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

Abstract

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 5 20.5525). 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 lowdensity 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 smallmolecule 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.

An in Vitro Assay Using Cultured Kupffer Cells Can Predict the Impact of Drug Conjugation on in Vivo Antibody Pharmacokinetics

“…Although it has been recognized for many years that ADC design can profoundly impact pharmacokinetics, antitumor activity, and tolerability, the ADC field has lacked in vitro assays to predict in vivo pharmacokinetics . This lack of early discovery ADME tools has put researchers in the ADC field at a disadvantage relative to their colleagues who develop small molecules, in light of the long history of in vitro systems to predict the intrinsic metabolic clearance of these drugs .…”

“…Although it has been recognized for many years that ADC design can profoundly impact pharmacokinetics, antitumor activity, and tolerability, the ADC field has lacked in vitro assays to predict in vivo pharmacokinetics. 25 This lack of early discovery ADME tools has put researchers in the ADC field at a disadvantage relative to their colleagues who develop small molecules, in light of the long history of in vitro systems to predict the intrinsic metabolic clearance of these drugs. 3 Previously, we reported that the apparent hydrophobicity of an ADC, as judged by its retention time from a hydrophobic interaction chromatography (HIC) column, correlated reasonably well with its plasma clearance in a rat pharmacokinetic study.…”

Pharmacokinetic and Drug Metabolism Properties of Novel Therapeutic Modalities

“…In vitro assays that can provide information on the in vivo pharmacokinetic behavior of a molecule prior to conducting in vivo studies are crucial to informing the design of new modalities and can increase the speed at which new modalities reach clinical trials (Xu and Vugmeyster, 2012;Pearson and Rock, 2015). One such assay described in this issue is a novel in vitro assay by Foti et al that informs how new chemical entities such as peptide-antibody conjugates will behave in vivo and provides a basis on which to prioritize molecules for in vivo studies (Foti et al, 2019). Advancements in the sensitivities of bioanalytical techniques have also enhanced the ability to measure drug at the site of action (Foti et al, 2015).…”