6 mL/min/kg; n = 3 in all species except hamster microsomes); these data are consistent with the low whole body blood clearance in the animal models. In hamster microsomes the CLintr was
2.5 ± 0.2 mL/min/g liver (low to moderate), an observation consistent with its moderate in vivo blood clearance (40% of hepatic blood flow) in that species. The CLintr of verapamil and diclofenac exceeded 5 mL/min/g liver, and Sotrastaurin in vitro that of the cocktail of substrates used in hepatocytes matched historical in-house values, indicating that all the preparations were metabolically active. DNDI-VL-2098 was stable in the tested recombinant human CYPs using 50 pmol and 100 pmol CYP content (T½ > 60 min for all isozymes, except CYP2C19 100 pmol where T½ = 43 min); this observation is consistent with its high stability Everolimus chemical structure in microsomes and hepatocytes. The t½ values of concomitantly run positive-controls matched historical in-house values (7-ethoxyresorufin: 2.3 min, diclofenac: 3.8 min, omeprazole: 2.0 min, dextromethorphan: 0.8 min, testosterone: 11.5 min at 50 pmol CYP content). DNDI-VL-2098 showed moderate to high binding (Table 5). The unbound fraction was determined to be 3–6% across the
species tested. Results for the concomitantly run highly bound compound diclofenac (percentage unbound 0.23 ± 0.10) matched the historical in-house values in this assay. DNDI-VL-2098 did not inhibit CYP1A2, CYP2C9, CYP2D6 and CYP3A4 at concentrations up to 12.5 μM (triplicate IC50 studies). It did however inhibit CYP2C19 with an IC50 value of 0.47 ± 0.24 μM. second IC50 values for concomitantly run positive control inhibitors α-napthoflavone, sulfaphenazole, N-3-benzylnirvanol, quinidine and ketoconazole (0.004 μM, 0.32 μM, 0.56 μM, 0.050 μM
and 0.011 μM, respectively) matched the historical in-house values in this assay. A minor monooxygenation metabolite (M-I, 19.44 min) was detected in mouse, rat and dog liver microsomes (<0.2% for mouse, <0.1% for rat and <0.5% for dog assuming similar ionization) based on peak area comparison of metabolite to parent peak, but it was not detected in incubations with human liver microsomes. The likely site of monooxygenation is in the trifluoromethoxyphenyl ring (Fig. 1) based on the fragmentation pattern. The metabolite was not detectable in mouse, rat, dog and human hepatocyte incubations nor in circulating blood samples from mouse (oral 50 mg/kg), rat (oral 500 mg/kg) and dog (oral 50 mg/kg). These results are consistent with studies in liver microsomes and hepatocytes indicating that DNDI-VL-2098 is stable in vitro. PA-824, a novel 4-nitroimidazole is currently in phase II clinical trial for tuberculosis (TB) and a structural analog of DNDI-VL-2098, produces 4 metabolites when incubated with human S9 fraction including a major des-nitro metabolite, and seven metabolites with purified Ddn (deazaflavin F420 dependent nitroreductase) and mycobacterium tuberculosis ( Dogra et al., 2011).