Daniel R. Mudra

Absorption barriers in the rat intestinal mucosa. 3: Effects of polyethoxylated solubilizing agents on drug permeation and metabolism.

Modern drug discovery chemical libraries contain a large number of molecular entities exhibiting low aqueous solubility, often necessitating the inclusion of solubilizing agents in preclinical models of absorption or metabolism. The objective of the present study was to investigate the effects of several commonly used polyethoxylated solubilizing agents on P450 (CYP) 3A and P-glycoprotein (P-gp) in the rat intestinal mucosa. Atenolol and verapamil were administered in the in situ perfused rat intestine or incubated with rat intestinal microsomes in the presence or absence of polyethylene glycol (PEG) 400 (2% or 20%, v/v) D-alpha-tocopheryl polyethylene glycol-1000 succinate (TPGS; 100 microg/mL), Cremophor EL (47.5 microg/mL) or polysorbate (Tween) 80 (25 microg/mL). Effects on the absorption of unchanged drug were minimal, with the exception of Tween 80 which caused a 5.0-fold increase in paracellular absorption. Rat intestinal CYP3A was significantly inhibited by PEG-400 and in situ, exceeded inhibition observed with ketoconazole. Cremophor and TPGS increased the fraction of norverapamil in the plasma, consistent with excipient-mediated inhibition of P-gp. These results suggest that caution be exercised when these solubilizing agents are included in preclinical oral dosing solutions as the perturbation of drug absorption barriers may heighten the risk of incorrectly classifying drug candidate PK-parameters.

Absorption barriers in the rat intestinal mucosa: 1. application of an in situ perfusion model to simultaneously assess drug permeation and metabolism

Modulation of intestinal drug absorption barriers can have a profound impact on the bioavailability of orally administered compounds. With its commonality of use as an absorption model, it is valuable to assess the role of such barriers in the rat intestinal mucosa. In the present study, atenolol and verapamil were concomitantly delivered in the in situ perfused rat intestine in the presence or absence of inhibitors to simultaneously assess the function and modulation of passive diffusion barriers, cytochrome P450 (CYP)3A metabolism and P-glycoprotein (P-gp) efflux. A high performance liquid chromatography-tandem mass spectrometry method measured atenolol, verapamil and the CYP3A-mediated metabolite, norverapamil, with linearity (r(2) > 0.99), precision (CV <or=7.5%) and accuracy (+/-17%). Absorption of parent drug was independent of verapamil concentration; however the formation and disposition of norverapamil were concentration-dependent and saturable. Norverapamil formation decreased (up to 80%) in the presence of CYP3A inhibitors and the fraction of norverapamil observed in the plasma was increased (4.5- to 7.2-fold) in the presence of P-gp inhibitors. These results suggest that in this model of the rat intestinal mucosa, atenolol serves as a marker for diffusion barriers whereas norverapamil formation and disposition are markers of CYP3A and P-gp, respectively.

Novel Autotaxin Inhibitors for the Treatment of Osteoarthritis Pain: Lead Optimization via Structure-Based Drug Design

In an effort to develop a novel therapeutic agent aimed at addressing the unmet need of patients with osteoarthritis pain, we set out to develop an inhibitor for autotaxin with excellent potency and physical properties to allow for the clinical investigation of autotaxin-induced nociceptive and neuropathic pain. An initial hit identification campaign led to an aminopyrimidine series with an autotaxin IC50 of 500 nM. X-ray crystallography enabled the optimization to a lead compound that demonstrated favorable potency (IC50 = 2 nM), PK properties, and a robust PK/PD relationship.

Absorption Barriers in the Rat Intestinal Mucosa: 2. Application of Physiologically Based Mathematical Models to Quantify Mechanisms of Drug Permeation and Metabolism

The absorption of drug molecules is often investigated using in vitro or in situ models of the intestinal mucosa; however, few studies have quantified the kinetics that limit absorption. The objective of this study was to quantify kinetic rates of rat intestinal absorption, metabolism, and efflux using nonlinear mixed effects modeling. A multicompartment model accurately described the absorption and distribution of atenolol and verapamil as well as the metabolism of verapamil and distribution of the metabolite, norverapamil. The accurate description of atenolol data required inclusion of an intermediate compartment in addition to paracellular clearance, whereas verapamil and norverapamil were modeled in the absence of paracellular clearance. The absorption of verapamil was well characterized by linear kinetics, whereas the formation and distribution of norverapamil were well characterized by Michaelis-Menten kinetics. The model identified EDTA as a modulator of physical barriers, ketoconazole as an inhibitor of cytochrome P450 3A and P-glycoprotein (P-gp), and PSC-833 and GF-120918 as specific P-gp inhibitors. These results demonstrate the utility of a physiologically based model to characterize (i) the drug distribution across the in situ perfused rat intestine and (ii) the effect of chemical modulators in this biological system.

Identification and biological activity of 6-alkyl-substituted 3-methyl-pyridine-2-carbonyl amino dimethyl-benzoic acid EP4 antagonists.

Continued SAR optimization of a series of 3-methylpyridine-2-carbonyl amino-2,4-dimethyl-benzoic acid led to the selection of compound 4f for clinical studies. Compound 4f showed an IC50 of 123nM for inhibition of PGE2-induced TNFα reduction in an ex vivo LPS-stimulated human whole blood assay (showing >10-fold increase over clinical compound CJ-023,423). Pharmacokinetic profile, selectivity and in vivo efficacy comparing 4f to NSAID diclofenac in the monoiodoacetic acid (MIA) pain model and adjuvant induced arthritis (AIA) inflammatory model are included.

Use of Osmotic Pumps to Establish the Pharmacokinetic–Pharmacodynamic Relationship and Define Desirable Human Performance Characteristics for Aggrecanase Inhibitors

The development of reliable relationships between in vivo target engagement, pharmacodynamic activity, and efficacy in chronic disease models is beneficial for enabling hypothesis-driven drug discovery and facilitating the development of patient-focused candidate selection criteria. Toward those ends, osmotic infusion pumps can be useful for overcoming limitations in the PK properties of proof-of-concept (POC) compounds to accelerate the development of such relationships. In this report, we describe the application of this strategy to the development of hydantoin-derived aggrecanase inhibitors (eg, 3) for the treatment of osteoarthiritis (OA). Potent, selective inhibitors were efficacious in both chemical and surgical models of OA when exposures were sustained in excess of 10 times the plasma IC50. The use of these data for establishing patient-focused candidate selection criteria is exemplified with the characterization of compound 8, which is projected to sustain the desired level of target engagement at a dose of 45 mg qd.

Discovery of potent aryl-substituted 3-[(3-methylpyridine-2-carbonyl) amino]-2,4-dimethyl-benzoic acid EP4 antagonists with improved pharmacokinetic profile.

Two new series of EP4 antagonists containing a 3-methylaryl-2-carbonyl core have been identified. One series has a 3-substituted-phenyl core, while the other one incorporates a 3-substituted pyridine. Both series led to compounds with potent activity in functional and human whole blood (hWB) assays. In the pyridine series, compound 7a was found to be a highly potent and selective EP4 antagonist, with suitable rat and dog pharmacokinetic profiles.

Discovery of substituted-2,4-dimethyl-(naphthalene-4-carbonyl)amino-benzoic acid as potent and selective EP4 antagonists.

A novel series of EP4 antagonists, based on a quinoline scaffold, has been discovered. Medicinal chemistry efforts to optimize the potency of the initial hit are described. A highly potent compound in a clinically relevant human whole blood assay was identified. Selectivity and pharmacokinetic profiles of this compound are discussed.

A Highly Selective Hydantoin Inhibitor of Aggrecanase-1 and Aggrecanase-2 with a Low Projected Human Dose

Aggrecanase-1 and -2 (ADAMTS-4 and ADAMTS-5) are zinc metalloproteases involved in the degradation of aggrecan in cartilage. Inhibitors could provide a means of altering the progression of osteoarthritis. We report the identification of 7 which had good oral pharmacokinetics in rats and showed efficacy in a rat chemical model of osteoarthritis. The projected human dose required to achieve sustained plasma levels ≥10 times the hADAMTS-5 IC50 is 5 mg q.d.

Discovery and characterization of [(cyclopentyl)ethyl]benzoic acid inhibitors of microsomal prostaglandin E synthase-1.

We describe a novel class of acidic mPGES-1 inhibitors with nanomolar enzymatic and human whole blood (HWB) potency. Rational design in conjunction with structure-based design led initially to the identification of anthranilic acid 5, an mPGES-1 inhibitor with micromolar HWB potency. Structural modifications of 5 improved HWB potency by over 1000×, reduced CYP2C9 single point inhibition, and improved rat clearance, which led to the selection of [(cyclopentyl)ethyl]benzoic acid compound 16 for clinical studies. Compound 16 showed an IC80 of 24nM for inhibition of PGE2 formation in vitro in LPS-stimulated HWB. A single oral dose resulted in plasma concentrations of 16 that exceeded its HWB IC80 in both rat (5mg/kg) and dog (3mg/kg) for over twelve hours.