A simple and sensitive HPLC-FL method for bioanalysis of velpatasvir, a novel hepatitis C virus NS5A inhibitor, in rat plasma: Investigation of factors determining its oral bioavailability

https://doi.org/10.1016/j.jchromb.2022.123399Get rights and content

Highlights

  • A new bioanalytical HPLC-FL method to determine velpatasvir (VEL).

  • The method offered sufficient sensitivity, comparable to LC-MS/MS methods.

  • VEL exhibited moderate intestinal permeability and significant biliary excretion.

  • VEL was significantly metabolized in the liver, but not in the intestine.

  • Gut absorption and hepatic first-pass metabolism mainly determined the oral bioavailability of VEL.

Abstract

Velpatasvir is a novel inhibitor of hepatitis C virus nonstructural protein 5A that received US Food and Drug Administration approval for the treatment of patients with chronic hepatitis C virus genotypes 1–6. In the present study, a sensitive bioanalytical method for velpatasvir was developed using high-performance liquid chromatography coupled with a fluorescence detector system, which was applied to elucidate the factors determining the oral bioavailability and disposition of velpatasvir. This method offered sufficient sensitivity, with a lower limit of quantification of 0.5 ng/mL, which is comparable to previously reported methods using liquid chromatography coupled with tandem mass spectrometry. Velpatasvir exhibited low oral bioavailability, moderate intestinal permeability, and significant biliary excretion in rats. It was also found to be significantly metabolized in the liver, with a low-to-moderate extraction ratio; however, its intestinal metabolism and enterohepatic circulation did not occur. Thus, our present results demonstrate that the oral bioavailability of velpatasvir is primarily dependent on gut absorption and hepatic first-pass metabolism. The fractions of velpatasvir dose unabsorbed from the gut and eliminated by the liver before reaching the systemic circulation following oral administration were estimated to be 32.8%–58.6% and 4.74%–30.54% of the oral dose, respectively. To our knowledge, this is the first systematic study to investigate the contributory roles of biopharmaceutical and pharmacokinetic factors on the oral bioavailability of velpatasvir, together with a new bioanalytical method for velpatasvir.

Introduction

Hepatitis C virus (HCV), a member of the Flaviviridae family, is a positive-sense, single-stranded RNA virus that has six different genotypes [1]. HCV encodes structural (core, E1, E2, and P7) and non-structural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins that have been recognized as potential antiviral targets against HCV [2], [3]. In particular, HCV NS5A is a pleiotropic phosphoprotein that interacts with several cellular and viral proteins [4]. It plays crucial roles in HCV replication, virion assembly, and modulation of host cell physiology [2], [3]. Velpatasvir (Fig. 1A) is a novel HCV NS5A inhibitor that received US Food and Drug Administration (FDA) approval in 2016 for the treatment of patients with chronic HCV genotypes 1–6 [5], [6]. It exhibits potent antiviral activity, with the EC50 of 0.002–0.024 nM against HCV replicons in all six genotypes [7].

The solubility of velpatasvir decreases as pH increases; velpatasvir is soluble at pH 1.2 and is practically insoluble above pH 4 [8]. It is ≥ 99.5% bound to human plasma proteins; binding is independent of drug concentrations over the range of 0.09 μg/mL to 1.8 μg/mL [9]. Furthermore, velpatasvir undergoes slow metabolism as mediated by cytochrome P450 (CYP) 2B6, 2C8, and 3A4 [9]. Following a single oral administration of velpatasvir in humans, ≥ 98% of the drug present in the plasma was found to be intact (unchanged) form [10]. Velpatasvir is excreted into the bile to a significant extent in rats and dogs; however its urinary excretion is negligible [9], [10]. The mean oral absolute bioavailability (F) of velpatasvir has been reported to be 27.7% in rats and 25.0% in dogs, in which both exhibit relatively high variations [11]. However, these reports mainly provided descriptive pharmacokinetic data, which were insufficient for a comprehensive insight into the fate of velpatasvir in the body after administration. To the best of our knowledge, there have been no systematic or quantitative studies on the contribution of several kinetic factors to the oral absorption and disposition of velpatasvir.

To date, a few analytical methods using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) have been developed to quantify velpatasvir in the plasma [12], [13], [14]. Although these methods provide sufficient sensitivity, with a lower limit of quantitation (LLOQ) of 1–10 ng/mL, LC-MS/MS system generally require expensive and complex instrumentation, facilities, and maintenance procedures with highly skilled technical expertise. In this regard, liquid chromatography coupled with ultraviolet (UV) or fluorescence (FL) detectors can be considered a potential alternative. However, previously reported high-performance liquid chromatography (HPLC) and thin layer chromatography (HPTLC) methods exhibited low sensitivity (LLOQ = 50–697 ng/mL) in determining plasma velpatasvir despite requiring a large sample volume (0.5–1 mL), which therefore may not be a suitable analytical tool for supporting pre-clinical and clinical pharmacokinetic studies [15], [16], [17]. Thus, there is still a need for more accessible and cost-effective analytical methods that can provide sufficient performance for the bioanalysis of velpatasvir.

Therefore, based on the fact that FL detectors generally offer better sensitivity and selectivity than UV detectors in HPLC analysis for many fluorescent compounds [18], [19], [20], [21], we attempted to develop a sensitive HPLC-FL method for the bioanalysis of velpatasvir. The sensitivity and reproducibility of the method were fully validated according to the FDA guidelines. We aimed to investigate the contributory roles of several biopharmaceutical and pharmacokinetic factors in determining the F and systemic disposition of velpatasvir. To this end, in vitro (gut luminal stability, intestinal membrane permeation, and intestinal/hepatic metabolism) and in vivo (biliary excretion, enterohepatic circulation, and plasma pharmacokinetics) studies were comprehensively conducted; the results were then interpreted based on mass balance principles and well-stirred organ clearance concepts.

Section snippets

Materials

Velpatasvir (purity > 99%) was purchased from MedKoo Bioscience Inc. (Morrisville, NC, USA). Celecoxib (Fig. 1A; purity ≥ 98%; used as an internal standard (IS)) was purchased from the Tokyo Chemical Industry Co. (Tokyo, Japan). Polyethylene glycol 400 (PEG 400), dimethyl sulfoxide, magnesium chloride, carboxymethyl cellulose (CMC), and ethanol were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Rat liver S9 (RLS9) and human liver S9 (HLS9) fractions were purchased from BD Genetech

Bioanalytical method development and optimization

Velpatasvir exhibited a relatively strong native fluorescence peak intensity at the excitation/emission wavelength pair of 338 nm/386 nm (Fig. 1B). A few analytical columns, such as YMC-Triart Diol-HILIC column (250 × 2 mm, 1.9 μm, 120 Å; YMC Co., Ltd, Kyoto, Japan), Kinetex® C18 column, SUPELCOSIL™ LC-Diol column (250 × 4.6 mm, 5 μm, 120 Å; Bellefonte, PA, USA), and Zorbax Eclipse XDB-C18 column (125 × 4.6 mm, 5 μm, 80 Å; Agilent, Waldbronn, Germany), were preliminarily tested using a

Discussion

This study aimed to develop a sensitive bioanalytical HPLC-FL method for velpatasvir as well as to elucidate the biopharmaceutical and pharmacokinetic factors determining the F of velpatasvir. Several bioanalytical conditions, such as the stationary phase, mobile phase, IS, and deproteinizing solvent, were optimized to achieve acceptable analyte separation from endogenous plasma interferences. The method validation data presented herein indicate that the proposed method is precise, accurate,

Conclusions

For the first time, a sensitive bioanalytical method for velpatasvir was developed using the HPLC-FL system and applied to elucidate the factors determining the F of velpatasvir. The advantages of the present HPLC-FL method include a simple pretreatment procedure, small sample volume, and sufficient sensitivity which is comparable to that of previously reported LC-MS/MS methods. Velpatasvir exhibited low F and moderate Fabs, and underwent significant biliary excretion in rats. It was revealed

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by a 2-Year Research Grant of Pusan National University.

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