Pharmacokinetics and Drug Metabolism, Amgen Research, Cambridge, Massachusetts (R.S.F., X.B., L.B., D.H., L.H.); Therapeutic Discovery (K.B., J.A., Y.C., J.R.F., C.G., B.H., T.I., J.L., L.P.M., J.M., C.N., T.E.N., K.S., C.M.T., B.W., L.Y.), Neuroscience (B.M.), and Pharmacokinetics and Drug Metabolism (H.L., M.S., L.T.), Amgen Research, Thousand Oaks, California; and Pharmacokinetics and Drug Metabolism, Amgen Research, South San Francisco, California (K.C., D.A.R.)
Pharmacokinetics and Drug Metabolism, Amgen Research, Cambridge, Massachusetts (R.S.F., X.B., L.B., D.H., L.H.); Therapeutic Discovery (K.B., J.A., Y.C., J.R.F., C.G., B.H., T.I., J.L., L.P.M., J.M., C.N., T.E.N., K.S., C.M.T., B.W., L.Y.), Neuroscience (B.M.), and Pharmacokinetics and Drug Metabolism (H.L., M.S., L.T.), Amgen Research, Thousand Oaks, California; and Pharmacokinetics and Drug Metabolism, Amgen Research, South San Francisco, California (K.C., D.A.R.).
Drug Metab Dispos. 2019 Oct;47(10):1111-1121. doi: 10.1124/dmd.119.087742. Epub 2019 Aug 6.
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.
近年来，人们对寻找非阿片类药物替代物来治疗慢性疼痛产生了浓厚的兴趣。最近，有报道称工程设计了一系列 Nav1.7 抑制肽-抗体缀合物，本文描述了鉴定新型方法来描述肽缀合物药代动力学特性的临床前研究工作。设计了冷冻保存的 plated mouse hepatocyte 测定法来测量肽-抗体缀合物从培养基中的耗竭情况，并且观察到在介质中剩余的百分比与体内清除率之间存在相关性（Pearson r = -0.5525）。生成了物理化学性质（电荷和疏水性）、受体结合[新生 Fc 受体（FcRn）]和体内药代动力学数据，并与我们的体外肝细胞测定结果进行了比较，该测定法被假设包含了所有上述特性。观察到疏水性、FcRn 结合、肝细胞测定中耗竭率之间存在相关性；最终与体内清除率存在相关性。随后的研究确定了低密脂蛋白和甘露糖/半乳糖受体在 Nav1.7 肽缀合物与小鼠肝细胞之间的关联中的潜在作用，尽管体内研究表明 FcRn 仍然是决定肽缀合物药代动力学的主要受体。最终，使用冷冻保存的肝细胞测定法以及 FcRn 结合和疏水性相互作用色谱法提供了一种快速筛选前体分子的有效且集成的方法，同时减少了体内药代动力学研究的数量。意义：尽管在小分子药物发现中有多种体外和计算工具可用，但蛋白质治疗剂的药代动力学特征仍然高度依赖于在临床前物种中进行体内研究。目前的工作展示了在进行体内研究之前，使用冷冻保存的肝细胞、疏水性相互作用色谱法和新生 Fc 受体结合来鉴定一系列 Nav1.7 肽-抗体缀合物，从而提供了一种快速评估新型蛋白质治疗平台的方法，同时减少了在临床前物种中进行的体内研究数量。
