Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/12—Drugs for disorders of the urinary system of the kidneys
• • 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
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/06—Antianaemics

Definitions

• the invention relates to the use of Factor H for the manufacture of a medicament intended for the treatment of Hemolytic Uremic Syndrome (HUS), a method of purifying Factor H from fresh frozen plasma and the factor H obtained by this method.
• HUS Hemolytic Uremic Syndrome
• Hemolytic uremic syndrome is defined by the association of microangiopathic hemolytic anemia, thromb ⁇ penia and kidney damage. It is the main cause of acute renal failure in children under 3 years of age.
• HUS There are two forms of HUS.
• HUS occurs during the summer period after an episode of diarrhea, often bloody.
• the typical HUS is secondary to an infection, in the majority of cases an infection with enteropathogenic Escherichia coli, in particular the serotype 0157: H7, producer of verotoxins.
• HUS HUS-like fatty liver disease
• Atypical HUS can occur at any age. It represents only 5% of cases of HUS in children.
• the clinical signs of the syndrome are due to the development of rich micro-cells platelets in small vessels. This particularly affects the gloraerules of the kidney causing acute kidney damage.
• Atypical HUS can be sporadic but it is often familial. In these two situations, the disease generally presents a recurrent development by pushing. His prognosis is poor.
• HUS may be associated with hypocomplementemia.
• the complement plays an essential role in the defense of the organism against infectious agents and in the inflammatory process.
• Plasma complement proteins are approximately 20 and function either as enzymes, as binding proteins, or as regulators (inhibitors or activators).
• the complement can be activated by two different routes: the conventional route and the alternative route.
• Factor H is a 155 kDa protein found in plasma at a concentration of 110-615 ⁇ g / ml. It is synthesized in the liver, macrophages, fibroblasts, endothelial cells and platelets. The secreted form of the protein is made up of 20 repeating units of 60 amino acids.
• the H factor is the central regulator of the alternative pathway of complement. It participates in the regulation of the level of immune complexes in the blood and therefore in the balance between the processes resulting in their generation or degradation ⁇ With factor I, factor H inactivates free or bound C3b molecules on the surface of cells. Thus, the immune complexes composed of an antigen-antibody complex complexed with the component of complement C3b can no longer activate the subsequent cascade of complement (components C5-C9).
• the I Factor H behaves first of all as a co-factor of the factor I.
• the Factor H and the Pactedr I proceed to the transformation of the protein C3b of the complement into C3bi (inactive molecule) by a cleavage of the • chain of the C3b protein.
• the C3b protein thus inactivated can no longer fulfill its role in the functioning of the complement, and no longer participates in the formation of C3 convertase;
• the 1 Factor H is involved in the endothelial and platelet mechanisms is finally involved in the dissociation of the preformed C3 convertase (C3bBb), in the alternative way of the activation of the complement.
• the latter activity depends directly on the molecular integrity of Factor H, and is more particularly dependent on the presence of an intact asn323-asn324 link in Factor H.
• Recurrence after transplantation in patients with an atypical form of HUS is observed in approximately 25% of cases.
• the prognosis for recurrence is poor; graft loss related to recurrence is the rule,
• the first-line treatment consisting of infusions of fresh frozen plasma with or without plasma exchange was started empirically in the 1970s long before the role of complement in HUS was known.
• Today, fresh frozen plasma infusions with or without plasma exchange are the basis for HUS therapy, however, the amounts and frequency of fresh frozen plasma infusions are still empirically determined.
• fresh frozen plasma contains anti-A or anti-B hemolysins and must be reserved for patients of the same ABO group, or at least for patients lacking A or B antigens corresponding to hemolysins (rule of reverse compatibility with that of red blood cells). Failure to comply with these rules exposes the recipient to post-transfusion hemolysis of red blood cells due to ABO incompatibility, In addition, in order to avoid any risk of immuno-immunization vis-à-vis the D antigen of the Rhesus system, it is necessary, especially in patients at risk (girls, women of childbearing age, multiririsfies ) to carry out infusions where the patient and the donor have the same characteristics with regard to this antigen,
• PFC can cause hyperhosphatemia in HUS patients because the phosphate concentration in the PPC, in particular the viro-attenuated plasma (PVA), is very high (9 to 12 ntirool / lj) and the patient HUS suffers from kidney failure.
• PVA viro-attenuated plasma
• Another treatment is a kidney transplant.
• the risk of recurrence after transplantation is very high,
• Treatment of recurrence consists of infusions of fresh frozen plasma, plasma exchanges with or without plasma infusions with [very inconsistent results. These inconsistent results can be explained by the number and volume of PFC infusions, each infusion representing a pool of donations from multiple donors and
• liver transplant As factor H is synthesized in the liver, it appears; logical to propose a liver transplant or even a combined liver-kidney transplant. !
• Factor H for example in the form of a concentrate of factor H from fresh frozen plasma, makes it possible to restore factor H deficiency in patients with HUS while reducing the volumes injected and the injection times with an effective, stable and safe product.
• factor H in the immediate post-liver transplant period makes it possible to compensate for the low production of factor H by the transplanted liver and thus for the immediate relapse and rejection of the graft.
• the present invention also relates to a factor H purification process comprising the steps of;
• the main object of the present invention is the use of Factor H for the manufacture of a medicament intended for the treatment of Hemolytic Uremic Syndrome (HUS), in particular of the typical form of HUS or of the atypical form of HUS.
• HUS Hemolytic Uremic Syndrome
• a preferred form of the invention is the use of Facteuir H for the manufacture of a medicament intended for the treatment of Hemolytic Uremic Syndrome, Factor H being purified from fresh human plasma! or plasma fractions from purification by conventional methods well known to those skilled in the art.
• This purification is well known to those skilled in the art. It can be carried out by chromatography using a lysine-sepharose column, QAE-Sephadex, DEAE-Toyopearl, Sephacryl S-300 and hydroxyapatite.
• Factor H resulting from purification from plasma; fresh frozen is for example in the form of a factor H concentrate.
• Hemoly, ⁇ re ⁇ ique tick the Factor H being obtained by genetics, by the expression of its gene in a cellulje chosen in the group made up of bacteria, yeast
• a particular embodiment of the invention consists in the use of Factor H for the manufacture of a medicament intended for the treatment of hememic hemorrhagic syndrome, the medicament thus obtained being BOUS lyophilized form.
• An additional embodiment of the invention consists in the use of Factor H for the manufacture of a medicament intended for the treatment of the Uremic Hemolytic Syndrome, the medicament thus obtained having undergone at least one method of elimination or iinactivation of '' at least one infectious agent.
• viruses and ATNCs unconventional transmissible agents
• prion protein a virus that has a high degree of virus.
• viruses and ATNCs conventional transmissible agents
• the drug may be inactivated viral 'lying.
• viral inactivated is understood to mean that the medicament has undergone at least one method of viral inactivation, known to a person skilled in the art by treatment with china products, for example by solvent / detergent / and / or heat, for example by dry heating or pasteurization, and / or by nanofiltration.
• HIV human acquired immunodeficiency virus
• HAV hepatitis A virus
• HBV hepatitis B virus
• CMV cytomegalovirus
• BVDV bovine viral diarrhea virus
• Another subject of the invention is a lyophilized and virally inactivated pharmaceutical composition, for example as described above, and comprising factor H and pharmaceutically acceptable excipients and / or vehicles.
• Another object of the present invention relates to a method for purifying Factor H comprising the steps i consisting in:
• step 3 adjudicate the pH of the fraction not retained after the chroma; tography of step 3 to allow the binding of the Fector H to a gel / resin of chromajtographic support comprising a grafted ligand of the Heparijne type,
• the chromatographic support onto which a heparin ligand from step 3) is grafted is a heparin Sepharose gel / resin.
• the chromatographic support onto which a heparin ligand from step 4) is grafted is a heparin Sepharose gel / resin.
• the gel / resin chromatography of the cation exchanger type of the strong acid type of step 6) is a ch.romatograpb.ie of the SP Sepharose type.
• the chromatography on a gel / resin of anion exchanger type of strong acid type of step 8) is a Q Sepharose FF type chromatography or equivalent.
• the pH of the fraction not retained in step 4) is adjusted to be within the range from pH 5.5 to pH 6.5 and preferably to be equal to pH 6.0.
• the pH of the fraction diluted in step 8) is adjusted to be within the range from pH 6.5 to pH 7.5.
• the purification process of the invention is the only one known process for the purification of a factor H from plasma which proves to be realize ⁇ sable, and which makes it possible to obtain a concentrated factor H purified in the absence of inhibitors of chemical or synthetic proteases, thus leaving no trace of cesj inhibitors in the final product.
• protease inhibitors inhibit the action of trypsin-like proteins, present in serum and plasma, which are responsible for the cleavage of the protein link joining the amino acids asn323 and asn 324 of the H factor molecule.
• addition of protease inhibitors contributes to the decrease in the proteolysis of this factor and therefore improves its stability.
• pjotease inhibitors are often highly toxic compounds, making them unsuitable for an industrial process of production of factor H intended for therapeutic use.
• the method of the invention has a significant advantage in that it makes it possible to obtain a factor H concentrate of which the 3 main types of activities are conserved, which none of the H factors described in the state of technique does have.
• the Facteujr H obtained by the process of the invention can therefore fill its activity as central regulator of the alternative pathway to complement, an activity which is revealed! deficient in HUS patients, especially atypical HUS.
• the Factor H produced by the process of the invention advises its dissociation activity of the preformed C3 convertase in the alternative route of the complement and appears capable of being used in the treatment of HUS thanks to its activity
• AS O.8 to 0.9
• Mistletoe although therapeutically effective, has many disadvantages, as was written in the introduction to the present application.
• the administration of plasma introduces into the body additional proteins which are useless for the treatment of HUS (albumin, fibrinogen, etc.) which, on the other hand, can trigger undesirable reactions linked to protein overload or cause reactions allergic, known as "serial illness".
• the inactivation of transmissible viruses present in the plasma is generally more difficult and less efficient than that implemented to inactivate the viruses present in blood derivatives.
• the Factor H concentrate obtained by the process of the invention can therefore benefit from recognized and proven treatments providing documented viral safety.
• the procedure used to purify the Factor H is shown in schematic form in Figure 1.
• the fresh frozen human plasma is thawed at a temperature of between 1 ° C and 6 ° C, then the plastic supernatant of the cryoprecipitate is separated from the insoluble traction of the cryoprecipitate by centriifugation.
• the plasma supernatant of the cryoprecipitate obtained is subjected to chromatography on resin / gel of anion exchanger type (for example , a gel / resin of the DEAE Sepha & ex type), in order to separate the Factors dependent on vitamin K from the plasma supernatant by retention of these Factors on the resin / gel.
• resin / gel of anion exchanger type for example , a gel / resin of the DEAE Sepha & ex type
• fraction A The fraction of plasma supernatant not retained (fraction A), the concentration of Factor H is in a range from about 400 to about 500 i ⁇ g of Factor H / liter, is then subjected to a! affinity chromatography on a gel / resin of ty & e Heparin Sepharose FF, in order to separate
• PH 1id the fraction A not retained (fraction B), the concentration of Factor H of which is included in a ga ⁇ time
• Fraction C The eluted fraction containing Factor H (Fraction C) is diluted, then subjected to a chromatography on gel / resin of the cation exchanger type of strong acid type! for example an SP type gel / resin
• the Factor H retained on the gel / resin is then eluted using a buffer having a higher ionic strength than that of the buffer used for i
• Fraction D The eluted fraction containing Factor H (Fraction D) is then subjected to a viral inactivation step by treatment with a solvent of detergent type, for example Polysorbate 80 and TnBP.
• a solvent of detergent type for example Polysorbate 80 and TnBP.
• Fraction D is then diluted, and the pH of this fraction is adjusted to be within a range from pH 6.5 to pH 7.5.
• the fraction D is then diluted, and the pH of this fraction is adjusted to be within a range from pH 6.5 to pH 7.5.
• the fraction D is then diluted, and the pH of this fraction is adjusted to be within a range from pH 6.5 to pH 7.5.
• the fraction D is then diluted, and the pH of this fraction is adjusted to be within a range from pH 6.5 to pH 7.5.
• a gel / resin of the anion exchanger type of strong acid type for example a gel / resin of the Q type Sepharose FF or an equivalent.
• the Factor H retained on the gel / resin is eluted using a buffer having an ionic strength greater than that of the tamipon used to balance the gel / resin.
• the agents previously introduced to carry out viral inadivation by treatment with a solvent of detergent type are eliminated during this tographic chroma 1 step, and the degree of purity of Factor H is increased.
• Fraction E The eluted fraction containing Factor H (Fraction E) is then subjected to a viral elimination step by nanofiltration on a filter having a porosity of approximately 15 nm. This viral elimination treatment effectively eliminates viruses, and
• the resulting solution (fraction F) is finally concentrated and adjusted by ultrafiltration then filtered on a 0.22 ⁇ m filter.
• Example 2 Method for Assaying the Factor H Activity
• the wells of an ELISA plate (of the 96 well type) are covered with a solution of purified C3b protein, having a concentration of 2.5 g / ml (Calbiochem: ref . 341274) in 0.2 M sodium carbonate buffer. To do this, 100 ⁇ l of solution are introduced into the wells 'and the plates are incubated for 1 hour ' at 37 ° C and 1 night at 4 ° C.
• washes of 300 ⁇ l / well are carried out with a 10 mM sodium phosphate buffer solution, 25 mM NaCl, 0.1% Tween 20, pH 7.2.
• the aspecific sites are then saturated by an incubation of one hour at 37 ° C with 300 ⁇ l / well of a 10 mM sodium phosphate buffer solution, 25 mM NaCl, pH 7.2, Tween 20 0.05%, and containing 1% BSA.
• washing the wells is carried out with the layering solution described above.
• 10 ⁇ gi / ml, 1 ⁇ g / ml, 0.25 ⁇ g / ml, 0.0625 ⁇ g / ml, 0.015625 ⁇ g / mli 0.00390625 ⁇ g / ml and 0.001 ⁇ g / ml is prepared. 100 ⁇ il of each solution are placed in a different well and an incubation of 30 min at 37 ° C is carried out. Three successive washes of 300 ⁇ l / well are then carried out with a 10 mM sodium phosphate buffer solution, 25 mM NaCl, 0.1% Tween 20, pH 7.2.
• a solution of human anti-factor E goat antibody (Calbio ⁇ hem ref: 341272) is diluted to 1/2000 in a PBS buffer (Sigma P-3813), pH 7.4, containing
• the substrate of OPD peroxidase (Sigma), at a concentration of 5 mg / 10 ml in a solution of sodium citrate, is added to the wells, as well as 10 ⁇ l of H 3 O 3 , at a rate of 100 ⁇ l / well in the end.
• the reaction mixture is left in contact with the wells for approximately 10 minutes before stopping the reaction by adding 50 ⁇ l of 4N H-SO 4 per well.
• the absorbance of the solution contained in the wells is then measured at a wavelength of 492 nm.
• the corresponding results are presented in FIG. 2.
• the graphical representations appearing in FIG. 2 give the value of the absorbance measured as a function of the concentration of Factor H or as a function of the concentration of protein (SAH).

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• 2005
  • 2005-12-07 FR FR0512404A patent/FR2894145B1/fr not_active Expired - Fee Related
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