Use of Cryopreserved Hepatocytes as Part of an Integrated Strategy to Characterize In Vivo Clearance for Peptide-Antibody Conjugate Inhibitors of Nav1.7 in Preclinical Species.

The identification of nonopioid alternatives to treat chronic pain has received a great deal of interest in recent years. Recently, the engineering of a series of Nav1.7 inhibitory peptide-antibody conjugates has been reported, and herein, the preclinical efforts to identify novel approaches to characterize the pharmacokinetic properties of the peptide conjugates are described. A cryopreserved plated mouse hepatocyte assay was designed to measure the depletion of the peptide-antibody conjugates from the media, with a correlation being observed between percentage remaining in the media and in vivo clearance (Pearson r = -0.5525). Physicochemical (charge and hydrophobicity), receptor-binding [neonatal Fc receptor (FcRn)], and in vivo pharmacokinetic data were generated and compared with the results from our in vitro hepatocyte assay, which was hypothesized to encompass all of the aforementioned properties. Correlations were observed among hydrophobicity; FcRn binding; depletion rates from the hepatocyte assay; and ultimately, in vivo clearance. Subsequent studies identified potential roles for the low-density lipoprotein and mannose/galactose receptors in the association of the Nav1.7 peptide conjugates with mouse hepatocytes, although in vivo studies suggested that FcRn was still the primary receptor involved in determining the pharmacokinetics of the peptide conjugates. Ultimately, the use of the cryopreserved hepatocyte assay along with FcRn binding and hydrophobic interaction chromatography provided an efficient and integrated approach to rapidly triage molecules for advancement while reducing the number of in vivo pharmacokinetic studies. SIGNIFICANCE STATEMENT: Although multiple in vitro and in silico tools are available in small-molecule drug discovery, pharmacokinetic characterization of protein therapeutics is still highly dependent upon the use of in vivo studies in preclinical species. The current work demonstrates the combined use of cryopreserved hepatocytes, hydrophobic interaction chromatography, and neonatal Fc receptor binding to characterize a series of Nav1.7 peptide-antibody conjugates prior to conducting in vivo studies, thus providing a means to rapidly evaluate novel protein therapeutic platforms while concomitantly reducing the number of in vivo studies conducted in preclinical species.

Applications of parallel synthetic lead hopping and pharmacophore-based virtual screening in the discovery of efficient glycine receptor potentiators.

Glycine receptors (GlyRs) are pentameric glycine-gated chloride ion channels that are enriched in the brainstem and spinal cord where they have been demonstrated to play a role in central nervous system (CNS) inhibition. Herein we describe two novel classes of glycine receptor potentiators that have been developed using similarity- and property-guided scaffold hopping enabled by parallel synthesis and pharmacophore-based virtual screening strategies. This effort resulted in the identification of novel, efficient and modular leads having favorable in vitro ADME profiles and high CNS multi-parameter optimization (MPO) scores, exemplified by azetidine sulfonamide 19 and aminothiazole sulfone (ent2)-20.

Species differences in distribution and prediction of human V(ss) from preclinical data.

Prediction of human volume of distribution at steady state (V(ss)) before first administration of a new drug candidate to humans has become an important part of the drug development process. This study examines the assumptions behind interspecies scaling techniques used to predict human V(ss) from preclinical data, namely the equivalency of V(ss,u) and/or f(ut) across species. In addition, several interspecies scaling techniques are evaluated side by side using a set of 67 reference compounds where observed V(ss) from rats, dogs, monkeys, and humans were compiled from the literature and where plasma protein binding was determined across species using an ultracentrifugation technique. Species similarity in V(ss,u) or f(ut) does not appear to be the norm among rats, dogs, monkeys, or humans. Despite this, interspecies scaling from rats, dogs, and monkeys is useful and can provide reasonably accurate predictions of human V(ss), although some interspecies scaling approaches were better than others. For example, the performance of the common V(ss,u) or f(ut) equivalency approaches using average V(ss,u) or f(ut) across three preclinical species was superior to allometric scaling techniques. In addition, considering data from several preclinical species, using the equivalency approach, was superior to scaling from any single species. Although the mechanistic tissue composition equations available in the Simcyp population-based pharmacokinetic simulator did not necessarily provide the most accurate predictions, and the equations used likely need refinement, they still provide the best opportunity for a mechanistic understanding and prediction of human V(ss).

Esterase activities in the blood, liver and intestine of several preclinical species and humans.

Species and tissue differences in the activity of three major classes of esterases, carboxylesterase (CE), butyrylcholinesterase (BChE) and paraoxonase (PON), were studied. Substantial species differences in activity of these esterases were observed between the mouse, rat, dog monkey and human. Such species differences must be considered when using these preclinical species to optimize the pharmacokinetic properties of ester compounds intended for human use.

Evaluation of a series of naphthamides as potent, orally active vascular endothelial growth factor receptor-2 tyrosine kinase inhibitors.

We have previously shown N-arylnaphthamides can be potent inhibitors of vascular endothelial growth factor receptors (VEGFRs). N-Alkyl and N-unsubstituted naphthamides were prepared and found to yield nanomolar inhibitors of VEGFR-2 (KDR) with an improved selectivity profile against a panel of tyrosine and serine/threonine kinases. The inhibitory activity of this series was retained at the cellular level. Naphthamides 3, 20, and 22 exhibited good pharmacokinetics following oral dosing and showed potent inhibition of VEGF-induced angiogenesis in the rat corneal model. Once-daily oral administration of 22 for 14 days led to 85% inhibition of established HT29 colon cancer and Calu-6 lung cancer xenografts at doses of 10 and 20 mg/kg, respectively.