JP2007043985A - Method for judging efficacy of treatment method including simultaneous use of interferon/ribavirin against hepatitis c - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for judging the efficacy of a treatment method including the simultaneous use of an interferon/ribavirin before starting the treatment or at an initial stage after starting the treatment. <P>SOLUTION: This method for judging the efficacy of the simultaneous use of the interferon/ribavirin for the treatment of hepatitis C comprises an investigation whether the arginine at 70th position of amino acid sequence in its core region of the hepatitis C virus contained in a specimen collected from a patient infected with hepatitis C virus genotype 1b type is varied to another amino acid or not, and also whether the amino acid at the 91st position is methionine or not. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for determining the effectiveness of interferon / ribavirin combination therapy for hepatitis C caused by genotype (genotype) 1b hepatitis C virus.

  Hepatitis C is caused by infection with hepatitis C virus (hereinafter sometimes referred to as “HCV”), and about 70% of humans infected with HCV for the first time become carriers and become chronic. 30-50% of patients with chronic hepatitis due to HCV (chronic hepatitis C) develop cirrhosis on average about 20 years after infection, and 60-80% of patients with cirrhosis transition to liver cancer on average about 30 years after infection .

  Interferon (hereinafter sometimes referred to as “IFN”) has been used as a treatment method for chronic hepatitis C, but in recent years, IFN and Ribavirin (hereinafter referred to as “RBV”), which is one of antiviral drugs, have been used. ”), And the therapeutic effect is improved even in intractable cases. In other words, when the amount of HCV RNA in the serum (HCV is RNA virus) is 100 kIU / mL or more, IFN monotherapy has only about 5% of effective cases, but IFN and RBV combined therapy Even when the serum HCV RNA level is 200 kIU / mL or more, the effective ratio is about 20%. Polyethylene glycolated IFN (hereinafter referred to as “PEG”) that has increased the blood half-life of IFN by binding polyethylene glycol. -IFN ") and RBV combination therapy improved the percentage of effective cases to about 40% (Non-patent Document 1).

  However, the proportion of effective cases is still less than half even with PEG-IFN and RBV combination therapy. On the other hand, side effects have been reported in combination therapy of IFN (including PEG-IFN) and RBV. If it is possible to determine whether IFN and RBV combination therapy is effective before treatment or at the beginning of treatment, give IFN / RBV combination therapy with side effects to patients who do not respond to IFN / RBV combination therapy. Can be avoided, and other treatment methods can be applied to the patient. Furthermore, it is possible to avoid placing an expensive burden on patients and society by administering these expensive drugs. Therefore, a method capable of determining whether or not an IFN / RBV combination therapy is effective before treatment or at an early stage of treatment is desired.

  Conventionally, a method for determining the effectiveness of treatment of chronic hepatitis C by IFN by examining the amino acid sequence from 2209th to 2248th in the NS5A region of HCV is known (Non-patent Document 2). 3). However, at the time this method was invented, IFN / RBV combination therapy was not performed, and it is unclear whether this method can be used to determine the effectiveness of IFN / RBV combination therapy.

Akio Takagi, et al., Internal Medicine 93 (3): 425-429, 2004 Enomoto N et al., Comparison of full-length sequences of interferon sensitive and resistant hepatitis C virus 1b. J Clin Invest 1995; 96: 224230. Enomoto N et al., Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection.N Engl J Med 1996; 334: 7781. (Chayama K et al., Genotypic subtyping of hepatitis C virus.J Gastroenterol Hepatol 1993; 8: 150156) Kato N et al., Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis.Proc Natl Acad Sci USA 1990; 87: 95249528.

  Accordingly, an object of the present invention is to provide a method by which the effectiveness of an IFN / RBV combination therapy can be determined before or after the start of treatment.

  As a result of diligent research, the inventors of the present application have found that in cases where IFN / RBV combination therapy is ineffective, a specific amino acid in the core region of HCV is significantly mutated to another amino acid. The present invention was completed by experimentally confirming that the effectiveness of the IFN / RBV combination therapy can be determined with a high probability by examining the amino acid sequence of the HCV core region contained in the collected specimen. Furthermore, it was found that the effectiveness of IFN / RBV combination therapy can be determined more accurately by adding to the criteria whether or not the amount of HCV RNA is significantly reduced in the initial stage after the start of IFN / RBV combination therapy. .

  That is, according to the present invention, the 70th arginine of the amino acid sequence in the core region of hepatitis C virus contained in a specimen collected from a patient infected with genotype 1b hepatitis C virus is other amino acid. A method for determining the effectiveness of an interferon / ribavirin combination therapy for the treatment of hepatitis C, which comprises examining whether or not the 91st amino acid is methionine. In addition to the above-described method of the present invention, the present invention further includes measuring the gene amount of hepatitis C virus contained in a sample collected before and after the start of the interferon / ribavirin combination therapy. The present invention provides a method for determining the effectiveness of interferon / ribavirin combination therapy for the treatment of hepatitis C.

  The present invention provides for the first time a method by which the effectiveness of an IFN / RBV combination therapy can be determined before or after the start of treatment. According to the present invention, since the effectiveness of the IFN / RBV combination therapy can be determined before or after the start of treatment, the IFN / RBV combination therapy is effective for patients who are not effective. Initiation or continuation of RBV combination therapy can be avoided and other treatment methods can be given to the patient. Furthermore, it is possible to avoid placing an expensive burden on patients and society by administering these expensive drugs. Therefore, the present invention is expected to greatly contribute to the treatment of chronic hepatitis C.

  The patient to which the method of the present invention is applied is a patient infected with HCV of genotype (genotype) 1b. HCV causing chronic hepatitis C is known to have genotype 1b and genotype 2. Genotype 1b is more common, and the refractory rate is higher. A method for discriminating between genotype 1b and genotype 2 is well known (Non-Patent Document 4), and can be easily discriminated based on the nucleotide sequence of the NS5 region of HCV RNA.

  The treatment method for which the effectiveness is determined by the method of the present invention is a combination therapy of IFN and RBV (hereinafter, simply referred to as “combination therapy”). The type of IFN is not particularly limited, and may be any IFN used for the treatment of chronic hepatitis C. Usually, IFNα, particularly IFNα2b and IFNα2a are used, but not limited thereto. In addition, in order to increase the blood half-life of IFN, PEG-IFN in which polyethylene glycol is bound to IFN is also used, but “interferon” in the present specification and claims is unless otherwise specified. And unless otherwise indicated in context, also includes IFN derivatives used to treat chronic hepatitis C, such as PEG-IFN. In addition, according to the current guidelines for the treatment of chronic hepatitis C with genotype 1b, combination therapy of PEG-IFNα2b and RBV is recommended for 48 weeks when the serum HCV RNA concentration is 100 kIU / mL or higher. In non-combination therapy, IFN (non-PEGylated) is supposed to be administered for 2 years, and the serum HCV RNA concentration is 100 to 500 kIU / mL. In addition, PEG-IFNα2a can be administered alone. On the other hand, when the serum HCV RNA concentration is a low viral load of less than 100 kIU / mL, IFN (non-PEGylated) is administered for 24 weeks or PEG-IFNα2a is administered for 24-48 weeks. In the case of re-administration, in the case of a high viral load, it is the same as the initial administration described above, and in the case of a low viral load, combination therapy of IFNα2b (non-PEGylated) and RBV for 24 weeks or PEG-IFNα2a alone 48 weeks of administration or 48 weeks of single administration of IFN (non-PEGylated). It should be noted that the combination therapy whose effectiveness is determined by the method of the present invention is not necessarily limited to the combination therapy according to this guideline, and may be other IFN / RBV combination therapy.

  The specimen provided for the method of the present invention is collected from a patient infected with HCV, and is not limited in any way as long as it contains HCV, such as a body fluid such as blood, a liver biopsy specimen, etc. It may be. Of these, blood samples that are easy to collect and have good sensitivity (in addition to whole blood, blood components such as serum and plasma) are preferable.

  In the method of the present invention, it is determined whether or not the 70th arginine in the amino acid sequence in the core region of HCV contained in the sample is mutated to another amino acid and whether or not the 91st amino acid is methionine. including. The genomic sequence of HCV and the amino acid sequence encoded by it are already known. For example, Non-Patent Document 5 and GenBank Accession No. D90208 describe a prototype (prototype) sequence (HCVJ) of HCV genome. The nucleotide sequence of the core region of HCVJ is shown together with the amino acid sequence encoded by SEQ ID NO: 1 in the sequence listing. In the method of the present invention, whether or not the 70th amino acid in the core region (arginine in HCVJ) is mutated to another amino acid, and whether or not the 91st amino acid is methionine (the 91st amino acid is Although HCVJ is also methionine, leucine is often found in HCV in sera of patients with high viral load chronic hepatitis C, as specifically shown in the Examples below, and the 91st amino acid is In the case of methionine (as is the case with HCVJ), the number of invalid cases is significantly higher). As specifically shown in the Examples below, in cases where combination therapy is ineffective, the possibility that the 70th arginine is mutated to another amino acid and / or the 91st amino acid is methionine is significant. Very expensive. Therefore, by examining these amino acids, it is possible to determine whether the combination therapy is invalid or not with high probability. As specifically shown in the examples below, in the ineffective case in which the 70th arginine is mutated, arginine is mutated to glutamine or histidine, and in most cases, it is mutated to glutamine. The 91st amino acid is methionine or leucine, and HCV in the serum of patients with high viral load chronic hepatitis C is often leucine, but combination therapy is ineffective when this amino acid is methionine. There is a significant probability.

  As described above, since the genome sequence of HCV is already known, what is the 70th and 91st amino acids in the core region can be examined by a method known per se. For example, it can be examined by performing reverse transcription PCR (RT-PCR) using HCV genomic RNA contained in a specimen as a template and determining the base sequence of the amplification product. The amplification region may be a region including at least the region encoding the 70th to 91st amino acids. As PCR primers, complementary oligonucleotides can be used at both ends of the amplification region, and the sequence of these oligonucleotides can be easily set based on the known sequence described in SEQ ID NO: 1. PCR can be performed by a conventional method using these primers and a PCR device such as a commercially available kit and a thermal cycler (trade name). Specific examples of RT-PCR are also shown in the following examples. It is described in detail. In addition, as described in the following examples, in order to increase the accuracy of measurement, nested PCR in which PCR is performed in two stages (the amplification product of the first stage PCR is used as a template and the region in the amplification product is in the second stage). Or a hemoinested PCR (the same primer used in the first stage is used as one of the primers used in the second stage PCR).

  In addition to the above-described method of the present invention, further, whether or not the amount of HCV RNA in the sample is significantly reduced at the initial stage of combination therapy is examined, and the result is also added to the judgment material, thereby further improving the accuracy of the judgment. Can do. That is, when HCV has the mutation described above and the amount of HCV RNA in the sample is not significantly reduced at the beginning of the combination therapy, the probability that the combination therapy is ineffective is further increased. The accuracy of this determination method can be further increased. Here, the initial stage of the combination therapy is not particularly limited. For example, it is 24 hours to 72 hours after the first administration, and the significant decrease is, for example, the amount of HCV RNA in the specimen during this period. Can be based on whether 1/5 to 1/20 or less, and specific criteria can be set as appropriate. The following examples are based on whether or not the serum HCV RNA concentration is 1/10 or less (ie, the common logarithm of the HCV RNA concentration is reduced by 1.0 or more) 48 hours after the first administration. It is not limited to. The amount of HCV RNA can be measured by a conventional quantitative RT-PCR method, and a kit for measuring the amount of HCV RNA is also commercially available, and can be easily measured using this kit. .

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

1. Patients and combination therapy The experiment involved 50 patients who were infected with HCV and received combination therapy. The age ranged from 20 to 65 years old, the median was 53 years old, 31 men and 19 women. Of the 50, 34 (68.0%) received 1.5 μg / kg PEG-IFNα2b subcutaneously once weekly and continued daily oral administration of 600-800 mg / day of RBV for 48 weeks. The remaining 16 (32.0%) patients received 6 million units of IFNα-2b (non-PEGylated) intramuscularly for 48 weeks (six times per week for the first 2 weeks, followed by 46 Weekly was 3 times per week) and daily oral administration of RBV of 600-800 mg / day was continued for 48 weeks. The dose of RBV was adjusted according to body weight (600 mg / day when the body weight is 60 kg or less, 800 mg / day when the body weight exceeds 60 kg). Of the 50 patients, 14 (28.0%) had reduced RBV doses because hemoglobin levels decreased during treatment. At the end of combination therapy or during combination therapy, patients with serum HCV RNA below the detection limit by qualitative PCR (described later) are effective cases (virological response cases, VR), at the end of combination therapy Patients who were positive for HCV RNA in serum by quantitative PCR (described later) and / or qualitative PCR were regarded as invalid cases (virological ineffective cases, NVR).

2. HCV typing and HCV RNA measurement
The HCV genotype was determined based on the nucleotide sequence of the NS5 region of HCV RNA by a conventional method (Non-patent Document 4). The HCV RNA concentration in serum was first determined by quantitative PCR (Amplicor HCV-RNA kit (trade name), version 2.0, manufactured by Roche Diagnostics). The detection limit of this measurement method is 0.5 kIU / mL. When HCV RNA was not measured by quantitative PCR, it was also measured by qualitative PCR (Amplicor (trade name), manufactured by Roche Diagnostics) with a lower detection limit. The detection limit for qualitative PCR is 100 copies / mL.

3. Determination of the nucleotide sequence of the core region and NS5A region of HCV RNA By determining the nucleotide sequence of the following region in the HCV RNA in the serum before the start of combination therapy, the amino acid sequence from the 1st to 191st of the core region and the NS5A region The amino acid sequence of the 2209th to 2248th region (ISDR) was determined. RNA was extracted from serum samples by a conventional method, and cDNA was synthesized using random primers and MMLV reverse transcriptase. Next, using the obtained cDNA as a template, the region in the core region and the region encoding ISDR are amplified by two-step heminested PCR (core region) or nested PCR (ISDR) using the following primer set: did.

(1) Primer set used for core region amplification
(i) First stage PCR
Forward primer (CC11): 5'-GCC ATA GTG GTC TGC GGA AC-3 '
Reverse primer (c14): 5'-GGA GCA GTC CTT CGT GAC ATG-3 '
(ii) Second stage PCR
Forward primer (CC9): 5'-GCT AGC CGA GTA GTG TT-3 '
Reverse primer: c14

(2) Primer set used for ISDR amplification
(i) First stage PCR
Forward primer (ISDR1): 5'-ATG CCC ATG CCA GGT TCC AG-3 '
Reverse primer (ISDR2): 5'-AGC TCC GCC AAG GCA GAA GA-3 '
(ii) Second stage PCR
Forward primer (ISDR3): 5'-ACC GGA TGT GGC AGT GCT CA-3 '
Reverse primer (ISDR4): 5'-GTA ATC CGG GCG TGC CCA TA-3 '

  For PCR to amplify any region, the PCR reaction mixture was first denatured at 95 ° C. for 15 minutes, followed by denaturation at 94 ° C. for 1 minute, annealing step at 55 ° C. for 2 minutes, and 72 ° C. for 3 minutes. The cycle consisting of the extension step was repeated 35 cycles, and finally, the extension step was further performed at 72 ° C. for 7 minutes. 1 μL of the reaction mixture after completion of the first stage PCR was added to the second stage reaction mixture, and the second stage PCR was performed under the same conditions as the first stage (however, the primers used were different as described above). The amplification product was subjected to agarose gel electrophoresis by a conventional method, then purified using a QIA quick PCR purification kit (trade name), and then subjected to direct sequencing (direct base sequencing). Direct sequencing was performed by dideoxynucleotide termination sequencing using a Big Dye Deoxy Terminator Cycle Sequencing kit (Perkin-Elmer). The amino acid sequence encoded by the determined base sequence was compared with the corresponding consensus amino acid sequence to identify the substituted amino acid. The consensus amino acid sequence was obtained by comparing the HCVJ sequence (SEQ ID NO: 1) described in Non-Patent Document 5 (GenBank Accession No. D90208) and the HCV sequences in 50 samples examined in this Example. Determined (see FIG. 1).

4). Statistical processing For analysis of amino acid substitutions in the core region and NS5A region, non-parametric tests including Mann-Whitney U test, χ ² test and Fisher exact probability test were used. Univariate and multivariate logistic regression analysis was used to determine the factors that significantly contributed to invalid cases. In addition, odds ratios and 95% confidence intervals (95% CI) were calculated. There was a statistically significant difference in all cases where the p-value by two-sided test was less than 0.05. Variables that achieved statistical significance (p <0.05) or boundary significance (p <0.10) in univariate analysis were entered into multiple logistic regression analysis to identify significant independent factors.

5. Results As a result of statistical processing comparing the effective case and the ineffective case, among the amino acid substitutions, the mutation of the 70th arginine and the mutation of the 91st leucine in the core region (mutations when compared with the consensus sequence) are It became clear that it was statistically significant. There were no statistically significant amino acid substitutions at other sites (including within the ISDR). The 61st to 110th amino acid sequences in the core region are shown in FIG. In FIG. 1, the amino acid sequence described at the top is the consensus sequence of this region, the second sequence is the amino acid sequence of this region of HCVJ, and below that in the samples collected from 50 patients The amino acid sequence of this region of HCV is described for all 50 samples. In addition, the amino acid shown by "-" in a sequence means that it is the same as the amino acid of the top consensus sequence.

  Table 1 below shows the number of cases, the ratio (in parentheses), and the p-value (Fischer's exact test) with mutations in the 70th and 91st amino acids (mutations compared to the consensus sequence). In Table 1, for example, the 70th amino acid is represented as “aa70”.

  As shown in Table 1, the mutations of the 70th amino acid and the 91st amino acid (mutations when compared with the consensus sequence) are effective cases and ineffective cases, either alone or in combination. There is a statistically significant difference between them (p <0.05). In particular, when at least one of these amino acids (that is, the 70th amino acid and / or the 91st amino acid) is mutated, the statistically significant difference from p <0.001 may be particularly large. Recognize. In the invalid case, at least one of these amino acids has been mutated with a probability of 100%. I understand that there is.

  For the same patient as in Example 1, whether the HCV RNA concentration in the serum after 48 hours became 1/10 or less of the HCV RNA concentration before the start of combination therapy (that is, the common logarithm of the HCV RNA concentration was 1.0 or more) Whether or not it was lowered) was also added to the judgment material. Of the 50 cases, one case was excluded because it had no specimen for 48 hours and could not be evaluated, and the remaining 49 cases were examined. The results are shown in FIG.

  As shown in FIG. 2, when there is no mutation in both the 70th and 91st, it is a remarkable example with a probability of 100% (described above). In addition, even when there is a mutation in at least one of the 70th and 91st, if the serum HCV RNA concentration is reduced to 1/10 or less after 48 hours, it is reduced (n = The ratio of 14) effective cases (n = 13) was 92.9%, and when it did not decrease (n = 13), the effective case (n = 2) was 15.4%. In other words, if there is no mutation in both the 70th and 91st mutations or the serum HCV RNA concentration decreases to 1/10 or less after 48 hours even if there is a mutation, 35/36 (97.2 %) Is a prominent example. From this, it can be seen that the accuracy of determination can be further improved by further adding the measurement of the gene amount of HCV in the early stage of combination therapy.

A combination therapy comparing the 61st to 110th amino acid sequences in the core region of HCV in the serum of high viral load chronic hepatitis C investigated in the examples of the present invention with the consensus sequence and the prototype sequence It is a figure shown with effectiveness of. The presence or absence of mutation of the 70th amino acid and / or the 91st amino acid in the core region of HCV in serum of chronic hepatitis C with high viral load investigated in the Examples of the present invention, and serum after 48 hours It is a figure which shows the result which divided the effective case and the invalid case into cases based on whether the HCV RNA concentration in the inside became 1/10 or less of the HCV RNA concentration before the start of combination therapy.

Claims (6)

  Whether or not the 70th arginine of the amino acid sequence in the core region of hepatitis C virus contained in a sample collected from a patient infected with genotype 1b hepatitis C virus has been mutated to another amino acid And determining whether or not the 91st amino acid is methionine, the effectiveness of interferon / ribavirin combination therapy for the treatment of hepatitis C.   The method according to claim 1, wherein it is determined whether or not the 70th arginine is mutated to glutamine and whether the 91st amino acid is methionine.   The gene sequence of a region including at least the region encoding the 70th to 91st amino acids in the core region of the hepatitis C virus gene contained in the sample is determined. The method described.   The method according to claim 3, which is carried out by performing reverse transcription PCR using a hepatitis C virus gene contained in the specimen as a template and determining the base sequence of the amplification product.   The method according to any one of claims 1 to 4, further comprising measuring a gene amount of hepatitis C virus contained in a sample collected before and after the start of the interferon / ribavirin combination therapy. . The method according to claim 1, wherein the sample is a blood sample.

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