title: Metabolites in safety testing assessment in early clinical development: a case study with a glucokinase activator.
abstract: The present article summarizes Metabolites in Safety Testing (MIST) studies on a glucokinase activator, N,N-dimethyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide (PF-04937319), which is under development for the treatment of type 2 diametes mellitus. Metabolic profiling in rat, dog, and human hepatocytes revealed that PF-04937319 is metabolized via oxidative (major) and hydrolytic pathways (minor). N-Demethylation to metabolite M1 [N-methyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide] was the major metabolic fate of PF-04937319 in human (but not rat or dog) hepatocytes, and was catalyzed by CYP3A and CYP2C isoforms. Qualitative examination of circulating metabolites in humans at the 100- and 300-mg doses from a 14-day multiple dose study revealed unchanged parent drug and M1 as principal components. Because M1 accounted for 65% of the drug-related material at steady state, an authentic standard was synthesized and used for comparison of steady-state exposures in humans and the 3-month safety studies in rats and dogs at the no-observed-adverse-effect level. Although circulating levels of M1 were very low in beagle dogs and female rats, adequate coverage was obtained in terms of total maximal plasma concentration (∼7.7× and 1.8×) and area under the plasma concentration-time curve (AUC; 3.6× and 0.8× AUC) relative to the 100- and 300-mg doses, respectively, in male rats. Examination of primary pharmacology revealed M1 was less potent as a glucokinase activator than the parent drug (compound PF-04937319: EC50 = 0.17 μM; M1: EC50 = 4.69 μM). Furthermore, M1 did not inhibit major human P450 enzymes (IC50 > 30 μM), and was negative in the Salmonella Ames assay, with minimal off-target pharmacology, based on CEREP broad ligand profiling. Insights gained from this analysis should lead to a more efficient and focused development plan for fulfilling MIST requirements with PF-04937319.
authors.affiliation: Pfizer Inc., Groton, Connecticut (R.S., K.A., H.E., J.C.P., T.C.G., L.D.), San Diego, California (D.K.D.), and Cambridge, Massachusetts (J.L., N.B.A., W.S.D., E.L., J.A.P., A.S.K.).
authors.forename: Raman
authors.initials: R
authors.lastname: Sharma
authors.affiliation: Pfizer Inc., Groton, Connecticut (R.S., K.A., H.E., J.C.P., T.C.G., L.D.), San Diego, California (D.K.D.), and Cambridge, Massachusetts (J.L., N.B.A., W.S.D., E.L., J.A.P., A.S.K.).
authors.forename: John
authors.initials: J
authors.lastname: Litchfield
authors.affiliation: Pfizer Inc., Groton, Connecticut (R.S., K.A., H.E., J.C.P., T.C.G., L.D.), San Diego, California (D.K.D.), and Cambridge, Massachusetts (J.L., N.B.A., W.S.D., E.L., J.A.P., A.S.K.).
authors.forename: Karen
authors.initials: K
authors.lastname: Atkinson
authors.affiliation: Pfizer Inc., Groton, Connecticut (R.S., K.A., H.E., J.C.P., T.C.G., L.D.), San Diego, California (D.K.D.), and Cambridge, Massachusetts (J.L., N.B.A., W.S.D., E.L., J.A.P., A.S.K.).
authors.forename: Heather
authors.initials: H
authors.lastname: Eng
authors.affiliation: Pfizer Inc., Groton, Connecticut (R.S., K.A., H.E., J.C.P., T.C.G., L.D.), San Diego, California (D.K.D.), and Cambridge, Massachusetts (J.L., N.B.A., W.S.D., E.L., J.A.P., A.S.K.).
authors.forename: Neeta B
authors.initials: NB
authors.lastname: Amin
authors.affiliation: Pfizer Inc., Groton, Connecticut (R.S., K.A., H.E., J.C.P., T.C.G., L.D.), San Diego, California (D.K.D.), and Cambridge, Massachusetts (J.L., N.B.A., W.S.D., E.L., J.A.P., A.S.K.).
authors.forename: William S
authors.initials: WS
authors.lastname: Denney
authors.affiliation: Pfizer Inc., Groton, Connecticut (R.S., K.A., H.E., J.C.P., T.C.G., L.D.), San Diego, California (D.K.D.), and Cambridge, Massachusetts (J.L., N.B.A., W.S.D., E.L., J.A.P., A.S.K.).
authors.forename: John C
authors.initials: JC
authors.lastname: Pettersen
authors.affiliation: Pfizer Inc., Groton, Connecticut (R.S., K.A., H.E., J.C.P., T.C.G., L.D.), San Diego, California (D.K.D.), and Cambridge, Massachusetts (J.L., N.B.A., W.S.D., E.L., J.A.P., A.S.K.).
authors.forename: Theunis C
authors.initials: TC
authors.lastname: Goosen
authors.affiliation: Pfizer Inc., Groton, Connecticut (R.S., K.A., H.E., J.C.P., T.C.G., L.D.), San Diego, California (D.K.D.), and Cambridge, Massachusetts (J.L., N.B.A., W.S.D., E.L., J.A.P., A.S.K.).
authors.forename: Li
authors.initials: L
authors.lastname: Di
authors.affiliation: Pfizer Inc., Groton, Connecticut (R.S., K.A., H.E., J.C.P., T.C.G., L.D.), San Diego, California (D.K.D.), and Cambridge, Massachusetts (J.L., N.B.A., W.S.D., E.L., J.A.P., A.S.K.).
authors.forename: Esther
authors.initials: E
authors.lastname: Lee
authors.affiliation: Pfizer Inc., Groton, Connecticut (R.S., K.A., H.E., J.C.P., T.C.G., L.D.), San Diego, California (D.K.D.), and Cambridge, Massachusetts (J.L., N.B.A., W.S.D., E.L., J.A.P., A.S.K.).
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Pfefferkorn
authors.affiliation: Pfizer Inc., Groton, Connecticut (R.S., K.A., H.E., J.C.P., T.C.G., L.D.), San Diego, California (D.K.D.), and Cambridge, Massachusetts (J.L., N.B.A., W.S.D., E.L., J.A.P., A.S.K.).
authors.forename: Deepak K
authors.initials: DK
authors.lastname: Dalvie
authors.affiliation: Pfizer Inc., Groton, Connecticut (R.S., K.A., H.E., J.C.P., T.C.G., L.D.), San Diego, California (D.K.D.), and Cambridge, Massachusetts (J.L., N.B.A., W.S.D., E.L., J.A.P., A.S.K.) amit.kalgutkar@pfizer.com.
authors.forename: Amit S
authors.initials: AS
authors.lastname: Kalgutkar
mesh_terms: D000818:Animals; D019540:Area Under Curve; D001572:Benzofurans / Q000097:blood / Q000493:pharmacokinetics*; D004285:Dogs; D020536:Enzyme Activators / Q000097:blood / Q000493:pharmacokinetics*; D005260:Female; D005941:Glucokinase / Q000378:metabolism*; D006801:Humans; D011743:Pyrimidines / Q000097:blood / Q000493:pharmacokinetics*; D051381:Rats
chemical_list: D001572:Benzofurans; D020536:Enzyme Activators; C000598526:N,N-dimethyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide; D011743:Pyrimidines; D005941:Glucokinase