Etavopivat, a Pyruvate Kinase Activator in Red Blood Cells, for the Treatment of Sickle Cell Disease.

Etavopivat is an investigational, oral, small molecule activator of erythrocyte pyruvate kinase (PKR) in development for the treatment of sickle cell disease (SCD) and other hemoglobinopathies. PKR activation is proposed to ameliorate the sickling of SCD red blood cells (RBCs) through multiple mechanisms, including reduction of 2,3-diphosphoglycerate (2,3-DPG), which consequently increases hemoglobin (Hb)-oxygen affinity; increased binding of oxygen reduces sickle hemoglobin polymerization and sickling. In addition, PKR activation increases adenosine triphosphate (ATP) produced via glycolytic flux, which helps preserve membrane integrity and RBC deformability. We evaluated the pharmacodynamic response to etavopivat in nonhuman primates (NHPs) and in healthy human subjects and evaluated the effects in RBCs from patients with SCD after ex vivo treatment with etavopivat. A single dose of etavopivat decreased 2,3-DPG in NHPs and healthy subjects. Hb-oxygen affinity was significantly increased in healthy subjects after 24 hours. After daily dosing of etavopivat over 5 consecutive days in NHPs, ATP was increased by 38% from baseline. Etavopivat increased Hb-oxygen affinity and reduced sickling in RBCs collected from patients with SCD with either homozygous hemoglobin S or hemoglobin S and C disease. Collectively, these results demonstrate the ability of etavopivat to decrease 2,3-DPG and increase ATP, resulting in increased Hb-oxygen affinity and improved sickle RBC function. Etavopivat is currently being evaluated in clinical trials for the treatment of SCD. SIGNIFICANCE STATEMENT: Etavopivat, a small molecule activator of the glycolytic enzyme erythrocyte pyruvate kinase, decreased 2,3-diphosphoglycerate in red blood cells (RBCs) from nonhuman primates and healthy subjects and significantly increased hemoglobin (Hb)-oxygen affinity in healthy subjects. Using ex vivo RBCs from donors with sickle cell disease (SCD) (homozygous hemoglobin S or hemoglobin S and C genotype), etavopivat increased Hb-oxygen affinity and reduced sickling under deoxygenation. Etavopivat shows promise as a treatment for SCD that could potentially reduce vaso-occlusion and improve anemia.

A membrane vesicle-based assay to enable prediction of human biliary excretion.

1. Prediction of biliary excretion is a challenge due to the lack of in vitro assays. Our laboratory previously demonstrated a highly significant correlation between in vitro IC50 values against mrp2 using rat canalicular liver plasma membrane vesicles and in vivo biliary excretion (Colombo et al., 2012). This study explores the possibility of predicting in vivo biliary excretion in human using membrane vesicles prepared from MDCKII cells transfected with human ABCC2. 2. In vitro MRP2 activity was determined by measuring the ATP-dependent uptake of 5(6)-carboxy-2',7'-dichlorofluorescein (CDCF) in inside-out membrane vesicles isolated from MDCK-ABCC2 cells. CDCF uptake was time- and concentration-dependent (Km of 4.0 ± 1.2 µM and a Vmax of 7.8 ± 0.9 pmol/mg/min) and inhibited by benzbromarone and MK-571 with IC50 values of 1.2 and 7.6 µM, respectively. 3. A significant linear correlation (r(2 )= 0.790) between the in vitro IC50 values from the described MRP2 assay and in vivo biliary excretion in humans was observed using 11 well-documented drugs covering low to high biliary excretions. 4. This study demonstrates, for the first time, that inhibition of CDCF uptake in MDCKII-ABCC2 vesicles not only provides a screening assay to assess MRP2 drug-drug interaction potential, but is also predictive of human MRP2-mediated biliary excretion.

A simplified approach to predict CYP3A-mediated drug-drug interactions at early drug discovery: validation with clinical data.

1. The present study evaluates which factors should be incorporated into a simplified approach to reasonably predict CYP3A-mediated drug-drug interaction (DDI) at an early drug discovery stage. 2. CYP3A IC50 values were obtained using human liver microsomes (HLM) and hepatocytes. Plasma and microsomal protein binding and in vitro hepatocyte partition coefficient (Kp) were also determined for 10 drugs. Therapeutic human maximum plasma concentrations (Cmax) were retrieved from the literature. DDI predictions were performed using an equation incorporating the fraction of the substrate metabolized by CYP3A with the total or free plasma Cmax, with or without correction for hepatocyte Kp. 3. Based on the Ki data from HLM, the use of total Cmax provided a prediction of DDI within 2-fold of the observed clinical values for 9 out of 10 drugs. 4. In comparison, free drug corrections for both Cmax and Ki values from HLM led to an underprediction of DDI (>3-fold error for five drugs). 5. Data from hepatocytes showed, in general, lower prediction accuracy than data from HLM. 6. CYP3A-mediated DDIs can be predicted with a high level of accuracy based on Ki estimates from HLM data and the total therapeutic plasma Cmax of the inhibitors. This approach should be widely applicable to the assessment of clinically significant DDIs risk in early drug discovery programs.

Assessment of CYP3A-mediated drug-drug interaction potential for victim drugs using an in vivo rat model.

The present study aims to determine if an in vivo rat model of drug-drug interaction (DDI) could be useful to discriminate a sensitive (buspirone) from a 'non-sensitive' (verapamil) CYP3A substrate, using ketoconazole and ritonavir as perpetrator drugs. Prior to in vivo studies, ketoconazole and ritonavir were shown to inhibit midazolam hydroxylation with IC50 values of 350 ± 60 nm and 11 ± 3 nm, respectively, in rat liver microsomes (RLM). Buspirone and verapamil were also shown to be substrates of recombinant rat CYP3A1/3A2. In the rat model, the mean plasma AUC0-inf of buspirone (10 mg/kg, p.o.) was increased by 7.4-fold and 12.8-fold after co-administration with ketoconazole and ritonavir (20 mg/kg, p.o.), respectively. The mean plasma AUC0-inf of verapamil (10 mg/kg, p.o.) was increased by 3.0-fold and 4.8-fold after co-administration with ketoconazole and ritonavir (20 mg/kg, p.o.), respectively. Thus, the rat DDI model correctly identified buspirone as a sensitive CYP3A substrate (>5-fold AUC change) in contrast to verapamil. In addition, for both victim drugs, the extent of DDI when co-administered was greater with ritonavir compared with ketoconazole, in line with their in vitro CYP3A inhibition potency in RLM. In conclusion, our study extended the rat DDI model applicability to two additional victim/perpetrator pairs. In addition, we suggest that use of this model would increase our confidence in estimation of the DDI potential for victim drugs in early discovery.

A clinical study to assess CYP1A2 and CYP3A4 induction by AZD7325, a selective GABA(A) receptor modulator - an in vitro and in vivo comparison.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: • AZD7325 is an orally administered, potent, selective gamma-amino-butyric acid (GABA(A) ) α2,3 receptor modulator intended for the treatment of anxiety. • The induction effects of AZD7325 on CYP1A2 and CYP3A4 have not been systematically studied. WHAT THIS STUDY ADDS: • The in vitro studies showed that AZD7325 was a moderate CYP1A2 inducer and potent CYP3A4 inducer. • The follow-up clinical studies in healthy volunteers demonstrated that the expected efficacious daily dose of AZD7325 only weakly induced the pharmacokinetics of the CYP3A4 sensitive substrate, midazolam, and had no effect on the pharmacokinetics of the CYP1A2 substrate, caffeine. There was no apparent change in AZD7325 exposure following co-administration of midazolam or caffeine compared with AZD7325 alone. • The study demonstrated that clinical exposure of the inducer plays a critical role in the determination of cytochrome P450 induction risk of a drug candidate. AIM(S): To investigate the potential of AZD7325 to induce CYP1A2 and CYP3A4 enzyme activities. METHODS: Induction of CYP1A2 and CYP3A4 by AZD7325 was first evaluated using cultured human hepatocytes. The effect of multiple doses of 10 mg AZD7325 on the pharmacokinetics of midazolam and caffeine was then examined in healthy subjects. RESULTS: The highest CYP1A2 and CYP3A4 induction responses were observed in human hepatocytes treated with 1 or 10 µm of AZD7325, in the range of 17.9%-54.9% and 76.9%-85.7% of the positive control responses, respectively. The results triggered the further clinical evaluation of AZD7325 induction potential. AZD7325 reached a plasma C(max) of 0.2 µm after 10 mg daily dosing to steady-state. AZD7325 decreased midazolam geometric mean AUC by 19% (0.81-fold, 90% CI 0.77, 0.87), but had no effect on midazolam C(max) (90% CI 0.82, 0.97). The mean CL/F of midazolam increased from 62 l h(-1) (midazolam alone) to 76 l h(-1) when co-administered with AZD7325. The AUC and C(max) of caffeine were not changed after co-administration of AZD7325, with geometric mean ratios (90% CI) of 1.17 (1.12, 1.23) and 0.99 (0.95, 1.03), respectively. CONCLUSIONS: While AZD7325 appeared to be a potent CYP3A4 inducer and a moderate CYP1A2 inducer from in vitro studies, the expected efficacious dose of AZD7325 had no effect on CYP1A2 activity and only a weak inducing effect on CYP3A4 activity. This comparison of in vitro and in vivo results demonstrates the critical role that clinical exposure plays in evaluating the CYP induction risk of a drug candidate.

Safety, Pharmacokinetics, and Pharmacodynamics of Etavopivat (FT-4202), an Allosteric Activator of Pyruvate Kinase-R, in Healthy Adults: A Randomized, Placebo-Controlled, Double-Blind, First-in-Human Phase 1 Trial.

Etavopivat (FT-4202) is an orally administered, small-molecule allosteric activator of erythrocyte pyruvate kinase-R (PKR) in clinical development for the treatment of sickle cell disease and other hemoglobin disorders. This randomized, placebo-controlled, double-blind, first-in-human combination single-ascending dose and multiple-ascending dose phase 1 trial (NCT03815695) evaluated the safety and pharmacokinetics/pharmacodynamics of etavopivat in 90 healthy adult subjects. In 4 single-ascending dose cohorts, 8 participants were randomized 3:1 to a single oral dose of either etavopivat (n = 6) or placebo (n = 2). In four 14-day multiple-ascending dose cohorts, 12 participants were randomized 3:1 to 14 days of etavopivat (n = 9) or placebo (n = 3). In these studies, most treatment-emergent adverse events were of mild severity (grade 1) and none led to study discontinuation. Etavopivat exhibited a linear and time-independent pharmacokinetic profile (at doses ≤400 mg) and elicited the expected pharmacodynamic effects of PKR activation (decreased 2,3-diphosphoglycerate and increased adenosine triphosphate) and evidence of improved hemoglobin-oxygen affinity. In addition, pharmacodynamic responses were durable with effects continuing for 48 to 72 hours after the last dose, thereby supporting once-daily dosing. Food appeared to have no clinically meaningful effects on etavopivat exposure, thus facilitating administration with or without food. In conclusion, the evaluation of etavopivat in healthy subjects demonstrated proof of mechanism (PKR activation) without significant adverse events. This study also allowed for the selection of dose levels, projected to have an acceptable safety profile and provide therapeutic benefit, for evaluation in future trials in patients with sickle cell disease.

Development and SAR of functionally selective allosteric modulators of GABAA receptors.

Positive modulators at the benzodiazepine site of α2- and α3-containing GABA(A) receptors are believed to be anxiolytic. Through oocyte voltage clamp studies, we have discovered two series of compounds that are positive modulators at α2-/α3-containing GABA(A) receptors and that show no functional activity at α1-containing GABA(A) receptors. We report studies to improve this functional selectivity and ultimately deliver clinical candidates. The functional SAR of cinnolines and quinolines that are positive allosteric modulators of the α2- and α3-containing GABA(A) receptors, while simultaneously neutral antagonists at α1-containing GABA(A) receptors, is described. Such functionally selective modulators of GABA(A) receptors are expected to be useful in the treatment of anxiety and other psychiatric illnesses.

A strategy to reduce biliary clearance in early drug discovery.

INTRODUCTION: Biliary excretion can modulate the pharmacokinetic profile of drug candidates, and may represent a liability for drug-drug interactions. This study proposes a strategy to reduce biliary clearance using the efflux ratio in Caco-2 cells in parallel to an abbreviated pharmacokinetic study in bile duct-cannulated rats (BDC). METHODS: Apical to basolateral (A to B) and basolateral to apical (B to A) permeability of 20 new chemical entities (NCEs) were determined in a 24-well permeability assay. In parallel, biliary excretion was determined in an abbreviated format in BDC rats. Test compounds were administered via an intravenous dose of 1 mg/kg and the percentage (%) of parent compound excreted in the bile in the first 3 hours after dosing was determined by LC-MS/MS analysis. RESULTS: A reasonably good correlation (r(2)=0.635) between the in vitro efflux ratio from the Caco-2 assay and in vivo biliary excretion of parent compound in BDC rats was observed. All seven compounds with an efflux ratio of <5 had less than 25% of the parent excreted in rat bile. In contrast, 3 out of the 13 compounds with an efflux ratio >5 had less than 25% of the dose excreted in rat bile. DISCUSSION: This suggests that a compound with an efflux ratio of <5 is at lower risk of having significant biliary clearance and that Caco-2 efflux ratio obtained from a high throughput screening assay may be used as an early indicator of biliary excretion. Although, we propose to reduce the occurrence of false positive prediction for biliary clearance (23%) by performing abbreviated PK in BDC rats for compounds with high efflux ratio.

A new 4-(2-methylquinolin-4-ylmethyl)phenyl P1' group for the beta-amino hydroxamic acid derived TACE inhibitors.

A new P1' group for TACE inhibitors was identified by eliminating the oxygen atom in the linker of the original 4-(2-methylquinolin-4-ylmethoxy)phenyl P1' group. Incorporation of this 4-(2-methylquinolin-4-ylmethyl)phenyl group onto different beta-aminohydroxamic acid cores provided compound 18, which demonstrated potent porcine TACE (p-TACE) and human whole blood activity, excellent PK properties, and good selectivity against a variety of MMPs.

Effects of prototypical microsomal enzyme inducers on cytochrome P450 expression in cultured human hepatocytes.

Cultured human hepatocytes are a valuable in vitro system for evaluating new molecular entities as inducers of cytochrome P450 (P450) enzymes. The present study summarizes data obtained from 62 preparations of cultured human hepatocytes that were treated with vehicles (saline or dimethylsulfoxide, 0.1%), beta-naphthoflavone (33 microM), phenobarbital (100 or 250 microM), isoniazid (100 microM) and/or rifampin (20 or 50 microM), and examined for the expression of P450 enzymes based on microsomal activity toward marker substrates, or in the case of CYP2C8, the level of immunoreactive protein. The results show that CYP1A2 activity was markedly induced by beta-naphthoflavone (on average 13-fold, n = 28 preparations), and weakly induced by phenobarbital (1.9-fold, n = 25) and rifampin (2.3-fold, n = 22); CYP2A6 activity tended to be increased with phenobarbital (n = 7) and rifampin (n = 3) treatments, but the effects were not statistically significant; CYP2B6 was induced by phenobarbital (6.5-fold, n = 13) and rifampin (13-fold, n = 14); CYP2C8 was induced by phenobarbital (4.0-fold, n = 4) and rifampin (5.2-fold, n = 4); CYP2C9 was induced by phenobarbital (1.8-fold, n = 14) and rifampin (3.5-fold, n = 10); CYP2C19 was markedly induced by rifampin (37-fold, n = 10), but relatively modestly by phenobarbital (7-fold, n = 9); CYP2D6 was not significantly induced by phenobarbital (n = 5) or rifampin (n = 5); CYP2E1 was induced by phenobarbital (1.7-fold, n = 5), rifampin (2.2-fold, n = 5), and isoniazid (2.3-fold, n = 5); and, CYP3A4 was induced by phenobarbital (3.3-fold, n = 42) and rifampin (10-fold, n = 61), but not by beta-naphthoflavone. Based on these observations, we generalize that beta-naphthoflavone induces CYP1A2 and isoniazid induces CYP2E1, whereas rifampin and, to a lesser extent phenobarbital, tend to significantly and consistently induce enzymes of the CYP2A, CYP2B, CYP2C, CYP2E, and CYP3A subfamilies but not the 2D subfamily.

Rational design, synthesis and structure-activity relationships of a cyclic succinate series of TNF-alpha converting enzyme inhibitors. Part 2: lead optimization.

Modifications of the lead TACE inhibitor 1 (N-hydroxy-trans-2-[[4-(4-quinolinyloxymethyl)anilinyl]carbonyl]-1-cyclohexanecarboxamide) at the cyclohexyl ring and the quinoline moiety led to the identification of a series of piperidine containing TACE inhibitors with potent activity in the inhibition of TNF-alpha release in the whole blood assay (WBA). The most potent analogue IM491 [N-hydroxy-(5S,6S)-1-methyl-6-[[4-(2-methyl-4-quinolinylmethoxy)anilinyl]carbonyl]-5-piperidinecarboxamide] exhibited an IC(50) value of 20 nM in WBA with excellent selectivity over MMP-1, -2 and -9 and is orally bioavailable with an F value of 43% in beagle dogs.

Synthesis and structure-activity relationship of a novel sulfone series of TNF-alpha converting enzyme inhibitors.

Replacement of the amide functionality in IM491 (N-hydroxy-(5S,6S)-1-methyl-6-[[4-(2-methyl-4-quinolinylmethoxy)anilinyl]carbonyl]5-piperidinecarboxamide) with a sulfonyl group led to a new series of alpha,beta-cyclic and beta,beta-cyclic gamma-sulfonyl hydroxamic acids, which were potent TNF-alpha converting enzyme (TACE) inhibitors. Among them, inhibitor 4b (N-hydroxy-(4S,5S)-1-methyl-5-[[4-(2-methyl-4-quinolinylmethoxy)phenyl]sulfonylmethyl]-4-pyrrolidinecarboxamide) exhibited IC50 values of < 1 nM and 180 nM in porcine TACE (pTACE) and cell assays, respectively, with excellent selectivity over MMP-1, -2, -9 and -13 and was orally bioavailable with an F value of 46% in mice.