RU2440133C2 - Medication composition and method of its obtaining for treatment of chronic viral hepatitis b or c and aids - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine and pharmacology and represents lyophilised medication for treatment of chronic viral hepatitis B or C and AIDS, which contains alpha fetoprotein (AFP) 60-150 mcg, interferon α 3000000-6000000 U and dextran 2-6 mg.

EFFECT: invention ensures improvement of treatment results, reduction of complications and treatment terms.

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Description

The invention relates to medicine, namely to clinical hepatology, virology, immunology and pharmacology, and can be used for the production of a combination drug based on human alpha-fetoprotein and interferon and its use for the treatment of chronic viral hepatitis B or C and AIDS.

Currently, there are methods for the etiotropic treatment of chronic viral hepatitis B and C (HVGV and HVGS), which include the appointment of interferons, which are a group of proteins produced by eukaryotic cells in response to various stimuli important in the suppression of viral infections. The mechanism of its action of interferons on CVH B and C is to inhibit the replication and transcription of viruses, resulting in a decrease in the level of virus RNA in serum. It also enhances the activity of natural T-killers, T-helpers, cytotoxic T-lymphocytes, phagocytic activity, the intensity of differentiation of B-lymphocytes, the expression of MHC type I and II. As a result, the level of viremia in serum decreases [1, 2].

However, it is known that interferon in the body undergoes rapid decay and is eliminated from the body, thus the effectiveness of therapy is significantly reduced. Therefore, recently, for the treatment of CVH B, prolonged (pegylated) interferons (pegintron; pegasis) have been used, and in the case of CVH C, in combination with ribovirin or its analogues [3, 4].

Currently, over 200 possible combinations of antiretroviral therapy have been developed, but not one, which would be the best for all patients. With the goal of maximum effectiveness, three antiretroviral drugs are included in the treatment regimen for HIV infection. The most effective combination is two nucleoside analogs of a reverse transcriptase inhibitor and one protease inhibitor. The main problem in the treatment of HIV infection is the low bioavailability of drugs and their pronounced toxicity. Moreover, the drugs used in the treatment of HIV infection are expensive and mainly foreign-made drugs are used.

CVH B and C, as well as HIV - intracellular infection. And the main problem of antiviral therapy is the low bioavailability of drugs to viruses within the cell. They don’t get there! Vaccines do not work either, because immunoglobulins cannot penetrate either the hepatocyte or the lymphocyte. On the membranes of liver cells and lymphocytes there is not a single receptor to any of the known antiretroviral drugs. And penetration into the cell is always receptor-mediated.

Such receptors exist for the embryonic protein alpha fetoprotein (AFP) on the membranes of both hepatocytes and lymphocytes [6].

AFP - is a fetal glycoprotein with a molecular weight of about 70,000 D. AFPs are produced in large quantities by fetal liver cells. It is a protein of the albuminoid family with the same molecular weight as albumin and, like albumin, is a transport protein for transporting biological compounds to cells. But, it is 15-40 times more effective in transporting properties of albumin. Moreover, if there are no albumin receptors on the membranes of hepatocytes and lymphocytes, then they have AFP [6]. Abortive, umbilical and placental blood obtained during medical abortions from clinically healthy women who underwent a standard examination is the raw material for obtaining AFP.

Closest to the claimed composition for the treatment of CVH B and C is the drug (Albuferon®), consisting of interconnected human albumin and interferon - α [5].

Despite the increase in the duration of interferon activity when it is combined with human albumin, the composition with its use has the following disadvantages.

1. The immunogenicity of the drug, ie interferon and albumin in the patient’s body produce neutralizing antibodies that reduce the effectiveness of treatment and reduce tolerance.

2. Despite the transport properties of albumin, it cannot penetrate into the cell and cannot transport interferon, because there are no receptors for it.

3. Complications persist during prolonged interferon therapy (anemia, thrombocytopenia, leukopenia, depression, etc.).

4. Long-term treatment of 24-72 weeks.

The technical result to which the invention is directed is to improve treatment results by attaching interferon with AFP, reducing complications and treatment time.

By mixing or chemically cross-linking AFP (Profetal® preparation) with interferon, and subjecting the resulting combination to subsequent freeze drying, we obtain, thus, a covalent or non-covalent complex that is capable of delivering “targeted” intracellular “delivery” (receptor-dependent endocytosis) of interferon and enhancing antiviral effect (drawing).

It is known that AFP has a hepatoprotective property due to the membrane-stabilizing effect due to the transport of polyunsaturated fatty acids [6]. AFP is a natural immunosuppressant (almost no antibodies are produced on it) and blocks the formation of neutralizing antibodies against interferon [7].

The specified technical result is achieved as follows. AFP protein is extracted from retroplacental, umbilical cord or abortion blood using one of the methods [8-14].

Dextran stabilizer with a molecular weight of 40,000-8,000 kilodaltons and interferon in the following proportions are added to the obtained AFP protein.

1. AFP - from 40 μg / ml to 100 μg / ml.

2. Interferon-α - from 3,000,000 units / ml to 18,000,000 units / ml.

3. Dextran from 2 mg / ml to 6 mg / ml.

Thus obtained composition is subjected to sterilizing filtration, filling in ampoules or vials and lyophilized (dried).

After checking the quality of the drug, it is prescribed for treatment of patients with chronic viral hepatitis and AIDS. The drug is administered subcutaneously or intramuscularly 1 time per day for 30 days in doses of AFP from 0.5 μg / kg, interferon from 10000 U / kg.

Examples of specific tasks.

Example 1

In 10 l of human retroplacental blood, chloroform in the amount of 100 ml per 10 l and aprotinin in the amount of 5 g are added. Blood is defended in the refrigerator at a temperature of 2-6 ° C for 24 hours. Centrifuged at 6000 rpm for 40 minutes, decantation. Serum volume after centrifugation was 7680 ml. Filtration through 3 layers of cotton fabric and membrane filters 3.0-0.22 microns. Triton X-100 solution is added to the serum to 0.01% saturation.

Sorbent (Sepharose FF) with covalently sewn antibodies (15-30 mg / ml) against human AFP, 180 ml, washed with 5 volumes of a solution of 0.15 M sodium chloride.

Before applying the serum, the sorbent was treated with a 1.5 M solution of magnesium chloride: sorbent / MgCl ₂ = 1: 3, exposure for 30 minutes (2 times). Then the sorbent is washed with a 0.15 M sodium chloride solution (10 volumes).

Serum of cattle is heated to 37 ° C in a thermostat and combined with a sorbent. Blocking lasts for 30 minutes at a temperature of 18-20 ° C (in a ratio of 1: 3). After that, the sorbent is washed with a 0.15 M sodium chloride solution (10 volumes).

Sorption occurs at pH 9.0 for 60 minutes at room temperature and vigorously stirring with an overhead stirrer until the AFP content is completely depleted, followed by settling for 2 hours.

After settling, the serum is decanted and the sorbent is washed with a 1.0 M solution of magnesium chloride on a Schott filter with exposure for 30 minutes with stirring. Repeated non-specific elution is carried out with a 2.0 M solution of magnesium chloride under similar conditions. Then the sorbent is washed with 0.15 M sodium chloride solution (10 volumes).

The AFP elution is carried out twice (for 1.5 hours) with vigorous stirring at room temperature 2.8-4 M magnesium chloride with 0.5% Triton X-100, adjusted with 0.1 M Gly-HCl buffer to pH 2.0- 3.0 with constant monitoring of pH. The ratio of eluent to the volume of the sorbent is 1: 1. Then the sorbent is washed with 0.15 M sodium chloride solution to a pH of 7.0. The eluent is neutralized with sodium hydroxide solution to a pH of 6.8.

AFP concentrate having a total volume of not more than 0.25 L is passed through a prepared chromatographic column with Sephedex G-25 sorbent at a rate of 20.0 ml / min.

During chromatography, the buffer is replaced: the elution buffer with 0.02 M Tris-hydrochloric acid, pH 6.3. With increasing optical density, the solution at the outlet of the column is collected in a sterile container. The collection of the drug is continued until the optical density drops below 0.003 Au.

Ion exchange chromatography. The preparation of the chromatographic column for ion exchange chromatography is carried out under aseptic conditions. 60 ml of the sorbent, DEAE Sepharose, is washed with at least 1.2 L of a sterile solution of Tris-hydrochloric acid, pH 6.3 at a rate of 4.0 ml / min through a chromatograph. At the end of this process, check the pH of the solution at the outlet of the column.

AFP concentrate having a total volume of not more than 0.8 L is passed through a prepared chromatographic column at a rate of 4.0 ml / min.

Before applying to the ion-exchange column in the AFP solution, the pH is checked, which should be 6.3. If deviating, the solution is titrated with an appropriate solution (0.1 M NaOH or 0.1 M HCl).

Then a sterile solution of 0.02 M Tris-hydrochloric acid, pH 6.3 is connected and the column is washed to zero optical density (not less than 2.0 L). A check is made for the absence of protein in washing from the newt after the procedure.

Then, gradient elution of the AFP from the ion-exchange column with a sterile solution of 0.02 M Tris-hydrochloric acid in a 0.5 M solution of sodium chloride, pH 6.3, gradient length 400 ml, speed 4 ml / min.

To increase the optical density, the protein solution at the outlet of the column is collected in a sterile container.

In the resulting AFP solution, the concentration of total protein and the content of AFP are determined. The purity of the AFP is determined by SDS polyacrylamide gel electrophoresis and should be at least 95%.

To obtain the intermediate product, a 10% solution of reopoliglyukin is used. When calculating the volume of reopoliglyukin, the actual AFP content in the purified concentrate is taken into account. The calculated volume, measured using a cylinder, is added to the protein container.

;

;

;

;

,

,

Vp / p - the volume of the resulting intermediate product, ml;

Vp / g is the volume of reopoliglyukin, ml;

m is the total AFP content in the concentrate, mcg.

The total volume of the substance should be V _{p / p}

V _{p / p} = m / 80.

A solution of 0.15 M sodium chloride is added to the container with protein and the calculated volume of reopoliglyukin:

V _NaCl = V _{p / p} -V _{p / g} -V _AFP .

The resulting AFP substance is tested for the absence of antibodies to HIV, hepatitis B virus and antibodies to hepatitis C.

After the technological steps, the AFP stabilized by reopoliglyukin is mixed with interferon so that 1-6 ml of the solution contains 3-6 million units of interferon, 60-150 μg of AFP and 2-6 mg of reopoliglukin.

Next, the AFP preparation with interferon stabilized by reopoliglyukin is subjected to sterilizing filtration through membrane filters with a pore diameter of 0.22 microns, bottled in sterile bottles or ampoules and subjected to freeze drying.

Thus obtained drug is tested in accordance with the regulatory and technological documentation in the department of quality control of medicines. The lyophilized dried AFP preparation should contain 3-6 million units of interferen, 60-150 μg AFP and 2-6 mg of reopoliglyukin.

Example 2

Viral hepatitis b

Case history No. 4735. Patient Yu., 25 years old, was hospitalized in the treatment department of the NUZ OKB Art. Perm II, Perm, from December 3, 2007 to December 21, 2007.

Diagnosis: Chronic viral hepatitis B (HBsAg +), inactive, replication phase, low viral load.

Complaints upon admission to periodic severity in the right hypochondrium, weakness, fatigue.

Medical history: In the fall of 2006, HBsAg was found at a physical examination. Directed to the hepatocenter. A 3-week course of reaferon was carried out, then received pegintron according to the generally accepted scheme. During treatment, an increase in copies of the virus by 40 times (410000 copies of the virus / ml) was observed.

Clinical observation:

In the hospital, interferon therapy was prescribed - α at 3,000,000 ME in combination with AFP at 0.075 mg IM. Interferon and AFP preparations obtained by the technology presented in example 1 were administered subcutaneously 1 time per day. The patient received treatment for 17 days, daily.

On the 17th day of therapy according to PCR diagnostics - DNA HBV was not detected.

Example 3

Viral hepatitis C.

Case history No. 4658. Patient K. was treated in the therapy department of the NIH OKB of Perm Station II from 11/27/07 to 12/29/07 with a diagnosis of: Chronic viral hepatitis C, genotype 1b, replication phase, inactive, cholestasis syndrome, moderate viral load.

Concomitant: duodenal ulcer, remission.

Upon admission, he complained of a feeling of heaviness in the right hypochondrium, which occurs periodically, more often after a diet violation.

Anamnesis of the disease: In childhood, he was sick with viral hepatitis A. In 1976, the patient underwent blood transfusion (amputation of the lower leg after a car accident). In June 2007, he was undergoing spa treatment; examination revealed hyperbilirubinemia up to 85.0 mmol / l, an increase in transaminase activity to three norms. PCR revealed viral hepatitis C, genotype 1b, moderate viral load - 4.2 * 10 ⁵ / ml. He was consulted at the hepatocenter in November 2007. No treatment was carried out.

Clinical observation:

Assigned to interferon - α 3,000,000 ME in combination with AFP 0.075 mg, preparations obtained by the technology described in example 1 were administered subcutaneously 1 time per day for 27 days daily i / m.

After the course of therapy, positive dynamics were noted: a decrease in the level of total bilirubin from 87.4 to 39.1 mmol / L, yellowness of the skin decreased significantly, and the viral load decreased to zero.

Example 4

Viral hepatitis C.

Case history No. 1053. Patient G., 27 years old, was treated in the therapy department of the NIH OKB of St. Petersburg Perm II from 03.17.08 to 02.17.08 with a diagnosis of

Primary: Chronic viral hepatitis C, inactive, genotype 3a, replication phase.

Concomitant: chronic gastritis, remission.

Upon receipt of complaints actively did not show.

Anamnesis of the disease: At the next professional examination in December 2007, ELISA revealed antibodies to viral hepatitis C.

Clinical observation: General condition is satisfactory. Objective status without features. The viral load before treatment was 6.8 * 10 ⁶ / ml.

The level of transaminases is normal (ACT 26 mmol / L, ALT 28 mmol / L).

Interferon-α was prescribed at a dose of 3000000 ME and AFP 0.075 mg, preparations obtained by the technology described in Example 1 were injected subcutaneously 1 time per day for 30 days daily in oil.

As a result of the treatment performed during PCR examination, HCV RNA was not detected.

The level of transaminases remained within the normal range - ACT 21 mmol / L, ALT 22 mmol / L.

Example 5

Viral hepatitis C.

Case history No. 1426. Patient D., 54 years old, was treated at the Department of Therapy “NIH OKB Art. Perm-II JSC Russian Railways” from 10.04.08 to 05.09.08 with a diagnosis of

Primary: Chronic viral hepatitis C, genotype 2a, inactive, moderate viral load.

Concomitant: Chronic pyelonephritis, incomplete remission. Chronic prostatitis.

Upon receipt of complaints actively did not show.

Anamnesis of the disease: In November 2007, at the next professional examination at the place of work, ELISA revealed antibodies to viral hepatitis C.

Directed for further examination and the course of individual antiviral therapy.

Clinical observation: General condition is satisfactory. Objective status without features.

The viral load before treatment was 2.74 * 10 ⁵ / ml.

Transaminases are normal (ACT 26 mmol / L, ALT 26 mmol / L).

Interferon-α was prescribed at a dose of 3,000,000 ME in combination with AFP at a dose of 150 μg, the preparations obtained by the technology described in Example 1 were injected subcutaneously once a day, the preparations were administered intramuscularly daily for 30 days.

After the course of therapy, transaminases remained within normal limits. PCR-RNA HCV was not detected.

Example 6

Viral hepatitis C.

Case history No. 1743. Patient L., 54 years old, was treated in the treatment department of the NIH OKB st. Perm-II JSC Russian Railways from 05/05/08 to 06/03/08 with a diagnosis of

Primary: Chronic viral hepatitis C, genotype 1b, replication phase, inactive, low viral load.

Upon receipt of complaints actively did not show.

Anamnesis of the disease: In September 2007, at the next professional examination at the place of work, ELISA revealed antibodies to hepatitis C.

Directed for further examination and the course of individual antiviral therapy.

Clinical observation: General condition is satisfactory. Objective status without features. The viral load before treatment was 1.3 * 10 ³ / ml.

Transaminases are normal (ACT 25 mmol / L, ALT 28 mmol / L).

Interferon-α was prescribed at a dose of 3,000,000 ME in combination with AFP at a dose of 150 μg, preparations obtained by the technology described in Example 1 were injected subcutaneously 1 time per day for 30 days daily intramuscularly.

After the course of therapy, transaminases remained within normal limits. PCR-RNA HCV was not detected.

Example 7

Viral hepatitis C.

Medical history No. 2015. Patient T., 53 years old, was treated at the Department of Therapy “NUZ OKB St. Perm-II JSC Russian Railways” from 05.26.08 to 06.24.08 with a diagnosis of

Primary: Chronic viral hepatitis C, genotype 3a, replication phase, active cytolysis, moderate viral load.

Upon receipt of complaints actively did not show.

Medical history: In March 2007, at the next professional examination at the place of work, ELISA revealed antibodies to hepatitis C.

Directed for further examination and the course of individual antiviral therapy.

Clinical observation: General condition is satisfactory. Objective status without features. The viral load before treatment was 1.89 * 10 ⁵ / ml. ALT 169 mmol / L, ACT 82 mmol / L. Other biochemical parameters were normal.

Interferon was prescribed - and at a dose of 3,000,000 ME in combination with AFP at a dose of 150 μg, the preparations obtained by the technology described in Example 1 were injected subcutaneously 1 time per day for 30 days daily intramuscularly.

After a course of therapy, HCV RNA was not detected by PCR. A significant decrease in the level of transaminases ALT 58 mmol / L, ACT 34 mmol / L was also noted.

Example 8

Viral hepatitis C.

Patient R., 45 years old. The main diagnosis: Chronic viral hepatitis C, moderate activity, genotype 3a, replication phase. The disease is occupational.

Concomitant: chronic gastroduodenitis, exacerbation.

Complaints: general weakness, decreased performance, sleep disturbance, heartburn, discomfort in the epigastric region and right hypochondrium.

Medical history: Sick since 2002. Occupational disease. A surgeon during an operative treatment of a patient with viral hepatitis C injured his right hand.

After an additional examination, antibodies to HCV were found. Since 2002, he repeatedly conducted anti-viral therapy with α-interferon drugs without effect.

Clinical observation: General condition is satisfactory. Objective status without features. The viral load before treatment was 5.9 * 10 ⁵ copies / ml, genotype 3a. Transaminase levels: ACT 33 mmol / L, ALT 73 mmol / L.

Interferon was prescribed - and at a dose of 3000000 ME and AFP 0.075 mg, the preparations obtained by the technology described in Example 1 were injected subcutaneously 1 time per day for 30 days daily intramuscularly.

As a result of the treatment performed during PCR examination, HCV RNA was not detected. The level of transaminases within the normal range is ACT 18 mmol / L, ALT 22 mmol / L. The patient also notes an increase in efficiency, normalization of sleep, the disappearance of discomfort in the epigastric region and the right hypochondrium.

Viral hepatitis C + HIV infection.

Example 9

HIV + viral hepatitis C.

Patient P., 27 years old, was on outpatient treatment from 03.17.08 to 17.2.08 with a diagnosis of

Primary: Chronic viral hepatitis C, genotope 3a, inactive, HIV.

Concomitant: chronic gastritis, remission.

Upon receipt of complaints actively did not show.

Anamnesis of the disease: At the next professional examination by ELISA, antibodies to viral hepatitis C and HIV were detected.

Directed for examination and the course of individual antiviral therapy.

Clinical observation:

The viral load of hepatitis C was detected before treatment 6.8 * 10 ⁶ / ml, genotype 3a and HIV (240,000 copies / ml).

Assigned to interferon - α 3,000,000 ME and AFP 0,075 mg, drugs obtained by the technology presented in example 1 were administered subcutaneously 1 time per day for 30 days daily intramuscularly.

After a course of therapy, HCV RNA and HIV RNA by PCR were not detected.

The patient observation period is 12 months with monthly virological monitoring. Hepatitis C and HIV viruses are absent.

Literature

1. Manns M.P. et al. Lancet, 2001; 358: 958-965.

2. Fried M.W. et al. N. Engl. J. Med., 2002; 347: 975-982.

3. Hadziyannis S.J. et al. Ann Intern Med., 2004; 140: 346-355.

4. Zeuzem S. et al. J. Hepatol. 2004; 40: 993-999.

5. Rustgi V. et al. J., EASL 2006. Hepatol., 2006, 44 (suppl. 2), A. 113.

6. Chereshnev VA, Rodionov S.Yu., et al., Alpha-fetoprotein. Ekaterinburg: Ural Branch of the Russian Academy of Sciences, 2004 .-- 376 p.

7. Mizejewski G.J. Alpha-fetoprotein as a biologic response modifier: Relevance to domain and sub domain structure. Proc. Soc. Exp. Biol. Med. (N.Y.). 1997. Vol.215: P.333-362.

8. RF patent No. 2100031 "Method for producing alpha-fetoprotein" according to application No. 95120418 of 01/01/1995.

9. RF patent №2121350 "Method for the preparation of alpha-fetoprotein" according to application No. 96111118 from 06/03/1996.

10. RF patent No. 2163139 "Method for the preparation of alpha-fetoprotein" according to application No. 2001014564 of 02.23.2000.

11. RF patent №2169578 "Method for producing a preparation of alpha-fetoprotein" according to application No.2000104565 of 02.23.2000.

12. RF patent No. 2170586 "Method for the preparation of alpha-fetoprotein" according to the application No. 2000104566 of 02.23.2000.

13. RF patent No. 2283131 "Method for the preparation of the drug alpha-fetoprotein dry" on the application No. 2005109879 from 04/05/2005.

14. RF patent No. 2308286 "Method for the preparation of alpha-fetoprotein" according to the application No. 2006100948 of 01/10/2006.

Claims (4)

1. Lyophilized preparation for the treatment of chronic viral hepatitis B or C and AIDS, containing alpha fetoprotein (AFP) 60-150 μg, interferon α 3000000-6000000 U and dextran 2-6 mg. 2. The drug according to claim 1, characterized in that reopolyglukin is used as dextran. 3. The method of obtaining the drug for the treatment of chronic viral hepatitis and AIDS according to claim 1, characterized in that the stabilized reopoliglucin AFP is mixed with interferon so that 1-6 ml of the solution contains 3-6 million units of interferon, 60-150 μg AFP and 2- 6 mg of dextran, the resulting composition is subjected to sterilizing filtration and produced in sterile vials or ampoules, followed by freeze drying. 4. The method according to claim 3, characterized in that the filtration is carried out through membrane filters with a pore diameter of 0.22 μm.

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