WO2012063007A1

WO2012063007A1 - Factor h for treating autoimmune diseases of the nervous system

Info

Publication number: WO2012063007A1
Application number: PCT/FR2011/052625
Authority: WO
Inventors: Zéra TELLIER; Marie-Hélène ANDRE
Original assignee: Laboratoire Francais Du Fractionnement Et Des Biotechnologies
Priority date: 2010-11-10
Filing date: 2011-11-10
Publication date: 2012-05-18
Also published as: AR083835A1; FR2967071A1

Abstract

The invention relates to the use of factor H for treating an autoimmune disease of the nervous system, such as neuromuscular disorders and peripheral neuropathies, in particular myasthenias and Guillain-Barré syndrome.

Description

H-factor for the treatment of autoimmune diseases of the nervous system

The invention relates to the use of factor H for the manufacture of a medicament for the treatment of autoimmune diseases of the nervous system.

State of the art:

Autoimmune diseases of the nervous system are disorders caused by cellular or humoral immune responses initially directed against auto-antigens of the nervous system.

Among the autoimmune diseases of the nervous system, we can distinguish between limited damage to the central nervous system, neuromuscular diseases, including myasthenia, characterized by a disorder of the neuromuscular junction, and peripheral neuropathies (such as Guillain-Barré), characterized by inflammation of the peripheral nervous system.

The neuromuscular junction is particularly sensitive to antibody-mediated attacks (Lang B, Vincent A. Autoimmune disorders of neuromuscular junction, Curr Opin Pharmacol 2009 Jun; 9 (3): 336-40). In the case of myasthenia gravis, the malfunction of the neuromuscular synapse results from a T cell-mediated humoral immune response. It results in muscle fatigability, exacerbated by stress. In the affected patients, the muscular deficit can be extremely variable in intensity and depends mainly on the affected territories. The disease progressively evolves by pushing. It can be life threatening when it reaches for example the muscles of the swallowing or the respiratory muscles. (Gajdos P. Myasthenic Syndrome, Rev. Prat 2000, 50: 419-23). Myasthenia gravis can be seen at any age, regardless of sex, but the onset of the disease is influenced by age and sex; There are two frequency peaks: one between 20 and 40 years old with a female predominance and the other around 50 years old with male predominance (Meriggioli MN and Sanders DB) Autoimmune Myasthenia Gravis: Emerging Clinical and Biological Heterogeneity Lancet Neurol. 2009 May; 8 (5): 475-90.) Most patients with myasthenia gravis have polyclonal anti-acetylcholine (Anti-RACh) receptor antibodies responsible for loss of Acetylcholine receptors in three mechanisms: fixation Acetylcholine uptake, accelerated degradation of the membrane acetylcholine receptor (antigenic modulation) and destruction by the complement lytic complex (C5b-B9) of the postsynaptic membrane. (Eymard B. Antibody in Myasthenia Gravis Rev Neurol 2009; 165 (137-143).

In 20% of generalized myasthenia, no anti-RACh antibodies are detected. For some, it is anti-tyrosine kinase antibody directed against the muscle (MuSK). MuSK receptors induce hCAC aggregation under the action of agrin released by the nerve terminal (Eymard B. Antibody in myasthenia, Rev Neurol 2009; 165: 137-143).

Some patients have no anti-RACh or anti-MuSK antibodies. The presence of autoantibodies such as anti-striated, anti-nuclear, anti-thyroid, or rheumatoid factor antibodies, however, expresses a complex autoimmune disorder at the origin of the disease.

The main treatment of this disease, in first intention, consists of an administration of anticholinesterase. The use of corticosteroids and / or immunosuppressants is reserved for the chronic treatment of anticholinesterase-resistant forms. Plasma exchange or administration of intravenous immunoglobulins (IglV) are indicated in the treatment of seizures of disease. Peripheral neuropathies, in turn, are multiple causes. They can result from a local, diffuse, metabolic or toxic process, such as an inflammatory process affecting the roots and peripheral nerves, thus causing polyradiculo neuritis. The main symptom of Guillain-Barré Syndrome (GBS) is acute, ascending paralysis. GBS reaches women as well as men, regardless of age and ethnicity. The average age observed is 40 years old.

The predominant lesion observed in this pathology is segmental demyelination, disseminated along the peripheral nerves, causing conduction block at the level of the nerve fiber. Electrophysiologically, a decrease in the conduction velocity and an increase in distal latencies are observed. This explains the rapid onset of paralysis and their spontaneous resolution. These are probably another type of lesions, the axonal lesions, secondary to prolonged demyelination, which explain the observed motor sequelae. The processes that trigger the demyelination are not known, the hypothesis of an autoimmune pathogenesis involving both cellular and humoral mechanisms is generally accepted. Approximately 2/3 of cases of Guillain-Barré syndrome occur after infection with germs such as: C. jejuni, cytomegalovirus (CMV), Mycoplasma pneumoniae or influenza influenzae virus (Burns TM, Guillain-Barré Syndrome, Semin Neurol 2008 28 (2): 152-167).

These infectious agents have on their surface epitopes similar to those present on the surface of the peripheral nerves (eg, gangliosides, glyco lipids, etc.); thus, it seems that during a banal infection, complement-fixing antibodies (IgG1 or IgG3 instead of IgM) would also attach to the gangliosides of the peripheral nerves, thus inducing an autoimmune reaction of varying degree depending on the individual. Macrophage-dependent myelin involvement is also observed; it would be mediated by antibody and complement deposition on Schwann cells and myelin membranes (Burns TM, Guillain-Barré Syndrome, Semin Neurol 2008, 28 (2): 152-167).

The onset of the disease is sudden onset with relatively symmetrical distal paresthesia. Sensory disturbances are quickly accompanied or followed by progressive limb fatigue. The progression of symptoms is rapid, 50% of patients reach clinical nadir in 2 weeks and 90% in 4 weeks (Burns TM, Guillain-Barré Syndrome, Semin Neurol 2008, 28 (2): 152-167). About 80 to 90% of the affected subjects become non-ambulatory during the disease.

The clinical course of the disease is very heterogeneous, the maximum severity of the motor deficit is very variable. Respiratory muscle involvement, requiring mechanical ventilation for 2 to 6 weeks on average, appears in 20 to 30%> cases of GBS. Some injuries can cause disabling motor sequelae requiring help with walking or use of a chair; persistent lesions are reported in 20 to 30% of cases in adults. GBS can lead to death in the acute phase of the disease.

Therapeutic management is based on two types of complementary measures: multidisciplinary symptomatic measures such as respiratory resuscitation, rehabilitation and management of pain; and specific measures aimed at limiting the spread of paralysis, promoting motor recovery and reducing sequelae. In particular plasma exchanges and intravenous immunoglobulins (IglV) are advocated. There is still a great need for new therapeutic strategies to treat these conditions. Summary of the invention

The Applicant of the present invention proposes to use the Factor H for the treatment of an autoimmune disease of the nervous system such as neuromuscular disorders and peripheral neuropathies, in particular myasthenia and Guillain-Barré syndrome.

An object of the invention is therefore a factor H for use in the treatment of an autoimmune disease of the nervous system.

The invention also relates to the use of factor H in the manufacture of a medicament for the treatment of an autoimmune disease of the nervous system, which may be in particular (i) a neuromuscular disease, in particular a disease of the junction neuromuscular, such as myasthenia gravis, or (ii) peripheral neuropathy, such as Guillain-Barré syndrome.

The invention also describes a method of treatment in a patient suffering from an autoimmune disease of the nervous system, comprising administering to said patient an effective amount of the H-factor.

Preferably, the factor H is used in a purified form from fresh plasma or a plasma fraction. According to another embodiment, the Factor H is used in a form of recombinant Factor H, preferably produced by a transgenic animal.

Legend of the Figure:

The Figure is a graph which shows the overall clinical score of myasthenia grafts, as a function of time, after injection of mAb35 antibody inducing experimental autoimmune myasthenia, with or without administration of Factor H (CFH)

Detailed description of the invention

General Definitions

Factor H is the main regulator of the alternative complement pathway. It acts in the liquid phase as on the surface of the cells. Originally designated "Beta 1 H globulin", this 155 kDa serum glycoprotein is also called H 1, FH, CFH, or HF1 factor. Factor H is synthesized in the liver, macrophages, fibroblasts, endothelial cells and platelets. The secreted form of the protein is composed of 20 repetitive units (or "short consensus repeats", SCRs) of 60 amino acids.

By "Factor H" is meant here any protein having the amino acid sequence of

Human or other native H-factor (eg, bovine, porcine, canine, murine). The term also includes any recombinant, derivative or mutant having a sequence substantially homologous to native H-factor. The term "substantially homologous sequence" includes any sequence subject to one or more substitutions, additions and / or deletions, preferably conservative. The terms "conservative substitutions, additions and / or deletions" mean any replacement, addition or deletion of amino acid residue by another, without major alteration of the general conformation and / or biological activity of Factor H. The conservative substitution includes , but not limited to, replacement with an amino acid having similar properties (such as, for example, shape, polarity, hydrogen bonding potential, acidity, basicity, hydrophobicity and the like). Amino acids with similar properties are well known in the art.

The term "Factor H" further includes naturally occurring allelic variations and / or Factor H isoforms found naturally in individuals of the same species, and any form or degree of glycosylation or other post-translational modification. Also included are homologues or derivatives of Factor H which exhibit the same or higher biological activity with respect to the activity of the wild form and / or which have a sequence identity of at least 80%, preferably at least 85%. %, more preferably at least 90%.

The anti-complementary activity of Factor H is reflected in the regulation of the rate of immune complexes in the blood, it therefore contributes to the balance between the processes resulting in their generation or their degradation. Factor H reduces the half life of C3 convertase (C3bBb) alternately by binding C3b and dissociating Bb and serves as a cofactor for Factor I in C3b proteolysis, free or bound to cell surface, in C3bi. Thus, immune complexes composed of an antigen-antibody complex associated with the complement component C3b can no longer activate the subsequent cascade of complement (C5-C9 components). The term "biological activity" of Factor H therefore includes here the ability to inhibit C3 convertase and / or to act as a factor I co-factor, resulting in the inhibition of activation of the complement cascade. The activity of the factor H can be measured in various ways, well known to those skilled in the art.

The term "treatment" or "therapy" includes both curative and prophylactic treatment of the disease. A curative treatment is defined as a treatment resulting in a cure or treatment that alleviates, improves and / or eliminates, reduces and / or stabilizes the symptoms of an illness or the suffering it causes. Prophylactic treatment includes both treatment leading to disease prevention and treatment that reduces and / or delays the incidence or risk of disease.

Therapeutic indications

The subject of the invention is therefore the use of the factor H for the treatment of autoimmune diseases of the nervous system, also called immunoinduced nerve disorders.

The autoimmune disease of the nervous system may be a neuromuscular disease, such as myasthenia gravis (Myasthenia gravis) or Lambert-Eaton syndrome, or peripheral neuropathy, such as Guillain-Barré syndrome. In general, all autoimmune diseases of the central or peripheral nervous system are included.

In particular, factor H is useful for the treatment of disorders of the neuromuscular junction and peripheral neuropathies, particularly in the treatment of relapsing myasthenic attacks and Guillain-Barré syndrome.

Factor H via its inhibitory action on the alternative pathway of complement, is involved in the modulation of the immune response involved in the pathogenesis of these nerve damage and promotes the prevention and resorption of inflammatory phenomena observed in these pathologies. Factor H acts indirectly on complement system components involved in the antibody-mediated attack process. Obtaining Factor H preparations

Factor H, preferably human, may be prepared by any of the conventional purification techniques, by peptide synthesis, namely in particular by chemical synthesis, or genetic engineering. In a preferred embodiment, Factor H is obtained from serum, plasma fractions, or non-cryoprecipitable plasma fraction, preferably human, by standard purification methods well known to those skilled in the art. The purification can be carried out by chromatography using a lysine-sepharose, QAESephadex, DEAE-Toyopearl, Sephacryl S-300 and hydroxyapatite column. It is detailed in the following documents: Fearon, J Immunol, 119, 1248-1252 (1977); Crossley et al., Biochem J, 191, 173-182, (1980); Nagasawa et al., J. Immunol, 125, 578-582, (1980); Weiler et al, PNAS, 73, 3268-3272, (1976) and Whaley et al, J Exp Med, 144, 1147-1163 (1976). Factor H resulting from purification from fresh frozen plasma is for example in the form of a factor H concentrate. A purified form of Factor H is more than 95% pure, preferably more than 99% pure. .

Preferably, the factor H is stored in freeze-dried form. According to a preferred embodiment, the factor H is thus obtained from a lyophilized preparation.

The H-factor preparation useful in the invention has generally undergone at least one step of removing or inactivating at least one infectious agent. Infectious agents include viruses and ATNCs (unconventional transmissible agents) such as prions. Viral inactivation often includes treatment with chemicals, for example solvent, detergent and / or heat, for example by dry heating or pasteurization, and / or nanofiltration.

The factor H preparation can be obtained by purification according to the method described in the patent application WO2007 / 066017, comprising the steps of:

(i) preparing the supernatant of a plasma cryoprecipitate,

(ii) subjecting this supernatant to anion exchange gel / resin chromatography

(iii) subjecting the non-retained fraction to chromatography on a gel / resin comprising a Heparin-type graft ligand

(iv) adjusting the pH of the non-retained fraction after the chromatography of the previous step to allow the binding of the Factor H to a gel / chromatographic support resin comprising a heparin-type graft ligand,

(v) eluting the H factor by a buffer of ionic strength greater than that of the gel / resin equilibration buffer,

(vi) dilute the eluted fraction and then subject it to gel / resin chromatography of the strong acid type cation exchanger type, (vii) eluting the H factor with a buffer of ionic strength greater than that of the gel / resin equilibration buffer,

(viii) dilute the eluted fraction and then subject it to gel / resin chromatography of the strong acid type anion exchanger type,

(ix) wash the gel / resin and elute the factor H,

(x) prepare an H factor concentrate.

Advantageously, the pH of the non-retained fraction of step iv) is adjusted to be in the range from pH 5.5 to pH 6.5 and preferably to pH 6.0. Advantageously, the pH of the fraction diluted in step viii) is adjusted to be in the range of pH 6.5 to pH 7.5.

According to another embodiment, the factor H can be purified from plasma according to the method described in the patent application WO2008 / 113589.

Factor H can also be obtained by genetic engineering, by the expression of its gene in a cell selected from the group consisting of bacteria, yeasts, fungi or mammalian cells, or from transgenic animals. According to a preferred embodiment, the human Factor H is produced in the milk of nonhuman transgenic mammals, genetically modified to produce this protein. Preferably, it is the milk of a rabbit or a transgenic goat. The secretion of Factor H by the mammary glands, allowing its secretion into the milk of the transgenic mammal, involves the control of Factor H expression in a tissue-dependent manner. Such control methods are well known to those skilled in the art. The control of the expression is carried out by means of sequences allowing the expression of the protein towards a particular tissue of the animal. These include the promoter sequences WAP, beta-casein, beta-lactoglobulin and signal peptide sequences. The process for extracting the proteins of interest from the milk of transgenic animals is described in patent EP 0 264 166.

Formulation

In order to obtain stable therapeutic Factor H preparations, the obtained Factor H preparation is formulated with suitable excipients and stabilizers. They may be diluents, agents, cryoprotectants, lyoprotectants, etc.

Among the conventional lyoprotectants, mention may be made of sugars (sucrose, trehalose, glucose, lactose, etc.), polyols (mannitol, sorbitol) and amino acids (glycine, arginine, histidine, alanine) which can be used at a concentration between 0 and 10%. Surfactants of the polysorbate type (Tween 20 or Tween 80 for example), polyoxamer (for example poloxamer 188), polyethylene glycol may also be added.

Antioxidants (eg, methionine, monothioglycerol, glutathione, citric acid, ascorbic acid, sodium metabisulfite, and sodium sulfite) can be added.

Buffer substances may be further used, for example in the form of carbonate, phosphate, citrate, acetate, borate, trimethamine [(2-amino-2-hydroxymethyl-1,3-propanediol), TRIS], glycine and lysine. Modes of administration

Factor H is preferably formulated within a pharmaceutical composition, said pharmaceutical composition comprising Factor H and a carrier or one or more pharmaceutically acceptable excipients.

The pharmaceutical composition may comprise Factor H in combination with another therapeutic agent.

According to a preferred embodiment, the factor H is used in the form of a liquid composition comprising a concentration of factor H of between 0.5 and 50 g / l, preferably between 1 and 30 g / l, more preferably between 5 and 20 g / l. L, or between 10 and 20g / L.

Factor H can be administered by any known route of administration, including the following routes: systemic (parenteral, intravenous, etc.), topical, oral, rectal, subcutaneous, intraspinal and intranasal, in a quantity preferred by the practitioner.

According to a preferred embodiment, the Factor H is administered intravenously. The doses of Factor H used in vivo are adjusted in a conventional manner depending on the desired effect, in particular for modulating the inflammation, and the targeted pathology. An example of a suitable dose is a dose at a concentration of from 5 to 2000 mg per day, preferably from 5 to 1000 mg per day, preferably from 20 to 500 mg per day, more preferably from 50 to 100 mg per day. The figures and examples illustrate the invention without limiting its scope. EXAMPLES

Example 1: Factor H Study in a Model of Experimental Autoimmune Myasthenia Gravis (EAMG) in the Rat Materials and Methods:

Implementation of the model of experimental autoimmune myasthenia gravis in rats:

The animal model is established by injection of a monoclonal antibody directed against the nicotinic receptor to skeletal muscle acetylcholine (mAb35) passively inducing the disease in animals.

Female 6 to week old female Lewis rats weighing 170 g were used for this purpose (9 lots of 6 rats).

MAb35 antibody was obtained from ATCC (TIB-175®).

The antibody is amplified by cell culture and then purified by chromatography, before concentration to 0.1 mg / ml.

The chosen induction dose is 0.50 mg / kg, by single intraperitoneal (i.p.) administration.

The rats present a modified posture, and a state of fatigue revealed by a reflex test. The rats catch a grid 20-30 times and then observe their state of fatigue on a platform (Τϋζϋη E et al J Immunol 2003; 171: 3847-54; Liu R. Exp Neurol 2009; 220 (2): 366-73 ).

Clinical evaluation of the administration of increasing doses of factor H in the myasthenic rat model:

1 hour after administration of mAb35, Factor H (CFH) is administered by IP to 5 groups of 6 rats:

o 3 experimental groups: Escalation of CFH doses: 50, 100 and 200 mg / kg

o 2 "control" groups: positive: mAb35 and PBS and negative: PBS

only

Table 1 summarizes the doses administered. Table 1: Summary of the experimental phase

The rats are weighed daily, their clinical status is evaluated as described above (observation of the posture of the animal and evaluation of the gripping force).

An overall clinical score is given, according to the following evaluation grid:

0: normal state

1: fatigue with exercise

2: fatigue without exercise

3: fatigue without exercise + weight loss

4: moribund (EUTHANASIA)

5: Deaths (EUTHANASIA)

The evaluation is done blind. Results:

The figure shows the beneficial effect of Factor H administration on the clinical score of model myasthenia gravis.

The observed effect is dose-dependent.

Table 2 below shows the significance of the results: Table 2: Unpaired Serial Student "t" Test of the G2 and G3 Group means:

24h 42h 48h 72h

Value p 0.01012 0.013618 0.010808 0.020882

Claims

claims

1. H-Factor for use in the treatment of autoimmune disease of the nervous system.

2. The factor H according to claim 1, wherein the autoimmune disease of the nervous system is a neuromuscular disease, in particular neuromuscular junction disease.

3. Factor H according to claim 2, the neuromuscular disease being a myasthenia.

4. Factor H according to claim 1, the autoimmune disease of the nervous system being a peripheral neuropathy.

5. Factor H according to claim 4, the peripheral neuropathy being a Guillain-Barré syndrome.

6. Factor H according to one of claims 1 to 5, in a form purified from fresh plasma or a plasma fraction.

7. Factor H according to one of claims 1 to 6, which is a human factor H.

8. Factor H according to one of claims 1 to 5, in a form of recombinant factor H produced by a transgenic animal.

9. Factor H according to any one of claims 1 to 8, in a form suitable for intravenous administration.

10. Use of a factor H for the preparation of a medicament for the treatment of an autoimmune disease of the nervous system.

11. Use of a factor H according to claim 10, wherein the autoimmune disease of the nervous system is a neuromuscular disease, in particular myasthenia or peripheral neuropathy, such as Guillain-Barré syndrome.