RU2461834C1 - Method for prediction of formation of virological response in patients with chronic hepatitis c - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: blood plasma is examined for the relation of interleukin-18 to tumour necrosis factor-alpha (IL-18/TNF-α) prior to and after antiviral therapy. If the IL-18/TNF-α value is less than 2 prior to the therapy and increased up to 2 and more after the therapy, formation of virological response is predicted.

EFFECT: method enables prediction of stable virological response for the purpose of the following management.

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Description

The invention relates to medicine, namely to infectious diseases and immunology.

Predicting the effectiveness of treatment for patients with chronic hepatitis C (CHC) is an important practical task due to the high cost of antiviral drugs, the presence of numerous side effects during their use, and the lack of the ability to reliably eliminate the virus from the patient’s body [1].

Currently, reliable methods for predicting a positive response, in other words, the formation of a virological response to antiviral therapy for chronic hepatitis C, are essentially absent.

Known prognostic factors for a favorable response to standard therapy for patients with chronic hepatitis C are the low initial level of viremia (less than 2 million copies / ml), the “not 1” HCV genotype, as well as a combination of these signs. Among the predictors of a positive response to treatment are considered indicators such as the absence of histological signs of cirrhosis, low iron content in the blood serum, as well as a short duration of the disease, young age, female gender, absence of excess body weight and associated diseases [2, 3, 4] .

However, these forecast criteria have several drawbacks associated with the need to use special and expensive equipment (for PCR analysis) and, accordingly, the high cost of some research methods. In addition, this is the ability of the viral load to fluctuate and the inability to predict the effectiveness of treatment in patients with HCV in whom the outcome of treatment may be different [5, 6, 7, 8].

Other prognostic criteria for responding to antiviral therapy also have disadvantages: it is often the inability to determine the duration of infection, the difficulty in assessing the severity of fibrosis, the need to use an invasive method for diagnosing liver puncture biopsy. None of the proposed forecasting methods takes into account the individual patient response to antiviral treatment.

There is a method for predicting the results of treatment with chronic hepatitis C by determining the synthesis of IFN-γ and IL-4 by peripheral blood mononuclear cells in vitro before and 4.5 hours after the first dose of interferon is administered [9]. If a more than twofold increase in the synthesis of IFN-γ and the ratio of the synthesis of IFN-γ / IL-4 after the first injection of a patient with CVH C interferon is detected, the development of a virologic response during interferon therapy is detected. In the case of an increase in the synthesis of IFN-γ and the ratio of the synthesis of IFN-γ / IL-4 after the first administration of interferon - less than 2 times the formation of a virological response should not be expected. This method is taken as the closest analogue.

The objective of the invention is to develop an affordable method for predicting the formation of a stable virologic response (SVR) in patients with chronic hepatitis C during treatment with standard antiviral drugs.

A stable antiviral response is characterized by the absence of HCV RNA in the blood serum after 6 months of follow-up, in contrast to the primary antiviral response at the end of treatment - which is predicted in the closest analogue and can be considered as its lack.

The technical result of the invention is the ability to predict a stable virologic response (SVR) for the subsequent selection of patient management tactics.

The technical result is achieved by determining the ratio of cytokine levels: interleukin-18 (IL-18) and tumor necrosis factor-alpha (TNF-α) in serum before treatment with antiviral drugs and after treatment.

The essence of the method lies in the fact that in the blood serum before treatment and after treatment the level of cytokines interleukin-18 (IL-18) and tumor necrosis factor-alpha (TNF-α) is determined to determine their ratio and if its value is less than 2 before treatment and an increase of 2 or more after the end of treatment predict the formation of a virological response.

Interleukin-18 is called an IFN-γ inducer. The IL-18 gene cloned in 1995 is similar to the IL-1 family, and therefore, IL-1γ was named. Subsequently, it was found that it does not bind to type I receptors of IL-1, so it was renamed to IL-18. It is produced mainly by macrophages, as well as T and B lymphocytes, dendritic cells, osteoblasts, Kupffer cells. IL-18 stimulates the cytotoxic activity of NK cells, T-cell proliferation, production of IL-12 and IFN-γ, IFN-γ, Th-cells, and macrophages - IL-1, IL-8 and TNF-α, reduces the production of IL- 10, enhances FasL-mediated cytotoxicity of Th1 and NK cells, and also plays a significant role in liver damage.

TNF-α is one of the key cytokines, which has a powerful anti-inflammatory effect and plays an important role in the pathogenesis of tissue damage. The biological effects of TNF-α are largely dependent on its concentration. A low level of cytokine production is necessary to maintain antitumor and antiviral protection, limiting the size of the lymphocyte pool by inducing apoptosis. A significant increase in the level of TNF-α, as a rule, is accompanied by a reaction of damage and inflammation, and ultrahigh concentrations - the development of severe generalized disorders, multiple organ failure. Both independently and in collaboration with other cytokines, TNF-α is involved in the activation of cytotoxic processes in cells, enhances phagocytosis. TNF-α plays a significant role in coordinating the inflammatory response and the cytokine cascade, gives a hepatotoxic effect, and takes part in apoptosis of damaged (including virus) cells. According to some authors, a consistently high level of TNF-α is a criterion for the development and severity of fibrosis, and also indicates a violation of the mechanisms of cytokine regulation of the immune response, when the protective nature of the activation of the synthesis of mediators of the immune response becomes damaging.

These cytokines are one of the main mediators of cellular immunity, affect the course of hepatitis C and the effectiveness of treatment of patients with chronic hepatitis C.

We have conducted relevant clinical studies. The criterion for inclusion of patients in the groups was the presence of proven chronic viral hepatitis C. The diagnosis of chronic hepatitis C was established on the basis of an epidemiological history (indications of past surgical interventions, blood transfusions and other drugs, other parenteral interventions, visits to the dental office, intravenous drug injections), clinic Anamnestic data (the presence of symptoms of viral hepatitis for 6 months or more, determined by an objective examination of the physical bubbled methods - enlarged liver and spleen), lab results (characteristic changes in biochemical parameters and identification of hepatitis B virus markers C. IFA and PCR, morphological studies puncture biopsies of liver with patient consent). To strictly verify the diagnosis and exclusion of viral hepatitis of a different etiology, as well as HIV infection, all patients underwent a blood serum study to detect HBsAg, a-HBcor-IgM and IgG, a-HAV IgM, a-HDV IgM and IgG, a-HIV.

Exclusion criteria: use of alcohol and drugs, co-infection with hepatitis B virus, HIV infection, pregnancy.

When developing the method, 55 patients with chronic hepatitis C were examined. Conducted standard antiviral therapy with interferon (domestic α-2b-interferon, "Vector", Novosibirsk) in a daily dose of 3 million ME 3 times a week in combination with ribamidil in a daily dose of 800-1200 mg depending on body weight for a period of 24 -48 weeks depending on the HCV genotype. Evaluation of the effectiveness of antiviral therapy was carried out, first of all, taking into account the biochemical and antiviral response. Based on testing of HCV RNA, patients were divided into 2 groups:

the group responded to treatment with the formation of a stable virological response, which is expressed in the disappearance of HCV RNA and its absence in the blood serum 6 months after the end of treatment, normalization of ALT, and the group did not respond to treatment with the preservation of HCV RNA and normalization or decrease in ALT activity. A retrospective analysis of the indicators of IL-18 and TNF-α, as well as their ratio.

At the same time, in patients from both compared groups, no significant differences were found in the initial indicators of IL-18 and TNF-α from sex, age of patients, epidemiological history (drug use history), the presence of concomitant diseases, as well as their dynamics from clinical manifestations, activity transaminases and regimens.

An analysis of the data showed that a stable virological response to antiviral therapy for patients with chronic hepatitis C should be expected if the ratio of IL-18 / TNF-α before treatment is less than 2.0, and after treatment is increased and will be 2.0 or more. Conversely, if the ratio of these cytokines before treatment is equal to or greater than 2.0, and after treatment is less than 2.0, then a stable virological response is not formed. Thus, it is not only the ratio of these indicators that matters, but also their dynamics. That is, a positive response to antiviral therapy is formed with an increase in the ratio of IL-18 / TNF-α after treatment, and a negative result with a decrease.

The method is as follows. Indicators of the level of IL-18 and TNF-α are determined in the blood serum before treatment and after the end of antiviral therapy (any of the interferon and ribavirin preparations are used according to standard schemes). The level of cytokines is investigated by enzyme-linked immunosorbent assay (ELISA) using, for example, the test system "Protein circuit" (St. Petersburg). The normal content of IL-18 in blood serum is 0-30 pg / ml, TNF-α-0-50 pg / ml. If the ratio of IL-18 / TNF-α before treatment will be less than 2.0, and after treatment will increase and will be 2.0 or more, the formation of a virological response in patients with chronic hepatitis C is predicted.

Clinical example No. 1. Patient N., born in 1970 Since June 2003, weakness, sleep disturbance, depression, hair loss, and dark urine have been worrying. Not surveyed. By chance, in December 2004, a-HCV was detected and diagnosed with chronic viral hepatitis C and chronic cholecystitis. He turned to the hospital No. 2 on an outpatient basis to resolve the issue of etiotropic therapy. He had a history of surgical treatment in 1999. An additional examination revealed genotype 1c, moderate viral load - 2 * 10 ⁶ IU / ml, increased ALT to 3N (110 units / l). Clinical diagnosis of chronic viral hepatitis C in the replication stage, moderate degree of activity, HCV genotype 1c. Concomitant disease: diffuse goiter of the II degree without impaired function. From April 11, 2005, for 12 months, he received combination therapy with reaferon 3 million IU per day 3 times a week and ribamidil 800 mg per day. When examining the level of cytokines before treatment, elevated concentrations of IL-18 (214.7 pcg / ml) and TNF-α (138.1 pcg / ml) were detected; the ratio of IL-18 / TNF-α was 1.5, which allows us to predict the formation of a virological response after antiviral treatment. Before treatment, complaints of weakness, periodically dull pain in the right hypochondrium, decreased appetite, poor sleep. An objective examination: the skin and mucous membranes are normal in color, there is no telangiectasia and palmar erythema, the liver is palpated at the edge of the costal arch, the spleen is not enlarged. On the introduction of reaferon, an increase in body temperature to 38.5 ° C, chills, sweating, weakness, poor sleep was noted; such a reaction lasted for 4 weeks. Throughout the treatment, headache, double vision, pain in the right hypochondrium were recorded, and also from 6 months I noted memory problems (forgetfulness, difficulty remembering), daytime sleepiness. I lost 9 kg (~ 9%) during the treatment; after the treatment was over, the weight almost recovered. After 1 month of treatment, ALT normalization (19 units / L) and a significant decrease in the concentration of the virus in serum (up to <500 IU / ml) were recorded. Sustained biochemical and antiviral responses have been achieved. The ratio of IL-18 / TNF-α was 28.4 after treatment, which confirmed the data before treatment for predicting the formation of a virological response.

Clinical example No. 2. Patient R., born in 1980 In 1996, a-HCV was detected, not observed and not treated. In June 2003, he began to be examined - HCV RNA in serum was detected, HCV 3a genotype, increased transaminase activity. He had a history of intravenous drug use (heroin) in 1996, the last injection was then the same. Complaints before treatment of weakness, sometimes heaviness in the right hypochondrium. Upon examination, the skin and visible mucous membranes of normal color, the liver protrudes 2-3 cm below the costal margin, the spleen is palpated. In a blood test, a moderate increase in ALT activity to 2N (98), a moderate viral load of 1.3 * 10 ⁶ IU / ml. Clinical diagnosis: chronic viral hepatitis C in the replication stage, moderate activity, HCV genotype 3a. There were no concomitant diseases. When studying the state of cytokines before treatment, elevated levels of IL-18 (231.2 pg / ml) and TNF-α (91.02 pg / ml) were noted, and the ratio of IL-18 / TNF-α was 2.5, which suggested a possible formation of virological response to antiviral treatment. From 07.06.04, for 6 months, an antiviral treatment with viferon was carried out in a daily dose of 6 million ME in combination with ribamidil in a daily dose of 800 mg. No reaction to the administration of viferon was noted. The patient tolerated the treatment well; no side effects were reported. If at the beginning of therapy according to the "psychotest""subclinically expressed anxiety / depression" was noted, then by the end of treatment this indicator was normal. Positive clinical dynamics were noted. He lost 7 kg (8%) over 6 months, but gained weight after stopping therapy. The level of TSH, thyroid hormones, autoimmune antibodies during treatment remained within the normal range. The activity of transaminases (ALT) did not normalize during therapy, persistently moderate viral load was observed in the blood serum, that is, the patient did not have a stable biochemical and antiviral response. By the end of therapy, a decrease in IL-18 (≤45 pcg / ml) and TNF-α (59.88 pcg / ml) was recorded; the ratio of IL-18 / TNF-α was 0.7. Thus, the ratio of IL-18 / TNF-α before treatment is more than 2.0 and its decrease after treatment indicates the antiviral treatment is ineffective.

Summing up, we can state that both individual and average statistical ratios of the IL-18 / TNF-α ratio can predict a stable virological response in patients with chronic hepatitis C receiving antiviral (PVT) standard therapy: if this ratio before treatment is below 2.0 , and after it rises to 2 or more, then patients respond to therapy, that is, a stable virological response is formed. Accordingly, if the ratio of IL-18 / TNF-α before the start of antiviral therapy is equal to or higher than 2.0, then a stable virological response is not formed. In this case, it is necessary to appropriately change the treatment tactics and not to use expensive and toxic antiviral drugs.

Thus, the proposed method allows for the prediction of a stable virologic response (SVR) for the subsequent selection of patient management tactics.

Information sources

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Claims (1)

A method for predicting the formation of a virological response in patients with chronic hepatitis C, characterized in that the ratio of interleukin-18 to tumor necrosis factor-alpha (IL-18 / TNF-α) is determined in the blood serum before antiviral treatment and after its completion and when the ratio is IL-18 / TNF-α less than 2 before treatment and its increase to 2 or more after treatment predict the formation of a virological response.