Tropifexor plus cenicriviroc combination versus monotherapy in nonalcoholic steatohepatitis: Results from the phase 2b TANDEM study.

BACKGROUND AND AIMS: With distinct mechanisms of action, the combination of tropifexor (TXR) and cenicriviroc (CVC) may provide an effective treatment for NASH. This randomized, multicenter, double-blind, phase 2b study assessed the safety and efficacy of TXR and CVC combination, compared with respective monotherapies. APPROACH AND RESULTS: Patients (N = 193) were randomized 1:1:1:1 to once-daily TXR 140 µg (TXR 140 ), CVC 150 mg (CVC), TXR 140 µg + CVC 150 mg (TXR 140 + CVC), or TXR 90 µg + CVC 150 mg (TXR 90 + CVC) for 48 weeks. The primary and secondary end points were safety and histological improvement, respectively. Rates of adverse events (AEs) were similar across treatment groups. Pruritus was the most frequently experienced AE, with highest incidence in the TXR 140 group (40.0%). In TXR and combination groups, alanine aminotransferase (ALT) decreased from baseline to 48 weeks (geometric mean change: -21%, TXR 140 ; -16%, TXR 140 + CVC; -13%, TXR 90 + CVC; and +17%, CVC). Reductions in body weight observed at week 24 (mean changes from baseline: TXR 140 , -2.5 kg; TXR 140 + CVC, -1.7 kg; TXR 90 + CVC, -1.0 kg; and CVC, -0.1 kg) were sustained to week 48. At least 1-point improvement in fibrosis stage/steatohepatitis resolution without worsening of fibrosis was observed in 32.3%/25.8%, 31.6%/15.8%, 29.7%/13.5%, and 32.5%/22.5% of patients in the TXR 140 , CVC, TXR 140 + CVC, and TXR 90 + CVC groups, respectively. CONCLUSIONS: The safety profile of TXR + CVC combination was similar to respective monotherapies, with no new signals. TXR monotherapy showed sustained ALT and body weight decreases. No substantial incremental efficacy was observed with TXR + CVC combination on ALT, body weight, or in histological end points compared with monotherapy.

Utility of food pellets containing 1-aminobenzotriazole for longer term in vivo inhibition of cytochrome P450 in mice.

1. The utility of 1-aminobenzotriazole (ABT), incorporated in food, has been investigated as an approach for longer term inhibition of cytochrome P450 (P450) enzymes in mice. 2. In rats, ABT inhibits gastric emptying, to investigate this potential limitation in mice we examined the effect of ABT administration on the oral absorption of NVS-CRF38. Two hour prior oral treatment with 100 mg/kg ABT inhibited the oral absorption of NVS-CRF38, Tmax was 4 hours for ABT-treated mice compared to 0.5 hours in the control group. 3. A marked inhibition of hepatic P450 activity was observed in mice fed with ABT containing food pellets for 1 month. P450 activity, as measured by the oral clearance of antipyrine, was inhibited on day 3 (88% of control), week 2 (83% of control) and week 4 (80% of control). 4. Tmax values for antipyrine were comparable between ABT-treated mice and the control group, alleviating concerns about impaired gastric function. 5. Inclusion of ABT in food provides a minimally invasive and convenient approach to achieve longer term inhibition of P450 activity in mice. This model has the potential to enable pharmacological proof-of-concept studies for research compounds which are extensively metabolised by P450 enzymes.

Application of Osmotic Pumps for Sustained Release of 1-Aminobenzotriazole and Inhibition of Cytochrome P450 Enzymes in Mice: Model Comparison with the Hepatic P450 Reductase Null Mouse.

The effectiveness of controlled release 1-aminobenzotriazole (ABT) administration to inhibit cytochrome P450 (P450) enzymes has been evaluated in mice. To maximize the duration of P450 inhibition in vivo, ABT was administered via an osmotic pump. The degree of P450 inhibition was compared with that achieved with a single bolus dose of ABT. Two-hour prior subcutaneous treatment of mice with ABT (50 mg/kg) inhibited antipyrine clearance by 88%. A less pronounced inhibitory effect (29% reduction in clearance) was observed when ABT was administered 24-hours before antipyrine administration, indicating partial restoration of P450 activity during this longer pretreatment time. The duration of ABT in mice was very short (mean residence time = 1.7 hours) after subcutaneous bolus administration. When the inhibitor was delivered by an osmotic pump, maximum blood concentrations of the inhibitor were observed 24 hours after device implantation and were maintained at steady state for 6 days. Inhibition of P450 activity, as measured by antipyrine clearance, was confirmed at 24 hours and 120 hours after pump implantation, highlighting the utility of this method as a longer-term model for P450 inhibition in mice. The magnitude of P450 inhibition in ABT-treated mice was compared with that in hepatic P450 reductase null mice and both models were comparable. In vivo ABT administration by an osmotic pump offers an effective approach for longer-term P450 inhibition in mice and avoids the necessity for multiple dosing of the inhibitor.

Application of a deuterium replacement strategy to modulate 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, a novel CRF1 antagonist.

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.

1-Aminobenzotriazole modulates oral drug pharmacokinetics through cytochrome P450 inhibition and delay of gastric emptying in rats.

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.

Preclinical metabolism and pharmacokinetics of NVS-CRF38, a potent and orally bioavailable corticotropin-releasing factor receptor 1 antagonist.

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.

Transient targeting of phosphoinositide 3-kinase acts as a roadblock in mast cells' route to allergy.

BACKGROUND: Tissue mast cell numbers are dynamically regulated by recruitment of progenitors from the vasculature. It is unclear whether progenitors are recruited during allergic sensitization and whether recruitment promotes allergic responses. OBJECTIVE: We sought to (1) determine the effect of mast cell recruitment on acute allergic responses and (2) to define the role of phosphoinositide 3-kinase (PI3K) isoforms in sequential steps to allergic responses. METHODS: Gene-targeted mice for PI3Kγ or PI3Kδ or mice treated with isoform-specific PI3K inhibitors (a novel PI3Kγ-specific inhibitor [NVS-PI3-4] and the PI3Kδ inhibitor IC87114) were used to monitor IgE-mediated mast cell recruitment, migration, adhesion by means of intravital microscopy, degranulation, TNF-α release, and subsequent endothelial cell activation in vivo or in bone marrow-derived mast cells. RESULTS: Functional PI3Kγ, but not PI3Kδ, was crucial for mast cell accumulation in IgE-challenged skin, TNF-α release from IgE/antigen-stimulated mast cells, and mast cell/endothelial interactions and chemotaxis. PI3Kγ-deficient bone marrow-derived mast cells did not adhere to the endothelium in TNF-α-treated cremaster muscle, whereas PI3Kδ was not required. Depletion of TNF-α blocked IgE-induced mast cell recruitment, which links tissue mast cell-derived cytokine release to endothelial activation and mast cell recruitment. Interference with mast cell recruitment protected against anaphylaxis and was superior to blockage of tissue mast cell degranulation. CONCLUSIONS: Interference with mast cell recruitment to exacerbated tissues provides a novel strategy to alleviate allergic reactions and surpassed attenuation of tissue mast cell degranulation. This results in prolonged drug action and allows for reduction of drug doses required to block anaphylaxis, an important feature for drugs targeting inflammatory disease in general.

Discovery and characterization of NVP-QAV680, a potent and selective CRTh2 receptor antagonist suitable for clinical testing in allergic diseases.

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.

A Study to Evaluate Relative Bioavailability, Food Effect, and Pharmacodynamics of Tropifexor, a Farnesoid X Receptor Agonist, in Healthy Participants.

This open-label, randomized, 3-treatment, 3-period, 6-sequence, crossover study in healthy subjects compared the pharmacokinetic and pharmacodynamic properties of a lipid-based (soft gelatin capsule) prototype final market image (pFMI) formulation of tropifexor (90-µg) to its clinical service form (CSF) and assessed the food effect for the pFMI formulation. In the fasted state, drug exposure was higher for the pFMI. The geometric mean ratios for pFMI versus CSF of peak concentration and area under the concentration-time curve were 2.0 and 1.5, respectively. No food effect was apparent for the pFMI formulation, and the geometric mean ratios for pFMI fed versus pFMI fasted of peak concentration and area under concentration-time curve were 1.0 and 1.0 respectively. Despite having lower systemic exposure, the CSF formulation provided a higher pharmacological response for the gut biomarker fibroblast growth factor 19. Under fasted conditions, fibroblast growth factor 19 maximum change from baseline serum concentration after drug administration and area under the change from baseline serum concentration-time curve from time 0 to 24 hours were 36% for CSF and 12% for FMI. For a second biomarker, serum 7-alpha hydroxy-4-cholest-3-one, the pharmacological activity was comparable between CSF (fasted) and pFMI (both fasted and fed states). The pFMI offers advantages over the CSF in terms of insensitivity to food effect, lower intersubject variability, and overcoming solubility limitations.

A novel murine model of severe pulmonary arterial hypertension.

RATIONALE: The complex pathologies associated with severe pulmonary arterial hypertension (PAH) in humans have been a challenge to reproduce in mice due to the subtle phenotype displayed to PAH stimuli. OBJECTIVES: Here we aim to develop a novel murine model of PAH that recapitulates more of the pathologic processes, such as complex vascular remodeling and cardiac indices, that are not characteristic of alternative mouse models. METHODS: Inhibition of vascular endothelial growth factor receptor (VEGFR) with SU5416 combined with 3 weeks of chronic hypoxia was investigated. Hemodynamics, cardiac function, histological assessment of pulmonary vasculature, and molecular pathway analysis gauged the extent of PAH pathology development. MEASUREMENTS AND MAIN RESULTS: The combination of VEGFR inhibition with chronic hypoxia profoundly exacerbated all measures of PAH-like pathology when compared with hypoxia alone (> 45 mm Hg right ventricular pressure, > 0.35 right ventricular hypertrophy). The changes in pulmonary vascular remodeling in response to hypoxia were further enhanced on SU5416 treatment. Furthermore, hypoxia/SU5416 treatment steadily decreased cardiac output, indicating incipient heart failure. Molecular analysis showed a dysregulated transforming growth factor-ß/bone morphogenetic protein/Smad axis in SU5416- and/or hypoxia-treated mice as well as augmented induction of IL-6 and Hif-1α levels. These changes were observed in accordance with up-regulation of Tph1 and Pdgfr gene transcripts as well as a rise in platelet-rich serotonin. Biomarker analysis in response to VEGFR inhibition and/or hypoxia revealed distinct signatures that correlate with cytokine profiles of patients with idiopathic PAH. CONCLUSIONS: These data describe a novel murine model of PAH, which displays many of the hallmarks of the human disease, thus opening new avenues of investigation to better understand PAH pathophysiology.

Drug-Drug Interaction Studies to Evaluate the Effect of Inhibition of UGT1A1 and CYP3A4 and Induction of CYP3A4 on the Pharmacokinetics of Tropifexor in Healthy Subjects.

Tropifexor, a farnesoid X receptor agonist, is currently under clinical development for the treatment of nonalcoholic steatohepatitis. Tropifexor undergoes glucuronidation by uridine 5'-diphosphoglucuronosyltransferase (UGT) 1A1 and oxidation by cytochrome P450 (CYP) 3A4, as reported in in vitro studies. Here, we report the results from 2 drug-drug interaction studies. Study 1 enrolled 20 healthy subjects to investigate the effect of the UGT1A1 inhibitor atazanavir (ATZ) on tropifexor pharmacokinetics (PK). Study 2 had 2 cohorts with 16 healthy subjects each to investigate the effect of the strong CYP3A4 inhibitor itraconazole and strong CYP3A4 inducer rifampin on the PK of tropifexor. Coadministration of ATZ reduced the maximum plasma concentration (Cmax ) of tropifexor by 40%; however, it did not lead to increased exposure of tropifexor (both area under the plasma concentration-time curve [AUC] from time 0 to the last quantifiable concentration [AUClast ] and AUC from time 0 to infinity [AUCinf ] reduced by only 10%), suggesting minor relevance of the UGT1A1 pathway for clearance of tropifexor and no expected drug-drug interactions based on UGT1A1 inhibition. Inhibition of CYP3A4 by itraconazole increased the Cmax of tropifexor by only 9% and exposure (both AUClast and AUCinf ) by 47%, suggesting a weak effect of strong CYP3A4 inhibitors on tropifexor PK. Inducing CYP3A4 with rifampin decreased Cmax (55%) and AUC (AUClast by 79% and AUCinf by 77%). Coadministration of tropifexor with either ATZ, itraconazole, or rifampin was well tolerated.

Pharmacokinetics of Tropifexor, a Potent Farnesoid X Receptor Agonist, in Participants With Varying Degrees of Hepatic Impairment.

Tropifexor, a non-bile acid farnesoid X receptor (FXR) agonist, has dose-proportional pharmacokinetics and no obvious major enterohepatic circulation. This open-label study investigated the effect of hepatic impairment (HI), as determined by Child-Pugh grade, on tropifexor's pharmacokinetics, safety, and tolerability following a 200-µg dose in the fasted state. Blood samples were collected through 168 hours after dosing for quantification and plasma protein-binding determination. Total tropifexor exposure was comparable across participants with HI vs those with normal hepatic function. Tropifexor was highly protein bound (>99%) in human plasma across participants of all groups. The average unbound fractions (percentage free) were 0.14% in participants with normal hepatic function and mild HI, which increased to 0.17% and 0.24% in participants with moderate and severe HI, respectively. Similar unbound drug exposure was noted in participants with mild HI and normal hepatic function. Participants with moderate HI (N = 8) had a 1.6-fold increase in unbound exposure (area under the plasma concentration-time curve from time 0 to infinity [AUCinf,u ]) and a 1.3-fold increase in maximal exposure (Cmax,u ) vs those with normal hepatic function (geometric mean ratio: AUCinf,u , 1.64 [90%CI, 1.25-2.16]; Cmax,u , 1.30 [90%CI, 0.96-1.76]). Participants with severe HI (N = 8) had a 1.6-fold increase in AUCinf,u (1.61 [90%CI, 1.04-2.49]) and comparable Cmax,u (1.02 [90%CI, 0.60-1.72]) compared to participants with normal hepatic function. Tropifexor was well tolerated. The relative insensitivity of tropifexor to HI offers the potential to treat patients with severe liver disease without dose adjustment.

Discovery, Preclinical Characterization, and Early Clinical Activity of JDQ443, a Structurally Novel, Potent, and Selective Covalent Oral Inhibitor of KRASG12C.

Covalent inhibitors of KRASG12C have shown antitumor activity against advanced/metastatic KRASG12C-mutated cancers, though resistance emerges and additional strategies are needed to improve outcomes. JDQ443 is a structurally unique covalent inhibitor of GDP-bound KRASG12C that forms novel interactions with the switch II pocket. JDQ443 potently inhibits KRASG12C-driven cellular signaling and demonstrates selective antiproliferative activity in KRASG12C-mutated cell lines, including those with G12C/H95 double mutations. In vivo, JDQ443 induces AUC exposure-driven antitumor efficacy in KRASG12C-mutated cell-derived (CDX) and patient-derived (PDX) tumor xenografts. In PDX models, single-agent JDQ443 activity is enhanced by combination with inhibitors of SHP2, MEK, or CDK4/6. Notably, the benefit of JDQ443 plus the SHP2 inhibitor TNO155 is maintained at reduced doses of either agent in CDX models, consistent with mechanistic synergy. JDQ443 is in clinical development as monotherapy and in combination with TNO155, with both strategies showing antitumor activity in patients with KRASG12C-mutated tumors. SIGNIFICANCE: JDQ443 is a structurally novel covalent KRASG12C inhibitor with a unique binding mode that demonstrates potent and selective antitumor activity in cell lines and in vivo models. In preclinical models and patients with KRASG12C-mutated malignancies, JDQ443 shows potent antitumor activity as monotherapy and in combination with the SHP2 inhibitor TNO155. This article is highlighted in the In This Issue feature, p. 1397.

JDQ443, a Structurally Novel, Pyrazole-Based, Covalent Inhibitor of KRASG12C for the Treatment of Solid Tumors.

Rapid emergence of tumor resistance via RAS pathway reactivation has been reported from clinical studies of covalent KRASG12C inhibitors. Thus, inhibitors with broad potential for combination treatment and distinct binding modes to overcome resistance mutations may prove beneficial. JDQ443 is an investigational covalent KRASG12C inhibitor derived from structure-based drug design followed by extensive optimization of two dissimilar prototypes. JDQ443 is a stable atropisomer containing a unique 5-methylpyrazole core and a spiro-azetidine linker designed to position the electrophilic acrylamide for optimal engagement with KRASG12C C12. A substituted indazole at pyrazole position 3 results in novel interactions with the binding pocket that do not involve residue H95. JDQ443 showed PK/PD activity in vivo and dose-dependent antitumor activity in mouse xenograft models. JDQ443 is now in clinical development, with encouraging early phase data reported from an ongoing Phase Ib/II clinical trial (NCT04699188).

A physical properties based approach for the exploration of a 4-hydroxybenzothiazolone series of beta2-adrenoceptor agonists as inhaled long-acting bronchodilators.

The chiral synthesis of a 4-hydroxybenzothiazolone based series of beta(2)-adrenoceptor agonists is described. Using this methodology a library of N-substituted analogues were prepared for the rapid identification of leads with the potential to be fast onset and long-acting inhaled bronchodilators with improved therapeutic margins. The design of the library to achieve the targeted profile was based upon lipophilicity and metabolism based hypotheses. This approach identified beta-phenethyl, alpha-substituted cyclopentyl and monoterpene N-substituents to be of particular interest for further evaluation, as exemplified by structures 19, 29 and 33, respectively.

Discovery and Optimization of Novel SUCNR1 Inhibitors: Design of Zwitterionic Derivatives with a Salt Bridge for the Improvement of Oral Exposure.

G-protein-coupled receptor SUCNR1 (succinate receptor 1 or GPR91) senses the citric cycle intermediate succinate and is implicated in various pathological conditions such as rheumatoid arthritis, liver fibrosis, or obesity. Here, we describe a novel SUCNR1 antagonist scaffold discovered by high-throughput screening. The poor permeation and absorption properties of the most potent compounds, which were zwitterionic in nature, could be improved by the formation of an internal salt bridge, which helped in shielding the two opposite charges and thus also the high polarity of zwitterions with separated charges. The designed compounds containing such a salt bridge reached high oral bioavailability and oral exposure. We believe that this principle could find a broad interest in the medicinal chemistry field as it can be useful not only for the modulation of properties in zwitterionic compounds but also in acidic or basic compounds with poor permeation.

Prediction of drug clearance by glucuronidation from in vitro data: use of combined cytochrome P450 and UDP-glucuronosyltransferase cofactors in alamethicin-activated human liver microsomes.

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.

Evaluation of recombinant cytochrome P450 enzymes as an in vitro system for metabolic clearance predictions.

The aim of this study was to explore the potential of recombinant cytochrome P450 (P450) enzymes for human metabolic clearance prediction. The relative abundance and relative activity approaches were compared as methods to bridge the gap between catalytic activities in recombinant P450 enzymes and human liver microsomes (HLMs). Relative activity factors were measured by determining the intrinsic clearance (CL(int)) of probe substrates (bufuralol-CYP2D6, diclofenac-CYP2C9, midazolam-CYP3A4, and phenacetin-CYP1A2) in recombinant P450s and 16 HLM donors. Simultaneous determination of drug depletion and metabolite formation profiles has enabled a direct comparison of these methods for CL(int) determination. Of the 110 drugs tested, 66% were metabolized by one or more P450 enzymes; of these 44% of were metabolized by CYP3A4 (0.3-21 microl/min/pmol of P450), 41% by CYP2D6 (0.6-60 microl/min/pmol of P450), 26% by CYP2C19 (0.4-8.1 microl/min/pmol of P450), 9% by CYP1A2 (0.4-2.5 microl/min/pmol of P450), and 4% by CYP2C9 (0.9-6.4 microl/min/pmol of P450). Recombinant enzymes demonstrated improved prediction reliability relative to HLMs and hepatocytes. The most reliable correlations in terms of lowest bias and highest precision were observed by comparing in vivo CL(int), calculated using the parallel-tube model and incorporating fraction unbound in blood, with in vitro CL(int) determined using relative activity factors and adjusted for nonspecific binding. Predictions were less reliable using the relative abundance approach. For these drugs, recombinant P450 enzymes offer improved assay sensitivity compared with HLMs and cryopreserved hepatocytes for CL(int) determination using the drug depletion method.

7-Azaindole-3-acetic acid derivatives: potent and selective CRTh2 receptor antagonists.

High throughput screening identified a 7-azaindole-3-acetic acid scaffold as a novel CRTh2 receptor antagonist chemotype, which could be optimised to furnish a highly selective compound with good functional potency for inhibition of human eosinophil shape change in whole blood and oral bioavailability in the rat.

Structure-Based and Property-Driven Optimization of N-Aryl Imidazoles toward Potent and Selective Oral RORγt Inhibitors.

Retinoic acid receptor-related orphan receptor gamma-t (RORγt) is considered to be the master transcription factor for the development of Th17 cells that produce proinflammatory cytokines such as IL-17A. Overproportionate Th17 cell abundance is associated with the pathogenesis of many inflammatory conditions including psoriasis. In a high-throughput fluorescence resonance energy transfer (FRET) screen, we identified compound 1 as a hit with promising lipophilic efficiency (LipE). Using structure-based drug design based on a number of X-ray cocrystal structures, we morphed this hit class into potent imidazoles, exemplified by compound 3. To improve the poor absorption, distribution, metabolism, and excretion (ADME) properties of neutral imidazoles, we extended our ligands with carboxylic acid substituents toward a polar, water-rich area of the protein. This highly lipophilicity-efficient modification ultimately led to the discovery of compound 14, a potent and selective inhibitor of RORγt with good ADME properties and excellent in vivo pharmacokinetics. This compound showed good efficacy in an in vivo delayed-type hypersensitivity pharmacology model in rats.