Glucokinase (GK) activation as a potential strategy to treat type 2 diabetes (T2D) is well recognized. Compound 1, a glucokinase activator (GKA) lead that we have previously disclosed, caused reversible hepatic lipidosis in repeat-dose toxicology studies. We hypothesized that the hepatic lipidosis was due to the structure-based toxicity and later established that it was due to the formation of a thiourea metabolite, 2. Subsequent SAR studies of 1 led to the identification of a pyrazine-based lead analogue 3, lacking the thiazole moiety. In vivo metabolite identification studies, followed by the independent synthesis and profiling of the cyclopentyl keto- and hydroxyl- metabolites of 3, led to the selection of piragliatin, 4, as the clinical lead. Piragliatin was found to lower pre- and postprandial glucose levels, improve the insulin secretory profile, increase β-cell sensitivity to glucose, and decrease hepatic glucose output in patients with T2D.
1. A high throughput screening (HTS) method for the evaluation of the seven major human hepatic CYP isoform activities was developed on a 96-well format, with automation. The method utilized pooled human liver microsomes and seven probe substrates, generic conditions for incubation, reaction termination and metabolite extraction with solid phase extraction (SPE) plates. Metabolites from the seven reactions were pooled and quantified using a generic liquid chromatography and tandem mass spectrometry (LCMS/MS) method. 2. The HTS method was validated based on Km values obtained, which were in agreement with literature data. 3. The isoform inhibition profiles of ketoconazole, quinidine, sulfaphenazole, tranylcypromine, alpha-naphthoflavone, and 4-methylpyrazole against CYPs 3A4, 2D6, 2C9, 2A6 land 2C19), 1A2 and 2E1, respectively, were obtained by this HTS method. Graphically obtained IC50 values are in agreement with literature reported values. 4. The HTS method represents a significant efficiency and selectivity improvement over traditional methods, and can be used for CYP inhibition assay and can be extended for liver activity profiling.
The rapid loss of human CYP1A2 (cytochrome P450 1A2) activity caused by the 8-methylxanthine furafylline is investigated with the aim of determining whether a stable covalent adduct of the xanthine to the enzyme could be identified. Metabolic studies employing expressed CYP1A2 with radiolabeled furafylline and a close analogue, cyclohexylline, where the furan ring is replaced with cyclohexane, indicate that these xanthines are bound in a 1:1 ratio to CYP1A2 protein. This result, combined with earlier kinetic studies, verifies that these compounds are mechanism-based inhibitors of the enzyme. The 8'-methyl carbinols are the only metabolites formed by CYP1A2, and substantial (70-80%) incorporation of oxygen from the medium into the carbinols is observed. Carbinol formation is further characterized by high intramolecular isotope effects (kH/kD > 9) and low intermolecular isotope effects (DV/K < 2). Overall partition ratios are low (5.0 and 7.6, respectively), confirming our previous conclusion that furafylline is an efficient inactivator. By contrast, the N7-methyl-8-methylxanthines are good substrates for CYP1A2 but are not themselves inactivating agents. In addition to other metabolic products, the 8'-methyl carbinols of these N7-methyl-8-methylxanthines are formed in substantial amounts with equally high intramolecular isotope effects; however, the carbinol oxygen is derived exclusively from molecular oxygen. We conclude that oxidation of the 8-methyl group of furafylline and cyclohexylline, but not their N7-methyl analogues, by CYP1A2 promotes a major fraction of the inactivating xanthines to a two electron oxidized intermediate which either terminates enzyme activity by reaction with an active site amino acid or is decomposed by reaction with the medium to give carbinol.
The CYP3A4 enzyme is known for its atypical inhibition kinetics; ligand inhibition can differ depending upon the probe drug used. A high throughput-LCMS/MS CYP3A4 inhibition assay with four substrate drugs was developed to minimize the potential oversight of CYP3A4 inhibition. The assay uses a 96-well format, human liver microsomes, and four CYP3A4 substrate drugs, midazolam, testosterone, nifedipine and terfenadine. After incubation of the individual substrate with human liver microsomes, the reaction is stopped by solid phase extraction and the four probe metabolites produced are pooled and measured by LCMS/MS with multiple-ion-monitoring mode. Using this assay, the IC(50) values of fourteen compounds recognized as substrates/inhibitors of CYP3A4, were measured for the CYP3A4 catalyzed-metabolism of probe drugs. IC(50) values were also obtained for the common set of compounds by the microtiter plate fluorescent assays with cDNA-expressed CYP3A4. Comparison of the results from the two methods suggests that decision making should be cautiously executed to predict drug interaction potential caused by inhibition of CYP3A4 considering the gap between the two assays and various other factors.
Vemurafenib, a selective inhibitor of oncogenic BRAF kinase carrying the V600 mutation, is approved for treatment of advanced BRAF mutation–positive melanoma. This study characterized mass balance, metabolism, rates/routes of elimination, and disposition of 14C-labeled vemurafenib in patients with metastatic melanoma. Seven patients with metastatic BRAF-mutated melanoma received unlabeled vemurafenib 960 mg twice daily for 14 days. On the morning of day 15, patients received 14C-labeled vemurafenib 960 mg (maximum 2.56 MBq [69.2 μCi]). Thereafter, patients resumed unlabeled vemurafenib (960 mg twice daily). Blood, urine, and feces were collected for metabolism, pharmacokinetic, and dose recovery analysis. Within 18 days after dose, ∼95% of 14C-vemurafenib–related material was recovered from feces (94.1%) and urine (<1%). The parent compound was the predominant component (95%) in plasma. The mean plasma elimination half-life of 14C-vemurafenib–related material was 71.1 h. Each metabolite accounted for <0.5% and ≤6% of the total administered dose in urine and feces, respectively (0–96 h postdose). No new metabolites were detected. Vemurafenib was well-tolerated. Excretion of vemurafenib via bile into feces is considered the predominant elimination route from plasma with minor renal elimination (<1%).
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