TW200904468A - Concentrate of chikungunya-specific immunoglobulins as a medical product - Google Patents

Abstract

The invention concerns a new medicinal product for the treatment of chikungunya, i. e a concentrate of chikungunya-specific immunoglobulins, as well as its process of preparation.

Description

200904468 IX. Description of the Invention: [Technical Field] The present invention relates to a novel pharmaceutical product for treating chikungunya, that is, a pyknuckle-specific immunoglobulin concentrate, and a preparation method thereof. [Prior Art] Preface Qu Gong fever (abbreviated as CHIK) is an infectious tropical disease caused by arbovirus (Togaviridae alphavirus), which is transmitted by genus mosquitoes. The name comes from Bantu's meaning: a person who bends, rolls, or bends a person's disease because it causes very serious joint pain and stiffness, giving the infected patient a very characteristic curved appearance. The arthropod vector is classified as a genus virus under its cycle. The arbovirus is defined by the WHO as a virus that is inherently present in nature, and is substantially or largely transmitted through blood parasitic arthropods. Infected between susceptible vertebrate hosts; they multiply and provoke viremia in vertebrates' to proliferate in arthropod tissues and to infect other vertebrate animals via biting insects after an external incubation period. The viral infection of the adult host to the adult female mosquito is carried out through the blood when biting. The virus multiplies in the mosquito and passes over the animal's stomach barrier. Now the salivary glands. The pollution of healthy humans is done by the anticoagulant saliva of the mosquitoes, and is released before the biting of the blood vessels. The person who is the host of the viremia causes only a few days before the disease. 200904468 More than 950 species In addition to mosquitoes, several of them can infect Qugong, but only A. elegans Mei/u and white-lined mosquitoes (jeea / Z? 〇iciw 5·) have been identified as epidemic vectors because they are adapted to humans. Living environment. These same species are also involved in the transmission of other arboviruses: dengue fever, hemorrhagic dengue fever (HDF), yellow fever, etc. Clinical conditions are mainly high fever, similar to dengue fever (dengue is often misjudged as tsugeo heat, vice versa Also) combined with incompetent joint pain and inter- or skin rash, but there are serious forms that have been neglected at present: violent hepatitis, heart attack, sterile meningitis, etc. Several other arboviruses of the alphavirus genus (approximately 30-kD capsid and poly-adenylated RN A at 3'), such as Ross River, O'nyongnyong and Mayalo Virus (Mayaro) The incubation period of this disease lasts an average of 4 to 7 days. The virus appears in the blood and the viremia that may be transmitted is prolonged for more than about 5 days. The antibodies are then developed. They continue to exist in the blood. Obtain immunity for at least one year (compare the following Phase II trials.) Traditionally, there is no viricide treatment and no vaccine is approved for marketing. Treatment is only a symptomatic reduction in fever and pain reduction. The United States Army Medical Research Institute of Infectious Diseases conducted Phase I trials and Phase I trials of the Quebek fever vaccine.

Phase II trial (Edelman R et al. “Phase II safety and immunogenicity study of live chikungunya virus vaccine" TSI-GSD-218. June 2000; Am J 200904468

Trap Med Hyg, 62:681-5), a randomized, double-blind, placebo-controlled study of the safety and immunogenicity of live-purified CHIK vaccine on 73 healthy adult volunteers The study consists of. 59 volunteers were immunized subcutaneously with CHIK vaccine and 14 subjects were injected with placebo. Fifty-seven (98%) of the vaccine recipients developed anti-CHIK neutralizing antibodies on day 28, and 85% of the vaccine recipients remained seropositive after one year. The combination of two antiviral compounds, ribavirin and interferon alpha, was also tested in Briolant S et al. ("In vitro inhibition of Chikungunya and Semliki Forest viruses replication by antiviral compounds: synergistic effect of Interferon-alpha and ribavirin combination”, Antiviral Res., 2004 Feb.; 61(2): lll-7). This combination of IFNct2b and ribavirin exhibits a synergistic antiviral effect on piroxime, which is highly promising for treatment. However, such treatment should be extremely expensive and repetitive and should involve many known interferon side effects. SUMMARY OF THE INVENTION Summary of the Invention In the face of the lack of confirmatory treatment, the vaccine is not ready quickly, and the annoying antiviral treatment, the applicant has sought to provide a novel treatment against pyknosis. Surprisingly, the applicant in this case has demonstrated that the specific immunoglobulin concentrate administered to Qugong fever can be used to solve this technical problem. Definitions The term "concentrate" means the product obtained by removing certain ingredients. The concentrate of the immunoglobulin is obtained by removing certain components of the plasma to achieve a fraction enriched with the plasma of the 200904468 globulin. "Immunoglobulin" (Ig) - The word means a natural globulin mainly present in plasma, which has an antibody function and can be used for therapeutic or prophylactic treatment. An immunoglobulin is a heterodimer composed of a disulfide bridge connecting a 2 heavy chain and a 2 light chain. Each strand is composed at the N-terminal position by the antibody directly against a variable functional site or region of the antigen (by rearranging the V-J gene in the light chain and VDJ encoded in the heavy bond), and at the C-terminal position The single CL function is located in the light chain or the three functional parts (CHI, CH2 and CH3) in the heavy chain. The variable functional portions of the heavy and light chains, in combination with the CHi and CL functional sites, form a Fab portion that retains an accessible effector molecule, such as an Fc r R acceptor and Clq; wherein the Fab portion is linked to the Fc region via an extremely elastic hinge region, When the Fc region mediates the effector properties of the antibody, each Fab binds to the antigen target.

IgG is the largest amount of immunoglobulin (75 to 80% of circulating antibodies). They protect the body from bacteria, viruses and toxins that circulate in the blood and lymph. In addition, they quickly combine complement (one of the components of the immune system). They also participate in memory reactions, which are the basis of immunity and the basis of the vaccination mechanism. Ultimately, immunoglobulin G crosses the placental barrier and thus produces passive immunity in the fetus.

IgA is mainly found in secretions such as saliva, intestinal fluid, sweat and milk. The primary role of immunoglobulin A is to prevent the binding of pathogenic agents to cells, particularly cells that make up the mucosa and epidermis.

IgM is an immunoglobulin secreted by the body after it first contacts the antigen. They are the first type of immunoglobulin released by plasmocytes. In the blood 200904468 The presence of IgM indicates an infection. Proteolysis of the immunoglobulin by papain produces two identical fragments, which are known Fab (fragment antigen binding) and one Fc (crystallizable portion) fragment. The Fc fragment supports the effector function of immunoglobulins. F(ab')2 fragments were generated via proteolysis of pepsin, wherein the two Fab fragments were still bound by a disulfide bridge and the Fc fragment was cleaved into a peptide. The F(ab')2 fragment is formed from two Fab' fragments (a Fab' fragment consisting of a Fab and a hinge region), joined via an interchain disulfide bridge to form F(ab')2. The term "chromatographic analysis" means a method of separation of the components of a mixture that is retained by their choice based on the appropriate medium. [Embodiment] DETAILED DESCRIPTION OF THE INVENTION First, the present invention relates to a typhoid fever-specific immunoglobulin concentrate which is a pharmaceutical product. Since Cohn developed the ethanol sinking method (Cohn et al. 1946, J. Am. Chem. Soc. 68, 459; Oncley et al. 1949, J. Am. Chem. Soc. 71, 541), IJ knows the use of rich Human plasma fractions containing immunoglobulins treat a variety of infections and congenital defects. In particular, the concentrate according to the present invention is a concentrate of immunoglobulins A, G and sputum which is specific to typhoid fever virus as a pharmaceutical product, or a concentrate of proprietary immunoglobulin G, or a proprietary immunoglobulin. The concentrate consists of a concentrate. It is particularly preferred that the concentrate according to the invention comprises from 90 to 98% of immunoglobulin. The concentrate according to the present invention, in addition to the intact typhoid fever-specific immunoglobulin, can contain the Przetox virus-specific F(ab)'2 and/or Fab sheet 200,904,468 segments, particularly 5 to 50% The F(ab)'2 and/or Fab, especially at least 50 to 60 g/L of Ig and fragments are used in pharmaceutical formulations. These F(ab)'2 or Fab fragments containing the binding positions of the antibodies may have lost some of the properties derived from intact antibodies, such as the ability to bind to Fq receptors. The concentrate according to the present invention, in addition to the intact typhoid fever-specific immunoglobulin, can contain a specific F(ab)'2 or Fab fragment exclusively derived from IgG and IgM. According to the invention, from 1 to 10 mmol of magnesium and/or zinc may be added to the concentrate. Another object of the present invention is the use of the concentrate according to the present invention for the preparation of a medicament for the treatment of tiaphlog. This treatment is preventive or curative. It is used to confer passive immunity to people who are not infected in a prevalent area, or to care for patients who have been infected with the virus. The drug to be considered is administered via a topical, subcutaneous, oral, intramuscular or intravenous route. The effect is several weeks, about 21 days; and in addition, if the epidemic or symptoms persist, the cast must be repeated. The invention also relates to a process for the preparation of a concentrate according to the invention. The first step in this method is to create at least 1,000 plasma donor banks, each containing a sufficient amount of anti-compressed Ig. These donors are from people who have been exposed to the disease or who have developed the disease. Titration can be carried out according to C. van de Water et al., Journal of Immunological Methods, 166 (1993), 157-164. 200904468 To enhance the immunoglobulins of this plasma bank, other plasma components known as "lipids and proteinaceous contaminants" are precipitated in a single step. This purification by a single step of incubation can be carried out according to Steinbuch (Steinbuch M., Archiv. Biochem. Biophys., 134, 279-284) by diluting plasma with octanoic acid in the precipitation conditions and adding octanoic acid. It can also be passed through precipitants such as Rivanol, chlorinated chlorinated, cetylpyridinium chloride, octanoic acid, polyphosphoric acid, and adsorbents and tricalcium phosphate and bentonite. get on. The supernatant can constitute an immunoglobulin concentrate according to the invention. It therefore contains a mixture of IgG, A and hydrazine. For example, this can be re-acquired via centrifugation or filtration, via the addition of at least one filter additive. The supernatant can be subjected to a conventional virus deactivation method using a solvent/detergent (Triton XI 00). If the precipitate is a octanoic acid precipitate, the residual octanoic acid in the supernatant is eliminated via tricalcium phosphate. In order to obtain a concentrate of IgG, IgA or IgM, the method of the application of EP 0 272 768 8.0 can be applied. The supernatant is then subjected to an additional step of purification via chromatographic analysis. The anion exchanger is carried out in an alkaline P oxime. In particular, the pH of the supernatant was previously adjusted to be between 8.9 and 9.1, and the column was equilibrated with a pH of 8.9 to 9.1. The chromatographic analysis step enables the immunoglobulin to be retained and passed through the unretained protein to the effluent. Chromatographic analysis can be performed on a reticular polysaccharide or ethylene polymer gel grafted with DEAE 'T MAE or QAE groups. After removing the unretained protein by washing the column with the same buffer as the equilibration buffer, the immunoglobulin 200904468 white is eluted with a phosphate buffer having a pH between 4 and 7, preferably at pH 6.2. It can be used to collect IgA by further dissolving with 100 to 175 mM NaCl (preferably 150 mM) pH in the same phosphate buffer of 6 to 6.3. It can be used to collect IgM by adjusting the pH to 6 to 7 and supplementing 250 to 350 mM NaCl (preferably 300 mM) with a phosphate buffer followed by dissolution. Any type of mixture between IgA, IgG and IgM can be considered by mixing the concentrates as described above. The immunoglobulin thus solubilized and collected can be concentrated by ultrafiltration and conventional filtration, and then filtered through a filter having a porosity of 1 Torr to 15 nm. The concentrated and filtered immunoglobulin solution is added with a pharmaceutically acceptable stabilizer and then packaged under sterile conditions and can be frozen and lyophilized. Nanofiltration is used to eliminate viruses that are resistant to solvent/detergent treatment. To prepare a concentrate of genomic fever virus-specific Ig and F(ab)'2 or Fab fragments, a concentrate (1) of immunoglobulin as described above is prepared: a mixture of A, G and hydrazine or G and hydrazine Mixture, or only G, or only Μ; then in a second step, decompose part of the resulting Ig concentrate to give F(ab), 2 or Fab fragment (2); and in a third step, mix the concentrate (1) And (2). To obtain F(ab)'2 fragment', proteolysis was carried out at pH 4 and 35t of pepsin 1% by weight protein (IGLOO step procedure). To obtain the Fab fragment, proteolysis was carried out on papain. [Simple description of the map]

Claims (1)

200904468 X. Patent application scope: 1. A concentrate of chikungunya virus-specific immunoglobulin as a pharmaceutical product. 2. The concentrate of claim 1 of the patent application consisting of a concentrate of immunoglobulins A, G and sputum. 3. A concentrate as claimed in claim 1 which consists of a concentrate of immunoglobulin G. 4. The concentrate of claim 1 of the patent, which consists of a concentrate of immunoglobulin. 5. The concentrate of any one of claims 1 to 4, comprising from 90 to 98% of immunoglobulin. 6. The concentrate of any one of claims 1 to 5, which also contains a typhoid fever specific F(ab)'2 fragment. 7. The concentrate of any one of claims 1 to 6 which also contains a specific Fab fragment of the typhoid fever virus. 8. A concentrate according to claim 7 which contains 5 to 50% of F(ab)'2 and/or Fab. 9. The concentrate of any one of clauses 6, 7 and 8 wherein the F(ab)'2 or Fab fragment is an F(ab)'2 or Fab fragment of IgG and IgM. 1 〇 A concentrate of any one of claims 1 to 9 can be added with 1 to 10 mmol of magnesium. 1 1. A concentrate according to any one of claims 1 to 10, wherein 1 to 10 mmol of zinc can be added thereto. 1 2 . The use of a concentrate according to any one of claims 1 to 11 for the treatment of chikungunya. 1 3. The use of the scope of claim 12 for the preparation of a medicament for administration via a topical, subcutaneous, oral, intramuscular or intravenous route. 1 4. A method of preparing a concentrate according to claim 1 or 2, characterized by the following steps: - creating at least one bank of plasma donors from a person who has been exposed to the disease, or Patients who develop disease, - precipitate lipids and protein contaminants in a single step, - regain ig concentrate in the supernatant. A method for preparing a concentrate according to any one of claims 1 to 5, characterized by the following steps: - creating at least 100 banks of plasma donors from a disease that has been exposed to the disease Human, or a patient who has developed a disease, - precipitates lipids and protein contaminants in a single step, - the supernatant is chromatographed in an anion exchanger at alkaline pH, - at a pH between 4 and 7 The phosphate buffer is used to elute IgG, preferably at pH 6.2, - 100 to 175 mM NaCl (preferably 150 mM) may be added thereto, and the same phosphate buffer having a pH of 6 to 6.3 may be followed to dissolve the IgA, and the pH may be 6 to 7. And adding 250 to 350 mM NaCl in the same phosphate buffer followed by IgM, - IgG, IgA and IgM concentrates can be mixed. 16. The preparation method according to claim 15, wherein the pH of the supernatant is adjusted to be between 8.9 and 9.1 before the chromatography, and the color layer-14-200904468 is analyzed by the column ρ Η 8.9 Buffer balance to 9.1. A method for preparing a concentrate according to any one of claims 6, 7 and 8 which is characterized by the following steps: (1) preparing an Ig as claimed in claim 14 or 15 Concentrate, or Ig G as in claim 15 or 16 of the patent application, or IgM as in claim 15 or 16 of the patent application, (2) an earlier concentrate of the proteolytic portion to give F(ab), 2 or Fab fragments, (3) Mixed parts (1) and (2). 1 8 _ - A method for preparing a concentrate according to claim 9 of the patent application, characterized by the following steps: (1) preparing an I g G concentrate as claimed in claim 15 or 16, (2) Prepare an I g Μ concentrate as in claim 15 or 16 of the patent application, '(3) Mixing parts (1) and (2), (4) an earlier mixture of proteolytic moieties to obtain I g G and I g F(ab)'2 or Fab fragments, (5) mixed (3) and (4). 19. A process according to claim 17 or 18, characterized in that the protein is hydrolyzed to 1% by weight of pepsin at pH 4 and 35. (20) The method of preparation according to claim 17 or 18, characterized in that the papain is proteolytically obtained to obtain a Fab fragment. 21. As claimed in any one of claims 14 to 20 The preparation method is characterized in that the precipitate is a octanoic acid precipitate, and the residual octanoic acid in the supernatant is eliminated via the tricalcium phosphate. -15-200904468 2 2. The preparation method according to any one of claims 14 to 21, The method of preparing the precipitate by centrifugation after adding at least one filter additive. The preparation method according to any one of claims 14 to 22, wherein the supernatant is treated with a solvent/detergent. The preparation method according to any one of the items 15 to 23, characterized in that the immunoglobulin which is eluted is subjected to ultrafiltration and is subjected to conventional sterile filtration, and then passed through a filter having a porosity of 1 〇〇 to 15 nm. The method of preparation according to any one of claims 15 to 24, characterized in that the pharmaceutical acceptable stabilizer is added to the concentrated and filtered immunoglobulin solution, and then dissolved in a sterile manner. Packed under liquid conditions and can be frozen and lyophilized. 200904468 VII. Designated representative map: (1) The representative representative of the case is: No. (2) The symbol of the symbol of the representative figure is simple: te. 8. If there is a chemical formula in this case Please reveal the chemical formula that best shows the characteristics of the invention: Μ 〇j \ w -4 -