Lead optimization of cathepsin K inhibitors for the treatment of Osteoarthritis.

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.

Reaction Phenotyping of Low-Turnover Compounds in Long-Term Hepatocyte Cultures Through Persistent Selective Inhibition of Cytochromes P450.

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 (f m) of low-turnover compounds. Several low-turnover compounds were used to validate this CRP method by determining their hepatic intrinsic clearance and f m, 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.

Discovery of pyrazolopyrimidine phosphodiesterase 10A inhibitors for the treatment of schizophrenia.

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.

Discovery of MK-8189, a Highly Potent and Selective PDE10A Inhibitor for the Treatment of Schizophrenia.

PDE10A is an important regulator of striatal signaling that, when inhibited, can normalize dysfunctional activity. Given the involvement of dysfunctional striatal activity with schizophrenia, PDE10A inhibition represents a potentially novel means for its treatment. With the goal of developing PDE10A inhibitors, early optimization of a fragment hit through rational design led to a series of potent pyrimidine PDE10A inhibitors that required further improvements in physicochemical properties, off-target activities, and pharmacokinetics. Herein we describe the discovery of an isomeric pyrimidine series that addresses the liabilities seen with earlier compounds and resulted in the invention of compound 18 (MK-8189), which is currently in Phase 2b clinical development for the treatment of schizophrenia.

Discovery of tetrahydropyridopyrimidine phosphodiesterase 10A inhibitors for the treatment of schizophrenia.

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.

Characterization of the absorption, metabolism, excretion, and mass balance of gefapixant in humans.

Gefapixant (MK-7264) is a first-in-class, selective antagonist of the P2X3 purinergic receptor currently being investigated as a therapeutic agent for the treatment of refractory or unexplained chronic cough. In non-clinical studies, gefapixant was eliminated primarily by renal excretion of the parent drug. The objective of this study was to assess the disposition of gefapixant in humans. The absorption, metabolism, and excretion profiles of gefapixant were assessed after oral administration of a single dose of [14 C]gefapixant to six healthy adult males. Following a single-oral [14 C]gefapixant dose to healthy adult males, the mass balance was achieved, with 98.9% of the administered radioactivity recovered in urine and feces. Elimination of gefapixant occurred primarily via renal excretion of the intact drug (64%); metabolism was a minor pathway of elimination of gefapixant (12% and 2% recovered in urine and feces, respectively). Single-dose administration of [14 C]gefapixant 50 mg was generally well tolerated in healthy adult males. The fraction of the anticipated therapeutic oral dose of gefapixant absorbed is estimated to be at least 78%. Gefapixant is expected to be the major circulating drug-related material in plasma, and the majority of the dosed drug will be excreted unchanged in urine.

Assessment of Pharmacokinetic Interaction Between Gefapixant (MK-7264), a P2X3 Receptor Antagonist, and the OATP1B1 Drug Transporter Substrate Pitavastatin.

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.

Assessment of the Effect of Pyrimethamine, a Potent Inhibitor of Multidrug and Toxin Extrusion Protein 1/2K, on the Pharmacokinetics of Gefapixant (MK-7264), a P2X3 Receptor Antagonist.

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.

Novel cytochrome P450-mediated ring opening of the 1,3,4-oxadiazole in setileuton, a 5-lipoxygenase inhibitor.

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.

Variability in Human In Vitro Enzyme Kinetics.

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.

Eplerenone decreases inflammatory foci in spontaneously hypertensive rat hearts with minimal effects on blood pressure.

The blood pressure (BP)-lowering effects of mineralocorticoid receptor (MR) antagonists in salt-sensitive rat models of hypertension are well understood. However, studies in salt-independent models have yielded mixed results, and therefore, we measured the hemodynamic effects of MR blockade in spontaneously hypertensive rats. We treated spontaneously hypertensive rats for 8 weeks with 30-300 mg.kg.d eplerenone or 20 mg.kg.d losartan and monitored BP using radiotelemetry and performed histopathological analyses of the hearts. Eplerenone, in contrast to losartan, caused only a small reduction in systolic BP at the highest dose tested. Both reduced left ventricular wall thickness, although eplerenone was less effective than losartan. Only losartan decreased heart weight. We observed foci of cardiomyopathy characterized by combinations of infiltrating monocytes, necrotic myocytes, and interstitial fibrosis in hearts of control animals. The number of foci seemed to be decreased in hearts of losartan- and eplerenone-treated animals. In a second study, using quantitative histomorphometry, the number of foci was significantly reduced by 20 mg.kg.d losartan (by 68%) or by 300 mg.kg.d eplerenone (by 50%). Our data support the hypothesis that a direct BP-independent effect on the progression of cardiomyopathy in the heart may be one basis for the cardiac protection afforded by MR antagonism.

Discovery of GlyT1 inhibitors with improved pharmacokinetic properties.

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.

Suppression of cathepsin K biomarker in synovial fluid as a free-drug-driven process.

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.

CYP2C75-involved autoinduction of metabolism in rhesus monkeys of methyl 3-chloro-3'-fluoro-4'-{(1R)-1-[({1-[(trifluoroacetyl)amino]cyclopropyl}carbonyl)amino]ethyl}-1,1'-biphenyl-2-carboxylate (MK-0686), a bradykinin B1 receptor antagonist.

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.

Establishment of intracellular tenofovir-diphosphate as the key determinant for in vitro-in vivo translation of antiviral efficacy.

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.

Purification and cloning of a Delta class glutathione S-transferase displaying high peroxidase activity isolated from the German cockroach Blattella germanica.

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.

Prediction of Metabolic Clearance for Low-Turnover Compounds Using Plated Hepatocytes with Enzyme Activity Correction.

BACKGROUND AND OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Applicability of in vitro-in vivo translation of cathepsin K inhibition from animal species to human with the use of free-drug hypothesis.

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.

Preclinical Characterization of the Phosphodiesterase 10A PET Tracer [(11)C]MK-8193.

PURPOSE: A positron emission tomography (PET) tracer for the enzyme phosphodiesterase 10A (PDE10A) is desirable to guide the discovery and development of PDE10A inhibitors as potential therapeutics. The preclinical characterization of the PDE10A PET tracer [(11)C]MK-8193 is described. PROCEDURES: In vitro binding studies with [(3)H]MK-8193 were conducted in rat, monkey, and human brain tissue. PET studies with [(11)C]MK-8193 were conducted in rats and rhesus monkeys at baseline and following administration of a PDE10A inhibitor. RESULTS: [(3)H]MK-8193 is a high-affinity, selective PDE10A radioligand in rat, monkey, and human brain tissue. In vivo, [(11)C]MK-8193 displays rapid kinetics, low test-retest variability, and a large specific signal that is displaced by a structurally diverse PDE10A inhibitor, enabling the determination of pharmacokinetic/enzyme occupancy relationships. CONCLUSIONS: [(11)C]MK-8193 is a useful PET tracer for the preclinical characterization of PDE10A therapeutic candidates in rat and monkey. Further evaluation of [(11)C]MK-8193 in humans is warranted.

The Efficacy of the Wee1 Inhibitor MK-1775 Combined with Temozolomide Is Limited by Heterogeneous Distribution across the Blood-Brain Barrier in Glioblastoma.

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.