Domingo Gargallo-Viola

Improved Murine Model of Malaria Using Plasmodium falciparum Competent Strains and Non-Myelodepleted NOD-scid IL2Rγnull Mice Engrafted with Human Erythrocytes

ABSTRACT Murine models of Plasmodium falciparum malaria may become crucial tools in drug discovery. Here we show that non-myelodepleted NOD-scid IL2Rγnull mice engrafted with human erythrocytes support an infectious burden up to tenfold higher than that supported by engrafted NOD-scid β2microglobulinnull mice. The new model was validated for drug discovery and was used to assess the therapeutic efficacy of 4-pyridones, selective inhibitors of P. falciparum cytochrome bc1.

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.

Anticoagulants Interfere with PCR Used To Diagnose Invasive Aspergillosis

Invasive aspergillosis is a disease that affects immunodepressed individuals and has become one of the leading causes of death in many transplant centers ([1][1]). Owing to the difficulties encountered in diagnosis of the disease and to the high sensitivity of the PCR technique, PCR-based methods

Quantitative measurement of Plasmodium-infected erythrocytes in murine models of malaria by flow cytometry using bidimensional assessment of SYTO-16 fluorescence.

Flow cytometry is a powerful tool for measuring parasitemias in murine malaria models used to test new antimalarials. Measurement of the emission of the nonpermeable nucleic acid dye YOYO‐1 (at 530 and 585 nm after excitation at 488 nm) allowed the unambiguous detection of low parasitemias (≥0.01%) but required prolonged fixation and permeabilization of the sample. Thus, we tested whether this issue could be overcome by use of the cell‐permeant dye SYTO‐16 with this same bidimensional method. Blood samples from CD1 mice infected with Plasmodium yoelii, Plasmodium vinckei, or Plasmodium chabaudi or from NODscidβ2m‐/‐ engrafted with human erythrocytes and infected with P. falciparum were stained with SYTO‐16 in the presence or absence of TER‐119 mAb (for engrafted mice) in 96‐well plate format and acquired in Trucount™ tubes. Bidimensional analysis with SYTO‐16 was quantitatively equivalent to YOYO‐1. Moreover, by combining SYTO‐16 with the use of TER‐119‐PE antimouse erythrocyte mAb and Trucount tubes, the measurement of the concentration of P. falciparum‐infected erythrocytes over a range of five orders of magnitude was achieved. Bidimensional analysis using SYTO‐16 can be used to accurately measure the concentration of Plasmodium spp.‐infected erythrocytes in mice without complex sample preparation.

Improvement of detection specificity of Plasmodium-infected murine erythrocytes by flow cytometry using autofluorescence and YOYO-1.

Microscopic analysis of blood smears is currently the most frequently used method to measure parasitemias in experiments of drug efficacy in murine models of malaria. However, it is subjective and labour intensive, which preclude its utilization in large‐scale evaluation programs. Flow cytometry is an alternative method, but due to the limited specificity achieved with the currently available techniques, it has not been widely used in murine models of malaria during preclinical evaluation. We describe a new flow cytometric method based on the differences of autofluorescence and DNA content measured after staining with YOYO‐1 that are observed in infected erythrocytes compared with noninfected erythrocytes.

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.

Activities of Sordarins in Experimental Models of Candidiasis, Aspergillosis, and Pneumocystosis

ABSTRACT 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 μg/ml, 79.5 and 46 μg · 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 againstPneumocystis carinii was studied in an experimentalP. 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 μg/ml, 8.5 and 11.8 μg · 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 withAspergillus in other animal models is warranted.

Exploration of 4(1H)-pyridones as a novel family of potent antimalarial inhibitors of the plasmodial cytochrome bc1

A novel family of antimalarials based on the 4(1H)-pyridone scaffold is described. The compounds display potent antimalarial activity against Plasmodium falciparum in vitro and in vivo. Like atovaquone, 4(1H)-pyridones exert their antimalarial action by inhibiting selectively the electron-transport chain in P. falciparum at the cytochrome bc1 level (complex III). However, despite the similar mechanism of action, no cross-resistance with atovaquone has been found, suggesting that the binding mode of 4(1H)-pyridones might be different from that of atovaquone. The medicinal chemistry program, focused on improving potency and physicochemical properties, ultimately led to the discovery of GSK932121, which was progressed efficiently into first time in human studies. However, progression of GSK932121 was terminated when new toxicology results were obtained in the rat with a soluble phosphate prodrug of the candidate, indicating a potentially narrow therapeutic index.

In Vitro Pharmacodynamic Parameters of Sordarin Derivatives in Comparison with Those of Marketed Compounds against Pneumocystis carinii Isolated from Rats

ABSTRACT 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 (Emax), the drug concentrations to reach 50% of Emax(EC50), and the slope of the dose-response curve were then estimated by the Hill equation (Emax sigmoid model). Sordarin derivatives were the most potent agents againstP. carinii, with EC50s of 0.00025, 0.0007, 0.0043, and 0.025 μg/ml for GM 191519, GM 237354, GM 193663, and GM 219771, respectively. The EC50s of pentamidine, atovaquone, and TMP-SMX were 0.025, 0.16, and 26.7/133.5 μg/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 [ED50], 0.05 mg/kg/day) followed by GM 237354 and GM 219771 (ED50s, 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.