Measurement of total and unbound bictegravir concentrations in plasma and cerebrospinal fluid by UHPLC-MS/MS
Graphical abstract
Introduction
Bictegravir is a novel integrase strand transfer inhibitor (INSTI) with in vitro antiviral activity against wild-type human immunodeficiency virus-1 (HIV-1), HIV-1 with INSTI-resistance-associated mutations, and all HIV-subtypes [[1], [2], [3]]. Bictegravir (BIC) inhibits HIV-integrase by binding to the integrase active site and blocking the strand transfer step of retroviral DNA integration, which is essential for the HIV replication cycle [2]. Also, BIC displays a high resistance barrier and has an improved phenotypic resistance profile compared to other INSTIs as dolutegravir (DTG), raltegravir (RAL) and elvitegravir (EVG) [2,[4], [5], [6]]. BIC has been recently approved, in co-formulation with tenofovir alafenamide (TAF) and emtricitabine (FTC) (Bictarvy®), for the treatment of HIV-1 infection in antiretroviral naive-subjects, and in those subjects with suppressed viremia [[4], [5], [6]].
Bictegravir, (2R,5S,13aR)-7,9-dioxo-10-[(2,4,6-trifluorobenzyl)carbamoyl]-2,3,4,5,7,9,13,13a-octahydro-2,5-methanopyrido[1',2':4,5]pyrazino[2,1-b][1,3]oxazepin-8-olate (Fig.1), differs from other INSTI drugs because it contains a bridged bicyclic ring and a distinct benzyl tail with a 2,4,6-trifluorobenzyl chemical group. These chemical changes contribute to an increase in plasma protein binding (>99%) and a reduction of activation of pregnane X receptor minimizing BIC interactions with other drugs, decreasing its clearance, and increasing its solubility. These characteristics are crucial determinants of drug penetration into certain compartments in the human body, e.g. the central nervous system (CNS), where is a possible source of continued viral replication in persons who have undetectable levels of HIV-RNA in plasma [[1], [2], [3]]. The study of CNS via cerebrospinal fluid (CSF) sampling and the measurement of antiretroviral (ARV) drugs concentrations in this fluid can inform whether the pharmacokinetic (PK) and pharmacodynamic (PD) conditions may allow ongoing viral replication and avoid HIV-related neurocognitive disorders. Moreover, BIC is extensively bound to albumin and α1-acid glycoprotein. Thus, in populational PK/PD analysis of BIC using total drug concentrations, low or high plasma or CSF concentrations of albumin or α1-acid glycoprotein could be associated with low clearance of BIC and diminish its antiretroviral efficacy. Therefore, it might be important to measure concentrations of unbound BIC when investigating its PK and PD.
As far as we know, only three specific development and validation studies have been published for measurement of total BIC concentrations in human plasma by high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS/MS) [[7], [8], [9]]. Still, none has been published to measure unbound BIC concentrations in plasma, as well as total and unbound BIC concentrations in CSF.
This study aimed to develop and validate ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) procedures for the measurement of total and unbound BIC mass concentrations in plasma and CSF. The application of these measurement procedures was demonstrated processing plasma, ultrafiltrate-plasma, CSF, and ultrafiltrate-CSF samples from HIV-1 subjects undergoing Bictarvy®-based antiretroviral therapy.
Section snippets
Chemical, materials and reagents
Bictegravir reference materials (purity of 98%) and Bictegravir-15N d2 (purity of 96.0%, isotopic purity of 96.6%) were purchased from Toronto Research Chemicals (Ontario, Canada).
LC-MS-grade acetonitrile, dimethyl sulfoxide (DMSO), formic acid, methanol, water, and Centrifree® Ultrafiltration Devices (with an Ultracel YM-T regenerated cellulose membrane 30,000 NMWL units) were supplied by Merck Millipore Group (Darmstadt, Germany).
Bictegravir-blank plasma and CSF samples collection
Bictegravir-blank human plasma and CSF were obtained from blood
Application to biological samples and incurred sample reanalysis
The UHPLC-MS/MS procedures were developed and validated to measure total and unbound BIC in human plasma and CSF to support an investigational study about the CNS effects of the BIC in HIV-1 subjects receiving FTC/TAF/BIC (Biktarvy®) ARV therapy. This study has been recently carried on in a single-centre [15] according to the principles of the Declaration of Helsinki and after the approval of the local Ethics Committee of our hospital.
Sample pretreatment
Given that BIC has high plasma protein-binding and throughput reasons of our laboratory, we only considered using a protein precipitation extraction procedure although this type of extraction is not the most appropriate procedure to prevent the matrix effects. Despite this, in our evaluation, a simple protein precipitation with acetonitrile provided acceptable recoveries and matrix effect results. On the other hand, the low amount of subject sample (100 μL) required for measurement of BIC
Conclusions
In conclusion, we developed and validated UHPLC-MS/MS procedures to measure concentrations of total and unbound BIC in plasma and CSF. These procedures include an appropriate and relative fast ultrafiltration procedure to separate the protein-bound BIC from the unbound BIC in plasma and CSF, a simple sample preparation based on protein precipitation, and a short run-time that resulted in adequate performance characteristics. Thus, these procedures are suited for research purposes and, in
Conflict of Interest
The authors declare that they have no potential conflict of interest.
Acknowledgements
We thank all the patients who volunteered for this study.
References (20)
- et al.
Initial antiretroviral therapy in an integrase inhibitor era: can we do better?
Infect. Dis. Clin. North. Am.
(2019) - et al.
Co-formulated bictegravir, emtricitabine, and tenofovir alafenamide versus dolutegravir with emtricitabine and tenofovir alafenamide for initial treatment of HIV-1 infection: week 96 results from a randomised, double-blind, multicentre, phase 3, non-inferiority trial
Lancet HIV.
(2019) - et al.
Bictegravir, emtricitabine, and tenofovir alafenamide versus dolutegravir, abacavir, and lamivudine for initial treatment of HIV‐1 infection (GS‐US‐380‐1489): a double‐blind, multicentre, phase 3, randomised controlled non‐inferiority trial
Lancet.
(2017) - et al.
A mass balance approach for calculation of recovery and binding enables the use of ultrafiltration as a rapid method for measurement of plasma protein binding for even highly lipophilic compounds
J. Pharm. Biomed. Anal.
(2013) - et al.
Challenges regarding analysis of unbound fraction of highly bound protein antiretroviral drugs in several biological matrices: lack of harmonisation and guidelines
Drug Discov. Today
(2015) - et al.
Bictegravir
Curr. Opin. HIV AIDS.
(2018) - et al.
Antiviral activity of bictegravir (GS-9883), a novel potent HIV-1 integrase strand transfer inhibitor with an improved resistance profile
Antimicrob. Agents Chemother.
(2016) Bictegravir/Emtricitabine/Tenofovir Alafenamide: A review in HIV-1 infection
Drugs.
(2018)- et al.
Development and validation of LC–MS/MS method for quantification of bictegravir in human plasma and its application to an intracellular uptake study
Biomed. Chromatogr.
(2019) - et al.
Bioanalytical method development and validation for simultaneous determination of bictegravir, tenofovir and emtricitabine in human plasma by LC-MS/MS
World J. Pharm. Pharmac. Sci.
(2018)
Cited by (13)
Tandem mass spectrometry of small-molecule antiviral drugs: 1. HIV-related antivirals
2020, International Journal of Mass SpectrometryAntiretroviral drugs from multiple classes induce loss of excitatory synapses between hippocampal neurons in culture
2024, Frontiers in PharmacologyComprehensive Review on Different Analytical Techniques for HIV 1- Integrase Inhibitors: Raltegravir, Dolutegravir, Elvitegravir and Bictegravir
2024, Critical Reviews in Analytical ChemistryCentral nervous system disorders after use of dolutegravir: evidence from preclinical and clinical studies
2023, Pharmacological Reports