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.

From fragment screening to in vivo efficacy: optimization of a series of 2-aminoquinolines as potent inhibitors of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1).

Using fragment-based screening of a focused fragment library, 2-aminoquinoline 1 was identified as an initial hit for BACE1. Further SAR development was supported by X-ray structures of BACE1 cocrystallized with various ligands and molecular modeling studies to expedite the discovery of potent compounds. These strategies enabled us to integrate the C-3 side chain on 2-aminoquinoline 1 extending deep into the P2' binding pocket of BACE1 and enhancing the ligand's potency. We were able to improve the BACE1 potency to subnanomolar range, over 10(6)-fold more potent than the initial hit (900 µM). Further elaboration of the physical properties of the lead compounds to those more consistent with good blood-brain barrier permeability led to inhibitors with greatly improved cellular activity and permeability. Compound 59 showed an IC(50) value of 11 nM on BACE1 and cellular activity of 80 nM. This compound was advanced into rat pharmacokinetic and pharmacodynamic studies and demonstrated significant reduction of Aß levels in cerebrospinal fluid (CSF).