Pulmonary arterial hypertension (PAH) is a devastating rare disease, which despite currently available treatments, still represents a high unmet medical need. Specific E3 ubiquitin protein ligase 1 (SMURF1) is a HECT E3 ligase that ubiquitinates key signaling molecules from the TGFβ/BMP pathways, which are of great relevance in the pathophysiology of PAH. Herein, the design and synthesis of novel potent small-molecule SMURF1 ligase inhibitors are described. Lead molecule 38 has demonstrated good oral pharmacokinetics in rats and significant efficacy in a rodent model of pulmonary hypertension.

Further optimization of an initial DP2 receptor antagonist clinical candidate NVP-QAV680 led to the discovery of a follow-up molecule 2-(2-methyl-1-(4-(methylsulfonyl)-2-(trifluoromethyl)benzyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)acetic acid (compound 11, NVP-QAW039, fevipiprant), which exhibits improved potency on human eosinophils and Th2 cells, together with a longer receptor residence time, and is currently in clinical trials for severe asthma.

The liver is known to be the principal site of drug metabolism. Depending on the route of administration, especially in cases of topical and local delivery, evaluation of local drug metabolism in extrahepatic tissues is vital to assess fraction of the drug metabolized. This parameter becomes important from the point of view of drug availability or the contribution to overall clearance. Examples include fraction metabolized in the gut for oral drugs and contribution of pulmonary or renal clearance to total clearance of a drug. Diseases of the eye represent a rising unmet medical need and a number of therapeutics are currently being developed in the form of small molecules and biologics. Treatment of ocular diseases has expanded to explore various topical formulations and local short- and long-term therapies by ocular routes of administration. Until recently, metabolism in the eye for any species, including human, was not well documented, but this topic is gaining wide interest. Many in vitro-ex vivo models, each with separate pros and cons, are being used for studying ocular metabolism. This review is aimed at providing a perspective on the relevance and application of ocular metabolism, melanin binding, and the use of tissue-and cell-derived ocular models in discovery and preclinical development. (C) 2017 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.

Idiopathic pulmonary fibrosis is a chronic progressive disease of increasing prevalence for which there is no effective therapy. Increased oxidative stress associated with an oxidant-antioxidant imbalance is thought to contribute to disease progression. NADPH oxidases (Nox) are a primary source of reactive oxygen species within the lung and cardiovascular system. We demonstrate that the Nox4 isoform is up-regulated in the lungs of patients with IPF and in a rodent model of bleomycin-induced pulmonary fibrosis and vascular remodeling. Nox4 is constitutively active, and therefore increased expression levels are likely to contribute to disease pathology. Using a small molecule Nox4/Nox1 inhibitor, we demonstrate that targeting Nox4 results in attenuation of an established fibrotic response, with reductions in gene transcripts for the extracellular matrix components collagen 1α1, collagen 3α1, and fibronectin and in principle pathway components associated with pulmonary fibrosis and hypoxia-mediated vascular remodeling: transforming growth factor (TGF)-β1, plasminogen activator inhibitor-1, hypoxia-inducible factor, and Nox4. TGF-β1 is a principle fibrotic mediator responsible for inducing up-regulation of profibrotic pathways associated with disease pathology. Using normal human lung-derived primary fibroblasts, we demonstrate that inhibition of Nox4 activity using a small molecule antagonist attenuates TGF-β1-mediated up-regulation in expression of profibrotic genes and inhibits the differentiation of fibroblast to myofibroblasts, that is associated with up-regulation in smooth muscle actin and acquisition of a contractile phenotype. These studies support the view that targeting Nox4 may provide a therapeutic approach for attenuating pulmonary fibrosis.

The simultaneous effects of the cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) on inhibition of in vivo metabolism and gastric emptying were evaluated with the test compound 7-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-3-(4-methoxy-2-methylphenyl)-2,6-dimethylpyrazolo[5,1-b]oxazole(NVS-CRF38), a novel corticotropin releasing factor receptor 1 (CRF1) antagonist with low water solubility, and the reference compound midazolam with high water solubility in rats. Pretreatment of rats with 100 mg/kg oral ABT administered 2 hours before a semisolid caloric test meal markedly delayed gastric emptying. ABT increased stomach weights by 2-fold; this is likely attributable to a prosecretory effect because stomach concentrations of bilirubin were comparable in ABT and control groups. ABT administration decreased the initial systemic exposure of orally administered NVS-CRF38 and increased Tmax 40-fold, suggesting gastric retention and delayed oral absorption. ABT increased the initial systemic exposure of midazolam, however for orally (but not subcutaneously) administered midazolam, extensive variability in plasma-concentration time profiles was apparent. Careful selection of administration routes is recommended for ABT use in vivo, variable oral absorption of coadministered compounds can be expected due to a disturbance of gastrointestinal transit.

Deuterium isotope effects were evaluated as a strategy to optimize the pharmacokinetics of 7-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-3-(4-methoxy-2-methylphenyl)-2,6-dimethylpyrazolo[5,1-b]oxazole (NVS-CRF38), a novel corticotropin-releasing factor receptor 1 (CRF1) antagonist. In an attempt to suppress O-demethylation of NVS-CRF38 without losing activity against the CRF1 receptor, the protons at the site of metabolism were replaced with deuterium. For in vitro and in vivo studies, intrinsic primary isotope effects (KH/KD) were determined by the ratio of intrinsic clearance (CLint) obtained for NVS-CRF38 and deuterated NVS-CRF38. In vitro kinetic isotope effects (KH/KD) were more pronounced when CLint values were calculated based on the rate of formation of the O-desmethyl metabolite (KH/KD ∼7) compared with the substrate depletion method (KH/KD ∼2). In vivo isotope effects were measured in rats after intravenous (1 mg/kg) and oral (10 mg/kg) administration. For both administration routes, isotope effects calculated from in vivo CLint corresponding to all biotransformation pathways were lower (KH/KD ∼2) compared with CLint values calculated from the O-demethylation reaction alone (KH/KD ∼7). Comparative metabolite identification studies were undertaken using rat and human microsomes to explore the potential for metabolic switching. As expected, a marked reduction of the O-demethylated metabolite was observed for NVS-CRF38; however, levels of NVS-CRF38's other metabolites increased, compensating to some extent for the isotope effect.

Deuterium isotope effects were evaluated as a strategy to optimize the pharmacokinetics of NVS-CRF38, a novel CRF1 receptor antagonist. In an attempt to supress O-demethylation of NVS-CRF38 without losing activity against the CRF1 receptor, the protons at the site of metabolism were replaced with deuterium. For in vitro and in vivo studies intrinsic primary isotope effects (KH/KD) were determined by the ratio of CLint obtained from the nondeuterated and deuterated substrate. In vitro isotope effects were more pronounced when CLint values were calculated based on the rate of formation of the O-desmethyl metabolite (KH/KD ~7), compared to the substrate depletion method (KH/KD ~2). In vivo isotope effects were measured in the rat after intravenous (1 mg/kg) and oral (10 mg/kg) administration. For both administration routes isotope effects calculated from in vivo CLint corresponding to all biotransformation pathways were lower (KH/KD ~2) compared to CLint values calculated from the O-demethylation reaction alone (KH/KD ~7). Comparative metabolite identification studies were undertaken using rat and human microsomes to explore the potential for metabolic switching. As expected a marked reduction of the O-demethylated metabolite was observed for the nondeuterated substrate, however levels of NVS-CRF38’s other metabolites increased, compensating to some extent for the isotope effect.

1. The pharmacokinetic properties and metabolism of NVS-CRF38 [7-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-3-(4-methoxy-2-methylphenyl)-2,6-dimethylpyrazolo[5,1-b]oxazole], a novel corticotropin-releasing factor receptor 1 (CRF1) antagonist, were determined in vitro and in animals. 2.  NVS-CRF38 undergoes near complete absorption in rats and dogs. In both species the compound has low hepatic extraction and is extensively distributed to tissues. 3. In rat and human hepatic microsomes and cryopreserved hepatocytes from rat, dog, monkey and human, NVS-CRF38 was metabolised to form O-desmethyl NVS-CRF38 (M7) and several oxygen adducts (M1, M3, M4, M5 and M6). In hepatocytes further metabolites were observed, specifically the carboxylic acid (M2) and conjugates (sulphate and glucuronide) of M7. 4.  Formation of primary metabolites in hepatocytes was blocked by the cytochrome P450 enzyme (P450) suicide inhibitor 1-aminobenzotriazole, implicating P450 enzymes in the primary metabolism of this compound. 5. NVS-CRF38 is weakly bound to plasma proteins from rat (fub = 0.19), dog (fub = 0.25), monkey (fub = 0.20) and humans (fub = 0.23). Blood-to-plasma partition for NVS-CRF38 approaches unity in rat and human blood. 6.  The hepatic clearance of NVS-CRF38 in humans is predicted to be low (extraction ratio ∼ 0.2) based on scaling from drug depletion profiles in hepatic microsomes.

Optimization of a 7-azaindole-3-acetic acid CRTh2 receptor antagonist chemotype derived from high throughput screening furnished a highly selective compound NVP-QAV680 with low nM functional potency for inhibition of CRTh2 driven human eosinophil and Th2 lymphocyte activation in vitro. The molecule exhibited good oral bioavailability in the rat, combined with efficacy in rodent CRTh2-dependent mechanistic and allergic disease models and was suitable for clinical development.

Background and purpose: α5IA (3‐(5‐methylisoxazol‐3‐yl)‐6‐[(1‐methyl‐1,2,3‐triazol‐4‐yl)methyloxy]‐1,2,4‐triazolo[3,4‐ a ]phthalazine) is a triazolophthalazine with subnanomolar affinity for α1‐, α2‐, α3‐ and α5‐containing GABA A receptors. Here we have evaluated the relationship between plasma α5IA concentrations and benzodiazepine binding site occupancy in rodents and primates (rhesus monkey). Experimental approach: In awake rats, occupancy was measured at various times after oral dosing with α5IA (0.03–30 mg·kg −1 ) using an in vivo {[ 3 H]flumazenil (8‐fluoro 5,6‐dihydro‐5‐methyl‐6‐oxo‐4 H ‐imidazo[1,5‐ a ][1,4]benzodiazepine‐3‐carboxylic acid ethyl ester)} binding assay. In anaesthetized rhesus monkeys, occupancy was measured using {[ 123 I]iomazenil (ethyl 5,6‐dihydro‐7‐iodo‐5‐methyl‐6‐oxo‐4 H ‐imidazo[1,5‐ a ][1,4]benzodiazepine‐3‐carboxylic acid ethyl ester)} γ‐scintigraphy and a bolus/infusion paradigm. In both rat and rhesus monkey, the plasma drug concentration corresponding to 50% occupancy (EC 50 ) was calculated. Key results: In rats, α5IA occupancy was dose‐ and time‐dependent with maximum occupancy occurring within the first 2 h. However, rat plasma EC 50 was time‐independent, ranging from 42 to 67 ng·mL −1 over a 24 h time course with the average being 52 ng·mL −1 (i.e. occupancy decreased as plasma drug concentrations fell). In rhesus monkeys, the EC 50 for α5IA displacing steady‐state [ 123 I]iomazenil binding was 57 ng·mL −1 . Conclusions and implications: Rat plasma EC 50 values did not vary as a function of time indicating that α5IA dissociates readily for the GABA A receptor in vivo . These data also suggest that despite the different assays used (terminal assays of [ 3 H]flumazenil in vivo binding in rats and [ 123 I]iomazenil γ‐scintigraphy in anaesthetized rhesus monkeys), these techniques produced similar plasma α5IA EC 50 values (52 and 57 ng·mL −1 respectively) and that the plasma–occupancy relationship for α5IA translates across these two species.

Glucuronidation via UDP-glucuronosyltransferase (UGT) is an increasingly important clearance pathway. In this study intrinsic clearance (CL(int)) values for buprenorphine, carvedilol, codeine, diclofenac, gemfibrozil, ketoprofen, midazolam, naloxone, raloxifene, and zidovudine were determined in pooled human liver microsomes using the substrate depletion approach. The in vitro clearance data indicated a varying contribution of glucuronidation to the clearance of the compounds studied, ranging from 6 to 79% for midazolam and gemfibrozil, respectively. The CL(int) was obtained using either individual or combined cofactors for cytochrome P450 (P450) and UGT enzymes with alamethicin activation and in the presence and absence of 2% bovine serum albumin (BSA). In the presence of combined P450 and UGT cofactors, CL(int) ranged from 2.8 to 688 microl/min/mg for zidovudine and buprenorphine, respectively; the clearance was approximately equal to the sum of the CL(int) values obtained in the presence of individual cofactors. The unbound intrinsic clearance (CL(int, u)) was scaled to provide an in vivo predicted CL(int); the data obtained in the presence of combined cofactors resulted in 5-fold underprediction on average. Addition of 2% BSA to the incubation with both P450 and UGT cofactors reduced the bias in the clearance prediction, with 8 of 10 compounds predicted within 2-fold of in vivo values with the exception of raloxifene and gemfibrozil. The current study indicates the applicability of combined cofactor conditions in the assessment of clearance for compounds with a differential contribution of P450 and UGT enzymes to their elimination. In addition, improved predictability of microsomal data is observed in the presence of BSA, in particular for UGT2B7 substrates.

The GABAA receptor α2/α3 subtype-selective compound 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023; also known as MK-0777) is a triazolopyridazine that has similar, subnanomolar affinity for the benzodiazepine binding site of α1-, α2-, α3-, and α5-containing GABAA receptors and has partial agonist efficacy at the α2 and α3 but not the α1 or α5 subtypes. The purpose of the present study was to define the relationship between plasma TPA023 concentrations and benzodiazepine binding site occupancy across species measured using various methods. Thus, occupancy was measured using either in vivo [3H]flumazenil binding or [11C]flumazenil small-animal positron emission tomography (microPET) in rats, [123I]iomazenil γ-scintigraphy in rhesus monkeys, and [11C]flumazenil PET in baboons and humans. For each study, plasma-occupancy curves were derived, and the plasma concentration of TPA023 required to produce 50% occupancy (EC50) was calculated. The EC50 values for rats, rhesus monkeys, and baboons were all similar and ranged from 19 to 30 ng/ml, although in humans, the EC50 was slightly lower at 9 ng/ml. In humans, a single 2-mg dose of TPA023 produced in the region of 50 to 60% occupancy in the absence of overt sedative-like effects. Considering that nonselective full agonists are associated with sedation at occupancies of less than 30%, these data emphasize the relatively nonsedating nature of TPA023.

Tiagabine is an anticonvulsant drug which may also have sleep-enhancing properties. It acts by inhibiting reuptake at the gamma-aminobutyric acid (GABA) transporter (GAT-1).