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Supplementary Figure S5. MALDI-TOF MS analyses of MK-1775 (m/z 501.2)in brain and flank GBM22 tumor samples after treatment with placebo or MK-1775

Supplementary Methods and Legend

Purpose: Wee1 regulates key DNA damage checkpoints, and in this study, the efficacy of the Wee1 inhibitor MK-1775 was evaluated in glioblastoma multiforme (GBM) xenograft models alone and in combination with radiation and/or temozolomide.Experimental Design: In vitro MK-1775 efficacy alone and in combination with temozolomide, and the impact on DNA damage, was analyzed by Western blotting and γH2AX foci formation. In vivo efficacy was evaluated in orthotopic and heterotopic xenografts. Drug distribution was assessed by conventional mass spectrometry (MS) and matrix-assisted laser desorption/ionization (MALDI)-MS imaging.Results: GBM22 (IC50 = 68 nmol/L) was significantly more sensitive to MK-1775 compared with five other GBM xenograft lines, including GBM6 (IC50 >300 nmol/L), and this was associated with a significant difference in pan-nuclear γH2AX staining between treated GBM22 (81% cells positive) and GBM6 (20% cells positive) cells. However, there was no sensitizing effect of MK-1775 when combined with temozolomide in vitro. In an orthotopic GBM22 model, MK-1775 was ineffective when combined with temozolomide, whereas in a flank model of GBM22, MK-1775 exhibited both single-agent and combinatorial activity with temozolomide. Consistent with limited drug delivery into orthotopic tumors, the normal brain to whole blood ratio following a single MK-1775 dose was 5%, and MALDI-MS imaging demonstrated heterogeneous and markedly lower MK-1775 distribution in orthotopic as compared with heterotopic GBM22 tumors.Conclusions: Limited distribution to brain tumors may limit the efficacy of MK-1775 in GBM. Clin Cancer Res; 21(8); 1916–24. ©2015 AACR.

Gefapixant (MK-7264, AF-219), a first-in-class P2X3 antagonist, is being developed as oral treatment for refractory or unexplained chronic cough. Based on in vitro data, gefapixant exerts inhibitory activity on the organic anion transporter (OAT) P1B1 transporter. Therefore, a drug-drug interaction study evaluating the potential effects of gefapixant on the OATP1B1 drug transporter, using pitavastatin as a sensitive probe substrate, was conducted. An open-label, 2-period, fixed-sequence study in 20 healthy adults 18 to 55 years old was conducted. In period 1, a 1-mg oral dose of pitavastatin was administered to each participant. After a ≥4-day washout, in period 2 participants received a 45-mg oral dose of gefapixant twice daily on days 1 through 4. On day 2 of period 2, pitavastatin was coadministered with the morning dose of gefapixant. Pitavastatin exposures following single-dose administration with and without multiple doses of gefapixant were similar: geometric mean ratio (90% confidence interval) of pitavastatin area under the plasma concentration-time curve from time 0 to infinity (AUC0-∞ ) (pitavastatin + gefapixant/pitavastatin alone) was 0.97 (0.93-1.02). The ratio of pitavastatin lactone AUC0-∞ to pitavastatin AUC0-∞ was also comparable between treatments. Administration of gefapixant and pitavastatin was generally well tolerated, with no safety findings of concern. These results support that gefapixant has a low potential to inhibit the OATP1B1 transporter.

Cathepsin K (Cat K) is a cysteine protease involved in bone remodeling. In addition to its role in bone biology, Cat K is upregulated in osteoclasts, chondrocytes and synoviocytes in osteoarthritic (OA) disease states making it a potential therapeutic target for disease-modifying OA. Starting from a prior preclinical compound, MK-1256, lead optimization efforts were carried out in the search for potent Cat K inhibitors with improved selectivity profiles with an emphasis on cathepsin F. Herein, we report the SAR studies which led to the discovery of the highly selective oxazole compound 23, which was subsequently shown to inhibit cathepsin K in vivo as measured by reduced levels of urinary C-telopeptide of collagen type I in dog.

There are many factors which are known to cause variability in human in vitro enzyme kinetic data. Factors such as the source of enzyme and how it was prepared, the genetics and background of the donor, how the in vitro studies are designed, and how the data are analyzed contribute to variability in the resulting kinetic parameters. It is important to consider not only the factors which cause variability within an experiment, such as selection of a probe substrate, but also those that cause variability when comparing kinetic data across studies and laboratories. For example, the artificial nature of the microsomal lipid membrane and microenvironment in some recombinantly expressed enzymes, relative to those found in native tissue microsomes, has been shown to influence enzyme activity and thus can be a source of variability when comparing across the two different systems. All of these factors, and several others, are discussed in detail in the chapter below. In addition, approaches which can be used to visualize the uncertainty arising from the use of enzyme kinetic data within the context of predicting human pharmacokinetics are discussed.

Recognizing the challenges of determining the relative contribution of different drug metabolizing enzymes to the metabolism of slowly metabolized compounds, a cytochrome P450 reaction phenotyping (CRP) method using cocultured human hepatocytes (HEPATOPAC) has been established. In this study, the emphasis on the relative contribution of different cytochrome P450 (P450) isoforms was assessed by persistently inhibiting P450 isoforms over 7 days with human HEPATOPAC. P450 isoform-selective inhibition was achieved with the chemical inhibitors furafylline (CYP1A2), tienilic acid (CYP2C9), (+)-N-3-benzylnirvanol (CYP2C19), paroxetine (CYP2D6), azamulin (CYP3A), and a combination of 1-aminobenzotriazole and tienilic acid (broad spectrum inhibition of P450s). We executed this CRP method using HEPATOPAC by optimizing for the choice of P450 inhibitors, their selectivity, and the temporal effect of inhibitor concentrations on maintaining selectivity of inhibition. In general, the established CRP method using potent and selective chemical inhibitors allows to measure the relative contribution of P450s and to calculate the fraction of metabolism (fm) of low-turnover compounds. Several low-turnover compounds were used to validate this CRP method by determining their hepatic intrinsic clearance and fm, with comparison with literature values. We established the foundation of a robust CRP for low-turnover compound test system which can be expanded to include inhibition of other drug metabolizing enzymes. This generic CRP assay, using human long-term hepatocyte cultures, will be an essential tool in drug development for new chemical entities in the quantitative assessment of the risk as a victim of drug-drug interactions. SIGNIFICANCE STATEMENT An ongoing trend is to develop drug candidates which have limited metabolic clearance. The current studies report a generic approach to conducting reaction phenotyping studies with human HEPATOPAC, focusing on P450 metabolism of low-turnover compounds. Potent and selective chemical inhibitors were used to assess the relative contribution of the major human P450s. Validation was achieved by confirming hepatic intrinsic clearance and fraction of metabolism for previously reported low-turnover compounds. This approach is adaptable for assessment of all drug metabolizing enzymes.

Gefapixant (MK-7264, AF-219) is a first-in-class P2X3 antagonist in development for refractory or unexplained chronic cough. Gefapixant is primarily cleared by renal excretion. To assess the importance of the multidrug and toxin extrusion protein 1 (MATE1) and MATE2K transporters in the elimination of gefapixant, a drug-drug interaction study was conducted evaluating the effect of coadministration of a single dose of pyrimethamine, a competitive inhibitor of MATE1 and MATE2K, on the single-dose pharmacokinetics of gefapixant in healthy participants. Safety and tolerability were also assessed. In this open-label, 2-period, fixed-sequence study, a 45-mg dose of gefapixant was administered to 12 participants in period 1. After a 7-day washout, a 50-mg dose of pyrimethamine was administered 3 hours before a 45-mg dose of gefapixant in period 2. Compared with the administration of gefapixant alone, concomitant dosing of gefapixant with pyrimethamine increased the total gefapixant plasma exposure (area under the plasma concentration-time curve from time 0 to infinity) by 24%, reduced gefapixant renal clearance by 30%, and increased gefapixant mean terminal half-life from 7.7 to 10.3 hours. The most frequently reported adverse events were dysgeusia, hypogeusia, and dry mouth; all adverse events were considered of mild intensity and resolved by the end of the study. These results support that MATE1 and/or MATE2K contribute to the renal clearance of gefapixant, but the effect of inhibition of these transporters on gefapixant pharmacokinetics is not considered clinically meaningful.

Gefapixant (MK-7264) is a P2X3 receptor antagonist in development for refractory or unexplained chronic cough. In vitro, gefapixant is a substrate of the renal efflux transporters MATE1 and -2K and an inhibitor of the hepatic uptake transporter OATP1B. The potential for gefapixant drug-drug interactions (DDI) was assessed using the probe compounds, pyrimethamine (PYR, a MATE inhibitor) and pitavastatin (PITA, an OATP1B substrate). Two Phase 1 open-label fixed-sequence trials were conducted to assess the single-dose pharmacokinetics (PK) of gefapixant and PITA, respectively. In Trial 1, 12 participants received 45 mg gefapixant alone in Period 1 and, in Period 2, received gefapixant three hours after a single dose of 50 mg PYR. In Trial 2, 20 participants received 1 mg PITA alone in Period 1 and, in Period 2, received 45 mg gefapixant for 4 days; PITA was coadministered with the third dose of gefapixant. Intensive PK samples, including urine in Trial 1, were collected after each dose of the respective victim drugs. Gefapixant and PITA PK (geometric mean ratios [GMR]) are summarized as follows. Coadministration of PYR and gefapixant resulted in clinically insignificant increases in gefapixant, attributable to reduced renal clearance. Gefapixant coadministration did not affect PITA exposure. These trials indicate that gefapixant has minimal potential for clinical DDI involving the interrogated transporters.

In vitro evaluation of tenofovir disproxil fumarate (TDF) and tenofovir alafenamide (TAF) revealed comparable antiviral effects with respect to the tenofovir-diphosphate (TFV-DP) level in human peripheral blood mononuclear cells (PBMCs), despite the EC50 values determined based on prodrug concentrations were nearly two orders of magnitude apart. In vivo EC50 obtained from meta-analyses were in good agreement with the in vitro results, indicating intracellular TFV-DP can be employed for in vitro-in vivo translation of viral inhibition for tenofovir prodrugs. Current analysis indicated that the intracellular concentrations of TFV-DP achieving maximal antiviral effect in vitro can be directly translatable in the clinic to accomplish maximal viral load suppression attainable by tenofovir-prodrugs.

The correlation of in vitro inhibition of cathepsin K (CatK) activity and in vivo suppression of collagen I biomarkers was examined with three selective CatK inhibitors to explore the potential translatability from animal species to human. These inhibitors exhibited good in vitro potencies toward recombinant CatK enzymes across species, with IC50 values ranging from 0.20 to 6.1 nM. In vivo studies were conducted in animal species following multiple-day dosing of the CatK inhibitors to achieve steady-state plasma drug concentration-time profiles. Measurement of urinary bone resorption biomarkers (cross-linked N-terminal telopeptide and helical peptide of type I collagen) revealed drug concentration-dependent suppression of biomarkers, with EC50 values estimated to be 12 to 160 nM. Marked improvement in the correlation between in vitro and in vivo CatK activities was observed with the application of unbound (free) fraction in plasma, consistent with the conditions stipulated by the free-drug hypothesis. These results indicate that the in vitro-in vivo translation of CatK inhibition observed in animal species can translate to humans when the unbound fraction of the inhibitor is considered. Interestingly, residual levels of urinary bone resorption marker were detected as the suppression reached saturation (at an average of 82% inhibition), an apparent phenomenon observed regardless of the species, biomarker, or compound examined. Since cathepsin enzymes other than CatK were reported to catalyze cleavage of collagen I, it is hypothesized that CatK-mediated degradation of collagen I in bone represents ~82% of overall collagen I turnover in the body.

Prediction of metabolic clearance has been a challenge for compounds exhibiting minimal turnover in typical in vitro stability experiments. The aim of the current study is to evaluate the utilization of plated human hepatocytes to predict intrinsic clearance of low-turnover compounds. The disappearance of test compounds was determined for up to 48 h while enzyme activities in plated hepatocytes were monitored concurrently in a complimentary experiment. Consistent with literature reports, marked time-dependent loss of cytochrome P450 (CYP) enzyme activities was observed during the 48-h incubation period. To account for the loss of enzyme activities, a term “fraction of activity remaining” was calculated based on area-under-the-curve derived from the average rate of activity loss (k avg), and then applied as a correction factor for intrinsic clearance determination. Twelve compounds were selected in this study to cover phase I and phase II biotransformation pathways, with in vivo intrinsic clearance values, representing metabolic clearance only, ranging from 0.66 to 47 ml/min/kg. Determination of in vitro intrinsic clearance using three individual preparations of hepatocytes revealed a reasonably good agreement (generally within threefold) between the predicted and the observed metabolic clearance for all 12 compounds tested. The current results indicated that plated hepatocytes can be utilized to provide clearance predictions for compounds with low-turnover in humans when corrected for the loss in enzyme activities.

Cathepsin K (CatK) inhibitors exhibited chondroprotective and pain-reducing effects in animal models, however, improvements were relatively modest at dose levels achieving maximal suppression of CatK biomarkers in urine. In this report, a previously characterized CatK inhibitor (MK-1256) is utilized to explore the potential of reduced target engagement and/or suboptimal exposure (free drug) as limiting factors to the pharmacological potential of CatK inhibitors in the knee joint. Following oral administration of MK-1256 at a dose level achieving maximal inhibition of urinary biomarker (helical peptide) in dogs, full suppression of the biomarker in synovial fluid was observed. Subsequent tissue distribution studies conducted in dogs and rabbits revealed that MK-1256 levels in synovial fluid and cartilage were consistent with the free-drug hypothesis. Reasonable projection (within twofold) of drug levels in these tissues can be made based on plasma drug concentration with adjustments for binding factors. These results indicate that the previously observed efficacies in the animal models were not limited by compound distribution or target engagement in the knee tissues.

Background FTO gene variants have been associated with obesity phenotypes in sedentary and obese populations, but rarely with skeletal muscle and elite athlete phenotypes. Methods In 1089 participants, comprising 530 elite rugby athletes and 559 non-athletes, DNA was collected and genotyped for the FTO rs9939609 variant using real-time PCR. In a subgroup of non-resistance trained individuals (NT; n = 120), we also assessed structural and functional skeletal muscle phenotypes using dual energy x-ray absorptiometry, ultrasound and isokinetic dynamometry. In a subgroup of rugby athletes (n = 77), we assessed muscle power during a countermovement jump. Results In NT, TT genotype and T allele carriers had greater total body (4.8% and 4.1%) and total appendicular lean mass (LM; 3.0% and 2.1%) compared to AA genotype, with greater arm LM (0.8%) in T allele carriers and leg LM (2.1%) for TT, compared to AA genotype. Furthermore, the T allele was more common (94%) in selected elite rugby union athletes (back three and centre players) who are most reliant on LM rather than total body mass for success, compared to other rugby athletes (82%; P = 0.01, OR = 3.34) and controls (84%; P = 0.03, OR = 2.88). Accordingly, these athletes had greater peak power relative to body mass than other rugby athletes (14%; P = 2 x 10 -6 ). Conclusion Collectively, these results suggest that the T allele is associated with increased LM and elite athletic success. This has implications for athletic populations, as well as conditions characterised by low LM such as sarcopenia and cachexia.

Glycine transporter 1 (GlyT1) represents a novel target for the treatment of schizophrenia via the potentiation of glutamatergic NMDA receptors. The discovery of 4,4-disubstituted piperidine inhibitors of GlyT1 which exhibit improved pharmacokinetic properties, including oral bioavailability, is discussed.

After oral treatment (once daily) for 4 weeks with the potent bradykinin B(1) receptor antagonist methyl 3-chloro-3'-fluoro-4'-{(1R)-1-[({1-[(trifluoroacetyl)amino]cyclopropyl}carbonyl)-amino]ethyl}-1,1'-biphenyl-2-carboxylate (MK-0686), rhesus monkeys (Macaca mulatta) exhibited significantly reduced systemic exposure of the compound in a dose-dependent manner, suggesting an occurrence of autoinduction of MK-0686 metabolism. This possibility is supported by two observations. 1) MK-0686 was primarily eliminated via biotransformation in rhesus monkeys, with oxidation on the chlorophenyl ring as one of the major metabolic pathways. This reaction led to appreciable formation of a dihydrodiol (M11) and a hydroxyl (M13) product in rhesus liver microsomes supplemented with NADPH. 2) The formation rate of these two metabolites determined in liver microsomes from MK-0686-treated groups was > or = 2-fold greater than the value for a control group. Studies with recombinant rhesus P450s and monoclonal antibodies against human P450 enzymes suggested that CYP2C75 played an important role in the formation of M11 and M13. The induction of this enzyme by MK-0686 was further confirmed by a concentration-dependent increase of its mRNA in rhesus hepatocytes, and, more convincingly, the enhanced CYP2C proteins and catalytic activities toward CYP2C75 probe substrates in liver microsomes from MK-0686-treated animals. Furthermore, a good correlation was observed between the rates of M11 and M13 formation and hydroxylase activities toward probe substrates determined in a panel of liver microsomal preparations from control and MK-0686-treated animals. Therefore, MK-0686, both a substrate and inducer for CYP2C75, caused autoinduction of its own metabolism in rhesus monkeys by increasing the expression of this enzyme.

Herein, we present the identification of a novel class of pyrazolopyrimidine phosphodiesterase 10A (PDE10A) inhibitors. Beginning with a lead molecule (1) identified through a fragment-based drug discovery (FBDD) effort, lead optimization was enabled by rational design, X-ray crystallography, metabolic and off-target profiling, and fragment scaffold-hopping. We highlight the discovery of PyP-1, a potent, highly selective, and orally bioavailable pyrazolopyrimidine inhibitor of PDE10A. PyP-1 exhibits sub-nanomolar potency (PDE10A Ki=0.23nM), excellent pharmacokinetic (PK) and physicochemical properties, and a clean off-target profile. It displays dose-dependent efficacy in numerous pharmacodynamic (PD) assays that measure potential for anti-psychotic activity and cognitive improvement. PyP-1 also has a clean preclinical profile with respect to cataleptic potential in rats, prolactin secretion, and weight gain, common adverse events associated with currently marketed therapeutics. Further, PyP-1 displays in vivo preclinical target engagement as measured by PET enzyme occupancy in concert with [(11)C]MK-8193, a novel PDE10A PET tracer.

Sulfation of ethinyl estradiol (EE) is a major pathway of first pass metabolism in both the intestine and liver. Consequently, we sought to identify the human sulfotransferases (SULTs) involved in the 3-O-sulfation of EE (EE-SULT). Based on the results described herein, cDNA-expressed human cytosolic SULT1A3 and SULT1E1 were identified as low Km isoforms (18.9 and 6.7 nM, respectively) mediating the sulfation of EE. In contrast, the EE-SULT catalyzed by other recombinant SULTs (SULT1A1 and 2A1) was a relatively high Km process (Km > or = 230 nM). The kinetics of EE-SULT in human intestine (Km1 = 24 nM; Km2 = 1206 nM) and liver (Km1 = 8 nM; Km2 = 2407 nM) cytosol was biphasic and conformed to a two-Km model with both low and high Km components. At a low EE concentration (3 nM), inhibition of EE-SULT activity (intestinal) was characterized with 2,6-dichloro p-nitrophenol (DCNP) (IC50 = 15.6 microM) and quercetin (IC50 = 0.4 microM). When these IC50 values were compared with those derived from expressed enzyme, inhibition of EE-SULT was consistent with the SULT1E1 (DCNP, IC50 = 20 microM; quercetin, IC50 = 0.6 microM), but not SULT1A3 (DCNP, IC50 = 12.4; quercetin, IC50 = 7 microM). Moreover, when estrone (which selectively inhibits expressed SULT1E1 and SULT1A3) was included in intestinal incubations, the high-affinity component of the Eadie-Hofstee plot for EE sulfation was inhibited, converting the plot from biphasic to monophasic. Collectively, these data are consistent with SULT1E1 as the primary sulfotransferase involved in EE sulfation at clinically relevant concentrations (

In vitro studies were performed to identify the human cytochrome P450 enzyme(s) involved in the hydroxylation (isopropyl moiety) of a previously reported endothelin ET(A) receptor antagonist, compound A [(+)-(5S,6R,7R)-2-isopropylamino-7-(4-methoxy-2-[(2R)-3-methoxy-2-methylpropyl])-5-(3,4-methylenedioxyphenyl)cyclopenteno(1,2-b) pyridine 6-carboxylic acid]. Several lines of evidence indicated that the reaction was mainly catalyzed by CYP2C8. Of the 10 recombinant cytochrome P450 isoforms tested, only CYP2C8 exhibited hydroxylase activity. In agreement, inhibitory antibodies selective for CYP2C8 attenuated (95%) the hydroxylase activity in human liver microsomes, whereas antibodies and chemical inhibitors selective for other cytochrome P450 isoforms had a minor or no effect on the reaction. In addition, the formation of the hydroxy metabolite correlated well with CYP2C8-selective paclitaxel 6alpha-hydroxylation (r(2) approximately 0.92; p0.0001) and amodiaquine N-de-ethylation (r(2) approximately 0.91; p0.0001) in a bank of human liver microsomes (n = 15 organ donors). Finally, compound A hydroxylase activity conformed to Michaelis-Menten kinetics, and the K(m) (Michaelis constant) in human liver microsomes was similar to that of CYP2C8 ( approximately 10 microM). It is concluded that the hydroxylation of compound A is mainly catalyzed by CYP2C8, and thus the reaction can possibly serve as an alternative marker assay for CYP2C8 in human liver microsomes.

Sulfation is an important reaction in the biotransformation of steroid hormones, neurotransmitters, drugs, and other xenobiotics, yet little is known about the effects of organic solvents on sulfotransferase (SULT) activities in vitro. Initial experiments found that surprisingly low levels of solvent had dramatic effects on sulfotransferase activity. Consequently, we evaluated the effects of five commonly used solvents (methanol, ethanol, acetonitrile, dimethyl sulfoxide, and dimethyl formamide) on activities of cDNA-expressed sulfotransferase isozymes 1A1 (4-nitrophenol sulfation), 1A3 (dopamine sulfation), 1E1 (ethynylestradiol sulfation), and 2A1 (dehydroepiandrosterone sulfation). In addition, 1-hydroxypyrene was used as a general fluorescent probe for all four sulfotransferase isoforms examined. When substrates were present at their respective isoform-specific Km values, methanol and ethanol (0.4%, v/v) generally had less effect than acetonitrile, dimethyl sulfoxide, and dimethyl formamide on sulfotransferase activities. Acetonitrile, a commonly used solvent in cytochrome P450 studies, inhibited SULT1A1 activities (approximately 40%) at 0.4% (v/v), but activated SULT1E1-mediated 1-hydroxypyrene sulfation approximately 2.6-fold. Assuming a two-site kinetic model, studies revealed that solvent affected Vmax1, Vmax2, and the Ki value of 1-hydroxypyrene sulfation mediated by SULT1E1. In contrast, the Km value was not affected, suggesting that solvent may potentially alter binding interactions of the second substrate molecule, but not the first. Additional experiments with expressed SULT1A1, supplemented with control protein, revealed that the inhibitory effect of solvent (0.4%, v/v) was reduced to15% for all solvents examined. Thus, it is recommended that ethanol is used as the preferred solvent vehicle and that incubations with expressed enzyme contain12 microg/ml total protein.

Aims To identify the cytochrome P450 (CYP) isoforms responsible for the metabolism of simvastatin hydroxy acid (SVA), the most potent metabolite of simvastatin (SV).

Compound A [(+)-(5S,6R,7R)-2-isopropylamino-7-[4-methoxy-2-((2R)-3-methoxy-2-methylpropyl)-5-(3,4-methylenedioxyphenyl) cyclopenteno [1,2-b] pyridine 6-carboxylic acid] is a new and selective endothelin ET(A) receptor antagonist. It underwent significant acyl glucuronidation and acyl glucosidation in human liver microsomes supplemented with UDP-glucuronic acid (UDPGA) and UDP-glucose (UDPG). These two conjugations were observed in a panel of human liver microsomal samples (n = 16) that gave rise to varying activities but with no significant correlation with each other in the native and activator-treated microsomal preparations (r(2)or= 0.4, p0.05). The lack of correlation may be explained by the involvement of multiple UDP-glucuronosyltransferases (UGTs; UGT1A1, 1A3, 1A9, 2B7 and 2B15) in the glucuronidation but essentially solely UGT2B7 in the glucosidation. Both reactions conformed to monophasic Michaelis-Menten kinetics in human liver microsomes. The glucuronidation reaction exhibited apparent K(m) values (mean +/- S.E.) for compound A and UDPGA of 8.4 +/- 0.6 and 605 +/- 35 microM, respectively, whereas the values for the glucosidation reaction were 10.2 +/- 1.5 and 670 +/- 120 microM, respectively. In both pooled human liver microsomes and expressed UGT2B7, UDPG and UDPGA competitively inhibited their counterpart conjugations with K(i) values close to their K(m) values, indicating a comparable affinity of the enzyme toward these two nucleotide sugars. We herein report a drug acyl glucoside formed in human liver microsomes at a considerable turnover rate and provide the evidence for a UGT isoform (UGT2B7) capable of transferring both glucuronic acid and glucose from UDPGA and UDPG to an aglycone.

Purpose: Wee1 regulates key DNA damage checkpoints, and in this study, the efficacy of the Wee1 inhibitor MK-1775 was evaluated in glioblastoma multiforme (GBM) xenograft models alone and in combination with radiation and/or temozolomide. Experimental Design: In vitro MK-1775 efficacy alone and in combination with temozolomide, and the impact on DNA damage, was analyzed by Western blotting and γH2AX foci formation. In vivo efficacy was evaluated in orthotopic and heterotopic xenografts. Drug distribution was assessed by conventional mass spectrometry (MS) and matrix-assisted laser desorption/ionization (MALDI)-MS imaging. Results: GBM22 (IC50 = 68 nmol/L) was significantly more sensitive to MK-1775 compared with five other GBM xenograft lines, including GBM6 (IC50 >300 nmol/L), and this was associated with a significant difference in pan-nuclear γH2AX staining between treated GBM22 (81% cells positive) and GBM6 (20% cells positive) cells. However, there was no sensitizing effect of MK-1775 when combined with temozolomide in vitro. In an orthotopic GBM22 model, MK-1775 was ineffective when combined with temozolomide, whereas in a flank model of GBM22, MK-1775 exhibited both single-agent and combinatorial activity with temozolomide. Consistent with limited drug delivery into orthotopic tumors, the normal brain to whole blood ratio following a single MK-1775 dose was 5%, and MALDI-MS imaging demonstrated heterogeneous and markedly lower MK-1775 distribution in orthotopic as compared with heterotopic GBM22 tumors. Conclusions: Limited distribution to brain tumors may limit the efficacy of MK-1775 in GBM. Clin Cancer Res; 21(8); 1916–24. ©2015 AACR.

A series of studies were conducted to explore the mechanism of the pharmacokinetic interaction between simvastatin (SV) and gemfibrozil (GFZ) reported recently in human subjects. After administration of a single dose of SV (4 mg/kg p.o.) to dogs pretreated with GFZ (75 mg/kg p.o., twice daily for 5 days), there was an increase (approximately 4-fold) in systemic exposure to simvastatin hydroxy acid (SVA), but not to SV, similar to the observation in humans. GFZ pretreatment did not increase the ex vivo hydrolysis of SV to SVA in dog plasma. In dog and human liver microsomes, GFZ exerted a minimal inhibitory effect on CYP3A-mediated SVA oxidation, but did inhibit SVA glucuronidation. After i.v. administration of [(14)C]SVA to dogs, GFZ treatment significantly reduced (2-3-fold) the plasma clearance of SVA and the biliary excretion of SVA glucuronide (together with its cyclization product SV), but not the excretion of a major oxidative metabolite of SVA, consistent with the in vitro findings in dogs. Among six human UGT isozymes tested, UGT1A1 and 1A3 were capable of catalyzing the glucuronidation of both GFZ and SVA. Further studies conducted in human liver microsomes with atorvastatin (AVA) showed that, as with SVA, GFZ was a less potent inhibitor of the CYP3A4-mediated oxidation of this drug than its glucuronidation. However, with cerivastatin (CVA), the glucuronidation as well as the CYP2C8- and CYP3A4-mediated oxidation pathways were much more susceptible to inhibition by GFZ than was observed with SVA or AVA. Collectively, the results of these studies provide metabolic insight into the nature of drug-drug interaction between GFZ and statins, and a possible explanation for the enhanced susceptibility of CVA to interactions with GFZ.

We describe the discovery of potent and orally bioavailable tetrahydropyridopyrimidine inhibitors of phosphodiesterase 10A by systematic optimization of a novel HTS lead. Lead compound THPP-1 exhibits nanomolar potencies, excellent pharmacokinetic properties, and a clean off-target profile. It displays in vivo target engagement as measured by increased rat striatal cGMP levels upon oral dosing. It shows dose-dependent efficacy in a key pharmacodynamic assay predictive of antipsychotic activity, the psychostimulant-induced rat hyperlocomotion assay. Further, THPP-1 displays significantly fewer preclinical adverse events in assays measuring prolactin secretion, catalepsy, and weight gain, in contrast to the typical and atypical antipsychotics haloperidol and olanzapine.

Setileuton [4-(4-fluorophenyl)-7-[({5-[(1S)-1-hydroxy-1-(trifluoromethyl)propyl]-1,3,4-oxadiazol-2-yl}amino)methyl]-2H-1-benzopyran-2-one] is a selective inhibitor of the 5-lipoxygenase enzyme, which is under investigation for the treatment of asthma and atherosclerosis. During the development of setileuton, a metabolite (M5) was identified in incubations with rat, dog, and human liver microsomes that represented the addition of 18 Da to the 1,3,4-oxadiazole portion of the molecule. Based on mass spectral data, a ring opened structure was proposed and confirmed through comparison with a synthetic standard. The metabolic ring opening was examined in vitro in rat liver microsomes and was determined to be mediated by cytochrome P450s (P450s). Upon examination of the specific P450s involved using cDNA-expressed rat P450s, it was shown that CYP1A2 likely was the major isoform contributing to the formation of M5. Studies using stable labeled molecular oxygen and water demonstrated that the oxygen was incorporated from molecular oxygen, rather than water, and confirmed that the metabolic formation was oxidative. An alternative, comparatively slow pathway of chemical hydrolysis also was identified and described. Three potential mechanisms for the two-step metabolic ring opening of the 1,3,4-oxadizole are proposed.

In vitro metabolism studies were conducted to determine the human cytochrome P450 enzyme(s) involved in the biotransformation of 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3b]pyridazine (TPA023), a selective agonist of human gamma-aminobutyric acid(A) receptor alpha2 and alpha3 subunits. Incubation of TPA023 with NADPH-fortified human liver microsomes resulted in the formation of t-butyl hydroxy TPA023, N-desethyl TPA023, and three minor metabolites. Both t-butyl hydroxylation and N-deethylation reactions were greatly inhibited (85%) in the presence of CYP3A-selective inhibitory antibodies and chemical inhibitors, indicating that members of the CYP3A subfamily play an important role in TPA023 metabolism. Eadie-Hofstee plots of t-butyl hydroxylation and N-deethylation in pooled CYP3A5-rich human liver microsomes revealed a low K(m) (3.4 and 4.5 microM, respectively) and a high K(m) (12.7 and 40.0 microM, respectively) component. For both metabolites, the high K(m) component was not observed with a pool of microsomal preparations containing minimal levels of CYP3A5. Preincubation of liver microsomes with mifepristone (selectivity for CYP3A4CYP3A5) greatly inhibited both t-butyl hydroxylation and N-deethylation (75%); however, the residual activities were significantly higher in the pooled CYP3A5-rich liver microsomes (p0.0005). In addition, elevated levels of residual t-butyl hydroxylase and N-deethylase activities were observed in the presence of both CYP3A5-rich and CYP3A5-deficient preparations when the substrate concentration increased from 4 to 40 microM. In agreement, metabolite formation catalyzed by recombinant CYP3A5 was described by a biphasic model. It is concluded that CYP3A4 plays a major role in TPA023 metabolism, and CYP3A5 may also contribute at higher concentrations of the compound.

A highly active glutathione S-transferase was purified from adult German cockroaches, Blattella germanica. The purified enzyme appeared as a single band of 24 kDa by SDS/PAGE, and had a different electrophoretic mobility than, a previously identified Sigma class glutathione S-transferase (Bla g 5). Kinetic study of 1-chloro-2,4-dinitrobenzene conjugation revealed a high catalytic rate but common substrate-binding and cosubstrate-binding affinities, with V(max), k(cat), K(m) for 1-chloro-2,4-dinitrobenzene and K(m) for glutathione estimated to be 664 micromol x mg(-1) x min(-1), 545 s(-1), 0.33 mm and 0.76 mm, respectively. Interestingly, this enzyme possessed the highest activity for cumene hydroperoxide among insect glutathione S-transferases reported to date. Along with the ability to metabolize 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane and 4-hydroxynonenal, this glutathione S-transferase may play a role in defense against insecticides as well as oxidative stress. On the basis of the amino acid sequences obtained from Edman degradation and MS analyses, a 987-nucleotide cDNA clone encoding a glutathione S-transferase (BggstD1) was isolated. The longest ORF encoded a 24 614 Da protein consisting of 216 amino acid residues. The sequence had close similarities ( approximately 45-60%) to that of Delta class glutathione S-transferases, but had only 14% identity to Bla g 5. The putative amino acid sequence contained matching peptide fragments of the purified glutathione S-transferase. ELISA showed that BgGSTD1 bound to serum IgE obtained from patients with cockroach allergy, indicating that the protein may be a cockroach allergen.

The cytotoxic effects of blattellaquinone (BTQ), a sex pheromone produced by adult female German cockroaches, have been studied using human lung adenocarcinoma A549 cells. 1,4-Benzoquinone (BQ), a toxic chemical implicated in benzene toxicity, was used as a reference compound. Both BQ and BTQ showed comparable toxicity toward A549 cells, with LD50 values estimated to be 14 and 19 microM, respectively. These two compounds increased the formation of an oxidized fluorescent probe, 2',7'-dichlorofluorescein, but had no effect on the cellular GSSG level. Interestingly, BTQ increased the level of 8-epi-prostaglandin F2alpha and was 4-fold more efficient in depleting cellular GSH content than BQ. Of the five GSH adducts of BTQ isolated, three were identified as mono-GSH conjugates, and the other two were di-conjugates. Mass spectrometric and NMR analyses of the di-conjugates showed that the second GSH molecule displaced the isovaleric acid moiety, potentially via a nucleophilic substitution reaction. The ability of BTQ to conjugate a second GSH molecule without quinone regeneration indicated that it may be a more effective cross-linking agent than BQ. Future experiments may be needed to evaluate the overall safety of BTQ before the commercialization of the compound as a cockroach attractant.

Following the finding that UGT2B7 catalyzes the transfer of the glycosyl group from both UDP-glucuronic acid (UDP-GlcA) and UDP-glucose (UDP-Glc) to an endothelin ET(A) receptor antagonist, Compound A [(+)-(5S,6R,7R)-2-isopropylamino-7-[4-methoxy-2-((2R)-3-methoxy-2-methylpropyl)-5-(3,4-methylenedioxyphenyl)cyclopenteno[1,2-b] pyridine 6-carboxylic acid], to form an acyl glucuronide and a glucoside (Tang et al., 2003), two additional nucleotide sugars [UDP-galactose (UDP-gal) and UDP-N-acetyl glucosamine (UDP-GlcNAc)] were examined as cosubstrates in human liver microsomes. It was found that UDP-gal, but not UDP-GlcNAc, also served as a sugar donor primarily through catalysis by UGT2B7, although at a significantly reduced catalytic rate. These three UDP-sugars showed pH-dependent kinetics and appeared to compete with each other, with IC50 values parallel to their respective apparent K(m) values. In contrast, only UDP-GlcA served as the sugar donor for the conjugation of diclofenac, a known UGT2B7 substrate, with an apparent K(m) for UDP-GlcA of 96 +/- 17 microM. UDP-Glc and UDP-gal, two futile sugar donors for diclofenac, were found to competitively inhibit the glucuronidation of this aglycone. Different from the case with Compound A, UDP-Glc and UDP-gal displayed K(i) values of 2054 +/- 108 microM and 1277 +/- 149 microM,10-fold greater than the K(m) for UDP-GlcA, indicating that these two nucleotide sugars were also capable of binding to the enzyme but with a lower affinity. The findings of this study indicate that the selectivity of UGT2B7 toward UDP-sugars is aglycone-dependent. With Compound A as the acceptor substrate, human UGT2B7 becomes more accommodative in the transfer of the glycosyl group from UDP-sugars beyond UDP-GlcA. The mechanism may involve enzyme conformational changes associated with Compound A binding to the enzyme.

Compound A (3-{2-oxo-3-[3-(5,6,7,8-tetrahydro-[1,8]napthyridin-2-yl)propyl]-imidazolidin-1-yl}-3(S)-(6-methoxy-pyridin-3-yl)propionic acid), a hydrophilic zwitter-ion, is a potent and selective alphavbeta3 integrin antagonist currently under clinical development for the treatment of osteoporosis. The mechanism of renal excretion of compound A was investigated using a combination of in vivo and in vitro approaches. In rats, renal excretion of compound A involved tubular secretion; ratios between renal clearance, corrected for unbound fraction in plasma (CLr,u) and glomerular filtration rate (GFR) were greater than unity (2-5). The tubular secretion of compound A was saturable at high plasma levels (26 microM), and was inhibited significantly, although modestly (about twofold) by relatively high plasma concentrations of the organic anion PAH (160 microM) and the cation cimetidine (about 400 microM), but not by the P-gp inhibitor quinidine (about 50 microM). However, compound A (about 100 microM) had a minimal effect on CLr/GFRs for cimetidine and PAH. In rhesus monkeys, renal elimination of compound A also involved tubular secretion, with a CLr,u/GFR ratio of about 30. The renal secretion of compound A was not affected by either cimetidine (about 120 microM) or PAH (about 80 microM). Similarly, compound A (about 40 microM) had a minimal effect on the renal tubular secretion of both cimetidine and PAH. At the doses studied, neither rat nor monkey plasma protein binding of compound A, cimetidine or PAH was affected in the presence of each other. In vitro transport studies showed that compound A was not a substrate for P-gp in the Caco-2, human MDR1 and mouse mdr1a transfected LLC-PK1 cell lines. In an uptake study using rOAT1 and rOAT3 transfected HEK cell lines, compound A was shown to be a substrate for rat OAT3 (Km= 15 microM), but not rat OAT1. The results suggest that the tubular secretion of compound A is not mediated by P-gp, but rather is mediated, at least in part, via the organic anion transporter OAT3, the renal transporter shown to be capable of transporting both the organic anion PAH and the organic cation cimetidine. Although there is a possibility for pharmacokinetic interactions between compound A and substrates or inhibitors of OAT3, at the renal excretion level, the magnitude of interaction would likely be modest in humans at clinically relevant doses.