Pharmacokinetics, Mass Balance, Safety, and Tolerability of Obeldesivir in Healthy Participants.

There is an unmet need for safe and efficacious oral therapies for COVID-19 with low potential for drug-drug interactions. Obeldesivir is an orally administered nucleoside prodrug that has shown antiviral potency in nonclinical studies against SARS-CoV-2 and its circulating variants. Obeldesivir is metabolized to the active nucleoside triphosphate (GS-443902), which acts as an inhibitor of the SARS-CoV-2 RNA-dependent RNA polymerase, thereby inhibiting viral RNA synthesis. Here, we report the safety, tolerability, and pharmacokinetics from a first-in-human, randomized, placebo-controlled, phase I study following oral administration of obeldesivir and a phase I, open-label absorption, distribution, metabolism, and excretion study following oral administration of [14C]-obeldesivir. Overall, obeldesivir was safe and well tolerated at single and multiple doses between 100 and 1,600 mg, with low potential for QT prolongation as assessed by QT-concentration analysis. The exposures to GS-441524 increased dose proportionally in the 100-900-mg dose range. GS-441524 accumulated by 35% after twice-daily and 12% after once-daily dosing for 5 days. Dose-proportional increases in the intracellular concentration of GS-443902 were also observed in peripheral blood mononuclar cells. Plasma exposure of GS-441524 was not significantly altered by food intake. Following oral administration of [14C]-obeldesivir (500 mg; 100 µCi), the mean cumulative [14C]-dose recovery was 90.7% with 58.5% in urine and 32.2% in feces. GS-441524 was the predominant plasma component (90% of 14C-area under the concentration-time curve) and was primarily eliminated via renal excretion. Collectively, data from these studies support selection of the obeldesivir 350 mg twice-daily dosing regimen for further evaluation in phase III studies for COVID-19.

Metabolism, pharmacokinetics and excretion of [14C]dimethyl fumarate in healthy volunteers: an example of xenobiotic biotransformation following endogenous metabolic pathways.

Delayed-release dimethyl fumarate (DMF), Tecfidera®, is approved globally for treating relapsing-remitting multiple sclerosis. The disposition of DMF was determined in humans after administration of a single oral dose of [14C]DMF, and the total recovery was estimated to be between 58.4% to 75.0%, primarily through expired air.The absorption of [14C]DMF-derived radioactivity was rapid, with Tmax at 1h postdose. Glucose was the predominant circulating metabolite, accounting for ∼60% of the total extractable radioactivity. Cysteine and N-acetylcysteine conjugates of mono- or di-methyl succinate were found to be the major urinary metabolites.In vitro studies showed that [14C]DMF was mainly metabolised to MMF, and fumarase exclusively converted fumaric acid to malic acid and did not catalyse the conversion of fumaric acid esters to malic acid. DMF was observed to bind with human serum albumin through Michael addition to the Cys-34 residue when exposed to human plasma.These findings indicate that DMF undergoes metabolism via hydrolysis, GSH conjugation, and the TCA cycle, leading to the formation of citric acid, CO2, and water. These ubiquitous and well-conserved metabolism pathways minimise the risk of drug-drug interactions and reduce variability related to pharmacogenetics and ethnicity.

Correction to: Use of Physiologically Based Pharmacokinetic (PBPK) Modeling for Predicting Drug-Food Interactions: an Industry Perspective.

An Erratum to this paper has been published: https://doi.org/10.1208/s12248-020-00535-z.

Use of Physiologically Based Pharmacokinetic (PBPK) Modeling for Predicting Drug-Food Interactions: an Industry Perspective.

The effect of food on pharmacokinetic properties of drugs is a commonly observed occurrence affecting about 40% of orally administered drugs. Within the pharmaceutical industry, significant resources are invested to predict and characterize a clinically relevant food effect. Here, the predictive performance of physiologically based pharmacokinetic (PBPK) food effect models was assessed via de novo mechanistic absorption models for 30 compounds using controlled, pre-defined in vitro, and modeling methodology. Compounds for which absorption was known to be limited by intestinal transporters were excluded in this analysis. A decision tree for model verification and optimization was followed, leading to high, moderate, or low food effect prediction confidence. High (within 0.8- to 1.25-fold) to moderate confidence (within 0.5- to 2-fold) was achieved for most of the compounds (15 and 8, respectively). While for 7 compounds, prediction confidence was found to be low (> 2-fold). There was no clear difference in prediction success for positive or negative food effects and no clear relationship to the BCS category of tested drug molecules. However, an association could be demonstrated when the food effect was mainly related to changes in the gastrointestinal luminal fluids or physiology, including fluid volume, motility, pH, micellar entrapment, and bile salts. Considering these findings, it is recommended that appropriately verified mechanistic PBPK modeling can be leveraged with high to moderate confidence as a key approach to predicting potential food effect, especially related to mechanisms highlighted here.

Direct protein-protein interactions and substrate channeling between cellular retinoic acid binding proteins and CYP26B1.

Cellular retinoic acid binding proteins (CRABPs) bind all-trans-retinoic acid (atRA) tightly. This study aimed to determine whether atRA is channeled directly to cytochrome P450 (CYP) CYP26B1 by CRABPs, and whether CRABPs interact directly with CYP26B1. atRA bound to CRABPs (holo-CRABP) was efficiently metabolized by CYP26B1. Isotope dilution experiments showed that delivery of atRA to CYP26B1 in solution was similar with or without CRABP. Holo-CRABPs had higher affinity for CYP26B1 than free atRA, but both apo-CRABPs inhibited the formation of 4-OH-RA by CYP26B1. Similar protein-protein interactions between soluble binding proteins and CYPs may be important for other lipophilic CYP substrates.

A Novel Plated Hepatocyte Relay Assay (PHRA) for In Vitro Evaluation of Hepatic Metabolic Clearance of Slowly Metabolized Compounds.

OBJECTIVE: Development and validation of a novel assay, the Plated Hepatocyte Relay Assay (PHRA), for the determination of the metabolic fates of slowly metabolized compounds. METHOD: Cryopreserved human hepatocytes were cultured for 4 h followed by incubation with slowly metabolized compounds for 24 h (initial incubation). On the next day, the incubated media were collected and added to hepatocytes was similarly prepared on the day of incubation (48 h incubation; 1(st) relay). The procedures were repeated on the next days (72 h (2(nd) relay), 96 h (3rd relay), and 120 h (4(th) relay) incubations). RESULTS: A proof-of-concept study with two low clearance compounds, diazepam and tolbutamide, and a validation study with 15 ultra-low clearance compounds (CLnon-renal < 1 mL/min/kg) and low clearance compounds (CLnon-renal 1- 5.1 mL/min/kg) were performed. Linear time-dependent disappearance of the parent compounds was observed for all compounds. Application of published free fraction values in combination with a correction factor with in vitro hepatic clearance results obtained with the PHRA accurately predicted in vivo hepatic clearance. CONCLUSION: PHRA represents a useful experimental system for the evaluation of the metabolic fates of low clearance compounds in drug development.

Discovery of biaryl carboxylamides as potent RORγ inverse agonists.

RORγt is a pivotal regulator of a pro-inflammatory gene expression program implicated in the pathology of several major human immune-mediated diseases. Evidence from mouse models demonstrates that genetic or pharmacological inhibition of RORγ activity can block the production of pathogenic cytokines, including IL-17, and convey therapeutic benefit. We have identified and developed a biaryl-carboxylamide series of RORγ inverse agonists via a structure based design approach. Co-crystal structures of compounds 16 and 48 supported the design approach and confirmed the key interactions with RORγ protein; the hydrogen bonding with His479 was key to the significant improvement in inverse agonist effect. The results have shown this is a class of potent and selective RORγ inverse agonists, with demonstrated oral bioavailability in rodents.

Effect of CYP3A5 expression on the inhibition of CYP3A-catalyzed drug metabolism: impact on modeling CYP3A-mediated drug-drug interactions.

The purpose of this study was to determine the impact of CYP3A5 expression on inhibitory potency (Ki or IC50 values) of CYP3A inhibitors, using recombinant CYP3A4 and CYP3A5 (rCYP3A4 and rCYP3A5) and CYP3A5 genotyped human liver microsomes (HLMs). IC50 ratios between rCYP3A4 and rCYP3A5 (rCYP3A5/rCYP3A4) of ketoconazole (KTZ) and itraconazole (ITZ) were 8.5 and 8.8 for midazolam (MDZ), 4.7 and 9.1 for testosterone (TST), 1.3 and 2.8 for terfenadine, and 0.6 and 1.7 for vincristine, respectively, suggesting substrate- and inhibitor-dependent selectivity of the two azoles. Due to the difference in the IC50 values for CYP3A4 and CYP3A5, nonconcordant expression of CYP3A4 and CYP3A5 protein can significantly affect the observed magnitude of CYP3A-mediated drug-drug interactions in humans. Indeed, the IC50 values of KTZ and ITZ for CYP3A-catalyzed MDZ and TST metabolism were significantly higher in HLMs with CYP3A5*1/*1 and CYP3A5*1/*3 genotypes than in HLMs with the CYP3A5*3/*3 genotype, showing CYP3A5 expression-dependent IC50 values. Moreover, when IC50 values of KTZ and ITZ for different HLMs were kinetically simulated based on CYP3A5 expression level and enzyme-specific IC50 values, a good correlation between the simulated and the experimentally measured IC50 values was observed. Further simulation analysis revealed that both the Ki ratio (for inhibitors) and Vmax/Km ratio (for substrates) between CYP3A4 and CYP3A5 were major factors for CYP3A5 expression-dependent IC50 values. In conclusion, the present study demonstrates that CYP3A5 genotype and expression level have a significant impact on inhibitory potency for CYP3A-catalyzed drug metabolism, but that the magnitude of its effect is inhibitor-substrate pair specific.

Stereospecific metabolism of itraconazole by CYP3A4: dioxolane ring scission of azole antifungals.

Itraconazole (ITZ) is a mixture of four cis-stereoisomers that inhibit CYP3A4 potently and coordinate CYP3A4 heme via the triazole nitrogen. However, (2R,4S,2'R)-ITZ and (2R,4S,2'S)-ITZ also undergo stereoselective sequential metabolism by CYP3A4 at a site distant from the triazole ring to 3'-OH-ITZ, keto-ITZ, and N-desalkyl-ITZ. This stereoselective metabolism demonstrates specific interactions of ITZ within the CYP3A4 active site. To further investigate this process, the binding and metabolism of the four trans-ITZ stereoisomers by CYP3A4 were characterized. All four trans-ITZ stereoisomers were tight binding inhibitors of CYP3A4-mediated midazolam hydroxylation (IC(50) 16-26 nM), and each gave a type II spectrum upon binding to CYP3A4. However, instead of formation of 3'-OH-ITZ, they were oxidized at the dioxolane ring, leading to ring scission and formation of two new metabolites of ITZ. These two metabolites were also formed from the four cis-ITZ stereoisomers, although not as efficiently. The catalytic rates of dioxolane ring scission were similar to the dissociation rates of ITZ stereoisomers from CYP3A4, suggesting that the heme iron is reduced while the triazole moiety coordinates to it and no dissociation of ITZ is necessary before catalysis. The triazole containing metabolite [1-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethanone] also inhibited CYP3A4 (IC(50) >15 µM) and showed type II binding with CYP3A4. The dioxolane ring scission appears to be clinically relevant because this metabolite was detected in urine samples from subjects that had been administered the mixture of cis-ITZ isomers. These data suggest that the dioxolane ring scission is a metabolic pathway for drugs that contain this moiety.

The kinetic mechanism for cytochrome P450 metabolism of type II binding compounds: evidence supporting direct reduction.

The metabolic stability of a drug is an important property that should be optimized during drug design and development. Nitrogen incorporation is hypothesized to increase the stability by coordination of nitrogen to the heme iron of cytochrome P450, a binding mode that is referred to as type II binding. However, we noticed that the type II binding compound 1 has less metabolic stability at sub-saturating conditions than a closely related type I binding compound 3. Three kinetic models will be presented for type II binder metabolism; (1) Dead-end type II binding, (2) a rapid equilibrium between type I and II binding modes before reduction, and (3) a direct reduction of the type II coordinated heme. Data will be presented on reduction rates of iron, the off rates of substrate (using surface plasmon resonance) and the catalytic rate constants. These data argue against the dead-end, and rapid equilibrium models, leaving the direct reduction kinetic mechanism for metabolism of the type II binding compound 1.

The effects of type II binding on metabolic stability and binding affinity in cytochrome P450 CYP3A4.

One goal in drug design is to decrease clearance due to metabolism. It has been suggested that a compound's metabolic stability can be increased by incorporation of a sp(2) nitrogen into an aromatic ring. Nitrogen incorporation is hypothesized to increase metabolic stability by coordination of nitrogen to the heme-iron (termed type II binding). However, questions regarding binding affinity, metabolic stability, and how metabolism of type II binders occurs remain unanswered. Herein, we use pyridinyl quinoline-4-carboxamide analogs to answer these questions. We show that type II binding can have a profound influence on binding affinity for CYP3A4, and the difference in binding affinity can be as high as 1200-fold. We also find that type II binding compounds can be extensively metabolized, which is not consistent with the dead-end complex kinetic model assumed for type II binders. Two alternate kinetic mechanisms are presented to explain the results. The first involves a rapid equilibrium between the type II bound substrate and a metabolically oriented binding mode. The second involves direct reduction of the nitrogen-coordinated heme followed by oxygen binding.

Cytochrome P450 2C9 type II binding studies on quinoline-4-carboxamide analogues.

CYP2C9 is a significant P450 protein responsible for drug metabolism. With the increased use of heterocyclic compounds in drug design, a rapid and efficient predrug screening of these potential type II binding compounds is essential to avoid adverse drug reactions. To understand binding modes, we use quinoline-4-carboxamide analogues to study the factors that determine the structure-activity relationships. The results of this study suggest that the more accessible pyridine with the nitrogen para to the linkage can coordinate directly with the ferric heme iron, but this is not seen for the meta or ortho isomers. The pi-cation interaction of the naphthalene moiety and Arg 108 residue may also assist in stabilizing substrate binding within the active-site cavity. The type II substrate binding affinity is determined by the combination of steric, electrostatic, and hydrophobicity factors; meanwhile, it is enhanced by the strength of lone pair electrons coordination with the heme iron.

Modeling and synthesis of novel tight-binding inhibitors of cytochrome P450 2C9.

Cytochrome P450 2C9 (2C9) is one of the three major drug metabolizing cytochrome P450 enzymes in human liver. Although the crystal structure of 2C9 has been solved, the important physicochemical properties of substrate-enzyme interactions remain difficult to be determined. This is due in part to the conformational flexibility of mammalian P450 enzymes. Therefore, probing the active-site with high-affinity substrates is important in further understanding substrate-enzyme interactions. Three-dimensional quantitative structure-activity relationships (3D-QSAR) and docking experiments have been shown to be useful tools in correlating biological activity with structure. In particular we have previously reported that the very tight-binding inhibitor benzbromarone can provide important information about the active-site of 2C9. In this study we report the binding affinities and potential substrate-enzyme interactions of 4H-chromen-4-one analogs, which are structurally similar to benzbromarone. The chromenone structures are synthetically accessible inhibitors and give inhibition constants as low as 4.2 nM, comparable with the very tightest-binding inhibitors of 2C9. Adding these compounds to our previous 2C9 libraries for CoMFA models reinforces the important electrostatic and hydrophobic features of substrate binding. These compounds have also been docked in the 2C9 crystal structure and the results indicate that Arg 108 plays significant roles in the binding of chromenone substrates.

High confidence predictions of drug-drug interactions: predicting affinities for cytochrome P450 2C9 with multiple computational methods.

Four different models are used to predict whether a compound will bind to 2C9 with a K(i) value of less than 10 microM. A training set of 276 compounds and a diverse validation set of 50 compounds were used to build and assess each model. The modeling methods are chosen to exploit the differences in how training sets are used to develop the predictive models. Two of the four methods develop partitioning trees based on global descriptions of structure using nine descriptors. A third method uses the same descriptors to develop local descriptions that relate activity to structures with similar descriptor characteristics. The fourth method uses a graph-theoretic approach to predict activity based on molecular structure. When all of these methods agree, the predictive accuracy is 94%. An external validation set of 11 compounds gives a predictive accuracy of 91% when all methods agree.