Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/06—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies from serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/10—RNA viruses
  • C07K16/116—Togaviridae (F); Matonaviridae (F); Flaviviridae (F)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the invention relates to a novel medicament for the treatment of chikungunya, namely a concentrate of chikungunya-specific immunoglobulins as well as a process for their preparation.
• Chikungunya (abbreviated CHIK), is a tropical infectious disease, caused by an arbovirus (alphavirus family Togaviridae), transmitted by mosquitoes of the genus Aedes.
• arbovirus alphavirus family Togaviridae
• the name is of Bantu origin and means: who bends, who curls up, or curved man's disease because it causes very strong articular pains associated with stiffness, which gives the infected patients a very characteristic curved attitude.
• the viruses that involve in their cycle arthropod vectors are grouped under the general term of arbovirus.
• Arboviruses are defined by WHO as viruses that persist in nature essentially or largely through biological transmission between susceptible vertebrate hosts by blood-sucking arthropods; they multiply and cause viremia in vertebrates, proliferate in arthropod tissues and are transmitted to a new vertebrate by biting arthropod after an extrinsic incubation period.
• Virus transmission from a viremic host to a female adult mosquito occurs through the blood sucked in during the bite. The virus multiplies in the mosquito, it crosses the stomach border of the animal and is found in its salivary glands. Contamination of a healthy man is achieved by the anti-coagulant saliva of released mosquito just before the sting in a blood vessel.
• the window during which a person is a viremic host before becoming ill is only a few days old.
• many of them are likely to transmit chikungunya, but only Aedes aegypti and Aedes albopictu ⁇ have so far been identified as epidemic vectors, because of their adaptation to areas of human habitat.
• These same species are also involved in the transmission of other arboviruses: dengue fever, dengue haemorrhagic fever (DHF), yellow fever, etc.
• the incubation of the disease lasts from four to seven days on average.
• the viremia that is to say the period of presence of the virus in the blood and therefore of possible transmission, is spread over about five days.
• the antibodies are then declared. They stay in the blood. Immunity is therefore normally acquired for life or at least one year (see below the Phase II trial).
• Phase I and Phase II were conducted in the United States for an anti-chikungunya vaccine by the US Army Medical Research Institute of Infectious Diseases.
• Phase II (Edelman R et al., "Phase II Safety and immunogenicity study of live chikungunya virus vaccine” TSI-GSD-218, June 2000, Am J Trop Med Hyg, 62: 681-5) randomized, double-blind, versus placebo, consisted of studying the safety and immunogenicity of a plaque - purified live chikungunya (CHIK) vaccine in 73 healthy adult volunteers. 59 volunteers were immunized once subcutaneously with CHIK vaccine and 14 were immunized with placebo. Fifty-eight (98%) of the 58 vaccinated patients developed anti-CHIK neutralizing antibodies at day 28, and 85% of those vaccinated were HIV-positive one year later.
• CHIK plaque - purified live chikungunya
• the Applicant sought to propose a new treatment against chikungunya.
• the Applicant has surprisingly shown that the administration of a chikungunya specific immunoglobulin concentrate makes it possible to solve this technical problem.
• concentrate means a product obtained by elimination of certain constituents.
• An immunoglobulin concentrate is obtained by removing certain constituents of the plasma to a plasma fraction enriched in immunoglobulins.
• Immunoglobulin is a natural globulin present mainly in the plasma, having antibody functions and used for curative or preventive purposes.
• Immunoglobulins are heterodimers consisting of 2 heavy chains and 2 light chains, linked together by disulfide bridges. Each chain consists, in "position N-terminal, a variable region or domain (encoded by rearranged V-J genes for the light chain and the heavy chain VDJ) specific for the antigen against which the antibody is directed, and in the C-terminal position, of a constant region consisting of a single CL domain for the light chain or of 3 domains (CH1, CH2 and CH3) for the heavy chain.
• the heavy and light chains CHi and CL domains form the Fab parts, which are connected to the Fc region by a very flexible hinge region allowing each Fab to bind to its antigenic target while the Fc region mediates the effector properties of the antibody remains accessible to effector molecules such as Fc ⁇ R receptors and CIq.
• IgG is the most abundant immunoglobulin
• immunoglobulins G cross the placenta and, as a result, cause passive immunity in the fetus.
• IgA is found mainly in secretions such as saliva, intestinal juice, sweat and breast milk.
• the essential role of IgA is to prevent pathogens bind to the cell and more specifically to recovery of cells constituting the mucosal and I 1 epidemic.
• IgM are immunoglobulins secreted during the first contact of the body with an antigen. This is the first class of immunoglobulin released by plasma cells. The presence of IgM in the blood indicates an ongoing infection.
• fragment Fab fragment Antigen Binding
• Fc fragment Antigen Binding
• the Fc fragment is the support of the effector functions of immunoglobulins.
• pepsin proteolysis an F (ab ') 2 fragment is generated, where the two Fab fragments remain linked by two disulfide bridges, and the Fc fragment is cleaved into several peptides.
• the F (ab ') 2 fragment is formed of two Fab' fragments (an Fab 'fragment consisting of an Fab and a hinge region), linked by interchain disulfide bridges to form an F (ab') 2.
• chromatography A method of separating the constituents of a mixture based on their selective adsorption by a suitable support is called "chromatography".
• the invention relates to a concentrate of immunoglobulins specific for chikungunya virus as a medicament.
• the concentrate according to the invention consists of a concentrate of immunoglobulins A, G, and M, or a concentrate of immunoglobulin G exclusively, or a concentrate of immunoglobulin M exclusively, specific to the virus. chikungunya, as a medicine.
• the concentrate according to the invention contains at least 50% of IgG type immunoglobulins and 90 to 98% of proteins reacting with antibodies specifically directed against human immunoglobulins.
• the concentrate according to the invention may contain, in addition to complete immunoglobulins specific for chikungunya virus, fragments F (ab) '2 and / or Fab specific to chikungunya virus, in particular 5 to 50% F (ab) '2 and / or Fab, in particular at least 50 to 60 g / L Ig and fragments for a pharmaceutical preparation.
• Such F (ab) '2 or Fab fragments which contain the binding site of the antibody, may have lost a number of properties of the whole antibody from which they are derived, such as the ability to bind Fcgamma receptors.
• the concentrate according to the invention may contain, in addition to complete immunoglobulins specific for the chikungunya virus, F (ab) '2 or Fab fragments specific for the chikungunya virus derived exclusively from IgG and IgM.
• the concentrate according to the invention may be supplemented with 1 to 10 mmol of magnesium and / or zinc.
• Another object of the invention is the use of a concentrate according to the invention, for the manufacture of a medicament for the treatment of chikungunya.
• This treatment is prophylactic and / or curative. It can either transfer passive immunity to people who have not yet been affected in an epidemic region, or treat patients already affected by the virus.
• the drug is administered topically, subcutaneously, orally, mucosally, intramuscularly or intravenously.
• the invention also relates to a method for preparing a concentrate according to the invention.
• the l st step of this process comprises the formation of a batch of at least 1000 plasma donations, each donation having a sufficient titer of anti-Ig chikungunya.
• a serum having a sufficient titre corresponds, for example, to a serum remaining positive for the detection anti-Chikungunya antibody, after being diluted to 1/1000 , when the titre is measured by an Elisa-type method.
• These donations come from people who have been in contact with the disease, or even from patients who have developed the disease.
• the titration can be performed according to the protocol described in C. van de Water et al., Journal of Immunological Methods, 166 (1993), 157-164.
• the other constituents of the plasma called "lipid and protein contaminants" are precipitated in a single step.
• This one-step precipitation purification can be carried out by diluting the plasma under Steinbuch precipitation conditions (Steinbuch M., Archiv Biochem Biophys., 134, 279-284) and adding caprylic acid thereto. .
• the supernatant resulting from the precipitation may constitute the immunoglobulin concentrate according to the invention. It then contains a mixture of IgG, A and M. This supernatant is recovered, for example by centrifugation or filtration, optionally by adding at least one filter aid.
• the supernatant resulting from the centrifugation or filtration can then undergo a viral inactivation treatment, for example a conventional viral inactivation treatment, solvent / detergent (Triton X100). If the precipitation made was a "caprylic" precipitation as described above, the caprylic acid residues in the supernatant are removed by PO4 calcium.
• a viral inactivation treatment for example a conventional viral inactivation treatment, solvent / detergent (Triton X100). If the precipitation made was a "caprylic" precipitation as described above, the caprylic acid residues in the supernatant are removed by PO4 calcium.
• patent EP1385886 can be applied, in particular the protocols relating to the adjustment of the pH, to the adsorption on a pre-equilibrated column, to the adsorption of the supernatant containing immunoglobulins and accompanying proteins on the column, column washing and sequential elution of the different classes of immunoglobulins, for example IgG, A or M.
• the supernatant then undergoes an additional purification step by chromatography on an anion exchanger performed at alkaline pH.
• the pH of the supernatant is adjusted beforehand to a pH of from pH "8.9 to pH 9.1 and the column is equilibrated with a buffer having a pH ranging from pH 8,9 to 9,1.
• Step chromatography allows the adsorption of immunoglobulins on the column and the passage of unadsorbed proteins in the effluent
• the chromatography can be carried out, for example, on a crosslinked polysaccharide gel or on a vinyl polymer, on which groups have been grafted. DEAE, TMAE or QAE.
• the immunoglobulins G are eluted with phosphate buffer at a pH ranging from pH 4 to 7, preferably at pH 6.2.
• Immunoglobulins thus eluted and collected can be concentrated by ultrafiltration and subjected, e.g., to conventional sterilizing filtration and then to filtration through nanometric filters of decreasing porosity of from 100 to 15 nanometers.
• the concentrated and filtered immunoglobulin solution is added with a pharmaceutically acceptable stabilizing agent such as those described in application WO 2004/091656, and then this solution is
• an immunoglobulin concentrate (1) ie a mixture of Ig A, G and M, or a mixture of IgG and M, or exclusively IgG, or exclusively Ig M, is prepared as described above, then in a second step, a portion of the Ig concentrate obtained is proteolyzed to obtain fragments F (ab) '2 or Fab (2). Finally, in a third step the concentrates (1) and (2) are mixed.
• the proteolysis is carried out at pH 4.0, at 35 ° C., with 1% of pepsin, the percentage corresponding to the weight of pepsin relative to the total weight of proteins of the concentrate.
• the proteolysis is carried out with 1% papain, the percentage corresponding to the weight of papain relative to the total weight of proteins of the concentrate.
• the proteolysis of immunoglobulins G, A and / or M can also be carried out using plasmin and / or trypsin, proteases whose mode of implementation is well known to those skilled in the art.
• One liter of plasma rich in anti-Chikungunya antibodies is collected from volunteer donors recently infected with Chikungunya virus and cured symptoms of the disease.
• the antibody titer is determined by an Elisa method of attaching virus antigens to a microtiter plate and then revealing specific antibodies using a horseradish peroxidase labeled anti-immunoglobulin reagent.
• the samples which have displayed a positive test at a dilution of at least 1 / 1000th in the context of a "specific" type Elisa method are retained.
• the plasma pool resulting from step 1.1 is cooled to -3 ° C. and added during the cooling of a volume of ethanol sufficient to obtain a final concentration of ethanol of 8%.
• the precipitate formed is removed.
• the pH of the supernatant is then adjusted to pH 5.9 by adding acetate buffer, for example, cooled to -5 ° C. and supplemented with sufficient ethanol to give a final ethanol concentration of 19%.
• the precipitate formed is collected by centrifugation, for example, and redissolved in an acetate buffer, for example, so as to obtain a pH value of 4.7 to 4.9.
• Octanoic acid is then added at 20 ° C., with vigorous stirring, until a final octanoic acid concentration of 20 g / l is obtained.
• the precipitate formed is separated by centrifugation or alluvial filtration and removed. Tricalcium phosphate or activated charcoal is added to the supernatant, and the mixture is clarified by deep filtration.
• the supernatant resulting from the clarification and containing the immunoglobulins is adjusted to pH 9 by the addition of a NaOH / glycine buffer, for example, and applied on an anion exchange column (Fractogel TMAE, for example) equilibrated at pH 9 with glycine / NaCl buffer at pH 9.
• a NaOH / glycine buffer for example
• an anion exchange column Frazier TMAE, for example
• a balancing buffer wash is carried out until a 280 nm OD at the column outlet close to the OD 280 measured during the establishment of the baseline.
• the IgG elution is then carried out via a first Sodium phosphate buffer at pH 6.2.
• a second elution is carried out with a phosphate buffer supplemented with 300 mM NaCl.
• the corresponding eluate contains a portion of the Ig G4, Ig A and Ig M.
• the detailed procedure for this purification is found in EP 1385886.
• This mixture of immunoglobulin is concentrated to 50 g / l by membrane ultrafiltration with a cut-off threshold of 30 kD or less.
• the pH of the concentrated mixture is adjusted by diafiltration against a citrate buffer at pH 3.8 to 4.2, to reach an acid pH included in this range.
• the solution is then supplemented with pepsin (10,000 FlP / mg) so that the amount of pepsin is 1% of the total amount of the proteins contained in the concentrated mixture.
• pepsin 10,000 FlP / mg
• This solution is then sterilely filtered to 0.2 ⁇ m and incubated at 37 ° C. for 20 h.
• the proteolysate is neutralized, for example by the addition of sodium hydroxide, at pH 6.2 +/- 0.2. Diafiltration of the neutralized proteolysate is performed against a glycine buffer at pH 6.2 +/- 0.2, until an OD280 of about 0.005 is obtained, the OD280 being measured on the filtrate line of the cut-off membrane. less than or equal to 30 kD.
• Peptides resulting from pepsin proteolysis and having a size less than or equal to 30 kD are removed during passage over the cut-off membrane.
• the proteolysate obtained therefore contains Fab fragments, F (ab) ' 2 type fragments, but proves to be devoid of Fc type fragments.
• the resulting proteolysate is then mixed with the remaining 75% of the first IgG-containing eluate.
• the mixture is subsequently concentrated by ultrafiltration to reach a final concentration ranging from 50 to 160 g / l, depending on the selected mode of administration.
• the concentrate is titrated according to the method described in Edelman, R. et al. (American Journal of Tropical Medicine and Hygiene, 62 (6), 2000, pp. 681-685).
• the anti-Chikungunya specific antibody titre of the concentrate thus obtained is at least 3 to 10 times higher than that of the starting plasma.
• the concentrate resulting from step 1.3 is stabilized by mixing with a formulation comprising pharmaceutically acceptable excipients, such as, for example, glycine at a final concentration of 0.22M, or such as those described in WO 200 / 091,656.
• a formulation comprising pharmaceutically acceptable excipients, such as, for example, glycine at a final concentration of 0.22M, or such as those described in WO 200 / 091,656.
• the pH of the formulation added to the concentrate is compatible with obtaining a liquid mixture having a pH ranging from 4.2 to 5.6.
• the administration of the resulting liquid mixture can be carried out, for example, intravenously, subcutaneously or intramuscularly, depending on the phlebological state of the recipient.
• the dose administered is 0.2 to 0.8 ml / kg and may, in the event of an epidemic, be administered preventively every 3 weeks to particularly exposed persons, for example the elderly, pregnant women or newborns. .

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Medicinal Chemistry (AREA)
• Virology (AREA)
• General Health & Medical Sciences (AREA)
• Molecular Biology (AREA)
• Immunology (AREA)
• Genetics & Genomics (AREA)
• Pharmacology & Pharmacy (AREA)
• Biophysics (AREA)
• Biochemistry (AREA)
• Veterinary Medicine (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Oncology (AREA)
• Communicable Diseases (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Microbiology (AREA)
• Mycology (AREA)
• Epidemiology (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Peptides Or Proteins (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=37433721

Family Applications (1)

Country Status (11)

Families Citing this family (3)

Family Cites Families (11)

• 2006
  • 2006-03-31 FR FR0602802A patent/FR2899111B1/fr not_active Expired - Fee Related
• 2007
  • 2007-04-02 EP EP07731239A patent/EP2004233A1/de not_active Withdrawn
  • 2007-04-02 AU AU2007239415A patent/AU2007239415A1/en not_active Abandoned
  • 2007-04-02 BR BRPI0709448-5A patent/BRPI0709448A2/pt not_active IP Right Cessation
  • 2007-04-02 JP JP2009502155A patent/JP2009531401A/ja active Pending
  • 2007-04-02 CA CA002647506A patent/CA2647506A1/en not_active Abandoned
  • 2007-04-02 US US11/912,439 patent/US20080219969A1/en not_active Abandoned
  • 2007-04-02 CN CNA2007800111587A patent/CN101410138A/zh active Pending
  • 2007-04-02 WO PCT/FR2007/000561 patent/WO2007118987A1/fr not_active Ceased
  • 2007-04-02 KR KR1020087025548A patent/KR20080108556A/ko not_active Withdrawn
• 2008
  • 2008-09-01 IL IL193819A patent/IL193819A0/en unknown

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events