Discovery and Characterization of 2-Acylaminoimidazole Microsomal Prostaglandin E Synthase-1 Inhibitors.

As part of a program aimed at the discovery of antinociceptive therapy for inflammatory conditions, a screening hit was found to inhibit microsomal prostaglandin E synthase-1 (mPGES-1) with an IC50 of 17.4 µM. Structural information was used to improve enzyme potency by over 1000-fold. Addition of an appropriate substituent alleviated time-dependent cytochrome P450 3A4 (CYP3A4) inhibition. Further structure-activity relationship (SAR) studies led to 8, which had desirable potency (IC50 = 12 nM in an ex vivo human whole blood (HWB) assay) and absorption, distribution, metabolism, and excretion (ADME) properties. Studies on the formulation of 8 identified 8·H3PO4 as suitable for clinical development. Omission of a lipophilic portion of the compound led to 26, a readily orally bioavailable inhibitor with potency in HWB comparable to celecoxib. Furthermore, 26 was selective for mPGES-1 inhibition versus other mechanisms in the prostanoid pathway. These factors led to the selection of 26 as a second clinical candidate.

Disposition of [14C]ruboxistaurin in humans.

Ruboxistaurin is a potent and specific inhibitor of the beta isoforms of protein kinase C (PKC) that is being developed for the treatment of diabetic microvascular complications. The disposition of [(14)C]ruboxistaurin was determined in six healthy male subjects who received a single oral dose of 64 mg of [(14)C]ruboxistaurin in solution. There were no clinically significant adverse events during the study. Whole blood, urine, and feces were collected at frequent intervals after dosing. Metabolites were profiled by high performance liquid chromatography with radiometric detection. The total mean recovery of the radioactive dose was approximately 87%, with the majority of the radioactivity (82.6 +/- 1.1%) recovered in the feces. Urine was a minor pathway of elimination (4.1 +/- 0.3%). The major route of ruboxistaurin metabolism was to the N-desmethyl ruboxistaurin metabolite (LY338522), which has been shown to be active and equipotent to ruboxistaurin in the inhibition of PKC(beta). In addition, multiple hydroxylated metabolites were identified by liquid chromatography-mass spectrometry in all matrices. Pharmacokinetics were conducted for both ruboxistaurin and LY338522 (N-desmethyl ruboxistaurin, 1). These moieties together accounted for approximately 52% of the radiocarbon measured in the plasma. The excreted radioactivity was profiled using radiochromatography, and approximately 31% was structurally characterized as ruboxistaurin or N-desmethyl ruboxistaurin. These data demonstrate that ruboxistaurin is metabolized primarily to N-desmethyl ruboxistaurin (1) and multiple other oxidation products, and is excreted primarily in the feces.

The interactions of a selective protein kinase C beta inhibitor with the human cytochromes P450.

Studies were performed to determine the cytochromes P450 (P450) responsible for the biotransformation of (S)-13[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16,21-dimetheno-1H, 13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-dione (LY333531) to its equipotent metabolite, N-desmethyl LY333531, and to examine the ability of these two compounds to inhibit P450-mediated metabolism. Kinetic studies indicated that a single enzyme in human liver microsomes was able to form N-desmethyl LY333531 with an apparent K(M) value of approximately 1 microM. The formation rate of N-desmethyl LY333531 was correlated with markers of nine P450s in a bank of 20 human liver microsomes. The only significant correlation observed was with the form-selective activity for CYP3A. Of the nine cDNA-expressed P450s examined, only CYP3A4 and CYP2D6 formed N-desmethyl LY333531. However, CYP3A4 formed N-desmethyl LY333531 at a rate 57-fold greater than that observed with CYP2D6. In incubations with human liver microsomes, quinidine, an inhibitor of CYP2D6, demonstrated little inhibition of metabolite formation while ketoconazole, an inhibitor of CYP3A, demonstrated almost complete inhibition. Thus, CYP3A is responsible for the formation of N-desmethyl LY333531. LY333531 and N-desmethyl LY333531 were also examined for their ability to inhibit metabolism mediated by CYP2D6, CYP2C9, CYP3A, and CYP1A2. LY333531 and N-desmethyl LY333531 were found to competitively inhibit CYP2D6 with calculated K(i) values of 0.17 and 1.0 microM, respectively. Less potent inhibition by these compounds of metabolism mediated by the other three P450s examined was observed. In conclusion, CYP3A is primarily responsible for forming N-desmethyl LY333531. Therefore, alterations in the activity of this enzyme have the potential to affect LY333531 clearance. In addition, LY333531 and its metabolite are predicted to be potential inhibitors of CYP2D6-mediated reactions in vivo.