Simulating Intestinal Transporter and Enzyme Activity in a Physiologically Based Pharmacokinetic Model for Tenofovir Disoproxil Fumarate.

Tenofovir disoproxil fumarate (TDF), a prodrug of tenofovir, has oral bioavailability (25%) limited by intestinal transport (P-glycoprotein), and intestinal degradation (carboxylesterase). However, the influence of luminal pancreatic enzymes is not fully understood. Physiologically based pharmacokinetic (PBPK) modeling has utility for estimating drug exposure from in vitro data. This study aimed to develop a PBPK model that included luminal enzyme activity to inform dose reduction strategies. TDF and tenofovir stability in porcine pancrelipase concentrations was assessed (0, 0.48, 4.8, 48, and 480 U/ml of lipase; 1 mM TDF; 37°C; 0 to 30 min). Samples were analyzed using mass spectrometry. TDF stability and permeation data allowed calculation of absorption rates within a human PBPK model to predict plasma exposure following 6 days of once-daily dosing with 300 mg of TDF. Regional absorption of drug was simulated across gut segments. TDF was degraded by pancrelipase (half-lives of 0.07 and 0.62 h using 480 and 48 U/ml, respectively). Previously reported maximum concentration (Cmax; 335 ng/ml), time to Cmax (Tmax; 2.4 h), area under the concentration-time curve from 0 to 24 h (AUC0-24; 3,045 ng · h/ml), and concentration at 24 h (C24; 48.3 ng/ml) were all within a 0.5-fold difference from the simulated Cmax (238 ng/ml), Tmax (3 h), AUC0-24 (3,036 ng · h/ml), and C24 (42.7 ng/ml). Simulated TDF absorption was higher in duodenum and jejunum than in ileum (p<0.05). These data support that TDF absorption is limited by the action of intestinal lipases. Our results suggest that bioavailability may be improved by protection of drug from intestinal transporters and enzymes, for example, by coadministration of enzyme-inhibiting agents or nanoformulation strategies.

The Emerging Pathogen Candida auris: Growth Phenotype, Virulence Factors, Activity of Antifungals, and Effect of SCY-078, a Novel Glucan Synthesis Inhibitor, on Growth Morphology and Biofilm Formation.

Candidaauris, a new multidrug-resistant Candida spp. which is associated with invasive infection and high rates of mortality, has recently emerged. Here, we determined the virulence factors (germination, adherence, biofilm formation, phospholipase and proteinase production) of 16 C. auris isolates and their susceptibilities to 11 drugs belonging to different antifungal classes, including a novel orally bioavailable 1,3-ß-d-glucan synthesis inhibitor (SCY-078). We also examined the effect of SCY-078 on the growth, ultrastructure, and biofilm-forming abilities of C. auris Our data showed that while the tested strains did not germinate, they did produce phospholipase and proteinase in a strain-dependent manner and had a significantly reduced ability to adhere and form biofilms compared to that of Candida albicans (P = 0.01). C. auris isolates demonstrated reduced susceptibility to fluconazole and amphotericin B, while, in general, they were susceptible to the remaining drugs tested. SCY-078 had an MIC90 of 1 mg/liter against C. auris and caused complete inhibition of the growth of C. auris and C. albicans Scanning electron microscopy analysis showed that SCY-078 interrupted C. auris cell division, with the organism forming abnormal fused fungal cells. Additionally, SCY-078 possessed potent antibiofilm activity, wherein treated biofilms demonstrated significantly reduced metabolic activity and a significantly reduced thickness compared to the untreated control (P < 0.05 for both comparisons). Our study shows that C. auris expresses several virulence determinants (albeit to a lesser extent than C. albicans) and is resistant to fluconazole and amphotericin B. SCY-078, the new orally bioavailable antifungal, had potent antifungal/antibiofilm activity against C. auris, indicating that further evaluation of this antifungal is warranted.

Development of a Novel Formulation That Improves Preclinical Bioavailability of Tenofovir Disoproxil Fumarate.

Tenofovir disoproxil fumarate (TDF), the bisphosphonate ester prodrug of tenofovir (TFV), has poor bioavailability due to intestinal degradation and efflux transport. Reformulation using U.S. Food and Drug Administration-approved esterase and efflux inhibitors to increase oral bioavailability could provide lower dose alternatives and reduce costs for patients with HIV in resource-limited settings. Inhibition of mucosal and intracellular esterases was studied in human and rat intestinal extracts (S9), where TDF was protected by the carboxylesterase inhibitor bis-para-nitrophenylphosphate, the ester mix EM1, and the generally recognized-as-safe (GRAS) excipient propylparaben. Permeability studies using Madin-Darby canine kidney and Caco-2 cell monolayers demonstrated that TDF was a substrate for the permeability glycoprotein with permeability glycoprotein inhibitors reducing basolateral to apical transport of TDF. These studies also showed that transport was increased by esterase inhibitors. TDF, TFV, and tenofovir monophosphonate ester transport across Caco-2 monolayers with esterase and efflux inhibitors revealed a maximum 38.7-fold increase in apical to basolateral TDF transport with the potent non-GRAS combination of EM1 and GF120918. Transport was increased 22.8-fold by the GRAS excipients, propylparaben, and d-a-tocopheryl polyethylene glycol 1000 succinate (a vitamin E derivative). TFV pharmacokinetics in rats following oral administration of TDF and GRAS esterase and efflux inhibitors confirmed enhanced bioavailability. Area under the curve increased 1.5- to 2.1-fold with various combinations of parabens and d-a-tocopheryl polyethylene glycol 1000 succinate. This significant inhibition of TDF hydrolysis and efflux in vivo exhibits the potential to safely increase TDF bioavailability in humans.

Midazolam exhibits characteristics of a highly permeable P-glycoprotein substrate.

PURPOSE: The purpose of this study was to investigate whether midazolam exhibits characteristics of a highly permeable P-glycoprotein (P-gp) substrate and to evaluate the potential influence of P-gp inhibition on 1-OH midazolam formation during midazolam transport. METHODS: P-gp interaction was investigated by P-gp ATPase assay, efflux inhibition studies, and transport studies of midazolam across MDR1-MDCK and 1-alpha,25-dihydroxy vitamin D3-induced Caco-2 monolayers with and without the P-gp inhibitor GF120918. RESULTS: Midazolam was highly permeable and transport appeared essentially unpolarized. In MDR1-MDCK, the basolateral-to-apical (B-to-A) permeability was slightly higher (16%) than apical-to-basolateral (A-to-B) permeability (p = 0.04); GF120918 increased A-to-B permeability by 27% (p = 0.01), and increased cellular midazolam accumulation during A-to-B transport by 45% (p = 0.01). Midazolam (200 microM) decreased rhodamine123 and vinblastine B/A ratios 3-fold (p < 0.006), while increasing their cellular accumulation (p < 0.003). P-gp ATPase activation by midazolam was dose-dependent and saturable [Km = 11.5(+/- 4.0) microM; Vmax = 41.1(+/- 7.4) nmol/mg/min]. P-gp inhibition increased 1-OH midazolam formation in A-to-B studies 1.3-fold when midazolam donor > or = 10 microM (p < 0.03). In B-to-A studies, P-gp inhibition did not significantly increase metabolite formation (p = 0.06). Midazolam's extraction ratio was not influenced by P-gp (p = 0.2). CONCLUSION: The results indicate that midazolam exhibited characteristics of a highly permeable P-gp substrate. 1-OH midazolam formation during A-to-B midazolam transport increased slightly when P-gp was inhibited.