Discovery of a 2'-fluoro-2'-C-methyl C-nucleotide HCV polymerase inhibitor and a phosphoramidate prodrug with favorable properties.

A series of 2'-fluorinated C-nucleosides were prepared and tested for anti-HCV activity. Among them, the triphosphate of 2'-fluoro-2'-C-methyl adenosine C-nucleoside (15) was a potent and selective inhibitor of the NS5B polymerase and maintained activity against the S282T resistance mutant. A number of phosphoramidate prodrugs were then prepared and evaluated leading to the identification of the 1-aminocyclobutane-1-carboxylic acid isopropyl ester variant (53) with favorable pharmacokinetic properties including efficient liver delivery in animals.

Potent Inhibition of Pseudogymnoascus destructans, the Causative Agent of White-Nose Syndrome in Bats, by Cold-Pressed, Terpeneless, Valencia Orange Oil.

The causative agent of White-nose Syndrome (WNS), Pseudogymnoascus destructans, has been shown to be fatal to several species of bats in North America. To date, no compounds or chemical control measures have been developed which eliminates the growth of the fungus in the environment or in affected animals. In the current study, we evaluated the activity of cold-pressed, terpeneless orange oil (CPT) against multiple isolates of P. destructans in vitro. For all assays, a modified Kirby-Bauer disk diffusion assay was used. Standardized spore suspensions were prepared, adjusted to a specific optical density, and used to plate fungal lawns. Plates were incubated at either 15°C or 4°C for up to 6 months and checked at regular intervals for growth. Once controls had grown, zones of inhibition were measured (mm) on test plates and compared to those obtained using current antifungal drugs. All P. destructans isolates were completely inhibited by 100% CPT (10 µL) at 1 month of incubation regardless of temperature (4°C and 15°C). Complete inhibition persisted up to 6 months following a single exposure at this concentration. Of the standard antifungals, only amphotericin B demonstrated any activity, resulting in zone diameters ranging from 58 mm to 74 mm. CPT, at the highest concentration tested (100%), had no significant effect against a variety of other environmental organisms including various filamentous fungi, bacteria and aerobic actinomycetes. Given that CPT is relatively non-toxic, the possibility exists that the all-natural, mixture could be used as an environmental pre-treatment to eradicate P. destructans from bat habitats. Additional studies are needed to assess any undesirable effects of CPT on bat behavior and health and overall impacts on other members of the interconnected ecosystem(s).

First Human Case of Fungal Keratitis Caused by a Putatively Novel Species of Lophotrichus.

We report an aggressive fungal keratitis caused by a putatively novel species of Lophotrichus in a patient with traumatic injury to the cornea from a dog paw. The organism was isolated from the patient's necrotic cornea, which perforated despite coverage with hourly fortified broad-spectrum topical antibiotic therapy. This report represents the first case of human infection caused by this species.

Utility of measuring (1,3)-ß-d-glucan in cerebrospinal fluid for diagnosis of fungal central nervous system infection.

(1-3)-ß-d-Glucan (BDG) from cerebrospinal fluid (CSF) is a promising marker for diagnostic and prognostic aid of central nervous system (CNS) fungal infection, but its relationship to serum values has not been studied. Herein, we detected BDG from CSF at levels 2-fold lower than those in serum in patients without evidence of fungal disease but 25-fold higher than those in in serum in noncryptococcal CNS fungal infections. CSF BDG may be a useful biomarker in the evaluation of fungal CNS disease.

Rhodotorula keratitis: a rarely encountered ocular pathogen.

PURPOSE: To describe a case of fungal keratitis involving an atypical organism with confirmatory in vivo confocal microscopy and to review the literature on Rhodotorula keratitis. METHODS: Case report and review of the medical literature. RESULTS: A 22-year-old college student was struck in the left eye with a tree branch and subsequently developed pain, redness and photophobia. Upon presentation, visual acuity was 20/200 and there was a large epithelial defect with diffuse stromal inflammation involving the anterior one-third of the cornea. Cultures of the infiltrate were performed for bacterial, viral and fungal organisms that resulted in a positive culture for Rhodotorula mucilaginosa. Fungal elements were confirmed in vivo by confocal microscopy. The patient was treated with voriconazole initially and had amphotericin added once Rhodotorula infection was confirmed. The patient responded well clinically, and one month after therapy was initiated, the corneal infiltrate had resolved leaving mild anterior stromal haze. Upon completion of therapy at three months, the patient was asymptomatic, had a clear cornea and had a best corrected visual acuity of 20/20 in the involved eye. There was no measurable change in his manifest refraction. CONCLUSIONS: Prior cases of Rhodotorula keratitis most often required surgical intervention and were associated with poor outcomes. This case shows that Rhodotorula keratitis can be successfully treated with topical antifungal agents if diagnosed early and appropriate treatment is initiated promptly. We report the first case of Rhodotorula keratitis confirmed by in vivo confocal microscopy. This is also the first description of pseudomycelium formation that has not been previously described in vivo, a morphological structure that this organism rarely demonstrates. Finally, this case shows that confocal microscopy may aid in the early diagnosis and management of fungal keratitis involving this rare but potentially damaging organism.

Discovery of the first C-nucleoside HCV polymerase inhibitor (GS-6620) with demonstrated antiviral response in HCV infected patients.

Hepatitis C virus (HCV) infection presents an unmet medical need requiring more effective treatment options. Nucleoside inhibitors (NI) of HCV polymerase (NS5B) have demonstrated pan-genotypic activity and durable antiviral response in the clinic, and they are likely to become a key component of future treatment regimens. NI candidates that have entered clinical development thus far have all been N-nucleoside derivatives. Herein, we report the discovery of a C-nucleoside class of NS5B inhibitors. Exploration of adenosine analogs in this class identified 1'-cyano-2'-C-methyl 4-aza-7,9-dideaza adenosine as a potent and selective inhibitor of NS5B. A monophosphate prodrug approach afforded a series of compounds showing submicromolar activity in HCV replicon assays. Further pharmacokinetic optimization for sufficient oral absorption and liver triphosphate loading led to identification of a clinical development candidate GS-6620. In a phase I clinical study, the potential for potent activity was demonstrated but with high intra- and interpatient pharmacokinetic and pharmacodynamic variability.

Synthesis and characterization of 2'-C-Me branched C-nucleosides as HCV polymerase inhibitors.

A series of 2'-C-methyl branched purine and pyrimidine C-nucleosides were prepared. Their anti-HCV activity and pharmacological properties were profiled, and compared with known 2'-C-Me N-nucleoside counterparts. In particular, 2'-C-Me 4-aza-7,9-dideazaadenosine C-nucleoside (2) was found to have potent and selective anti-HCV activity in vitro as well as a favorable pharmacokinetic profile and in vivo potential for enhanced potency over the corresponding N-nucleoside.

Oral cancer screening in a high-risk underserved community--Vancouver Downtown Eastside.

The objective of this study was to evaluate a clinic-centered oral cancer screening initiative in one of the poorest communities in Canada, assessing the need for screening and the acceptance of screening and identifying hindrances to both screening and follow-up. The study group included 204 people in the Vancouver Downtown Eastside (DTES). This was shown to be a high-risk community based on risk factors, lack of access to care, and the high frequency of oral mucosal anomalies. Acceptance of screening was high (98%); however, acceptance of biopsy for abnormal findings and follow-up was low. Among the 12 patients with clinical leukoplakia who were biopsied, 10 showed cancer or precancer. In summary, these data show a need for screening in this population and an ability to recruit patients to screening. They support a future expansion of this initiative to create a more comprehensive strategy for outreach to this underserved community that extends to screening, diagnostic work-up, and treatment.