UPLC–MS/MS method with automated on-line SPE for the isomer-specific quantification of the first-generation anti-HCV protease inhibitors in peripheral blood mononuclear cells☆
Graphical abstract
Introduction
To date, HCV infection affects over 170 million people worldwide [1]. For many years, the dual therapy with Pegylated-Interferon α (Peg-IFN α) and ribavirin (RBV) has been the “standard of care”, achieving an overall efficacy close to 50%. However, different viral genotypes show a different response rate to this therapy: genotypes 1 and 4 are less susceptible yielding a 45% of sustained virological response (SVR).
Recently, the deeper knowledge of viral life-cycle allowed the development of new “Directly Acting Agents” (DAAs).
Since 2012, the protease inhibitors (PIs) boceprevir (BOC) and telaprevir (TVR) have been approved in Europe for use in combination with dual therapy and became the new standard of care for HCV-1 infection [2], [3], [4].
Although leading to a higher response (about 75%), these drugs also increased the incidence of adverse events and side effects compared to dual therapy: some of these adverse events can be ascribable to PIs (mainly of gastrointestinal nature), while some others seem to consist in an exacerbation of RBV side-effects (mainly hemolytic anemia).
In recent publications TVR has been demonstrated to increase RBV exposure in a concentration dependent manner, probably through a reduction of glomerular filtration rate, thus leading to anemia [5], [6]. Boceprevir is also associated with a higher rate of anemia, but with a delayed onset and no evidence of reduction in renal function [7].
Up to now, several works described chromatographic methods for their quantification in plasma [8], [9], [10], [11], [12] BOC and TVR plasma pharmacokinetics and [6], [9], highlighting their differences in concentration (BOC concentration seems to be 10 times lower than TVR one). However, the two drugs show a similar efficacy.
Another critical point on the pharmacokinetics of these drugs is the presence of two isomeric forms, which have a different antiviral activity [8], [9].
However, only a few methods can discriminate BOC and TVR isomers in plasma [9], [11], [13].
Furthermore, the real active concentration of the two drugs is that into hepatocytes, then plasma concentrations have to be considered as a “surrogate” to estimate the intracellular exposure. Unfortunately, the withdrawal of hepatocytes is too invasive to be routinely performed. For this reason, the adoption of a closer cellular model could be useful to obtain at least an overview of the compartmentalization of these drugs in an intracellular environment [14].
Then, being peripheral blood mononuclear cells (PBMCs) the easiest to reach in human body, in this work we aimed to develop a UPLC–MS/MS method to achieve a quantification of BOC and TVR isomers in this matrix.
The method has been validated according to FDA guidelines [15].
Section snippets
Chemicals
Acetonitrile HPLC grade and methanol HPLC grade were purchased from J.T. Baker (Deventer, Holland). HPLC grade water was produced with Milli-DI system coupled with a Synergy 185 system by Millipore (Milan, Italy). Boceprevir (BOC, 1:1 mixture of 2 diastereoisomers [active SCH 534128 and inactive SCH 534129]) were purchased from Sequoia Research Chemicals (Pangbourne, UK). Telaprevir’s S and R isomers (TEL-S and TEL-R) were kindly obtained from Janssen (Beers, Belgium). 6,7-Dimethyl-
Specificity, selectivity and carry-over
Typical chromatogram resulting from the injection of STD 9 is reported in Fig. 1.
No co-eluting interfering peaks from endogenous compounds resulted from analysis of blank samples. Just a very low carry-over has been observed, 20- and 10-folds lower than LLOQ for TVR and BOC, respectively (Fig. 2).
Accuracy, precision and limit of quantification
The intra- and inter-day precision and accuracy data for TVR and BOC quantification are summarized in Table 2.
LLOQs were 0.039 and 0.019 for TVR and BOC isomers, respectively, while the LOD were 0.019
Discussion and conclusions
The use of new DAAs for treatment of HCV infection opens the way to new issues which will be the object of many new works in the immediate future.
Up to now, with dual therapy, not enough interest has been given to the pharmacokinetics of anti-HCV drugs, mainly because of the absence of resistance onset by the virus and the inter-patient variability in terms of toxicity and outcome [26], [27].
The adoption of TVR and BOC highlighted the importance of drug pharmacokinetic studies, and then
Conflicts of interest
The authors disclose no conflicts.
Funding
This study was supported by internal founding.
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UNI EN ISO 9001:2008 Certificate Laboratory; Certificate No. IT-64386; Certification for: “Design, Development and Application of Determination Methods for Anti-infective Drugs. Pharmacogenetic Analyses.” www.tdm-torino.org
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Both authors equally contributed to this paper.