Yohannes Teffera

Ratios of biliary glutathione disulfide (GSSG) to glutathione (GSH): a potential index to screen drug-induced hepatic oxidative stress in rats and mice

Hepatotoxicity of drug candidates is one of the major concerns in drug screening in early drug discovery. Detection of hepatic oxidative stress can be an early indicator of hepatotoxicity and benefits drug selection. The glutathione (GSH) and glutathione disulfide (GSSG) pair, as one of the major intracellular redox regulating couples, plays an important role in protecting cells from oxidative stress that is caused by imbalance between prooxidants and antioxidants. The quantitative determination of the GSSG/GSH ratios and the concentrations of GSH and GSSG have been used to indicate oxidative stress in cells and tissues. In this study, we tested the possibility of using the biliary GSSG/GSH ratios as a biomarker to reflect hepatic oxidative stress and drug toxicity. Four compounds that are known to alter GSH and GSSG levels were tested in this study. Diquat (diquat dibromide monohydrate) and acetaminophen were administered to rats. Paraquat and tert-butyl hydroperoxide were administered to mice to induce changes of biliary GSH and GSSG. The biliary GSH and GSSG were quantified using calibration curves prepared with artificial bile to account for any bile matrix effect in the LC–MS analysis and to avoid the interference of endogenous GSH and GSSG. With four examples (in rats and mice) of drug-induced changes in the kinetics of the biliary GSSG/GSH ratios, this study showed the potential for developing an exposure response index based on biliary GSSG/GSH ratios for predicting hepatic oxidative stress.

Discovery of triazine-benzimidazoles as selective inhibitors of mTOR.

mTOR is part of the PI3K/AKT pathway and is a central regulator of cell growth and survival. Since many cancers display mutations linked to the mTOR signaling pathway, mTOR has emerged as an important target for oncology therapy. Herein, we report the discovery of triazine benzimidazole inhibitors that inhibit mTOR kinase activity with up to 200-fold selectivity over the structurally homologous kinase PI3Kα. When tested in a panel of cancer cell lines displaying various mutations, a selective inhibitor from this series inhibited cellular proliferation with a mean IC(50) of 0.41 μM. Lead compound 42 demonstrated up to 83% inhibition of mTOR substrate phosphorylation in a murine pharmacodynamic model.

Discovery and optimization of potent and selective imidazopyridine and imidazopyridazine mTOR inhibitors

mTOR is a critical regulator of cellular signaling downstream of multiple growth factors. The mTOR/PI3K/AKT pathway is frequently mutated in human cancers and is thus an important oncology target. Herein we report the evolution of our program to discover ATP-competitive mTOR inhibitors that demonstrate improved pharmacokinetic properties and selectivity compared to our previous leads. Through targeted SAR and structure-guided design, new imidazopyridine and imidazopyridazine scaffolds were identified that demonstrated superior inhibition of mTOR in cellular assays, selectivity over the closely related PIKK family and improved in vivo clearance over our previously reported benzimidazole series.

Whole‐body tissue distribution study of drugs in neonate mice using desorption electrospray ionization mass spectrometry imaging

RATIONALE Although Desorption Electrospray Ionization (DESI) Mass Spectrometry Imaging (MSI) is uniquely suited for whole-body (WB) tissue distribution study of drugs, success in this area has been difficult. Here, we present WB tissue distribution studies using DESI-MSI and a new histological tissue-friendly solvent system. METHODS Neonate pups were dosed subcutaneously (SC) with clozapine, compound 1, compound 2, or compound 3. Following euthanization by hypothermia, neonates underwent a transcardiac perfusion (saline) to remove blood. After cryosectioning, DESI-MSI was conducted for the WB tissue slides, followed sequentially by histological staining. RESULTS Whole-body tissue imaging showed that clozapine and its N-oxide metabolite were distributed in significant amounts in the brain, spinal cord, liver, heart (ventricle), and lungs. Compound 1 was distributed mainly in the liver and muscle, and its mono-oxygenated metabolite was detected by DESI-MSI exclusively in the liver. Compound 2 was distributed mainly in the muscle and fatty tissue. Compound 3 was distributed mainly in fatty tissue and its metabolites were also mainly detected in the same tissue. CONCLUSIONS The results demonstrate the successful application of DESI-MSI in whole-body tissue distribution studies of drugs and metabolites in combination with sequential histology staining for anatomy. The results also identified lipophilicity as the driving force in the tissue distribution of the three Amgen compounds.

Application of on‐line nano‐liquid chromatography/mass spectrometry in metabolite identification studies

Metabolite identification is an important part of the drug discovery and development process. High sensitivity is necessary to identify metabolic products in vitro and in vivo. The most common method utilizes standard high-performance liquid chromatography (4.6  mm i.d. column and 1  mL/min flow rate) coupled to tandem mass spectrometry (HPLC/MS/MS). We have developed a method that utilizes a nano-LC system coupled to a high-resolution tandem mass spectrometer to identify metabolites from in vitro and in vivo samples. Using this approach, we were able to increase the sensitivity of analysis by approximately 1000-fold over HPLC/MS. In vitro samples were analyzed after simple acetonitrile precipitation, centrifugation, and dilution. The significant improvement in sensitivity enabled us to conduct experiments at very low substrate concentrations (0.01  μM), and very low incubation volumes (20  μL). In vivo samples were injected after simple dilution without any pre-purification. All the metabolites identified by conventional HPLC/MS/MS were also identified using the nano-LC method. This study demonstrates a very sensitive approach to identifying phase I and II metabolites with throughput and separation equivalent to the standard HPLC/MS/MS method.

Discovery of novel 1,2,3,4-tetrahydroisoquinolines and 3,4-dihydroisoquinoline-1(2H)-ones as potent and selective inhibitors of KDR: Synthesis, SAR, and pharmacokinetic properties

1,2,3,4-Tetrahydroisoquinolines and 3,4-dihydroisoquinoline-1(2H)-ones were identified as potent and selective inhibitors of KDR. The discovery, synthesis, and structure-activity relationships of these novel inhibitors are reported. In vitro metabolism and pharmacokinetic profiles of the most interesting compounds are discussed.

Selective antagonism of TRPA1 produces limited efficacy in models of inflammatory- and neuropathic-induced mechanical hypersensitivity in rats

The transient receptor potential ankyrin 1 (TRPA1) channel has been implicated in pathophysiological processes that include asthma, cough, and inflammatory pain. Agonists of TRPA1 such as mustard oil and its key component allyl isothiocyanate (AITC) cause pain and neurogenic inflammation in humans and rodents, and TRPA1 antagonists have been reported to be effective in rodent models of pain. In our pursuit of TRPA1 antagonists as potential therapeutics, we generated AMG0902, a potent (IC90 of 300 nM against rat TRPA1), selective, brain penetrant (brain to plasma ratio of 0.2), and orally bioavailable small molecule TRPA1 antagonist. AMG0902 reduced mechanically evoked C-fiber action potential firing in a skin-nerve preparation from mice previously injected with complete Freund’s adjuvant, supporting the role of TRPA1 in inflammatory mechanosensation. In vivo target coverage of TRPA1 by AMG0902 was demonstrated by the prevention of AITC-induced flinching/licking in rats. However, oral administration of AMG0902 to rats resulted in little to no efficacy in models of inflammatory, mechanically evoked hypersensitivity; and no efficacy was observed in a neuropathic pain model. Unbound plasma concentrations achieved in pain models were about 4-fold higher than the IC90 concentration in the AITC target coverage model, suggesting that either greater target coverage is required for efficacy in the pain models studied or TRPA1 may not contribute significantly to the underlying mechanisms.

Impact of Hydrolysis Mediated Clearance on the Pharmacokinetics of Novel Anaplastic Lymphoma Kinase Inhibitors

Compound 1 [(E)-4-fluoro-N-(6-((4-(2-hydroxypropan-2-yl)piperidin-1-yl)methyl)-1-((1S,4S)-4-(isopropylcarbamoyl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-ylidene)benzamide], a new, potent, selective anaplastic lymphoma kinase (ALK) inhibitor with potential application for the treatment of cancer, was selected as candidate to advance into efficacy studies in mice. However, the compound underwent mouse-specific enzymatic hydrolysis in plasma to a primary amine product (M1). Subsequent i.v. pharmacokinetics studies in mice showed that compound 1 had high clearance (CL) and a short half-life. Oral dose escalation studies in mice indicated that elimination of compound 1 was saturable, with higher doses achieving sufficient exposures above in vitro IC50. Chemistry efforts to minimize hydrolysis resulted in the discovery of several analogs that were stable in mouse plasma. Three were taken in vivo into mice and showed decreased CL corresponding to increased in vitro stability in plasma. However, the more stable compounds also showed reduced potency against ALK. Kinetic studies in NADPH-fortified and unfortified microsomes and plasma produced submicromolar Km values and could help explain the saturation of elimination observed in vivo. Predictions of CL based on kinetics from hydrolysis and NADPH-dependent pathways produced predicted hepatic CL values of 3.8, 3.0, 1.6, and 1.2 l/h⋅kg for compound 1, compound 2 [(E)-3,5-difluoro-N-(6-((4-(2-hydroxypropan-2-yl)piperidin-1-yl)methyl)-1-((1s,4s)-4-(isopropylcarbamoyl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-ylidene)benzamide], compound 3 [(E)-3-chloro-5-fluoro-N-(6-((4-(2-hydroxypropan-2-yl)piperidin-1-yl)methyl)-1-((1s,4s)-4-(isopropylcarbamoyl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-ylidene)benzamide], and compound 4 [(E)-N-(6-((4-(2-hydroxypropan-2-yl)piperidin-1-yl)methyl)-1-((1s,4s)-4-(isopropylcarbamoyl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-ylidene)-3-(trifluoromethyl)benzamide], respectively. The in vivo observed CLs for compounds 1, 2, 3, and 4 were 5.52, 3.51, 2.14, and 2.66 l/h⋅kg, respectively. These results indicate that in vitro metabolism kinetic data, incorporating contributions from both hydrolysis and NADPH-dependent metabolism, could be used to predict the systemic CL of compounds cleared via hydrolytic pathways provided that the in vitro assays thoroughly investigate the processes, including the contribution of other metabolic pathways and the possibility of saturation kinetics.

Abstract 1795: Characterization of a novel series of potent, selective inhibitors of wild type and mutant/fusion anaplastic lymphoma kinase

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that has been implicated as a driving oncogene in a number of cancers, including non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma (ALCL), neuroblastoma and inflammatory myofibroblastic tumors (IMT). Numerous genetic aberrations at the ALK locus are observed in cancer including point mutations, amplifications, translocations and inversions. Inversions are exemplified by inv(2)(p21;p23), which leads to the constitutively active oncogenic fusion protein EML4-ALK present in ∼5% of NSCLC. Crizotinib, a dual cMet/ALK kinase inhibitor, was recently approved by the FDA for locally advanced or metastatic NSCLC that is ALK-positive, thereby validating ALK as therapeutic target. Here we describe the pharmacological characterization of a novel series of potent, selective and orally bioavailable ALK kinase inhibitors. Members of this series inhibit wild type ALK, NPM-ALK fusion and crizotinib resistant ALK[L1196M] kinase activity at sub-nanomolar concentrations, displaying up to ∼200 fold increased inhibitory activity over crizotinib. Kinase profiling indicate that members of this series display increased selectivity scores relative crizotinib. In Karpas-299 cells, selected compounds inhibited both pY1604 ALK activation (IC50 = 2 nM) and cell proliferation (IC50 = 1 nM). Members of this series were also evaluated in the EML4-ALK expressing NSCLC cell line H3122, and displayed equipotent inhibition of pY1604 ALK activation and inhibition of cell proliferation (both IC50 = 1 nM). Members of this class did not inhibit growth of an ALK negative lymphoma cell line (HT). The in vivo activity of this series was examined in the Karpas-299 ALCL xenograft model. Compound was dosed daily (PO) at 10, 30 and 60 mg/kg. Tumor growth inhibition was observed at all dose levels, and the highest dose level resulted in significant tumor regression (96%, p 80% pALK inhibition was observed at the lowest doses tested, and complete inhibition was seen at doses of 30 mg/kg and above. In a direct comparison, members of this series achieved ALK inhibition in these tumors at a ∼15 fold lower plasma concentration than crizotinib. A PK/PD time course study was performed in the Karpas-299 model. A single 60 mg/kg dose of an inhibitor was able to maintain >90% ALK inhibition in tumors up to 24 hours post-dose, indicating significant tumor penetration and sustained ALK kinase inhibition. In conclusion, the described compounds are potent and selective inhibitors of ALK kinase, possess an impressive efficacy profile and drug-like pharmacokinetic properties. These features together indicate the potential for significant advantages over crizotinib. There is a compelling case for their clinical evaluation in patients with ALK-driven cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1795. doi:1538-7445.AM2012-1795

Species Difference in Glucuronidation Formation Kinetics with a Selective mTOR Inhibitor

The mammalian target of rapamycin (mTOR) is a protein kinase that shows key involvement in age-related disease and promises to be a target for treatment of cancer. In the present study, the elimination of potent ATP-competitive mTOR inhibitor 3-(6-amino-2-methylpyrimidin-4-yl)-N-(1H-pyrazol-3-yl)imidazo[1,2-b]pyridazin-2-amine (compound 1) is studied in bile duct–cannulated rats, and the metabolism of compound 1 in liver microsomes is compared across species. Compound 1 was shown to undergo extensive N-glucuronidation in bile duct–catheterized rats. N-glucuronides were detected on positions N1 (M2) and N2 (M1) of the pyrazole moiety as well as on the primary amine (M3). All three N-glucuronide metabolites were detected in liver microsomes of the rat, dog, and human, while primary amine glucuronidation was not detected in cynomolgus monkey. In addition, N1- and N2-glucuronidation showed strong species selectivity in vitro, with rat, dog, and human favoring N2-glucuronidation and monkey favoring N1-glucuronide formation. Formation of M1 in monkey liver microsomes also followed sigmoidal kinetics, singling out monkey as unique among the species with regard to compound 1 N-glucuronidation. In this respect, monkeys might not always be the best animal model for N-glucuronidation of uridine diphosphate glucuronosyltransferase (UGT) 1A9 or UGT1A1 substrates in humans. The impact of N-glucuronidation of compound 1 could be more pronounced in higher species such as monkey and human, leading to high clearance in these species. While compound 1 shows promise as a candidate for investigating the impact of pan-mTOR inhibition in vivo, opportunities may exist through medicinal chemistry efforts to reduce metabolic liability with the goal of improving systemic exposure.