frequency (2 × 10−4 to 4 × 10−4). We show here that this high-frequency azole resistance (HFAR) acquired in vitro was due, at least in part, to the upregulation of CgCDR1. The CgCDR1 deletion mutant DSY1041 could still develop HFAR but in a medium containing fluconazole at 5 μg/ml. In the HFAR strain derived from DSY1041, a distinct ABC transporter gene similar to CgCDR1, called CgCDR2, was upregulated. This gene was slightly expressed in clinical isolates but was upregulated in strains with the HFAR phenotype. Deletion of both CgCDR1 and CgCDR2 suppressed the development of HFAR in a medium containing fluconazole at 5 μg/ml, showing that both genes are important mediators of resistance to azole derivatives in C. glabrata. We also show here that the HFAR phenomenon was linked to the loss of mitochondria in C. glabrata. Mitochondrial loss could be obtained by treatment with ethidium bromide and resulted in acquisition of resistance to azole derivatives without previous exposure to these agents. Azole resistance obtained in vitro by HFAR or by agents stimulating mitochondrial loss was at least linked to the upregulation of both CgCDR1 and CgCDR2.
PMCID: PMC90441  PMID: 11257032
4.  Antiviral Activity of β-l-2′,3′-Dideoxy-2′,3′-Didehydro-5-Fluorocytidine in Woodchucks Chronically Infected with Woodchuck Hepatitis Virus 
The l-nucleoside analog β-l-2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine (β-l-Fd4C) was first shown to exhibit potent activity against hepatitis B virus (HBV) in tissue culture and then to significantly inhibit viral spread during acute infection in the duck HBV model (F. Le Guerhier et al., Antimicrob. Agents Chemother. 44:111–122, 2000). We have therefore examined its antiviral activity in a mammalian model of chronic HBV infection, the woodchuck chronically infected with woodchuck hepatitis virus (WHV). Side-by-side comparison of β-l-Fd4C and lamivudine administered intraperitoneally during short-term and long-term protocols demonstrated a more profound inhibition of viremia in β-l-Fd4C-treated groups. Moreover, β-l-Fd4C induced a marked inhibition of intrahepatic viral DNA synthesis compared with that induced by lamivudine. Nevertheless, covalently closed circular (CCC) DNA persistence explained the lack of clearance of infected hepatocytes expressing viral antigens and the relapse of WHV replication after drug withdrawal. Liver histology showed a decrease in the inflammatory activity of chronic hepatitis in woodchucks receiving β-l-Fd4C. An electron microscopy study showed the absence of ultrastructural changes of hepatic mitochondria, biliary canaliculi, and bile ducts. However, a loss of weight was observed in all animals, whatever the treatment, as was a transient skin pigmentation in all woodchucks during β-l-Fd4C treatment. There was no evidence that lamivudine or β-l-Fd4C could prevent the development of hepatocellular carcinoma with the protocols used. These results indicate that β-l-Fd4C exhibits a more potent antiviral effect than lamivudine in the WHV model but was not able to eradicate CCC DNA and infected cells from the liver at the dosage and with the protocol used.
PMCID: PMC90426  PMID: 11257017
5.  Efficacy of 2-Amino-7-(1,3-Dihydroxy-2-Propoxymethyl)Purine for Treatment of Vaccinia Virus (Orthopoxvirus) Infections in Mice 
We have previously shown that the N-7 substituted acyclic nucleoside analog 2-amino-7-[1,3-dihydroxy-2-propoxy)methyl]purine (compound S2242) is, both in vitro and in animal models, a potent inhibitor of the replication of several herpesviruses (Neyts et al., Antimicrob. Agents Chemother. 39:56–60, 1995). Here we report on the potent and selective antiviral activity of S2242 against vaccinia virus (VV), an orthopoxvirus. The 50% effective concentrations for inhibition of VV-induced cytopathic effect and viral DNA synthesis in cell culture were 2.4 and 0.2 μg/ml, respectively. We next studied the efficacy of S2242 in VV-infected mice. Immunocompetent NMRI mice that had been inoculated intravenously with VV developed tail lesions. Mice that had been treated for 5 consecutive days via the subcutaneous (s.c.) route with 100 mg of the diacetate ester prodrug of S2242 (compound H961) per kg of body weight did not develop any lesions and demonstrated no adverse effects. Severe combined immunodeficient (SCID) mice that had been inoculated intraperitoneally with VV became sick and died within 1 month after infection. Following treatment with H961 at 100 mg/kg for 10 consecutive days (either via oral gavage or s.c. injection) VV-inoculated SCID mice were completely protected, for at least 3 months, against virus-induced morbidity and mortality. At that time, no virus could be recovered from the organs of these mice (as assessed by titration for infectious virus, a DNA hybridization assay, and a PCR for VV-specific sequences). Compound S2242 and its oral prodrug H961 could be useful in treatment of orthopoxvirus infections.
PMCID: PMC90244  PMID: 11120949
6.  Inducible Azole Resistance Associated with a Heterogeneous Phenotype in Candida albicans 
The development of azole resistance in Candida albicans is most problematic in patients with AIDS who receive long courses of drug for therapy or prevention of oral candidiasis. Recently, the rapid development of resistance was noted in other immunosuppressed patients who developed disseminated candidiasis despite fluconazole prophylaxis. One of these series of C. albicans isolates became resistant, with an associated increase in mRNA specific for a CDR ATP-binding cassette transporter efflux pump (K. A. Marr, C. N. Lyons, T. R. Rustad, R. A. Bowden, and T. C. White, Antimicrob. Agents Chemother. 42:2584–2589, 1998). Here we study this series of C. albicans isolates further and examine the mechanism of azole resistance in a second series of C. albicans isolates that caused disseminated infection in a recipient of bone marrow transplantation. The susceptible isolates in both series become resistant to fluconazole after serial growth in the presence of drug, while the resistant isolates in both series become susceptible after serial transfer in the absence of drug. Population analysis of the inducible, transiently resistant isolates reveals a heterogeneous population of fluconazole-susceptible and -resistant cells. We conclude that the rapid development of azole resistance occurs by a mechanism that involves selection of a resistant clone from a heterogeneous population of cells.
PMCID: PMC90239  PMID: 11120944
7.  Correlation between Antifungal Susceptibilities of Coccidioides immitis In Vitro and Antifungal Treatment with Caspofungin in a Mouse Model 
Caspofungin (Merck Pharmaceuticals) was tested in vitro against 25 clinical isolates of Coccidoides immitis. In vitro susceptibility testing was performed in accordance with the National Committee for Clinical Laboratory Standards document M38-P guidelines. Two C. immitis isolates for which the caspofungin MICs were different were selected for determination of the minimum effective concentration (MEC), and these same strains were used for animal studies. Survival and tissue burdens of the spleens, livers, and lungs were used as antifungal response markers. Mice infected with strain 98-449 (48-h MIC, 8 μg/ml; 48-h MEC, 0.125 μg/ml) showed 100% survival to day 50 when treated with caspofungin at ≥1 mg/kg. Mice infected with strain 98-571 (48-h MIC, 64 μg/ml; 48-h MEC, 0.125 μg/ml) displayed ≥80% survival when the treatment was caspofungin at ≥5 mg/kg. Treatment with caspofungin at 0.5, 1, 5, or 10 mg/kg was effective in reducing the tissue fungal burdens of mice infected with either isolate. When tissue fungal burden study results were compared between strains, caspofungin showed no statistically significant difference in efficacy in the organs of the mice treated with both strains. A better in vitro-in vivo correlation was noted when we used the MEC instead of the MIC as the endpoint for antifungal susceptibility testing. Caspofungin may have a role in the treatment of coccidioidomycosis.
PMCID: PMC90557  PMID: 11353637
8.  Mechanisms of Macrolide Resistance in Clinical Pneumococcal Isolates in France 
The genetic basis of macrolide resistance was investigated in a collection of 48 genotypically unrelated clinical isolates of Streptococcus pneumoniae obtained between 1987 and 1997 in France. All strains were resistant to erythromycin, clindamycin, and streptogramin B, exhibiting a macrolide-lincosamide-streptogramin B resistance phenotype, and harbored the erm(B) gene. None of the strains carried the mef(A) or erm(A) subclass erm(TR) gene.
PMCID: PMC90344  PMID: 11158772
10.  Use of Mutator Strains for Characterization of Novel Antimicrobial Agents 
PMCID: PMC90517  PMID: 11372639
12.  Levofloxacin-Resistant Streptococcus pneumoniae: Second Look 
PMCID: PMC90629  PMID: 11441827
13.  Cloning and Biochemical Characterization of FOX-5, an AmpC-Type Plasmid-Encoded β-Lactamase from a New York City Klebsiella pneumoniae Clinical Isolate 
Antimicrobial Agents and Chemotherapy  2001;45(11):3189-3194.
Klebsiella pneumoniae 5064, isolated in New York, carried plasmid-mediated resistance to multiple β-lactams and was unresponsive to clavulanic acid. The β-lactamase gene responsible for cephalosporin resistance encoded FOX-5, with 96 to 97% amino acid identities to other members of the FOX family of β-lactamases. The blaFOX-5 coding region was located next to a transposase gene from the Aeromonas salmonicida insertion element ISAS2.
PMCID: PMC90802  PMID: 11600376
14.  In Vitro Activities of Posaconazole (Sch 56592) Compared with Those of Itraconazole and Fluconazole against 3,685 Clinical Isolates of Candida spp. and Cryptococcus neoformans 
Antimicrobial Agents and Chemotherapy  2001;45(10):2862-2864.
Posaconazole is a new investigational triazole with broad-spectrum antifungal activity. The in vitro activities of posaconazole were compared with those of itraconazole and fluconazole against 3,685 isolates of Candida spp. (3,312 isolates) and C. neoformans (373 isolates) obtained from over 70 different medical centers worldwide. The MICs of the antifungal drugs were determined by broth microdilution tests performed according to the National Committee for Clinical Laboratory Standards method using RPMI 1640 as the test medium. Posaconazole was very active against all Candida spp. (MIC at which 90% of the isolates were inhibited [MIC90], 0.5 μg/ml; 97% of MICs were ≤1 μg/ml) and C. neoformans (MIC90, 0.5 μg/ml; 100% of MICs were ≤1 μg/ml). Candida albicans was the most susceptible species of Candida (MIC90, 0.06 μg/ml), and Candida glabrata was the least susceptible (MIC90, 4 μg/ml). Posaconazole was more active than itraconazole and fluconazole against all Candida spp. and C. neoformans. These results provide further evidence for the spectrum and potency of posaconazole against a large and geographically diverse collection of clinically important fungal pathogens.
PMCID: PMC90743  PMID: 11557481
15.  Update on Rifampin Resistance in the Legionellaceae 
PMCID: PMC90628  PMID: 11441826
16.  Azasordarins: Susceptibility of Fluconazole-Susceptible and Fluconazole-Resistant Clinical Isolates of Candida spp. to GW 471558 
The in vitro activity of the azasordarin GW 471558 was compared with those of amphotericin B, flucytosine, itraconazole, and ketoconazole against 177 clinical isolates of Candida spp. GW 471558 showed potent activity against Candida albicans, Candida glabrata, and Candida tropicalis, even against isolates with decreased susceptibility to azoles. Candida krusei, Candida parapsilosis, Candida lusitaniae, and Candida guilliermondii are resistant to GW 471558 in vitro (MICs, >128 μg/ml).
PMCID: PMC90570  PMID: 11353650
17.  Activities of BMS 284756 (T-3811) against Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae Isolates from SENTRY Antimicrobial Surveillance Program Medical Centers in Latin America (1999) 
The antimicrobial activity of BMS 284756, a novel des-F(6)-quinolone, was comparatively evaluated against 257 Streptococcus pneumoniae, 198 Haemophilus influenzae, and 88 Moraxella catarrhalis strains isolated in Latin America between July and September of 1999 as part of the SENTRY Antimicrobial Surveillance Program. Nearly 28.0% of S. pneumoniae strains were nonsusceptible to penicillin. The rank order of quinolone potency versus S. pneumoniae was BMS 284756 (MIC at which 90% of isolates were inhibited [MIC90], 0.12 μg/ml) > trovafloxacin (MIC90, 0.25 μg/ml) > gatifloxacin (MIC90, 0.5 μg/ml) > levofloxacin and ciprofloxacin (MIC90, 1 to 2 μg/ml). All S. pneumoniae strains that were not susceptible to other quinolones were inhibited by BMS 284756 at ≤2 μg/ml. The overall prevalence of β-lactamase production was 15.2% in H. influenzae and 98.9% in M. catarrhalis. BMS 284756 showed excellent potency and spectrum against this group of pathogens, inhibiting all isolates at ≤0.12 μg/ml. BMS 284756 exhibited activity similar to those displayed by the new fluoroquinolones, such as levofloxacin, trovafloxacin, or gatifloxacin, and could be a therapeutic option for empirical treatment of community-acquired respiratory tract infections.
PMCID: PMC90489  PMID: 11302811
19.  Molecular Analysis of Tn1546-Like Elements in Vancomycin-Resistant Enterococci Isolated from Patients in Europe Shows Geographic Transposon Type Clustering 
Resistance mechanism relatedness was studied in 18 clinical, European vanA vancomycin-resistant enterococci. Molecular analysis revealed 10 Tn1546-like elements, suggesting two evolutionary lineages. Lineage I dominated the European mainland, and lineage II dominated the United Kingdom and Israel. Geographic clustering reflected different types of meat consumption between countries, since each lineage is associated with colonization of different animals.
PMCID: PMC90411  PMID: 11181398
20.  In Vitro Uptake of SCH 27899 (Evernimicin) by Rat Alveolar Macrophages 
The in vitro uptake of [14C]evernimicin ([14C]SCH 27899) by primary cultures of rat alveolar macrophages and hepatocytes was determined. Both cell populations exhibited linear rates of uptake. However, the initial rate of drug uptake by alveolar macrophages was about threefold higher than that by hepatocytes. These findings demonstrate that [14C]evernimicin is taken up by rat alveolar macrophages, supporting the likelihood that the drug is able to reach sites of infection.
PMCID: PMC90404  PMID: 11181391
21.  Compartmental Pharmacokinetics of the Antifungal Echinocandin Caspofungin (MK-0991) in Rabbits 
The pharmacokinetics of the antifungal echinocandin-lipopeptide caspofungin (MK-0991) in plasma were studied in groups of three healthy rabbits after single and multiple daily intravenous administration of doses of 1, 3, and 6 mg/kg of body weight. Concentrations were measured by a validated high-performance liquid chromatography method and fitted into a three-compartment open pharmacokinetic model. Across the investigated dosage range, caspofungin displayed dose-independent pharmacokinetics. Following administration over 7 days, the mean peak concentration in plasma (Cmax) ± standard error of the mean increased from 16.01 ± 0.61 μg/ml at the 1-mg/kg dose to 105.52 ± 8.92 μg/ml at the 6-mg/kg dose; the mean area under the curve from 0 h to infinity rose from 13.15 ± 2.37 to 158.43 ± 15.58 μg · h/ml, respectively. The mean apparent volume of distribution at steady state (Vdss) was 0.299 ± 0.011 liter/kg at the 1-mg/kg dose and 0.351 ± 0.016 liter/kg at the 6-mg/kg dose (not significant [NS]). Clearance (CL) ranged from 0.086 ± 0.017 liter/kg/h at the 1-mg/kg dose to 0.043 ± 0.004 liter/kg/h at the 6-mg/kg dose (NS), and the mean terminal half-life was between 30 and 34 h (NS). Except for a trend towards an increased Vdss, there were no significant differences in pharmacokinetic parameters in comparison to those after single-dose administration. Caspofungin was well tolerated, displayed linear pharmacokinetics that fit into a three-compartment pharmacokinetic model, and achieved sustained concentrations in plasma that were multiple times in excess of reported MICs for susceptible opportunistic fungi.
PMCID: PMC90333  PMID: 11158761
22.  In Vivo Activity of Evernimicin (SCH 27899) against Methicillin-Resistant Staphylococcus aureus in Experimental Infective Endocarditis 
Currently, there exist few satisfactory alternatives to vancomycin for therapy of serious methicillin-resistant Staphylococcus aureus (MRSA) infections. We employed a rat model of aortic valve endocarditis to assess the potential efficacy of evernimicin (SCH 27899) compared with vancomycin against infection with a strain susceptible to both agents (MICs of 0.25 and 0.50 μg/ml, respectively). Infected animals were assigned to one of three groups: controls (no treatment), evernimicin at 60 mg/kg of body weight by intravenous (i.v.) infusion once daily, or vancomycin at 150 mg/kg of body weight per day by continuous i.v. infusion. Therapy was administered for 5.5 days. At the start of therapy, colony counts in vegetations were 6.63 ± 0.44 log10 CFU/g. In both treatment groups, bacterial density within vegetations was significantly reduced in comparison with control animals that had not been treated. Final colony counts were as follows (mean ± standard deviation): controls, 10.12 ± 1.51 log10 CFU/g of vegetation; evernimicin, 7.22 ± 2.91 log10 CFU/g of vegetation; vancomycin, 5.65 ± 1.76 log10 CFU/g of vegetation. The difference between the evernimicin and vancomycin groups was not significant. These results confirmed the bacteriostatic activity of evernimicin in vivo in an experimental model of severe MRSA infection.
PMCID: PMC90262  PMID: 11120967
24.  Staphylococcus aureus Mutants Isolated via Exposure to Nonfluorinated Quinolones: Detection of Known and Unique Mutations 
Antimicrobial Agents and Chemotherapy  2001;45(12):3422-3426.
The in vitro development of resistance to the new nonfluorinated quinolones (NFQs; PGE 9262932, PGE 4175997, and PGE 9509924) was investigated in Staphylococcus aureus. At concentrations two times the MIC, step 1 mutants were isolated more frequently with ciprofloxacin and trovafloxacin (9.1 × 10−8 and 5.7 × 10−9, respectively) than with the NFQs, gatifloxacin, or clinafloxacin (<5.7 × 10−10). Step 2 and step 3 mutants were selected via exposure of a step 1 mutant (selected with trovafloxacin) to four times the MICs of trovafloxacin and PGE 9262932. The step 1 mutant contained the known Ser80-Phe mutation in GrlA, and the step 2 and step 3 mutants contained the known Ser80-Phe and Ser84-Leu mutations in GrlA and GyrA, respectively. Compared to ciprofloxacin, the NFQs were 8-fold more potent against the parent and 16- to 128-fold more potent against the step 3 mutants. Mutants with high-level NFQ resistance (MIC, 32 μg/ml) were isolated by the spiral plater-based serial passage technique. DNA sequence analysis of three such mutants revealed the following mutations: (i) Ser84-Leu in GyrA and Glu84-Lys and His103-Tyr in GrlA; (ii) Ser-84Leu in GyrA, Ser52-Arg in GrlA, and Glu472-Val in GrlB; and (iii) Ser84-Leu in GyrA, Glu477-Val in GyrB, and Glu84-Lys and His103-Tyr in GrlA. Addition of the efflux pump inhibitor reserpine (10 μg/ml) resulted in 4- to 16-fold increases in the potencies of the NFQs against these mutants, whereas it resulted in 2-fold increases in the potencies of the NFQs against the parent.
PMCID: PMC90847  PMID: 11709318
25.  Antifungal Activities and Cytotoxicity Studies of Six New Azasordarins 
Antimicrobial Agents and Chemotherapy  2001;45(11):3132-3139.
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 (MIC90s) were 0.002 μg/ml with GW 479821, 0.015 μg/ml with GW 515716 and GW 587270, and 0.06 μg/ml with GW 471552, GW 471558, and GW 570009. The MIC90s 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 μg/ml, respectively, against C. tropicalis and 4, 0.25, 0.06, 0.25, 0.5, and 0.5 μg/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 MIC90s of 2, 4, 4, and 1 μg/ml, respectively. The compounds were extremely potent against P. carinii, showing 50% inhibitory concentrations of ≤0.001 μg/ml. However Cryptococcus neoformans was resistant to all compounds tested (MIC > 16 μg/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 μg/ml, those of GW 479821 and GW 515716 ranged from 0.12 to 2 μg/ml, and those of GW 570009 ranged from 0.12 to 4 μg/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 μg/ml. Against Aspergillus spp., the MICs of the compounds tested were higher than 16 μg/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 (Tox50s) 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 μg/ml, respectively. The cytotoxicity values of GW 471552 and GW 471558 were >100 μg/ml for all cell lines tested. Tox50s 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 μg/ml to 23 μg/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.
PMCID: PMC90794  PMID: 11600368

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