ABSTRACT
Approximately 75% of xenobiotics are primarily eliminated through metabolism; thus the accurate scaling of metabolic clearance is vital to successful drug development. Yet, when data is scaled from in vitro to in vivo, hepatic metabolic clearance, the primary source of metabolism, is still commonly underpredicted. Over the past decades, with biophysics used as a key component to restore aspects of the in vivo environment, several new cell culture settings have been investigated to improve hepatocyte functionalities. Most of these studies have focused on shear stress, i.e., flow mediated by a pressure gradient. One potential conclusion of these studies is that hepatocytes are naturally "mechanosensitive," i.e., they respond to a change in their biophysical environment. We demonstrate that hepatocytes also respond to an increase in hydrostatic pressure that, we suggest, is directly linked to the lobule geometry and vessel density. Furthermore, we demonstrate that hydrostatic pressure improves albumin production and increases cytochrome P-450 (CYP) 1A2 expression levels in an aryl hydrocarbon-dependent manner in human hepatocytes. Increased albumin production and CYP function are commonly attributed to the impacts of shear stress in microfluidic experiments. Therefore, our results highlight evidence of a novel link between hydrostatic pressure and CYP metabolism and demonstrate that the spectrum of hepatocyte mechanosensitivity might be larger than previously thought.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Cytochrome P-450 CYP1A2/genetics , Liver/metabolism , Mechanotransduction, Cellular/genetics , Receptors, Aryl Hydrocarbon/genetics , Cell Culture Techniques , Gene Expression Regulation/genetics , Hep G2 Cells , Hepatocytes/metabolism , Humans , Hydrostatic Pressure , Inactivation, Metabolic/genetics , Liver/drug effects , Signal Transduction/geneticsABSTRACT
Systematic optimisation of a poorly soluble lead series of isoxazole-3-carboxamides was conducted. Substitution of the 4-position with specific polar functionality afforded the requisite balance of potency, solubility and physicochemical properties. Compound 21a was found to be efficacious in the rat Capsaicin Hargreaves assay following oral administration.
Subject(s)
Cyclohexanols/chemistry , Isoxazoles/chemistry , TRPV Cation Channels/antagonists & inhibitors , Administration, Oral , Amides/chemistry , Amides/pharmacokinetics , Amides/therapeutic use , Animals , Capsaicin/toxicity , Cyclohexanols/pharmacokinetics , Cyclohexanols/therapeutic use , Disease Models, Animal , Drug Evaluation, Preclinical , Humans , Hyperalgesia/chemically induced , Hyperalgesia/drug therapy , Isoxazoles/pharmacokinetics , Isoxazoles/therapeutic use , Microsomes, Liver/metabolism , Rats , Structure-Activity Relationship , TRPV Cation Channels/metabolismABSTRACT
Optimization of a water soluble, moderately potent lead series of isoxazole-3-carboxamides was conducted, affording a compound with the requisite balance of potency, solubility and physicochemical properties for in vivo use. Compound 8e was demonstrated to be efficacious in a rat model of inflammatory pain, following oral administration.
Subject(s)
Isoxazoles/chemistry , TRPV Cation Channels/antagonists & inhibitors , Administration, Oral , Amides/chemical synthesis , Amides/chemistry , Amides/therapeutic use , Animals , Disease Models, Animal , Humans , Isoxazoles/chemical synthesis , Isoxazoles/therapeutic use , Pain/drug therapy , Rats , TRPV Cation Channels/metabolismABSTRACT
Optimisation of a screening hit incorporating both TRPV1 activity and solubility was conducted. Substitution of the isoxazole-3-carboxamide with the bespoke 1S, 3R-3-aminocyclohexanol motif afforded the requisite balance of potency and solubility. Compounds 32 and 40 were found to have antihyperalgesic effects in the rat CFA Hg assay and induce a mechanism based hyperthermia.