Clearance In Liver Microsomes Research Articles

Improved Preclinical Drug Metabolism and Pharmacokinetics of Pibothiadine (HEC121210), a Novel Hepatitis B Virus Capsid Assembly Modulator

ABSTRACT Pibothiadine (PBD; HEC121120) is a novel hepatitis B virus capsid assembly modulator based on GLS4 (morphothiadine) and has inhibitory activities against resistant strains. To assess the overall preclinical drug metabolism and pharmacokinetics (DMPK) properties of PBD, in vivo pharmacokinetics studies in rats and dogs have been performed along with a series of in vitro metabolism assays. The oral bioavailability of PBD in rats and dogs might be related to its medium permeability in Caco-2 cells and largely be impacted by the pH-dependent solubility. PBD was highly distributed to the liver where the local exposure was 16.4 fold of the system exposure. PBD showed relatively low metabolic rate in recombinant human cytochrome P450 enzymes, whereas low to moderate in vitro clearance in liver microsomes and low (dog) to moderate (rat) in vivo clearance. Furthermore, β-oxidation and dehydrogenation were proposed as the primary metabolic pathways of PBD in rats. Compared to GLS4, the higher systemic exposure of PBD might be attributed to its improved oral absorption and metabolic stability. In addition, the enhanced liver/plasma exposure ratio could further increase the local exposure around the target. These improved DMPK properties might indicate better development of PBD in the clinical phase.

Prediction of physicochemical and pharmacokinetic properties of botanical constituents by computational models.

Botanicals contain complex mixtures of chemicals most of which lack pharmacokinetic data in humans. Since physicochemical and pharmacokinetic properties dictate the in vivo exposure of botanical constituents, these parameters greatly impact the pharmacological and toxicological effects of botanicals in consumer products. This study sought to use computational (i.e., in silico) models, including quantitative structure-activity relationships (QSAR) and physiologically based pharmacokinetic (PBPK) modeling, to predict properties of botanical constituents. One hundred and three major constituents (e.g., withanolides, mitragynine, and yohimbine) in 13 botanicals (e.g., ashwagandha, kratom, and yohimbe) were investigated. The predicted properties included biopharmaceutical classification system (BCS) classes based on aqueous solubility and permeability, oral absorption, liver microsomal clearance, oral bioavailability, and others. Over half of these constituents fell into BCS classes I and II at dose levels no greater than 100 mg per day, indicating high permeability and absorption (%Fa > 75%) in the gastrointestinal tract. However, some constituents such as glycosides in ashwagandha and Asian ginseng showed low bioavailability after oral administration due to poor absorption (BCS classes III and IV, %Fa < 40%). These in silico results fill data gaps for botanical constituents and could guide future safety studies. For example, the predicted human plasma concentrations may help select concentrations for in vitro toxicity testing. Additionally, the in silico data could be used in tiered or batteries of assays to assess the safety of botanical products. For example, highly absorbed botanical constituents indicate potential high exposure in the body, which could lead to toxic effects.

Determination of enzymatic kinetics of metabolism of dimethoate and omethoate in rats and humans

1. Dimethoate is an organophosphate insecticide. The objective of this work was to determine the enzymatic kinetics of metabolism of dimethoate and its active metabolite omethoate in rats and humans and obtain key input parameters for physiologically based pharmacokinetic (PBPK) model. 2. First, the intrinsic clearance of dimethoate expressed as formation rate of omethoate was determined to be ∼42-fold lower in human liver microsomes (HLM) (0.39 µL/min/mg) than in rat liver microsomes (RLM) (16.6 µL/min/mg) by an LC/MS/MS method. Next, dimethoate clearance in liver microsomes was determined using parent depletion and total [14C]-metabolite formation methods. Results from both approaches showed slower clearance of dimethoate in HLM (1.1–3.3 µL/min/mg) than in RLM (12.7–17.4 µL/min/mg). 3. Investigation of in vitro enzymatic kinetics of omethoate demonstrated that the intrinsic clearance rates for omethoate in adult and juvenile RLM and HLM were similar. No significant turnover of dimethoate was apparent in rat cytosol or plasma. In contrast, degradation of omethoate in human plasma was slightly higher than in rat plasma. 4. Finally, toxicokinetics of dimethoate were determined in adult and juvenile rats. In both age groups, following oral dosing, absorption of dimethoate was rapid with formation of significant amounts of omethoate.

Synthesis and structure-activity relationships for a new class of tetrahydronaphthalene amide inhibitors of Mycobacterium tuberculosis

Drug resistant tuberculsosis (TB) is global health crisis that demands novel treatment strategies. Bacterial ATP synthase inhibitors such as bedaquiline and next-generation analogues (such as TBAJ-876) have shown promising efficacy in patient populations and preclinical studies, respectively, suggesting that selective targeting of this enzyme presents a validated therapeutic strategy for the treatment of TB. In this work, we report tetrahydronaphthalene amides (THNAs) as a new class of ATP synthase inhibitors that are effective in preventing the growth of Mycobacterium tuberculosis (M.tb) in culture. Design, synthesis and comprehensive structure-activity relationship studies for approximately 80 THNA analogues are described, with a small selection of compounds exhibiting potent (in some cases MIC90 <1 μg/mL) in vitro M.tb growth inhibition taken forward to pharmacokinetic and off-target profiling studies. Ultimately, we show that some of these THNAs possess reduced lipophilic properties, decreased hERG liability, faster mouse/human liver microsomal clearance rates and shorter plasma half-lives compared with bedaquiline, potentially addressing of the main concerns of persistence and phospholipidosis associated with bedaquiline.

Novel Sigma 1 Receptor Antagonists as Potential Therapeutics for Pain Management

The sigma 1 receptor (S1R) is a molecular chaperone protein located in the endoplasmic reticulum and plasma membranes and has been shown to play important roles in various pathological disorders including pain and, as recently discovered, COVID-19. Employing structure- and QSAR-based drug design strategies, we rationally designed, synthesized, and biologically evaluated a series of novel triazole-based S1R antagonists. Compound 10 exhibited potent binding affinity for S1R, high selectivity over S2R and 87 other human targets, acceptable in vitro metabolic stability, slow clearance in liver microsomes, and excellent blood-brain barrier permeability in rats. Further in vivo studies in rats showed that 10 exhibited negligible acute toxicity in the rotarod test and statistically significant analgesic effects in the formalin test for acute inflammatory pain and paclitaxel-induced neuropathic pain models during cancer chemotherapy. These encouraging results promote further development of our triazole-based S1R antagonists as novel treatments for pain of different etiologies.

Mechanistic insights on clearance and inhibition discordance between liver microsomes and hepatocytes when clearance in liver microsomes is higher than in hepatocytes

Human liver microsomes (HLM) and human hepatocytes (HHEP) are two common in vitro systems used in metabolic stability and inhibition studies. The comparison between the assays using the two systems can provide mechanistic insights on the interplay of metabolism, passive permeability and transporters. This study investigated the critical factors impacting the unbound intrinsic clearance (CLint,u) and IC50 of CYP3A inhibition between HLM and HHEP. The HLM/HHEP CLint,u ratio and HHEP/HLM IC50 ratio are inversely correlated to passive permeability, but have no correlation with P-gp efflux ratio. Cofactor-supplemented permeabilized HHEP (MetMax™) collapses the IC50 differences between HHEP and HLM. P-gp inhibitor, encequidar, shows minimal impact on CLint,u and IC50 in HHEP. This is the first study that is able to separately investigate the effects of passive permeability and efflux transport. These data collectively show that passive permeability plays a critical role in metabolism and enzyme inhibition in HHEP, while P-gp efflux has a minor role. This may be due to low functional P-gp activity in suspension HHEP under the assay conditions. Low passive permeability may limit metabolism and enzyme inhibition in HHEP, leading to lower CLint,u and higher IC50 in HHEP compared to HLM. When liver microsomes give higher CLint,u than hepatocytes, microsomes are more predictive of in vivo clearance than hepatocytes.

Absorption, distribution, metabolism, excretion (ADME), drug-drug interaction potential and prediction of human pharmacokinetics of SUVN-G3031, a novel histamine 3 receptor (H3R) inverse agonist in clinical development for the treatment of narcolepsy

SUVN-G3031 is a potent and selective inverse agonist of Histmine-3 (H3) receptor that is being investigated for the treatment of narcolepsy. SUVN-G3031 has high passive permeability, not a substrate for P-glycoprotein, has high plasma unbound fractions and was equally distributed between blood and plasma. Major routes of metabolism in vitro were cyclization (Metabolite A) in microsomes and dealkylation (Metabolite D) in hepatocytes. Intrinsic clearance in liver microsomes and hepatocytes was low as monitored by metabolite formation approach. CYP3A4 and MAO-A were the major enzymes involved in the formation of metabolite A and metabolite D respectively. The human hepatic clearance estimated by well-stirred model from hepatocytes was low (2.7 L.h −1) illustrating the importance of metabolite formation kinetics for prediction of human clearance for SUVN-G3031. Renal clearance in humans (9.7 L.h −1) was predicted from dog renal clearance and accounts for ~78% of the total clearance. SUVN-G3031 was neither an inhibitor nor inducer of the P450 enzymes at clinically relevant concentrations. SUVN-G3031 did not inhibit the major uptake transporters and was not a substrate for the uptake transporters. The potential of SUVN-G3031 as a victim and perpetrator of drug-drug interactions is remote. The predicted human pharmacokinetic parameters were consistent with those observed in the first-in-human study.

Towards the Development of an In vivo Chemical Probe for Cyclin G Associated Kinase (GAK).

SGC-GAK-1 (1) is a potent, selective, cell-active chemical probe for cyclin G-associated kinase (GAK). However, 1 was rapidly metabolized in mouse liver microsomes by cytochrome P450-mediated oxidation, displaying rapid clearance in liver microsomes and in mice, which limited its utility in in vivo studies. Chemical modifications of 1 that improved metabolic stability, generally resulted in decreased GAK potency. The best analog in terms of GAK activity in cells was 6-bromo-N-(1H-indazol-6-yl)quinolin-4-amine (35) (IC50 = 1.4 μM), showing improved stability in liver microsomes while still maintaining a narrow spectrum activity across the kinome. As an alternative to scaffold modifications we also explored the use of the broad-spectrum cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) to decrease intrinsic clearance of aminoquinoline GAK inhibitors. Taken together, these approaches point towards the development of an in vivo chemical probe for the dark kinase GAK.

Synthesis and SAR development of quinoline analogs as novel P2X7 receptor antagonists.

The P2X7 receptor (P2X7R) plays an important role in diverse conditions associated with tissue damage and inflammation, suggesting that the human P2X7R (hP2X7R) is an attractive therapeutic target. In the present study, the synthesis and structure-activity relationship (SAR) of a novel series of quinoline derivatives as P2X7R antagonists are described herein. These compounds exhibited mechanistic activity (YO PRO) in an engineered HEK293 expressing hP2X7R as well as a functional response (IL-1β) in human THP-1 (hTHP-1) cellular assays. Compound 19 was identified as the most promising compound in this series with excellent cellular potency, low liver microsomal clearance, good permeability and low efflux ratio. In addition, this compound also displayed good pharmacokinetic properties and acceptable brain permeability (Kp,uu of 0.37).

Efavirenz clearances in vitro and in vivo in six cynomolgus monkeys associated with polymorphic cytochrome P450 2C9 and simulated by individual physiologically based pharmacokinetic models.

Cynomolgus monkey cytochrome P450 2C9 (formerly known as P450 2C43) variation was reportedly associated with metabolic clearance of the antiretroviral drug efavirenz in vivo (in three wild-type, one heterozygote and two homozygote animals), being unlikely in the case of human P450 2B6-dependent efavirenz clearance. In this study, the liver microsomal elimination rates of efavirenz for the same individual animals previously treated with intravenous/oral administrations of efavirenz showed significant reductions associated with the P450 2C9 p.[(I112L)] genotype (p<0.05). Simulations of efavirenz clearance after oral administrations in individual cynomolgus monkeys were performed using individual simplified physiologically based pharmacokinetic (PBPK) modeling consisting of gut, liver and central compartments. The modeled hepatic intrinsic clearances were also significantly associated with the P450 2C9 genotypes, however, absorption rate constants or volumes of the systemic circulation were not likely determining factors for the individual efavirenz clearance variations in the six cynomolgus monkeys. This study provides important information to help simulate the clearances of efavirenz and related medicines associated with polymorphic P450 2C9 in individual cynomolgus monkeys, thereby facilitating the calculation of the fraction of liver microsomal clearance for estimating in vivo drug clearance with simplified PBPK modeling.

Optimization of a novel series of potent and orally bioavailable GPR119 agonists

We describe the discovery and optimization of a novel series of furo[3,2-d]pyrimidines as G protein-coupled receptor 119 agonists. Agonistic activity of 4 (EC50=129nM) was improved by replacing the intramolecular hydrogen bond between the fluorine atom and the aniline hydrogen in the head moiety with a covalent C-C bond to enhance conformational restriction, which consequently gave a lead compound 12 (EC50=53nM). Optimized compound 26, which was identified by the further optimization of 12, exhibited potent activity (EC50=42nM) with improved clearance in liver microsomes and induced a 33% reduction in blood glucose area under the curve at a dose of 10mg/kg in an oral glucose tolerance test in C57BL/6N mice.

Intrinsic Clearance of Xenobiotic Chemicals by Liver Microsomes: Assessment of Trophic Magnification Potentials.

The use of trophic magnification factors (TMFs) to characterize the bioaccumulation potentials of chemicals was encouraged; however, the method for the assessment of trophic magnification potentials is still lacking. We optimized the in vitro assays used for the measurement of intrinsic clearance in liver microsomes by incorporating benzo[a]pyrene (B(a)P) as a benchmark compound. The intrinsic clearance of 40 compounds was then measured in microsomes from fish (weevers) and birds (quail); the characteristics of the trophic transfer of these 40 compounds were previously investigated in an aquatic food web in Bohai in northern China. Chemicals that are biotransformed at a rate similar to or higher than that of B[a]P in the microsomes of both weevers and quail (in vitro intrinsic clearance values, CL; CL/CLB[a]P: 0.1 to 2.4) generally exhibited no significant trophic magnification or dilution in the food web (TMF ≈ 1 or < 1), whereas chemicals that are biotransformed at extremely slow rates compared with B[a]P (CL/CLB[a]P: 0 to 0.2) showed significant trophic magnification in the food web (TMF > 1). The in vitro intrinsic clearance values of the target chemicals were found to be consistent with their respective trophic transfer behavior in the aquatic food web. Significant negative correlations were also found between the TMFs and the intrinsic clearance values of all target chemicals obtained in microsomes from both weevers and quail. Multiple linear regression analysis showed that biotransformation rates (CL/CLB[a]P) are a more important factor compared with the lipophilicity of the chemicals (log Kow) in the assessment of the trophic magnification of chemicals in the aquatic food web.

Identification of saturated and unsaturated fatty acids released during microsomal incubations

1. In vitro clearance in liver microsomes is routinely measured in drug discovery and development for new chemical entities. Literature reports indicate that long chain fatty acids such as arachidonic, linoleic and oleic acids may be released over a period of time during microsomal incubations. Some fatty acids have been shown to interfere with oxidative and conjugative reactions in microsomes, thus potentially inhibiting microsomal clearance of compounds.2. The present study was aimed at deciphering the fatty acids present or released from microsomes. Analytical methods were developed to characterize and quantitatively assess the fatty acids without chemical derivatization in rat, monkey and human liver microsomes. Additionally, incubations with uridine-5′-diphosphoglucuronic acid (UDPGA) were utilized to trap the released fatty acids as their glucuronate esters, which were characterized and confirmed by high-resolution LC-MS/MS.3. Our results indicate for the first time that timnodonic, trans-eicosenoic, gondoic, behenic, and nervonic acid were released during microsomal incubations. Additionally, α- and γ-linolenic, timnodonic, palmitoleic, linoleic, arachidonic, palmitic, oleic, and stearic acid were identified as their corresponding acyl-glucuronides in rat, monkey and human liver microsomes, providing the first direct evidence that the released fatty acids are capable of forming glucuronides under incubation conditions.

Design, Synthesis, and Biological Evaluation of Indole-2-carboxamides: A Promising Class of Antituberculosis Agents

Indole-2-carboxamides have been identified as a promising class of antituberculosis agents from phenotypic screening against mycobacteria. One of the hits, indole-2-carboxamide analog (1), had low micromolar potency against Mycobacterium tuberculosis (Mtb), high mouse liver microsomal clearance, and low aqueous solubility. Structure-activity relationship studies revealed that attaching alkyl groups to the cyclohexyl ring significantly improved Mtb activity but reduced solubility. Furthermore, chloro, fluoro, or cyano substitutions on the 4- and 6-positions of the indole ring as well as methyl substitution on the cyclohexyl ring significantly improved metabolic stability. 39 and 41, the lead candidates, displayed improved in vitro activity compared to most of the current standard TB drugs. The low aqueous solubility could not be mitigated because of the positive correlation of lipophilicity with Mtb potency. However, both compounds displayed favorable oral pharmacokinetic properties in rodents and demonstrated in vivo efficacy. Thus, indole-2-carboxamides represent a promising new class of antituberculosis agents.

Design and Synthesis of Diazatricyclodecane Agonists of the G-Protein-Coupled Receptor 119

A series of GPR119 agonists based on a 2,6-diazatricyclo[3.3.1.1∼3,7∼]decane ring system is described. Also provided is a detailed account of the development of a multigram scale synthesis of the diazatricyclic ring system, which was achieved using a Hofmann-Löffler-Freytag reaction as the key step. The basis for the use of this complex framework lies in an attempt to constrain one end of the molecule in the "agonist conformation" as was previously described for 3-oxa-7-aza-bicyclo[3.3.1]nonanes. Optimization of carbamate analogues of the diazatricylic compounds led to the identification of 32i as a potent agonist of the GPR119 receptor with low unbound human liver microsomal clearance. The use of an agonist response weighted ligand lipophilic efficiency (LLE) termed AgLLE is discussed along with the issues of applying efficiency measures to agonist programs. Ultimately, solubility limited absorption and poor exposure reduced further interest in these molecules.

Systematic and Pairwise Analysis of the Effects of Aromatic Halogenation and Trifluoromethyl Substitution on Human Liver Microsomal Clearance

Fluorine- and chlorine-containing moieties have been strategically integrated into chemical structures to optimize the pharmacokinetic and metabolic properties of therapeutic agents, based partly on the concept that the addition of these substituents may lower microsomal clearance. A large-scale systematic mechanistic study of drug metabolic alteration by aromatic halogenation has hitherto not been possible due to the lack of either large clearance databases or adequate data mining tools. To address this, we systematically searched compound pairs in Pfizer's human liver microsomal clearance database of over 220,000 unique compounds to assess the effects of fluoro-, chloro- and trifluoromethyl-substitution on phenyl derivatives. Although the para-position fluorination and chlorination lowered the microsomal clearance statistically, the substitution at the ortho and meta positions for the studied fluorine- and chlorine-containing moieties dramatically increased the microsomal clearance. More importantly, we found that changes in physicochemical properties, electronic properties, and specific binding of substrates to drug metabolizing enzymes, for instance, cytochrome P450s, are all determining factors that drive the direction of microsomal clearance when a specific series of compounds are studied.

Metabolism of 4-Aminopiperidine Drugs by Cytochrome P450s: Molecular and Quantum Mechanical Insights into Drug Design

4-Aminopiperidines are a variety of therapeutic agents that are extensively metabolized by cytochrome P450s with CYP3A4 as a major isoform catalyzing their N-dealkylation reaction. However, its catalytic mechanism has not been fully elucidated in a molecular interaction level. Here, we applied theoretical approaches including the molecular mechanics-based docking to study the binding patterns and quantum mechanics-based reactivity calculations. They were supported by the experimental human liver microsomal clearance and P450 isoform phenotyping data. Our results herein suggested that the molecular interactions between substrates and CYP3A4 active site residues are essential for the N-dealkylation of 4-aminopiperidines. We also found that the serine 119 residue of CYP3A4 may serve as a key hydrogen-bonding partner to interact with the 4-amino groups of the studied drugs. The reactivity of the side chain α-carbon hydrogens drives the direction of catalysis as well. As a result, structure-based drug design approaches look promising to guide drug discovery programs into the optimized drug metabolism space.

The Development and Validation of a Computational Model to Predict Rat Liver Microsomal Clearance

As the cost of discovering and developing new pharmaceutically relevant compounds continues to rise, it is increasingly important to select the right molecules to prosecute very early in drug discovery. The development of high throughput in vitro assays of hepatic metabolic clearance has allowed for vast quantities of data generation; however, these large screens are still costly and remain dependant on animal usage. To further expand the value of these screens and ultimately aid in animal usage reduction, we have developed an in silico model of rat liver microsomal (RLM) clearance. This model combines a large amount of rat clearance data (n = 27,697) generated at multiple Pfizer laboratories to represent the broadest possible chemistry space. The model predicts RLM stability (with 82% accuracy and a kappa value of 0.65 for test data set) based solely on chemical structural inputs, and provides a clear assessment of confidence in the prediction. The current in silico model should help accelerate the drug discovery process by using confidence-based stability-driven prioritization, and reduce cost by filtering out the most unstable/undesirable molecules. The model can also increase efficiency in the evaluation of chemical series by optimizing iterative testing and promoting rational drug design.

Preclinical Drug Metabolism and Pharmacokinetic Evaluation of GW844520, A Novel Anti-Malarial Mitochondrial Electron Transport Inhibitor

GW844520 is a potent and selective inhibitor of the cytochrome bc1 complex of mitochondrial electron transport in P. falciparum, the parasite primarily responsible for the mortality associated with malaria worldwide. GW844520 is fully active against the parasite including resistance isolates, showing no cross resistance with agents in use. To evaluate full potential of this development candidate, we conducted drug metabolism and pharmacokinetic studies of this novel anti-malarial. GW844520 had low blood clearance of about 0.5–4% of hepatic blood flow and a steady-state volume of distribution of 2–4 times total body water in mouse, rat, dog, and monkey. Oral bioavailability was high (51–100%). Consistent with the in vivo data, GW844520 had low intrinsic clearance in liver microsomes and hepatocytes of animal and human origin, high passive cellular permeability and was not a P-glycoprotein substrate. GW844520 did not associate appreciably with blood cells but was highly bound to plasma proteins (>99%) in all species. GW844520 was a substrate and inhibitor of human CYP2D6 but not of CYP1A2, 2C9, 2C19, and 3A4. This conjunctive analysis supports continued evaluation of this compound in definitive pre-IND studies and exemplifies our strategy supporting the discovery of novel agents to treat diseases of the developing world.

Comparison of Intrinsic Clearance in Liver Microsomes and Hepatocytes from Rats and Humans: Evaluation of Free Fraction and Uptake in Hepatocytes

Apparent intrinsic clearance (CL(int,app)) of 7-ethoxycoumarin, phenacetin, propranolol, and midazolam was measured using rat and human liver microsomes and freshly isolated and cryopreserved hepatocytes to determine factors responsible for differences in rates of metabolism in these systems. The cryopreserved and freshly isolated hepatocytes generally provided similar results, although there was greater variability using the latter system. The CL(int,app) values in hepatocytes are observed to be lower than that in microsomes, and this difference becomes greater for compounds with high CL(int,app). This could partly be attributed to the differences in the free fraction (fu). The fu in hepatocyte incubations (fu,hep-inc) was influenced not only by the free fraction of compounds in the incubation buffer (fu,buffer) but also by the rate constants of uptake (k(up)) and metabolism (k(met)). This report provides a new derivation for fu,hep-inc, which can be expressed as fu,hep-inc = [k(up)/(k(met) + k(up))]/[1 + (C(hep)/C(buffer)) x (V(hep)/V(buffer))], where the C(hep), C(buffer), V(hep), and V(buffer) represent the concentrations of a compound in hepatocytes and buffer and volumes of hepatocytes and buffer, respectively. For midazolam, the fu,hep-inc was calculated, and the maximum metabolism rate in hepatocytes was shown to be limited by the uptake rate.