Federico Gómez de las Heras

A murine model of falciparum-malaria by in vivo selection of competent strains in non-myelodepleted mice engrafted with human erythrocytes.

To counter the global threat caused by Plasmodium falciparum malaria, new drugs and vaccines are urgently needed. However, there are no practical animal models because P. falciparum infects human erythrocytes almost exclusively. Here we describe a reliable falciparum murine model of malaria by generating strains of P. falciparum in vivo that can infect immunodeficient mice engrafted with human erythrocytes. We infected NODscid/β2m−/− mice engrafted with human erythrocytes with P. falciparum obtained from in vitro cultures. After apparent clearance, we obtained isolates of P. falciparum able to grow in peripheral blood of engrafted NODscid/β2m−/− mice. Of the isolates obtained, we expanded in vivo and established the isolate Pf3D70087/N9 as a reference strain for model development. Pf3D70087/N9 caused productive persistent infections in 100% of engrafted mice infected intravenously. The infection caused a relative anemia due to selective elimination of human erythrocytes by a mechanism dependent on parasite density in peripheral blood. Using this model, we implemented and validated a reproducible assay of antimalarial activity useful for drug discovery. Thus, our results demonstrate that P. falciparum contains clones able to grow reproducibly in mice engrafted with human erythrocytes without the use of myeloablative methods.

Synthesis and Structure-Activity Relationships of 4-Pyridones as Potential Antimalarials

A series of diaryl ether substituted 4-pyridones have been identified as having potent antimalarial activity superior to that of chloroquine against Plasmodium falciparum in vitro and murine Plasmodium yoelii in vivo. These were derived from the anticoccidial drug clopidol through a systematic study of the effects of varying the side chain on activity. Relative to clopidol the most active compounds show >500-fold improvement in IC50 for inhibition of P. falciparum in vitro and about 100-fold improvement with respect to ED50 against P. yoelii in mice. These compounds have been shown elsewhere to act selectively by inhibition of mitochondrial electron transport at the cytochrome bc1 complex.

Antifungal Activities and Cytotoxicity Studies of Six New Azasordarins

ABSTRACT 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 againstPneumocystis 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 againstP. 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. AgainstFusarium 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.

Candida albicans infection enhances immunosuppression induced by cyclophosphamide by selective priming of suppressive myeloid progenitors for NO production

Systemic infections caused by fungi after cytoreductive therapies are especially difficult to deal with in spite of currently available antimicrobials. However, little is known about the effects of fungi on the immune system of immunosuppressed hosts. We have addressed this by studying the in vitro T cell responses after systemic infection with Candida albicans in cyclophosphamide-treated mice. After cyclophosphamide treatment, a massive splenic colonization of the spleens, but not lymph nodes, by immature myeloid progenitor (Ly-6G(+)CD11b(+))cells is observed. These cells are able to suppress proliferation of T lymphocytes via a nitric oxide (NO)-dependent mechanism. Systemic infection with a sublethal dose of C. albicans did not cause immunosuppression per se but strongly increased NO-dependent suppression in cyclophosphamide-treated mice, by selective priming of suppressive myeloid progenitors (Ly-6G(+)CD11b(+)CD31(+)CD40(+)WGA(+)CD117(low/-)CD34(low/-)) for iNOS protein expression. The results indicate that systemic C. albicans infection can augment the effects of immunosuppressive therapies by promoting functional changes in immunosuppressive cells.

Antifungal Activities of Two New Azasordarins, GW471552 and GW471558, in Experimental Models of Oral and Vulvovaginal Candidiasis in Immunosuppressed Rats

ABSTRACT 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 × 108 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 107 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) theC. 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.

A new synthesis of 3,5-dihydroxy-7-(1-pyrrolyl)-6-heptenoic acids, a family of HMGCoA reductase inhibitors with antifungal activity

Abstract Starting from a 3,5-dihydroxyheptanoic acid derivative, a new synthesis of pyrrole statins that contain a double bond on the dihydroxyacid chain has been developed. Key steps are N-acylamino acid enamide formation through enamine trapping with aromatic acyl chlorides and subsequent munchnone cycloaddition with activated acetylenes.