WO2018172759A1

WO2018172759A1 - Method for identification of sofosbuvir resistant patients

Info

Publication number: WO2018172759A1
Application number: PCT/GB2018/050718
Authority: WO
Inventors: Graham Foster
Original assignee: Queen Mary University of London
Current assignee: Queen Mary University of London
Priority date: 2017-03-20
Filing date: 2018-03-20
Publication date: 2018-09-27
Anticipated expiration: 2019-09-20
Also published as: GB201704386D0

Abstract

The present invention provides a method for the identification of a subject infected with a Hepatitis C VPirus (HCV) and resistant to treatment with sofosbuvir, comprising determining the presence of one or more mutations in the NS5B protein of the hepatits C virus selected from the group consisting: A150V, I85V, K206E, K100R, A150S, G188D, T213N and N244I. The invention provides an oligonucleotide probe or set of oligonucleotide probes for use in such a method. Also provided are methods of treatment for a patient infected with HCV identified by a method of the invention and anti-HCV drugs for use in such methods.

Description

METHOD FOR IDENTIFICATION OF SOFOSBUVIR RESISTANT PATIENTS

The present invention relates to a method for the identification of patients receiving treatment for Hepatitis C Virus infection who are resistant to sofosbuvir.

Infection with genotype 3 Hepatitis C virus (HCV) is common, consisting of approximately 40% of HCV infections. The HCV genome is a positive sense single-stranded RNA molecule consisting of a 9600 base pair long single open-reading frame (ORF). Translation of the single ORF of HCV yields a single protein product which can then be processed to produce smaller active proteins that allow viral replication within a host cell or permit assembly of viral particles, for example core proteins E1 and E2, and non-structural proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B.

Sofosobuvir is an inhibitor of the hepatitis C virus polymerase and is the main therapeutic drug for the treatment of genotype 3 HCV. It is usually combined with another drug, usually an NS5A inhibitor such as daclatasvir or velpatasvir and around 90% of patients are cured. It is not clear why some patients do not respond to sofosbuvir based therapies and it is not clear whether re-treating such patients with extended duration of therapy will be beneficial or not. New, sofosbuvir free, antiviral therapies are in development and a test that would identify which patients were unlikely to respond to sofosbuvir would be of clinical value.

For patients with genotype 3 HCV who have not responded to sofosbuvir (many tens of thousands per year) testing prior to re-treatment is likely to be recommended given that therapy is around US$40,000- US$50,000. A pretreatment test costing US$1000 that avoided futile therapy would be financially very attractive and be highly cost effective.

According to a first aspect of the present invention, there is provided a method for the identification of a subject infected with a hepatitis C virus (HCV) and resistant to treatment with sofosbuvir, comprising determining the presence of one of more mutations in the NS5B protein of hepatits C virus selected from the group consisting: A150V, I85V, K206E, K100R, A150S, G188D, T213N and N244I.

Suitably, the determination may be performed on a biological sample from the subject. The sample may be physioloigcal fluid such as blood, plasma, saliva, urine, tears, milk, or semen, or a biopsy or cell sample, for example a blood cell such as erythrocytes, leucocytes, monocytes, macrophages or a sample of liver tissue. In other embodiments, the sample may be prepared by co-culturing a physioloigcal fluid sample containing HCV from the patient with a cell from a cell line, and then subsequently analysing the HCV captured by the cell. The HCV infection may be a genotype 3 HCV infection or co-infection with multiple genotypes of HCV.

The subject to be identified according to the methods of the invention may be a human subject. The subject according to the first aspect of the invention may also be referred to as a patient. The method of the invention may suitably be practiced in various embodiments. Some suitable mutations or combinations of mutations at least are as follows: (i) A150V;

(ii) I85V or in I85V combination with one or more of K206E, A150V, K100R, A150S, G188D. T213N and N244I);

(iii) K206E and A150V; or

(iv) K100R, A150S, G188D, T213N and N244I.

The mutations described herein use the preferred terminology for amino acid substitutions as set out in J. T. den Dunnen and S. E. Antonarakis, Hum. Genet 109(1 ), 121-124 (2001 )), in which the amino acids are identified using the single-letter code. The amino acid which is replaced is indicated first, then the residue number, then followed by the amino acid which is present instead in the mutation.

The mutations described herein in the NS5B protein of hepatits C virus can therefore also be identified as:

In another embodiment of this aspect, the invention may be a method for the identification of a subject infected with a hepatitis C virus (HCV) having one of more mutations in genes present in the NS5B protein of hepatits C virus selected from the group consisting: 185V, K206E, A150V, K100R, A150S, G188D. T213N and N244I.

A reference sequence for genotype 3 HCV may be the sequence shown at https :/7www. neb I . n I m . n i h .qov/n uccore/HW 121682.1 identified as GenBank Accession no. HW121682, Version HW121682.1 and deposited on 13 November 2013 (see Figure 7) and as published in WO 2013/031956.

The locations of the mutations in a HCV viral genome with accompanying protein sequence are shown in Figure 9.

The determination of the presence of one of more mutations in genes present in the NS5B protein of hepatits C virus in the subject selected from the group consisting: I85V, K206E, A150V, K100R, A150S, G188D, T213N and N244I may be completed by sequencing the genome of the HCV in the cells of an infected individual.

More straightforwardly, the invention can also be performed by probing for the presence of one of more of the mutations in genes present in the NS5B protein of hepatits C virus in the subject selected from the group consisting: I85V, K206E, A150V, K100R, A150S, G188D, T213N and N244I.

The mutations in the sequence of the NS5B protein of HCV may be identified using whole genome sequencing or PCR, for example nested PCR. Where PCR is used the oligonucleotide primers may be sequence specific primers selected according to one or more the mutations referred to above. The primer is a synthetic DNA sequence designed according to the considerations set out herein and may be referred to as a cDNA primer. The oligonucleotide may of from 18 to 22 base pairs in length and may be any contiguous sequence of 18 to 22 base pairs selected from the mutated nucleic acid sequences as shown in Figure 8(a), 8(b), 8(c) or Table 1 below (in which the mutated residue is indicated with an underline), or a sequence complementary or homologous thereto.

Table 1

"Identity" as known in the art is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness (homology) between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. While there exist a number of methods to measure identity between two polypeptides or two polynucleotide sequences, methods commonly employed to determine identity are codified in computer programs. Preferred computer programs to determine identity between two sequences include, but are not limited to, GCG program package (Devereux, et al., Nucleic acids Research, 12, 387 (1984), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215, 403 (1990).

Preferably, the nucleic acid sequence of the HCV isolate has at least 50% identity, using the default parameters of the BLAST computer program (Atschul et al., J. Mol. Biol. 215, 403-410 (1990) provided by HGMP (Human Genome Mapping Project), at the nucleic acid level to the sequences described herein. More preferably, the nucleic acid sequence may have at least 60%, 70%, 80%, 90% and still more preferably 95% (still more preferably at least 99%) identity, at the nucleic acid level.

The nucleic acid sequence may also comprise a sequence which has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% identity with a sequence as described herein using the default parameters of the BLAST computer program provided by HGMP.

A capture fusion HCV replication assay as described in WO 2013/064833 and Cunningham ef al (Hepatology, 61 , 1 192-1204 (2015)) can further be used as part of a method of the present invention to identify patients resistant to sofsobuvir.

In brief the capture fusion assay provides a method for replicating HCV virus in vitro which comprises the following steps: (i) fusing an HCV-infected white blood cell with a hepatocyte cell to produce a fused cell; and

(ii) culturing the fused cell so that HCV replication may occur. The HCV-infected white blood cell may be suitably isolated from an HCV patient. The hepatocyte may be from any suitable source of hepatocytes, including hepatocyte cell lines, for example the hepatoma cell line Huh7.5.

Alternatively, the HCV-infected white blood cell can be made by infecting a white blood cell with HCV virus in vitro. The white blood cell may be derivable from a white blood cell (e.g. a monocyte) cell line, for example THP-1 . The white blood cell may be pre-treated with at least one proinflammatory reagent prior to infection in vitro. The white blood cell may be pre-treated with a cocktail of pro-inflammatory reagents. The pro-inflammatory reagent may be, for example, interferon-γ (IFNy) and/or PMA. Alternatively, the pro-inflammatory reagent may be lipopolysaccharide and other Toll Like receptor agonists including flagellin, imiquimod and IL-1 . The white blood cell may be any white blood cell which has the capacity to capture HCV virus. Suitable white blood cell types include: monocytes, polymorphs, macrophages and B cells.

The method may comprise the following steps:

(i) culturing a white blood cell in vitro with serum from an HCV patient, so that the white blood cell is infected with HCV in the serum; and

(ii) fusing the HCV-infected white blood cell with a hepatocyte.

A fused cell or a plurality of cells prepared according to this method can then be used permit analysis of the genome of the HCV in accordance with a method of the present invention.

Detection of a mutated HCV sequence in accordance with the invention may be carried out by any suitable technique. For example, whole genome sequencing may be performed on an isolated viral genomic. However, techniques such as polymerase chain reaction (PCR) may be used conveniently also. Appropriate oligonucleotide probes can be designed to hybridise to the specific mutated HCV sequences referred to herein. Other suitable assays may include dot-blot, ELISA or other antibody based assays also if specific regions of the HCV DNA sequences containing the mutations are translated into protein.

In the methods of this aspect of the invention the subject may be further treated with an anti- Hepatitis C Virus (HCV) drug wherein the drug is not sofosbuvir. The drug may be selected from the group consisiting of daclatasvir, velpatasvir, elbasvir or grazoprevir, or a combination thereof.

According to a second aspect of the invention, there is provided an oligonucleotide probe or a set of oligonucleotide probes having nucleic acid sequences which hybridise to a nucleic acid sequence of Table 1 , or a sequence having 75% identity thereto. In some instances, more than one set of oligonucleotide probes can be used if more than one mutation is to be detected. The probe or set of probes can be used as a primer or set of primers in a method according to the first aspect. According to a third aspect of the invention, there is provided a kit comprising a set of oligonucleotide probes described above. In some instances, more than one set of oligonucleitide probes can be used if more than one mutation is to be detected. The kit of parts may additionally comprise aqueous media for sample preparation, such as isotonic saline.

According to a fourth aspect of the invention, there is provided a method of treatment for a patient infected with Hepatitis C Virus (HCV) comprising the step of administering an anti-HCV drug wherein the drug is not sofosbuvir and wherein the patient has previously been determined to be infected with an HCV strain having one or mutations in the NS5B protein of HCV selected from

(i) A150V;

(ii) I85V or I85V in combination with one or more of K206E, A150V, K100R, A150S, G188D. T213N and N244I);

(iii) K206E and A150V; or

(iv) K100R, A150S, G188D, T213N and N244I.

According to a fifth aspect of the invention, there is provided an anti-Hepatitis C Virus (HCV) drug for use in the treatment of HCV in a patient wherein the drug is not sofosbuvir and the patient is infected with a HCV strain having one or more mutations in the NS5B protein of HCV selected from

(i) A150V;

(i) I85V or I85V in combination with one or more of K206E, A150V, K100R, A150S, G188D. T213N and N244I;

(ii) K206E and A150V; or

(iii) K100R, A150S, G188D, T213N and N244I.

Suitably, the HCV infection is a genotype 3 Hepatitis C virus (HCV) infection. The anti-HCV-drug may be IFN, PEG-IFN, ribavirin, or any HCV direct-acting antiviral (DAA) agent. In the case of a genotype 3 Hepatitis C virus infection the HCV direct-acting antiviral (DAA) agent may be for example daclatasvir, velpatasvir, elbasvir or grazoprevir, or a combination thereof.

Methods in accordance with this aspect of the invention therefore include combinations of anti-HCV drugs which may be administered simultaneously, separately or sequentially to a patient. Such combinations of drugs may therefore extend to the provision of a kit of parts which may additionally comprise an administration vehicle including, but not limited to, tablets for oral administration, inhalers for lung administration and injectable solutions for intravenous administration.

The anti-HCV drug (or drugs) may be administered by any convenient route, for example intravenously, intradermally, intramuscularly, orally or by other routes. In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic. Oral forms of the active substance include controlled-release and/or sustained release table formulations which may be additionally enteric coated.

For administration to humans, it is expected that the daily dosage of the active agent will be from 0.01 mg/kg up to 10mg/kg body weight, typically around 1 mg/kg. The physician in any event will determine the actual dosage which will be most suitable for an individual which will be dependent on factors including the age, weight, sex and response of the individual. The above dosages are exemplary of the average case. There can, of course, be instances where higher or lower dosages are merited, and such are within the scope of this invention

In the examples of the present invention, the sequence of the virus was analysed in patients by full genome sequencing which identified clusters of mutations associated with treatment failure.

Note that of the patients who failed therapy 50% were 'sensitive' to sofosbuvir and previous attempts to identify viral variants associated with treatment failure have been unsuccessful - probably due to the high level of viral variation, the low number of treatment failures and the fact that many 'treatment failures' are not due to sofosbuvir resistance. By applying a novel phenotyping assay combined with sequencing in a method according to the present invention it has been possible to identify previously unrecognised drug resistant variants.

To confirm that the variants are associated with treatment failure a cohort of patients treated with sofosbuvir were examined which found a significant recution in response to therapy in patients whose virus contained the mutations described herein, specifically the A150V mutation. The other mutations were relatively rare and the combinations were present at such low frequency that a full analysis was not possible.

To further confirm that the clusters of mutations were associated with treatment failure the hepatitis C replicon (a model for HCV replication) was manipulated which confirmed that the clusters seen reduce the sensitivity of the virus to sofosbuvir.

The data presented herein show that sofosbuvir resistance is associated with the following mutations or combinations of mutations in the NS5B protein of HCV of at least:-

(i) A150V;

(ϋ) I85V or I85V in combination with one or more of K206E, A150V, K100R, A150S,

G188D. T213N and N244I;

(iii) A150V and K206E; or

(iv) K100R, A150S, G188D, T213N and N244I. Preferred features for the second and subsequent aspects of the invention as for the first aspect mutatis mutandis. An important utility of the present invention is in the treatment of patients who are resistant to sofosbuvir. The invention enables such resistant patients to be detected at an early stage so that the treatment regime can be amended suitably so that the patient then receives a treatment that will be effective. In one embodiment, the invention may comprise

(a) co-culturing a sample of blood plasma from a patient infected with HCV with a population of monocytes

(b) subsequently fusing the monocytes of (a) with cells of a hepatoma cell line

(c) analysing the genotype of the HCV present in the fused cells of (b) by PCR for the present of more mutations in the NS5B protein of HCV selected from:

(i) A150V;

(ii) I85V or I85V in combination with one or more of K206E, A150V, K100R, A150S, G188D, T213N and N244l;

(ii) A150V and K206E; or

(iii) K100R, A150S, G188D, T213N and N244I.

The present invention will now be further described by way of illustration with reference to the accompanying examples which are not to be construed as being limitations to the claimed invention.

Reference is also made to the following drawings in which:

FIGURE 1 shows NS5B Mutations Identified in Drug Insensitive samples. FIGURE 2 shows RBV and SOF sensitivity in G3 treatment non-responders to new DAA therapy. HCV sera from G3 patients (n=15) who achieved SVR or relapsed were used in capture fusion to assay sensitivity to SOF and RBV. Individual IC50 values for SOF (A) and RBV (B) were compared between the SVR and relapse groups. Capture fusion data for SOF and RBV was grouped by clinical outcome into patients who achieved SVR (C, D) or relapsed (E-H). Relapse samples were further divided in to SOF and RBV sensitive (E, F) and insensitive (G, H). IC50 values for each drug represent the average of the indicated number of patients in each group. Graphs show mean ± SEM.

FIGURE 3 shows analysis of NS5B sequences from drug sensitive and insensitive HCV isolates. Schematic of NS5B with indicated amino acid positions. Viral genomes from drug sensitive and insensitive relapse isolates were compared by sequence alignment.

Nucleotide changes causing non-synonymous mutations within NS5B were identified in sequences from drug-insensitive-relapse samples at the amino acid positions indicated. FIGURE 4 shows effect of mutations on SOF sensitivity in a transient replicon assay.

Indicated mutations introduced into the NS5B of the S52-G3 replicon were tested for their sensitivity to increasing concentrations of SOF. Sensitivity of each mutated replicon was compared to the un-modified, wild-type replicon (Wt) and a replicon containing an S282T mutation. The above is representative of 3 independent experiments performed in Huh7.5- SEC14L2 cells. Error bars depict mean ± SEM.

FIGURE 5 shows effect of mutations on RBV sensitivity in a transient replicon assay. Engineered mutations were also tested for sensitivity to RBV. Sensitivity of each replicon was compared to the un-modified, wild-type replicon (Wt) The above is representative of 3 independent experiments performed in Huh7.5-SEC14L2 cell. Error bars depict mean ±

SEM.

FIGURE 6 shows luciferase activity of mutated G3 replicons. Luciferase levels for each replicon were quantified 3 days post electroporation in a similar fashion to drug sensitivity experiments. Luciferase activity was normalised to a sample taken 4 hrs post electroporation. Error bars depict mean ± SEM.

FIGURE 7 shows the reference nucleic acid sequence for HCV from:

https://www.ncbi.nlm.niri.goV/nuccore/HW121682.1

FIGURE 8 shows the mutations found in HCV isolates from patients with resistance to sofosbuvir, Figure 8(a) shows I85V and K206E, Figure 8(b) shows A150V and K206E, and Figure 8(c) shows K100R, A150S, G188D, T213N, and N244I. FIGURE 9 shows the full length nucleic acid sequence and corresponding presumed amino acid coding regions for HCV genotype 3a fui!-iength genome (DBNSacc) with the mutations in the NS5B region identified and stop codons shown by

The S5B region starts at nucleotide residue 7633 and finishes at nucleotide residue 9404 in the genome sequence. The numbering of amino acid residue in the NS5B protein therefore starts from the corresponding initial residue in the DS5B region as shown.

Example 1 : Comparison of Sofosbuvir and ribavirin sensitivity

A comparison of Sofosbuvir and ribavirin sensitivity in pre-treatment samples from patients infected with genotype 3 HCV who did, or did not, achieve SVR was carried out. The 'capture-fusion' assay was used to assess sensitivity of HCV to sofosbuvir and ribavirin from patients with decompensated cirrhosis who relapsed following 12 weeks therapy with sofosbuvir and an NS5A inhibitor as part of the English Early Access Programme (Cheung ef al., Journal of hepatology 65, 741 -747 (2016); Foster et al., Journal of hepatology 64, 1224-1231 (2016)). Stored pre-treatment samples from patients who either relapsed or achieved a sustained virological response (SVR) were obtained from the HCV Research UK (HCVRUK) national biobank and were selected by the biobank on the basis of genotype and viral load >1 x10⁵ lU/ml. (Table 2). Fourteen samples were analysed in a blinded fashion using the 'capture-fusion' replication system as previously described (Cunningham ef al., Hepatology 61 (4), 1 192-1204, (2014)). Following unblinding the IC50s were compared to clinical outcome (Table 1 ). Samples from patients who relapsed were significantly less sensitive than samples from patients who achieved an SVR and two different sensitivity patterns were noted with 7 of 10 samples from patients who relapsed being 'insensitive' to sofosbuvir (arbitrarily defined as IC50 >0.1 M). All patients who were insensitive to sofosbuvir had a reduced sensitivity to ribavirin ((IC50 > 1 .25μΜ) (Figure 2 summarises the dose response curves). Example 2: NS5B Sequencing analysis of HCV samples used in capture fusion

High-throughout, next generation sequencing was performed on all samples analysed. Substitutions were ignored that were present in fewer than 15% of the sequencing reads and only considered sequences with a read frequency of >16%. No known sofosbuvir resistance associated substitutions (L159F, S282T or V321A (Lontok ef al., Hepatology, 62(5) 1623-1632 (2015)) were found. In hypothesis generating experiments it was noted that in the sofosbuvir insensitive samples (IC50 >0.1 μΜ) two distinct patterns were seen with either five (patient 9) or two amino acid changes (patients 1 1 -15). These patterns were not seen in patients who were 'sensitive' to sofosbuvir although individual substitutions, specifically A150V and K206E were observed (Table 1 which shows mutations identified in NS5B). All the RAS were located in Domains 1 and 2 of the polymerase protein (Figure 3).

Example 3: Effect of identified NS5B substitutions on drug sensitivity in a transient replicon assay

Each substitution and combination of substitutions, corresponding to the clinical samples was introduced by site-directed mutagenesis into the S52-SG G3 replicon (Saeed ef al., Antimicrob. Agents Chemother 56(10) 5365-5373 (2012)). Expression of HCV proteins in the mutated replicons was assessed by immunofluorescence of HCV antigens (Figure 4) and the impact on SOF and RBV sensitivity analysed in a transient replicon assay (Witteveldt ef al., Antimicrob. Agents Chemother 60(5) 2981 -2992 (2016)). SOF dose response curves for the mutated replicons were compared to the wild type replicon (Wt) and a control replicon containing the well-described S282T mutation (Table 2 which shows which effect of mutations on sofosbuvir sensitivity). Individually most of the mutations had only a modest effect on sofosbuvir sensitivity but in combination the effect was much more pronounced. Example 4: Prevalence and impact of NS5B mutations in a second patient cohort To further examine the impact of the substitutions, their frequency was examined in a second, independent cohort of patients with genotype 3 HCV receiving sofosbuvir based therapies. Sequences were studied from pre-treatment genotype 3 samples from the BOSON trial (Foster ef al., Gastroenterology 149, 1462-1470 (2015)). The frequency of the identified mutations in patients who did, or did not, achieve an SVR was examined (Table 3 which shows frequency of mutations in patients) shows the results. Individually, each of the identified variants associated with reduced sofosbuvir sensitivity, was rare with the exception of the A150V variant. This variant was present in 37% of patients and in the 204 samples with the V residue a SVR was achieved in 153 patients (75%) compared to a response rate of 168 of 191 (88%) patients with the A variant (p<0.001 ). The combination of A150V and K206E was uncommon (22 patients, 6%) and was associated with a SVR of 82%.

Table 3

Table 4

Table 5

Claims

1. A method for the identification of a subject infected with a hepatitis C virus (HCV) and resistant to treatment with sofosbuvir, comprising determining the presence of one or more mutations in the NS5B protein of the hepatits C virus selected from the group consisting: A150V, I85V, K206E, K100R, A150S, G188D, T213N and N244I.

2. The method of claim 1 wherein the mutations are A150V and K206E.

3. The method of claim 2 wherein the mutation is A150V.

4. The method of claim 1 wherein the mutations are I85V in combination with one or more selected from the list comprising A150V, K206E, K100R, A150S, G188D, T213N and N244I.

5. The method of claim 2 wherein the mutation is I85V.

6. The method of claim 1 wherein the mutations are selected from K100R, A150S, G188D, T213N and N244I.

7. An oligonucleotide probe having a nucleic acid sequence which hybridises to a nucleic acid sequence as follows:

or a sequence having 75% identity thereto.

8. A set of oligonucleotide probes having nucleic acid sequences which hybridise to a nucleic acid sequence as follows:

and one or more nucleic acid sequences selected from:

or a sequence having 75% identity thereto.

9. A set of oligonucleotide probes having nucleic acid sequences which hybridise to nucleic acid sequences as follows:

or a sequence having 75% identity thereto.

10. A set of oligonucleotide probes having nucleic acid sequences which hybridise to a nucleic acid sequence as follows:

or a sequence having 75% identity thereto.

1 1. A kit comprising an oligonucleotide probe of claim 7 or a set of oligonucleotide probes as claimed in any one of claims 8 to 10 and comprising sample preparation media.

12. A method of treatment for a patient infected with Hepatitis C Virus (HCV) comprising the step of administering an anti-HCV drug wherein the drug is not sofosbuvir and wherein the patient has previously been determined to be infected with a HCV strain having one or more mutations in the NS5B protein of HCV selected from

(i) A150V;

(ii) K206E and A150V; or

(iii) K100R, A150S, G188D, T213N and N244I.

13. An anti-Hepatitis C Virus (HCV) drug for use in the treatment of HCV in a patient wherein the drug is not sofosbuvir and the patient is infected with a HCV strain having one or more mutations in the NS5B protein of HCV selected from (i) A150V;

(ii) K206E and A150V; or

(iii) K100R, A150S, G188D, T213N and N244I.

14. An anti-Hepatitis C Virus (HCV) drug for use in the treatment of HCV according to claim 13 wherein the anti-HCV drug is selected from the group consisting: daclatasvir, velpatasvir, elbasvir or grazoprevir, or a combination thereof.