Investigation of pharmacokinetic drug interaction between clesacostat and DGAT2 inhibitor ervogastat in healthy adult participants.

Co-administration of clesacostat (acetyl-CoA carboxylase inhibitor, PF-05221304) and ervogastat (diacylglycerol O-acyltransferase inhibitor, PF-06865571) in laboratory models improved non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) end points and mitigated clesacostat-induced elevations in circulating triglycerides. Clesacostat is cleared via organic anion-transporting polypeptide-mediated hepatic uptake and cytochrome P450 family 3A (CYP3A); in vitro clesacostat is identified as a potential CYP3A time-dependent inactivator. In vitro ervogastat is identified as a substrate and potential inducer of CYP3A. Prior to longer-term efficacy trials in participants with NAFLD, safety and pharmacokinetics (PK) were evaluated in a phase I, non-randomized, open-label, fixed-sequence trial in healthy participants. In Cohort 1, participants (n = 7) received clesacostat 15 mg twice daily (b.i.d.) alone (Days 1-7) and co-administered with ervogastat 300 mg b.i.d. (Days 8-14). Mean systemic clesacostat exposures, when co-administered with ervogastat, decreased by 12% and 19%, based on maximum plasma drug concentration and area under the plasma drug concentration-time curve during the dosing interval, respectively. In Cohort 2, participants (n = 9) received ervogastat 300 mg b.i.d. alone (Days 1-7) and co-administered with clesacostat 15 mg b.i.d. (Days 8-14). There were no meaningful differences in systemic ervogastat exposures when administered alone or with clesacostat. Clesacostat 15 mg b.i.d. and ervogastat 300 mg b.i.d. co-administration was overall safe and well tolerated in healthy participants. Cumulative safety and no clinically meaningful PK drug interactions observed in this study supported co-administration of these two novel agents in additional studies exploring efficacy and safety in the management of NAFLD.

The Current Status and Use of Microphysiological Systems by the Pharmaceutical Industry: The International Consortium for Innovation and Quality Microphysiological Systems Affiliate Survey and Commentary.

Microphysiological systems (MPS) are comprised of one or multiple cell types of human or animal origins that mimic the biochemical/electrical/mechanical responses and blood-tissue barrier properties of the cells observed within a complex organ. The goal of incorporating these in vitro systems is to expedite and advance the drug discovery and development paradigm with improved predictive and translational capabilities. Considering the industry need for improved efficiency and the broad challenges of model qualification and acceptance, the International Consortium for Innovation and Quality (IQ) founded an IQ MPS working group in 2014 and Affiliate in 2018. This group connects thought leaders and end users, provides a forum for crosspharma collaboration, and engages with regulators to qualify translationally relevant MPS models. To understand how pharmaceutical companies are using MPS, the IQ MPS Affiliate conducted two surveys in 2019, survey 1, and 2021, survey 2, which differed slightly in the scope of definition of the complex in vitro models under question. The surveys captured demographics, resourcing, rank order for organs of interest, compound modalities tested, and MPS organ-specific questions, including nonclinical species needs and cell types. The major focus of this manuscript is on results from survey 2, where we specifically highlight the context of use for MPS within safety, pharmacology, or absorption, disposition, metabolism, and excretion and discuss considerations for including MPS data in regulatory submissions. In summary, these data provide valuable insights for developers, regulators, and pharma, offering a view into current industry practices and future considerations while highlighting key challenges impacting MPS adoption. SIGNIFICANCE STATEMENT: The application of microphysiological systems (MPS) represents a growing area of interest in the drug discovery and development framework. This study surveyed 20+ pharma companies to understand resourcing, current areas of application, and the key challenges and barriers to internal MPS adoption. These results will provide regulators, tech providers, and pharma industry leaders a starting point to assess the current state of MPS applications along with key learnings to effectively realize the potential of MPS as an emerging technology.

A Novel Milli-fluidic Liver Tissue Chip with Continuous Recirculation for Predictive Pharmacokinetics Applications.

A crucial step in lead selection during drug development is accurate estimation and optimization of hepatic clearance using in vitro methods. However, current methods are limited by factors such as lack of physiological relevance, short culture/incubation times that are not consistent with drug exposure patterns in patients, use of drug absorbing materials, and evaporation during long-term incubation. To address these technological needs, we developed a novel milli-fluidic human liver tissue chip (LTC) that was designed with continuous media recirculation and optimized for hepatic cultures using human primary hepatocytes. Here, we characterized the LTC using a series of physiologically relevant metrics and test compounds to demonstrate that we could accurately predict the PK of both low- and high-clearance compounds. The non-biological characterization indicated that the cyclic olefin copolymer (COC)-based LTC exhibited negligible evaporation and minimal non-specific binding of drugs of varying ionic states and lipophilicity. Biologically, the LTC exhibited functional and polarized hepatic culture with sustained metabolic CYP activity for at least 15 days. This long-term culture was then used for drug clearance studies for low- and high-clearance compounds for at least 12 days, and clearance was estimated for a range of compounds with high in vitro-in vivo correlation (IVIVC). We also demonstrated that LTC can be induced by rifampicin, and the culture age had insignificant effect on depletion kinetic and predicted clearance value. Thus, we used advances in bioengineering to develop a novel purpose-built platform with high reproducibility and minimal variability to address unmet needs for PK applications.

Considerations from an International Regulatory and Pharmaceutical Industry (IQ MPS Affiliate) Workshop on the Standardization of Complex In Vitro Models in Drug Development.

In May 2022, there is an International Regulatory and Pharmaceutical Industry (Innovation and Quality [IQ] Microphysiological Systems [MPS] Affiliate) Workshop on the standardization of complex in vitro models (CIVMs) in drug development. This manuscript summarizes the discussions and conclusions of this joint workshop organized and executed by the IQ MPS Affiliate and the United States Food and Drug Administration (FDA). A key objective of the workshop is to facilitate discussions around opportunities and/or needs for standardization of MPS and chart potential pathways to increase model utilization in the context of regulatory decision making. Participation in the workshop included 200 attendees from the FDA, IQ MPS Affiliate, and 26 global regulatory organizations and affiliated parties representing Europe, Japan, and Canada. It is agreed that understanding global perspectives regarding the readiness of CIVM/MPS models for regulatory decision making and potential pathways to gaining acceptance is useful to align on globally. The obstacles are currently too great to develop standards for every context of use (COU). Instead, it is suggested that a more tractable approach may be to think of broadly applicable standards that can be applied regardless of COU and/or organ system. Considerations and next steps for this effort are described.

Design of Next-Generation DGAT2 Inhibitor PF-07202954 with Longer Predicted Half-Life.

Diacylglycerol O-acyltransferase 2 (DGAT2) inhibitors have been shown to lower liver triglyceride content and are being explored clinically as a treatment for non-alcoholic steatohepatitis (NASH). This work details efforts to find an extended-half-life DGAT2 inhibitor. A basic moiety was added to a known inhibitor template, and the basicity and lipophilicity were fine-tuned by the addition of electrophilic fluorines. A weakly basic profile was required to find an appropriate balance of potency, clearance, and permeability. This work culminated in the discovery of PF-07202954 (12), a weakly basic DGAT2 inhibitor that has advanced to clinical studies. This molecule displays a higher volume of distribution and longer half-life in preclinical species, in keeping with its physicochemical profile, and lowers liver triglyceride content in a Western-diet-fed rat model.

Discovery of Ervogastat (PF-06865571): A Potent and Selective Inhibitor of Diacylglycerol Acyltransferase 2 for the Treatment of Non-alcoholic Steatohepatitis.

Discovery efforts leading to the identification of ervogastat (PF-06865571), a systemically acting diacylglycerol acyltransferase (DGAT2) inhibitor that has advanced into clinical trials for the treatment of non-alcoholic steatohepatitis (NASH) with liver fibrosis, are described herein. Ervogastat is a first-in-class DGAT2 inhibitor that addressed potential development risks of the prototype liver-targeted DGAT2 inhibitor PF-06427878. Key design elements that culminated in the discovery of ervogastat are (1) replacement of the metabolically labile motif with a 3,5-disubstituted pyridine system, which addressed potential safety risks arising from a cytochrome P450-mediated O-dearylation of PF-06427878 to a reactive quinone metabolite precursor, and (2) modifications of the amide group to a 3-THF group, guided by metabolite identification studies coupled with property-based drug design.

Quantitative prediction of breast cancer resistant protein mediated drug-drug interactions using physiologically-based pharmacokinetic modeling.

Quantitative assessment of drug-drug interactions (DDIs) involving breast cancer resistance protein (BCRP) inhibition is challenged by overlapping substrate/inhibitor specificity. This study used physiologically-based pharmacokinetic (PBPK) modeling to delineate the effects of inhibitor drugs on BCRP- and organic anion transporting polypeptide (OATP)1B-mediated disposition of rosuvastatin, which is a recommended BCRP clinical probe. Initial static model analysis using in vitro inhibition data suggested BCRP/OATP1B DDI risk while considering regulatory cutoff criteria for a majority of inhibitors assessed (25 of 27), which increased rosuvastatin plasma exposure to varying degree (~ 0-600%). However, rosuvastatin area under plasma concentration-time curve (AUC) was minimally impacted by BCRP inhibitors with calculated G-value (= gut concentration/inhibition potency) below 100. A comprehensive PBPK model accounting for intestinal (OATP2B1 and BCRP), hepatic (OATP1B, BCRP, and MRP4), and renal (OAT3) transport mechanisms was developed for rosuvastatin. Adopting in vitro inhibition data, rosuvastatin plasma AUC changes were predicted within 25% error for 9 of 12 inhibitors evaluated via PBPK modeling. This study illustrates the adequacy and utility of a mechanistic model-informed approach in quantitatively assessing BCRP-mediated DDIs.

Correction: A high-throughput microfluidic microphysiological system (PREDICT-96) to recapitulate hepatocyte function in dynamic, re-circulating flow conditions.

Correction for 'A high-throughput microfluidic microphysiological system (PREDICT-96) to recapitulate hepatocyte function in dynamic, re-circulating flow conditions' by Kelly Tan et al., Lab Chip, 2019, 19, 1556-1566, DOI: .

A thermoplastic microfluidic microphysiological system to recapitulate hepatic function and multicellular interactions.

Hepatic in vitro platforms ranging from multi-well cultures to bioreactors and microscale systems have been developed as tools to recapitulate cellular function and responses to aid in drug screening and disease model development. Recent developments in microfabrication techniques and cellular materials enabled fabrication of next-generation, advanced microphysiological systems (MPSs) that aim to capture the cellular complexity and dynamic nature of the organ presenting highly controlled extracellular cues to cells in a physiologically relevant context. Historically, MPSs have heavily relied on elastomeric materials in their manufacture, with unfavorable material characteristics (such as lack of structural rigidity) limiting their use in high-throughput systems. Herein, we aim to create a microfluidic bilayer model (microfluidic MPS) using thermoplastic materials to allow hepatic cell stabilization and culture, retaining hepatic functional phenotype and capturing cellular interactions. The microfluidic MPS consists of two overlapping microfluidic channels separated by a porous tissue-culture membrane that acts as a surface for cellular attachment and nutrient exchange; and an oxygen permeable material to stabilize and sustain primary human hepatocyte (PHH) culture. Within the microfluidic MPS, PHHs are cultured in the top channel in a collagen sandwich gel format with media exchange accomplished through the bottom channel. We demonstrate PHH culture for 7 days, exhibiting measures of hepatocyte stabilization, secretory and metabolic functions. In addition, the microfluidic MPS dimensions provide a reduced media-to-cell ratio in comparison with multi-well tissue culture systems, minimizing dilution and enabling capture of cellular interactions and responses in a hepatocyte-Kupffer coculture model under an inflammatory stimulus. Utilization of thermoplastic materials in the model and ability to incorporate multiple hepatic cells within the system is our initial step towards the development of a thermoplastic-based high-throughput microfluidic MPS platform for hepatic culture. We envision the platform to find utility in development and interrogation of disease models of the liver, multi-cellular interactions and therapeutic responses.

A high-throughput microfluidic microphysiological system (PREDICT-96) to recapitulate hepatocyte function in dynamic, re-circulating flow conditions.

Microphysiological systems (MPSs) are dynamic cell culture systems that provide micro-environmental and external cues to support physiologically relevant, organ-specific functions. Recent progresses in MPS fabrication technologies have enabled the development of advanced models to capture microenvironments with physiological relevance, while increasing throughput and reducing material-based artefacts. In addition to conventional cell culture systems, advanced MPSs are emerging as ideal contenders for disease modeling and incorporation into drug screening. Since liver is a central organ for drug metabolism, liver-on-chip models have been developed to recapitulate hepatic microenvironment with varying complexities, while allowing long-term culture. Recently, we have developed a novel thermoplastic, oxygen-permeable MPS for primary human hepatocyte (PHH) culture. Herein, we have adapted and extended the MPS to a) a 96 microfluidic array (PREDICT-96 array) and b) integrated a novel, ultra-low volume, re-circulating pumping system (PREDICT-96 pump) - collectively known as the PREDICT-96 platform. The PREDICT-96 platform conforms to the industrial standard 96-well footprint and enables media re-circulation. First, we demonstrate the introduction of PHHs into the PREDICT-96 array using standard handling procedures for multi-well plates and allow cells to stabilize in static conditions. Next, we introduce recirculating flow into the bottom channel (using PREDICT-96 pump) to mimic mass transport in vivo. Our results indicate an increase in metabolic and secretory functions of PHHs in the PREDICT-96 platform, and their maintenance over 10 days of flow. Furthermore, long-term culture with fluid flow allows for the periodic introduction of media components (e.g., fatty acids, cytokines) and capture cellular responses to chronic stimuli. The low-volume footprint of the pump and small media volume in the MPS allow for the interrogation of hepatic responses incorporating secretion feedback to a stimulus, which is essential for disease model development and drug interrogation. We envision future development of this liver model to incorporate key primary hepatic cells, multi-cellular co-cultures and adaptation, integration with high-throughput analytical tools.

Discovery and in Vitro Optimization of 3-Sulfamoylbenzamides as ROMK Inhibitors.

Inhibitors of the renal outer medullary potassium channel (ROMK) show promise as novel mechanism diuretics, with potentially lower risk of diuretic-induced hypokalemia relative to current thiazide and loop diuretics. Here, we report the identification of a novel series of 3-sulfamoylbenzamide ROMK inhibitors. Starting from HTS hit 4, this series was optimized to provide ROMK inhibitors with good in vitro potencies and well-balanced ADME profiles. In contrast to previously reported small-molecule ROMK inhibitors, members of this series were demonstrated to be highly selective for inhibition of human over rat ROMK and to be insensitive to the N171D pore mutation that abolishes inhibitory activity of previously reported ROMK inhibitors.

A cross-species translational pharmacokinetic-pharmacodynamic evaluation of core body temperature reduction by the TRPM8 blocker PF-05105679.

PF-05105679 is a moderately potent TRPM8 blocker which has been evaluated for the treatment of cold pain sensitivity. The TRPM8 channel is responsible for the sensation of cold environmental temperatures and has been implicated in regulation of core body temperature. Consequently, blockade of TRPM8 has been suggested to result in lowering of core body temperature. As part of the progression to human studies, the effect of PF-05105679 on core body temperature has been investigated in animals. Safety pharmacology studies showed that PF-05105679 reduced core body temperature in a manner that was inversely related to body weight of the species tested (greater exposure to PF-05105679 was required to lower temperature by 1°C in higher species). Based on an allometric (body weight) relationship, it was hypothesized that PF-05105679 would not lower core body temperature in humans at exposures that could exhibit pharmacological effects on cold pain sensation. On administration to humans, PF-05105679 was indeed effective at reversing the cold pain sensation associated with the cold pressor test in the absence of effects on core body temperature.

Examination of the Human Cytochrome P4503A4 Induction Potential of PF-06282999, an Irreversible Myeloperoxidase Inactivator: Integration of Preclinical, In Silico, and Biomarker Methodologies in the Prediction of the Clinical Outcome.

The propensity for CYP3A4 induction by 2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999), an irreversible inactivator of myeloperoxidase, was examined in the present study. Studies using human hepatocytes revealed moderate increases in CYP3A4 mRNA and midazolam-1'-hydroxylase activity in a PF-06282999 dose-dependent fashion. At the highest tested concentration of 300 µM, PF-06282999 caused maximal induction in CYP3A4 mRNA and enzyme activity ranging from 56% to 86% and 47% t0 72%, respectively, of rifampicin response across the three hepatocyte donor pools. In a clinical drug-drug interaction (DDI) study, the mean midazolam Cmax and area under the curve (AUC) values following 14-day treatment with PF-06282999 decreased in a dose-dependent fashion with a maximum decrease in midazolam AUC0-inf and Cmax of ∼57.2% and 41.1% observed at the 500 mg twice daily dose. The moderate impact on midazolam pharmacokinetics at the 500 mg twice daily dose of PF-06282999 was also reflected in statistically significant changes in plasma 4ß-hydroxycholesterol/cholesterol and urinary 6ß-hydroxycortisol/cortisol ratios. Changes in plasma and urinary CYP3A4 biomarkers did not reach statistical significance at the 125 mg three times daily dose of PF-06282999, despite a modest decrease in midazolam systemic exposure. Predicted DDI magnitude based on the in vitro induction parameters and simulated pharmacokinetics of perpetrator (PF-06282999) and victim (midazolam) using the Simcyp (Simcyp Ltd., Sheffield, United Kingdom) population-based simulator were in reasonable agreement with the observed clinical data. Since the magnitude of the 4ß-hydroxycholesterol or 6ß-hydroxycortisol ratio change was generally smaller than the magnitude of the midazolam AUC change with PF-06282999, a pharmacokinetic interaction study with midazolam ultimately proved important for assessment of DDI via CYP3A4 induction.

Determination of Unbound Partition Coefficient and in Vitro-in Vivo Extrapolation for SLC13A Transporter-Mediated Uptake.

Unbound partition coefficient (Kpuu) is important to an understanding of the asymmetric free drug distribution of a compound between cells and medium in vitro, as well as between tissue and plasma in vivo, especially for transporter-mediated processes. Kpuu was determined for a set of compounds from the SLC13A family that are inhibitors and substrates of transporters in hepatocytes and transporter-transfected cell lines. Enantioselectivity was observed, with (R)-enantiomers achieving much higher Kpuu (>4) than the (S)-enantiomers (<1) in human hepatocytes and SLC13A5-transfected human embryonic 293 cells. The intracellular free drug concentration correlated directly with in vitro pharmacological activity rather than the nominal concentration in the assay because of the high Kpuu mediated by SLC13A5 transporter uptake. Delivery of the diacid PF-06649298 directly or via hydrolysis of the ethyl ester prodrug PF-06757303 resulted in quite different Kpuu values in human hepatocytes (Kpuu of 3 for diacid versus 59 for prodrug), which was successfully modeled on the basis of passive diffusion, active uptake, and conversion rate from ester to diacid using a compartmental model. Kpuu values changed with drug concentrations; lower values were observed at higher concentrations possibly owing to a saturation of transporters. Michaelis-Menten constant (Km) of SLC13A5 was estimated to be 24 µM for PF-06649298 in human hepatocytes. In vitro Kpuu obtained from rat suspension hepatocytes supplemented with 4% fatty acid free bovine serum albumin showed good correlation with in vivo Kpuu of liver-to-plasma, illustrating the potential of this approach to predict in vivo Kpuu from in vitro systems.

Optimization of a Dicarboxylic Series for in Vivo Inhibition of Citrate Transport by the Solute Carrier 13 (SLC13) Family.

Inhibition of the sodium-coupled citrate transporter (NaCT or SLC13A5) has been proposed as a new therapeutic approach for prevention and treatment of metabolic diseases. In a previous report, we discovered dicarboxylate 1a (PF-06649298) which inhibits the transport of citrate in in vitro and in vivo settings via a specific interaction with NaCT. Herein, we report the optimization of this series leading to 4a (PF-06761281), a more potent inhibitor with suitable in vivo pharmacokinetic profile for assessment of in vivo pharmacodynamics. Compound 4a was used to demonstrate dose-dependent inhibition of radioactive [(14)C]citrate uptake in liver and kidney in vivo, resulting in modest reductions in plasma glucose concentrations.

Discovery and characterization of novel inhibitors of the sodium-coupled citrate transporter (NaCT or SLC13A5).

Citrate is a key regulatory metabolic intermediate as it facilitates the integration of the glycolysis and lipid synthesis pathways. Inhibition of hepatic extracellular citrate uptake, by blocking the sodium-coupled citrate transporter (NaCT or SLC13A5), has been suggested as a potential therapeutic approach to treat metabolic disorders. NaCT transports citrate from the blood into the cell coupled to the transport of sodium ions. The studies herein report the identification and characterization of a novel small dicarboxylate molecule (compound 2) capable of selectively and potently inhibiting citrate transport through NaCT, both in vitro and in vivo. Binding and transport experiments indicate that 2 specifically binds NaCT in a competitive and stereosensitive manner, and is recognized as a substrate for transport by NaCT. The favorable pharmacokinetic properties of 2 permitted in vivo experiments to evaluate the effect of inhibiting hepatic citrate uptake on metabolic endpoints.

Discovery and Optimization of Imidazopyridine-Based Inhibitors of Diacylglycerol Acyltransferase 2 (DGAT2).

The medicinal chemistry and preclinical biology of imidazopyridine-based inhibitors of diacylglycerol acyltransferase 2 (DGAT2) is described. A screening hit 1 with low lipophilic efficiency (LipE) was optimized through two key structural modifications: (1) identification of the pyrrolidine amide group for a significant LipE improvement, and (2) insertion of a sp(3)-hybridized carbon center in the core of the molecule for simultaneous improvement of N-glucuronidation metabolic liability and off-target pharmacology. The preclinical candidate 9 (PF-06424439) demonstrated excellent ADMET properties and decreased circulating and hepatic lipids when orally administered to dyslipidemic rodent models.

Discovery of Selective Small Molecule Inhibitors of Monoacylglycerol Acyltransferase 3.

Inhibition of triacylglycerol (TAG) biosynthetic enzymes has been suggested as a promising strategy to treat insulin resistance, diabetes, dyslipidemia, and hepatic steatosis. Monoacylglycerol acyltransferase 3 (MGAT3) is an integral membrane enzyme that catalyzes the acylation of both monoacylglycerol (MAG) and diacylglycerol (DAG) to generate DAG and TAG, respectively. Herein, we report the discovery and characterization of the first selective small molecule inhibitors of MGAT3. Isoindoline-5-sulfonamide (6f, PF-06471553) selectively inhibits MGAT3 with high in vitro potency and cell efficacy. Because the gene encoding MGAT3 (MOGAT3) is found only in higher mammals and humans, but not in rodents, a transgenic mouse model expressing the complete human MOGAT3 was used to characterize the effects of 6f in vivo. In the presence of a combination of diacylglycerol acyltransferases 1 and 2 (DGAT1 and DGAT2) inhibitors, an oral administration of 6f exhibited inhibition of the incorporation of deuterium-labeled glycerol into TAG in this mouse model. The availability of a potent and selective chemical tool and a humanized mouse model described in this report should facilitate further dissection of the physiological function of MGAT3 and its role in lipid homeostasis.