Initial Pharmacological Characterization of a Major Hydroxy Metabolite of PF-5190457: Inverse Agonist Activity of PF-6870961 at the Ghrelin Receptor.

Preclinical and clinical studies have identified the ghrelin receptor [growth hormone secretagogue receptor (GHSR)1a] as a potential target for treating alcohol use disorder. A recent phase 1a clinical trial of a GHSR1a antagonist/inverse agonist, PF-5190457, in individuals with heavy alcohol drinking identified a previously undetected major hydroxy metabolite of PF-5190457, namely PF-6870961. Here, we further characterized PF-6870961 by screening for off-target interactions in a high-throughput screen and determined its in vitro pharmacodynamic profile at GHSR1a through binding and concentration-response assays. Moreover, we determined whether the metabolite demonstrated an in vivo effect by assessing effects on food intake in male and female rats. We found that PF-6870961 had no off-target interactions and demonstrated both binding affinity and inverse agonist activity at GHSR1a. In comparison with its parent compound, PF-5190457, the metabolite PF-6870961 had lower binding affinity and potency at inhibiting GHSR1a-induced inositol phosphate accumulation. However, PF-6870961 had increased inhibitory potency at GHSR1a-induced ß-arrestin recruitment relative to its parent compound. Intraperitoneal injection of PF-6870961 suppressed food intake under conditions of both food restriction and with ad libitum access to food in male and female rats, demonstrating in vivo activity. The effects of PF-6870961 on food intake were abolished in male and female rats knockout for GHSR, thus demonstrating that its effects on food intake are in fact mediated by the GHSR receptor. Our findings indicate that the newly discovered major hydroxy metabolite of PF-5190457 may contribute to the overall activity of PF-5190457 by demonstrating inhibitory activity at GHSR1a. SIGNIFICANCE STATEMENT: Antagonists or inverse agonists of the growth hormone secretagogue receptor (GHSR)1a have demonstrated substantial potential as therapeutics for alcohol use disorder. We here expand understanding of the pharmacology of one such GHSR1a inverse agonist, PF-5190457, by studying the safety and pharmacodynamics of its major hydroxy metabolite, PF-6870961. Our data demonstrate biased inverse agonism of PF-6870961 at GHSR1a and provide new structure-activity relationship insight into GHSR1a inverse agonism.

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

(R)-2-(2-methylimidazo[2,1-b]thiazol-6-yl)-1-(2-(5-(6-methylpyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)-2,7-diazaspiro[3.5]nonan-7-yl)ethan-1-one (PF-5190457) 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 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 molybdenum-containing 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.

Nonalcoholic Fatty Liver Disease and Diabetes Are Associated with Decreased CYP3A4 Protein Expression and Activity in Human Liver.

Nonalcoholic fatty liver disease (NAFLD) is a major cause of chronic liver disease in the Western population. We investigated the association of nonalcoholic fatty liver disease (NAFLD) and diabetes mellitus on CYP3A4 activity in human liver tissue from brain dead donors ( n = 74). Histopathologically graded livers were grouped into normal ( n = 24), nonalcoholic fatty liver (NAFL, n = 26), and nonalcoholic steatohepatitis (NASH, n = 24) categories. The rate of conversion of midazolam to its 1-hydroxy metabolite was used to assess in vitro CYP3A4 activity in human liver microsomes (HLM). A proteomics approach was utilized to quantify the protein expression of CYP3A4 and related enzymes. Moreover, a physiologically based pharmacokinetic (PBPK) model was developed to allow prediction of midazolam concentration in NAFL and NASH livers. CYP3A4 activity in NAFL and NASH was 1.9- and 3.1-fold ( p < 0.05) lower than normal donors, respectively. Intrinsic clearance (CLint) was 2.7- ( p < 0.05) and 4.1-fold ( p < 0.01) lower in donors with NAFL and NASH, respectively. CYP3A4 protein expression was significantly lower in NAFL and NASH donors ( p < 0.05) and accounted for significant midazolam hydroxylation variability in a multiple linear regression analysis (ß = 0.869, r2 = 0.762, P < 0.01). Diabetes was also associated with decreased CYP3A4 activity and protein expression. Both midazolam CLint and CYP3A4 protein abundance decreased significantly with increase in hepatic fat accumulation. Age and gender did not exhibit any significant association with the observed alterations. Predicted midazolam exposure was 1.7- and 2.3-fold higher for NAFL and NASH, respectively, which may result in a longer period of sedation in these disease-states. Data suggests that NAFLD and diabetes are associated with the decreased hepatic CYP3A4 activity. Thus, further evaluation of clinical consequences of these findings on the efficacy and safety of CYP3A4 substrates is warranted.

Multiplex and Label-Free Relative Quantification Approach for Studying Protein Abundance of Drug Metabolizing Enzymes in Human Liver Microsomes Using SWATH-MS.

We describe a sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) based method for label-free, simultaneous, relative quantification of drug metabolism enzymes in human liver microsomes (HLM; n = 78). In-solution tryptic digestion was aided by a pressure cycling method, which allowed a 90 min incubation time, a significant reduction over classical protocols (12-18 h). Digested peptides were separated on an Acquity UHPLC Peptide BEH C18 column using a 60 min gradient method at a flow rate of 0.100 mL/min. The quadrupole-time-of-flight mass spectrometer (ESI-QTOFMS) was operated in positive electrospray ionization mode, and data were acquired by data-dependent acquisition (DDA) and SWATH-MSALL mode. A pooled HLM sample was used as a quality control to evaluate variability in digestion and quantification among different batches, and inter-batch %CV for various proteins was between 3.1 and 7.8%. Spectral library generated from the DDA data identified 1855 distinct proteins and 25 681 distinct peptides at a 1% global false discovery rate (FDR). SWATH data were queried and analyzed for 10 major cytochrome P450 (CYP) enzymes using Skyline, a targeted data extraction software. Further, correlation analysis was performed between functional activity, protein, and mRNA expression for ten CYP enzymes. Pearson correlation coefficient (r) between protein and activity for CYPs ranged from 0.314 (CYP2C19) to 0.767 (CYP2A6). A strong correlation was found between CYP3A4 and CYP3A5 abundance and activity determined using midazolam and testosterone (r > 0.600, p < 0.001). Protein-to-activity correlation was moderate (r > 0.400-0.600, p < 0.001) for CYP1A2, CYP2A6, CYP2B6, CYP2C9, and CYP2E1 and significant but poor (r < 0.400, p < 0.05) for CYP2C8, CYP2C19, and CYP2D6. The findings suggest the suitability of SWATH-MS based method as a valuable and relatively fast analytical technique for relative quantification of proteins in complex biological samples. We also show that protein abundance is a better surrogate than mRNA to predict the activity of CYP activity.

Development and validation of an assay for a novel ghrelin receptor inverse agonist PF-5190457 and its major hydroxy metabolite (PF-6870961) by LC-MS/MS in human plasma.

PF-5190457 is a selective and potent ghrelin receptor inverse agonist presently undergoing clinical trials to treat alcohol use disorder (AUD). We describe the development and validation of a selective and sensitive liquid chromatography-tandem mass spectrometry-based method for quantification of PF-5190457 and its recently discovered hydroxy metabolite PF-6870961 in human plasma. Analytes were extracted after simple protein precipitation using methanol (2.5 ng mL-1 tacrine as an internal standard). A gradient liquid chromatography method was used to separate the analytes on an Acquity UPLC BEH C18 analytical column. The separation was achieved at a flow rate of 0.25 mL min-1 and the total chromatographic runtime was 11.30 min. Positive electrospray ionization and multiple reaction monitoring mode were used for the quantification of all the analytes. The calibration curves from six validation runs were linear with a correlation coefficient of ≥0.996 for the concentration range of 1-1000 ng mL-1 and 2-250 ng mL-1 for PF-5190457 and PF-6870961, respectively. The retention time for PF-5190457, PF-6870961 and tacrine were 4.4, 3.8, and 4.6 min, respectively. The lower limit of quantification for PF-5190457 and PF-6870961 was 1 and 2 ng mL-1, respectively. The inter-assay precision and accuracy results obtained were within the Food and Drug Administration recommended ±15% limit of nominal values. All the analytes were found to be stable under varied stability conditions. The recovery of PF-5190457 and PF-6870961 ranged from 95 to 103%. Further, the application of the method was demonstrated by measuring the concentration of PF-5190457 and its hydroxy metabolite in patient plasma samples from 100 mg dose.

Characterization of Byproducts from Chemical Syntheses of Oligonucleotides Containing 1-Methyladenine and 3-Methylcytosine.

Oligonucleotides serve as important tools for biological, chemical, and medical research. The preparation of oligonucleotides through automated solid-phase synthesis is well-established. However, identification of byproducts generated from DNA synthesis, especially from oligonucleotides containing site-specific modifications, is sometimes challenging. Typical high-performance liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoresis methods alone are not sufficient for characterizing unexpected byproducts, especially for those having identical or very similar molecular weight (MW) to the products. We used a rigorous quality control procedure to characterize byproducts generated during oligonucleotide syntheses: (1) purify oligonucleotides by different HPLC systems; (2) determine exact MW by high-resolution MS; (3) locate modification position by MS/MS or exonuclease digestion with matrix-assisted laser desorption ionization-time of flight analysis; and (4) conduct, where applicable, enzymatic assays. We applied these steps to characterize byproducts in the syntheses of oligonucleotides containing biologically important methyl DNA adducts 1-methyladenine (m1A) and 3-methylcytosine (m3C). In m1A synthesis, we differentiated a regioisomeric byproduct 6-methyladenine, which possesses a MW identical to uncharged m1A. As for m3C, we identified a deamination byproduct 3-methyluracil, which is only 1 Da greater than uncharged m3C in the ∼4900 Da context. The detection of these byproducts would be very challenging if the abovementioned procedure was not adopted.