posaconazole and Disease-Models--Animal

Tuberculosis and parasitic infections continue to impose a significant threat to global public health and economic growth. There is an urgent need to develop new treatments to combat these diseases. Here, we report the

Topics: Animals; Chagas Disease; Disease Models, Animal; Mice; Mycobacterium tuberculosis; Nitroimidazoles; Nitroreductases; Trypanosoma cruzi; Tuberculosis

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is an increasing threat to global health. Available medicines were introduced over 40 years ago, have undesirable side effects, and give equivocal results of cure in the chronic stage of the disease. We report the development of two compounds, 6 and (S)-7, with PCR-confirmed curative activity in a mouse model of established T. cruzi infection after once daily oral dosing for 20 days at 20 mg/kg 6 and 10 mg/kg (S)-7. Compounds 6 and (S)-7 have potent in vitro activity, are noncytotoxic, show no adverse effects in vivo following repeat dosing, are prepared by a short synthetic route, and have druglike properties suitable for preclinical development.

Topics: Animals; Chagas Disease; Disease Models, Animal; Dose-Response Relationship, Drug; Male; Mice; Molecular Structure; Parasitic Sensitivity Tests; Pyrimidines; Structure-Activity Relationship; Trypanocidal Agents; Trypanosoma cruzi

We report the discovery of nontoxic fungicide fenarimol (1) as an inhibitor of Trypanosoma cruzi ( T. cruzi ), the causative agent of Chagas disease, and the results of structure-activity investigations leading to potent analogues with low nM IC(50)s in a T. cruzi whole cell in vitro assay. Lead compounds suppressed blood parasitemia to virtually undetectable levels after once daily oral dosing in mouse models of T. cruzi infection. Compounds are chemically tractable, allowing rapid optimization of target biological activity and drug characteristics. Chemical and biological studies undertaken in the development of the fenarimol series toward the goal of delivering a new drug candidate for Chagas disease are reported.

Topics: Animals; Cell Line; Chagas Disease; Disease Models, Animal; Gas Chromatography-Mass Spectrometry; Inhibitory Concentration 50; Male; Mice; Nuclear Magnetic Resonance, Biomolecular; Pyrimidines; Rats; Rats, Sprague-Dawley; Structure-Activity Relationship; Trypanocidal Agents; Trypanosoma cruzi

The in vivo efficacy of posaconazole against 4 clinical Aspergillus fumigatus isolates with posaconazole MICs ranging from 0.03 to 16 mg/liter, as determined by CLSI method M38A, was assessed in a nonneutropenic murine model of disseminated aspergillosis. The underlying resistance mechanisms of the isolates included substitutions in the cyp51A gene at codon 220 (M220I), codon 54 (G54W), and codon 98 (L98H). The latter was combined with a 34-bp tandem repeat in the gene promoter region (TR L98H). The control isolate exhibited a wild-type phenotype without any known resistance mechanism. Oral posaconazole therapy was started 24 h after infection and was given once daily for 14 consecutive days. Mice were treated with four different doses (1 to 64 mg/kg of body weight), and survival was used as the end point. Survival was dependent both on the dose and on the MIC. The Hill equation with a variable slope fitted the relationship between the dose/MIC ratio and 14-day survival well (R2, 0.92), with a 50% effective dose (ED50) of 29.0 mg/kg (95% confidence interval [CI], 15.6 to 53.6 mg/kg). This also applied to the relationship between the area under the plasma concentration-time curve (AUC)/MIC ratio and 14-day survival (50% effective pharmacodynamic index [EI50], 321.3 [95% CI, 222.7 to 463.4]). Near-maximum survival was reached at an AUC/MIC ratio of nearly 1,000. These results indicate that treatment of infections with A. fumigatus strains for which MICs are 0.5 mg/liter requires doses exceeding the present licensed doses. Increasing the standard dosing regimen may have some effect and may be clinically useful if no alternatives are available.

Topics: Administration, Oral; Animals; Antifungal Agents; Area Under Curve; Aspergillosis; Aspergillus fumigatus; Cytochrome P-450 Enzyme System; Disease Models, Animal; Drug Administration Schedule; Female; Fungal Proteins; Mice; Microbial Sensitivity Tests; Mutation; Triazoles

We have evaluated the efficacy of posaconazole, amphotericin B, and itraconazole in a murine model of disseminated infection by Fonsecaea monophora. Of these three antifungal drugs tested, posaconazole prolonged survival significantly and reduced the fungal load in most of the organs tested. Bioassay studies demonstrated the relationship between posaconazole levels and dose escalation in serum and brain tissue. Posaconazole may have a clinical role in the treatment of disseminated infections by F. monophora.

Topics: Animals; Antifungal Agents; Ascomycota; Brain; Disease Models, Animal; Kidney; Liver; Lung; Male; Mice; Mycoses; Triazoles

Invasive fusariosis is a highly aggressive fungal infection associated with high mortality in heavily immunocompromised patients. Although posaconazole is efficacious as salvage therapy against infections caused by Fusarium species, concerns remain regarding this agent in the setting of reduced potency. To evaluate the efficacy of posaconazole as treatment or prophylaxis against invasive fusariosis caused by Fusarium solani, we utilized a neutropenic murine model of disseminated disease. ICR mice were administered escalating doses of posaconazole (6.25, 12.5, 25, or 50 mg/kg of body weight twice daily [BID]) by oral gavage beginning 2 days prior to inoculation in the prophylaxis studies or beginning 12 h after inoculation as treatment. Therapy was continued until day 9 postinoculation, and animals were monitored off therapy until day 15 for survival. Fungal burden was assessed as CFU in the kidneys. A clear dose-response relationship was observed, as the highest dose of posaconazole (50 mg/kg) was the most effective in prolonging survival and reducing tissue fungal burden both as prophylaxis and as treatment. This dose response was associated with high posaconazole serum concentrations as measured by bioassay. However, the extent of efficacy was also dependent on the infecting inoculum, as greater increases in survival and reductions in fungal burden were observed with the lower inocula tested. In this model high dosages of posaconazole were effective as treatment and prophylaxis against disseminated fusariosis caused by F. solani.

Topics: Animals; Antifungal Agents; Chemoprevention; Disease Models, Animal; Drug Resistance, Fungal; Fusarium; Humans; Kidney; Mice; Mice, Inbred ICR; Microbial Sensitivity Tests; Mycoses; Neutropenia; Treatment Outcome; Triazoles

Principal mechanisms of resistance to azole antifungals include the upregulation of multidrug transporters and the modification of the target enzyme, a cytochrome P450 (Erg11) involved in the 14alpha-demethylation of ergosterol. These mechanisms are often combined in azole-resistant Candida albicans isolates recovered from patients. However, the precise contributions of individual mechanisms to C. albicans resistance to specific azoles have been difficult to establish because of the technical difficulties in the genetic manipulation of this diploid species. Recent advances have made genetic manipulations easier, and we therefore undertook the genetic dissection of resistance mechanisms in an azole-resistant clinical isolate. This isolate (DSY296) upregulates the multidrug transporter genes CDR1 and CDR2 and has acquired a G464S substitution in both ERG11 alleles. In DSY296, inactivation of TAC1, a transcription factor containing a gain-of-function mutation, followed by sequential replacement of ERG11 mutant alleles with wild-type alleles, restored azole susceptibility to the levels measured for a parent azole-susceptible isolate (DSY294). These sequential genetic manipulations not only demonstrated that these two resistance mechanisms were those responsible for the development of resistance in DSY296 but also indicated that the quantitative level of resistance as measured in vitro by MIC determinations was a function of the number of genetic resistance mechanisms operating in any strain. The engineered strains were also tested for their responses to fluconazole treatment in a novel 3-day model of invasive C. albicans infection of mice. Fifty percent effective doses (ED(50)s) of fluconazole were highest for DSY296 and decreased proportionally with the sequential removal of each resistance mechanism. However, while the fold differences in ED(50) were proportional to the fold differences in MICs, their magnitude was lower than that measured in vitro and depended on the specific resistance mechanism operating.

Topics: Animals; Antifungal Agents; Base Sequence; Candida albicans; Candidiasis; Disease Models, Animal; DNA Primers; DNA, Fungal; Drug Resistance, Fungal; Female; Fluconazole; Fungal Proteins; Genes, Fungal; Genetic Engineering; Humans; In Vitro Techniques; Membrane Transport Proteins; Mice; Mice, Inbred BALB C; Microbial Sensitivity Tests; Mutation

We have evaluated the in vitro activity of posaconazole (PSC) against 50 clinical strains of Rhizopus oryzae using a broth microdilution method, the Neo-Sensitabs tablet diffusion method, and minimal fungicidal concentration (MFC) determination. In general, PSC showed low MICs against this fungus, and the MICs correlated with the inhibition zone diameters. Most of the MFCs, however, were from 1 to 4 dilutions higher than their corresponding MICs. We then investigated the efficacies of several different doses of PSC in a murine model. All treatments began 24 h after challenge and lasted for 7 days. The drug was administered twice a day to mice infected with three strains that showed intermediate PSC susceptibility (MIC = 2 microg/ml) and three PSC-susceptible strains (MIC = 0.25 microg/ml). A dose of 10 mg/kg of body weight was ineffective, while doses of 20 and 30 mg/kg prolonged the survival of the mice. The 50 strains tested were segregated into two groups on the basis of the in vitro data. For the group with the most strains (85%), the strains had low PSC MICs, mice infected with the strains showed higher rates of survival (30 to 40%), and PSC was able to reduce the fungal load in the kidney and less regularly in the brain. For the second group (15% of the strains), the strains had intermediate PSC MICs, mice infected with the strains had lower survival rates (10 to 20%), and PSC treatment resulted in variable and no reductions in the fungal loads in the kidneys and brains, respectively.

Topics: Animals; Antifungal Agents; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Resistance, Fungal; In Vitro Techniques; Kaplan-Meier Estimate; Kidney; Mice; Mucormycosis; Rhizopus; Triazoles

Chagas' disease, the leading cause of heart failure in Latin America, is caused by the kinetoplastid protozoan Trypanosoma cruzi. The sterols of T. cruzi resemble those of fungi, both in composition and in biosynthesis. Azole inhibitors of sterol 14alpha-demethylase (CYP51) successfully treat fungal infections in humans, and efforts to adapt the success of antifungal azoles posaconazole and ravuconazole as second-use agents for Chagas' disease are under way. However, to address concerns about the use of azoles for Chagas' disease, including drug resistance and cost, the rational design of nonazole CYP51 inhibitors can provide promising alternative drug chemotypes. We report the curative effect of the nonazole CYP51 inhibitor LP10 in an acute mouse model of T. cruzi infection. Mice treated with an oral dose of 40 mg LP10/kg of body weight twice a day (BID) for 30 days, initiated 24 h postinfection, showed no signs of acute disease and had histologically normal tissues after 6 months. A very stringent test of cure showed that 4/5 mice had negative PCR results for T. cruzi, and parasites were amplified by hemoculture in only two treated mice. These results compare favorably with those reported for posaconazole. Electron microscopy and gas chromatography-mass spectrometry (GC-MS) analysis of sterol composition confirmed that treatment with LP10 blocked the 14alpha-demethylation step and induced breakdown of parasite cell membranes, culminating in severe ultrastructural and morphological alterations and death of the clinically relevant amastigote stage of the parasite.

Topics: Aminopyridines; Animals; Antiprotozoal Agents; Catalytic Domain; Chagas Disease; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Disease Models, Animal; Enzyme Inhibitors; Female; Humans; Indoles; Mice; Mice, Inbred C3H; Microscopy, Electron, Transmission; Models, Molecular; Protozoan Proteins; Recombinant Proteins; Sterols; Trypanosoma cruzi

We report structure-activity studies of a large number of dialkyl imidazoles as inhibitors of Trypanosoma cruzi lanosterol-14alpha-demethylase (L14DM). The compounds have a simple structure compared to posaconazole, another L14DM inhibitor that is an anti-Chagas drug candidate. Several compounds display potency for killing T. cruzi amastigotes in vitro with values of EC(50) in the 0.4-10 nM range. Two compounds were selected for efficacy studies in a mouse model of acute Chagas disease. At oral doses of 20-50 mg/kg given after establishment of parasite infection, the compounds reduced parasitemia in the blood to undetectable levels, and analysis of remaining parasites by PCR revealed a lack of parasites in the majority of animals. These dialkyl imidazoles are substantially less expensive to produce than posaconazole and are appropriate for further development toward an anti-Chagas disease clinical candidate.

Topics: Acute Disease; Animals; Chagas Disease; Cytochrome P-450 Enzyme Inhibitors; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Imidazoles; Mice; Mice, Inbred BALB C; Models, Molecular; Molecular Structure; Parasitic Sensitivity Tests; Sterol 14-Demethylase; Structure-Activity Relationship; Trypanosoma cruzi

Serum (1-->3)-beta-D-glucan concentrations were serially measured in the presence and absence of antifungal therapy in a murine model of invasive pulmonary aspergillosis. Serum (1-->3)-beta-D-glucan was detected early during the course of infection, and reductions in this biomarker were associated with improved survival in animals treated with antifungal agents.

Topics: Animals; Animals, Outbred Strains; Antifungal Agents; Aspergillosis; Aspergillus fumigatus; beta-Glucans; Biomarkers; Disease Models, Animal; Humans; Lung; Lung Diseases, Fungal; Mice; Mice, Inbred ICR; Proteoglycans; Treatment Outcome

Two clinical isolates of Aspergillus fumigatus, designated AT and DK, were recently obtained from patients failing caspofungin and itraconazole therapy, respectively. The isolates were tested by microdilution for susceptibility to itraconazole, voriconazole, posaconazole, ravuconazole, and caspofungin and by Etest for susceptibility to amphotericin B and caspofungin. Susceptibility testing documented that the DK isolate was azole resistant (itraconazole and posaconazole MICs, >4 microg/ml; voriconazole MIC, 2 microg/ml; ravuconazole MIC, 4 microg/ml), and the resistance was confirmed in a hematogenous mouse model, with mortality and the galactomannan index as the primary and secondary end points. Sequencing of the cyp51A gene revealed the M220K mutation, conferring multiazole resistance. The Etest, but not microdilution, suggested that the AT isolate was resistant to caspofungin (MIC, >32 microg/ml). In the animal model, this isolate showed reduced susceptibility to caspofungin. Sequencing of the FKS1 gene revealed no mutations; the enzyme retained full sensitivity in vitro; and investigation of the polysaccharide composition showed that the beta-(1,3)-glucan proportion was unchanged. However, gene expression profiling by Northern blotting and real-time PCR demonstrated that the FKS gene was expressed at a higher level in the AT isolate than in the susceptible control isolate. To our knowledge, this is the first report to document the presence of multiazole-resistant clinical isolates in Denmark and to demonstrate reduced susceptibility to caspofungin in a clinical A. fumigatus isolate with increased expression of the FKS gene. Further research to determine the prevalence of resistance in A. fumigatus worldwide, and to develop easier and reliable tools for the identification of such isolates in routine laboratories, is warranted.

Topics: Adult; Animals; Antifungal Agents; Aspergillosis; Aspergillus fumigatus; Azoles; Base Sequence; Caspofungin; Cytochrome P-450 Enzyme System; Disease Models, Animal; DNA, Fungal; Drug Resistance, Multiple, Fungal; Echinocandins; Fungal Proteins; Genes, Fungal; Glucosyltransferases; Humans; In Vitro Techniques; Itraconazole; Lipopeptides; Mice; Microbial Sensitivity Tests; Mutation

Three isolates of zygomycetes belonging to two different genera (Rhizopus oryzae and Absidia corymbifera) were used to produce a systemic infection in neutropenic mice. On days -2 and -1 and at 2 h prior to infection, the mice received either posaconazole (POS) at doses ranging from 20 to 80 mg/kg of body weight/day or amphotericin B (AMB) at 1 mg/kg/day. Antifungal drug efficacy was assessed by determination of the prolongation of survival, determination of the percentage of infected organs (brain, lung, spleen, and kidney), and histological examination for the number of infection foci and their sizes in brain and kidney tissues. AMB significantly prolonged the survival of mice infected with all isolates. POS significantly prolonged the survival of mice infected with zygomycetes. Cultured organs from mice infected with R. oryzae were all positive, while treated mice challenged with A. corymbifera generally showed lower percentages of infected organs compared with the percentages for the controls. Zygomycete isolates established an active infection (the presence of hyphae) in the brains and the kidneys of all controls. In mice challenged with R. oryzae, both antifungal drugs were effective at reducing the number and the size of infection foci in the kidneys. Only AMB reduced the numbers, but not the sizes, of infection foci in the brain. Finally, both drugs significantly reduced the numbers and the sizes of infection foci in both tissues of mice infected with A. corymbifera. Our data suggest that prophylaxis with POS has some potential to prevent zygomycosis.

Topics: Absidia; Amphotericin B; Animals; Antibiotic Prophylaxis; Antifungal Agents; Brain; Cells, Cultured; Disease Models, Animal; Kidney; Lung; Male; Mice; Microbial Sensitivity Tests; Mucormycosis; Rhizopus; Spleen; Time Factors; Triazoles