Role of Molybdenum-Containing Enzymes in the Biotransformation of the Novel Ghrelin Receptor Inverse Agonist PF-5190457: A Reverse Translational Bed-to-Bench Approach

Abstract

was identified as a potent and selective inverse agonist of the ghrelin receptor [growth hormone secretagogue receptor 1a (GHS-R1a)]. The present translational bed-to-bench work characterizes the biotransformation of this compound in vivo and then further explores in vitro metabolism in fractions of human liver and primary hepatocytes. Following oral administration of PF-5190457 in a phase 1b clinical study, hydroxyl metabolites of the compound were observed, including one that had not been observed in previously performed human liver microsomal incubations. PF-6870961 or (R)-1-(2-(5-(2-hydroxy-6-methylpyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)-2,7-diazaspiro[3.5]nonan-7-yl)-2-(2-methylimidazo[2,1-b]thiazol-6-yl)ethan-1-one was biosynthesized using liver cytosol, and the site of hydroxylation was shown to be on the pyrimidine using nuclear magnetic resonance spectroscopy. The aldehyde oxidase (AO) inhibitor raloxifene and the xanthine oxidase inhibitor febuxostat inhibited the formation of PF-6870961 in human liver cytosol, suggesting both enzymes were involved in the metabolism of the drug. However, greater inhibition was observed with raloxifene, indicating AO is a dominant enzyme in the biotransformation. The intrinsic clearance of the drug in human liver cytosol was estimated to be 0.002 ml/min per milligram protein. This study provides important novel information at three levels: 1) it provides additional new information on the recently developed novel compound PF-5190457, the first GHS-R1a blocker that has moved to development in humans; 2) it provides an example of a reverse translational approach where a discovery in humans was brought back, validated, and further investigated at the bench level; and 3) it demonstrates the importance of considering the molybdenumcontaining oxidases during the development of new drug entities. SIGNIFICANCE STATEMENT PF-5190457 is a novel ghrelin receptor inverse agonist that is currently undergoing clinical development for treatment of alcohol use disorder. PF-6870961, a major hydroxyl metabolite of the compound, was observed in human plasma, but was absent in human liver microsomal incubations. PF-6870961 was biosynthesized using liver cytosol, and the site of hydroxylation on the pyrimidine ring was characterized. Inhibitors of aldehyde oxidase and xanthine oxidase inhibited the formation of PF-6870961 in human liver cytosol, suggesting both enzymes were involved in the metabolism of the drug. This information is important for patient selection in subsequent clinical studies.

Metabolism by Aldehyde Oxidase: Drug Design and Complementary Approaches to Challenges in Drug Discovery

“…6 However, drug metabolism by XO has been reported, with some overlapping specificity with AO: recent examples include the mGlu5-negative allosteric modulators VU0409106 in human and rat liver S9 211 and VU0424238 in rat and mouse liver S9, 69 as well as the ghrelin receptor inverse agonist PF-5190457 in human liver cytosol. 67 Additionally, some compounds may be exclusive AO substrates in species that express the enzyme, but XO substrates in dog, which does not (as might be the case for the PI3Kδ inhibitor idelalisib, see section 7.1). To discriminate between the two enzymes, allopurinol has been extensively used as a selective XO inhibitor, 119 although more recently febuxostat has been shown to provide better potency and high selectivity toward XO.…”

“…To discriminate between the two enzymes, allopurinol has been extensively used as a selective XO inhibitor, 119 although more recently febuxostat has been shown to provide better potency and high selectivity toward XO. 67,212 Assays with recombinant AO have also been suggested, as they provide direct evidence for AO-mediated metabolism. 213 While initial attempts at recombinant expression of human AO in Escherichia coli struggled to yield adequate levels of active protein expression, probably due to low MoCo incorporation, 41,190,193 a ∼100-fold increase in holoprotein expression levels was recently attained through a method combining codon optimization of the construct with in vitro chemical sulfuration of the recombinant protein.…”

“…While very few drugs currently on the market are AO substrates or inhibitors, therefore not posing a significant risk for AO-mediated drug−drug interactions (DDI), as more AO drug substrates make their way into clinical trials, the risks of DDI due to AO activity might increase and may eventually become clinically significant, making the correct measurement of Fm AO even more necessary. 5,25,63,67,217,218 The current established approach for the determination of Fm AO is the time-dependent inhibition of AO activity by hydralazine, either in hepatocytes or liver S9 fractions. 210,219−222 This method is not without caveats, however, as hydralazine is known to inhibit some of the major hepatic CYPs at the concentrations needed for AO inhibition.…”

“…Assays run in liver S9 in the presence of H 2 18 O can be used to provide additional evidence for contributions of AO (and XO) versus CYPs, as AO and XO utilize oxygen from water rather than atmospheric oxygen to catalyze oxidations, and they can also provide information on the site of metabolism. , The role of XO, a closely related molybdo-flavoenzyme, is mostly endogenous and relates to purine catabolism . However, drug metabolism by XO has been reported, with some overlapping specificity with AO: recent examples include the mGlu5-negative allosteric modulators VU0409106 in human and rat liver S9 and VU0424238 in rat and mouse liver S9, as well as the ghrelin receptor inverse agonist PF-5190457 in human liver cytosol . Additionally, some compounds may be exclusive AO substrates in species that express the enzyme, but XO substrates in dog, which does not (as might be the case for the PI3Kδ inhibitor idelalisib, see section ).…”

“…Additionally, some compounds may be exclusive AO substrates in species that express the enzyme, but XO substrates in dog, which does not (as might be the case for the PI3Kδ inhibitor idelalisib, see section ). To discriminate between the two enzymes, allopurinol has been extensively used as a selective XO inhibitor, although more recently febuxostat has been shown to provide better potency and high selectivity toward XO. , …”