WO2017212018A1 - Procédés et compositions pharmaceutiques pour le traitement de maladies inflammatoires auto-immunes - Google Patents

Procédés et compositions pharmaceutiques pour le traitement de maladies inflammatoires auto-immunes Download PDF

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WO2017212018A1
WO2017212018A1 PCT/EP2017/064085 EP2017064085W WO2017212018A1 WO 2017212018 A1 WO2017212018 A1 WO 2017212018A1 EP 2017064085 W EP2017064085 W EP 2017064085W WO 2017212018 A1 WO2017212018 A1 WO 2017212018A1
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syndrome
autoimmune
disease
chronic
arthritis
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PCT/EP2017/064085
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English (en)
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Renato Monteiro
Sanae BEN MKADDEM
Eric DAUGAS
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université Paris Diderot - Paris 7
Assistance Publique-Hôpitaux De Paris (Aphp)
Centre National De La Recherche Scientifique (Cnrs)
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Publication of WO2017212018A1 publication Critical patent/WO2017212018A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/10—Protein-tyrosine kinases (2.7.10)
    • C12Y207/10002—Non-specific protein-tyrosine kinase (2.7.10.2), i.e. spleen tyrosine kinase

Definitions

  • the present invention relates to methods and pharmaceutical compositions for the treatment of autoimmune inflammatory diseases.
  • autoimmune and inflammatory renal diseases present as a major public health problem worldwide.
  • autoimmune and inflammatory renal diseases mostly glomerular diseases, are the leading cause of renal transplantation in France and the third leading cause of renal replacement therapy by dialysis after hypertension and diabetes (REIN, Rapport Annuel 2010 (Rein rapports 201 1 , 2012, www. entour-biomedecine.fr)).
  • GN glomerulonephritis
  • IgA-N or Berger's disease well-characterized IgA nephropathy
  • LN lupus nephritis
  • ANCA Anti-Neutrophil Cytoplasmic Antibodies
  • AAV Anti-Neutrophil Cytoplasmic Antibodies
  • Patients present with a relative silent evolution and late appearance of symptoms, independent of the type of immune involvement.
  • diagnosis is only possible through invasive investigations, mainly renal histology comprising evaluation of the presence of immunoglobulins and complement deposits in the glomeruli. There is thus a medical need for the treatment of autoimmune and inflammatory diseases.
  • the immune system is controlled by a finely tuned network of regulatory mechanism (1).
  • certains immunoreceptors have been shown to exert inhibitory and activating signal through ITAM motif (Yxx[L/I]x6-sYxx[L/I]) (2) depending on the valency of their respective ligand.
  • Low valency interactions were shown to induce anergy and an inhibitory crosstalk with heterologous receptors thereby reducing the susceptibility to autoimmune and inflammatory diseases (3-10).
  • high valency ligand interactions promote an activating signal launching inflammatory and immune cascades to fight the inflammatory insult and restore homeostasis, but in case of ill-regulation or chronic stimulation can also result in autoimmune and inflammatory diseases (1, 10).
  • the ITAM motif is found in the cytoplasmic domain of several transmembrane adapter molecules, such as the common ⁇ subunit of FcR (FcRy), the Iga and Ig subunits of the BCR, and the ⁇ , ⁇ , ⁇ and ⁇ subunits of the TCR-associated CD3 complex (1, 2), and in the cytoplasmic tail of other receptors, such as the FcyRIIA (11).
  • FcRy FcR
  • FcRy FcR
  • Iga and Ig subunits of the BCR and the ⁇ , ⁇ , ⁇ and ⁇ subunits of the TCR-associated CD3 complex (1, 2)
  • FcyRIIA FcyRIIA
  • the present invention relates to methods and pharmaceutical compositions for the treatment of autoimmune inflammatory diseases.
  • the present invention is defined by the claims.
  • Immunoreceptors play a crucial role in the regulation of immune homeostasis and inflammation. Depending on ligand valency they can promote either activating or inhibitory signals. The inventors investigated how these receptors translate outside ligand interactions into opposite signals.
  • the Src-family kinase Fyn was the crucial effector for inhibition of SHP-1
  • Lyn/Lck were required for its activation downstream of Fc, B-cell and T-cell antigen receptors.
  • a Fyn-PI3K-PKC axis turns off Lyn-mediated SHP-1 activation by shifting tyrosine into serine phosphorylation. Accordingly, while Lyn-deficient mice aggravate inflammatory diseases, Fyn-deficient mice were protected.
  • the first object of the present invention relates to a method of treating an autoimmune inflammatory disease in a subject in thereof comprising administering to the subject a therapeutically effective amount of a Fyn inhibitor.
  • treatment or “treat” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase "induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • loading regimen may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • the phrase "maintenance regimen” or “maintenance period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • continuous therapy e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.
  • intermittent therapy e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • the autoimmune inflammatory disease is selected from the group consisting of arthritis, rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic
  • the subject suffers from a renal autoimmune inflammatory disease.
  • the subject suffers from an allergic disorder.
  • an allergic disorder As used herein,
  • Allergic disorder refers to any disorder resulting from antigen activation of mast cells that results in an "allergic reaction” or state of hypersensitivity and influx of inflammatory and immune cells.
  • Those disorders include without limitation: systemic allergic reactions, systemic anaphylaxis or hypersensitivity responses, anaphylactic shock, drug allergies, and insect sting allergies; respiratory allergic diseases, such asthma, hypersensitivity lung diseases, hypersensitivity pneumonitis and interstitial lung diseases (ILD) (e.g.
  • idiopathic pulmonary fibrosis ILD associated with rheumatoid arthritis, or other autoimmune conditions
  • rhinitis hay fever, conjunctivitis, allergic rhinoconjunctivitis and vaginitis
  • skin and dermatological disorders including psoriasis and inflammatory dermatoses, such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, dermatitis herpetiforms, linear IgA disease, acute and chronic urticaria and scleroderma
  • vasculitis e.g.
  • necrotizing, cutaneous, and hypersensitivity vasculitis necrotizing, cutaneous, and hypersensitivity vasculitis
  • spondyloarthropathies e.g., intestinal reactions of the gastrointestinal system (e.g., inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, ileitis, enteritis, nontropical sprue and celiac disease).
  • the subject suffers from asthma.
  • asthma refers to an inflammatory disease of the respiratory airways that is characterized by airway obstruction, wheezing, and shortness of breath.
  • the subject suffers from anaphylaxis.
  • anaphylaxis refers to a life threatening allergic reaction characterized by decreased blood pressure, respiratory failure with bronchoconstriction, and skin rash due to release of mediators from cells such as mast cells.
  • the autoimmune inflammatory diseases is secondary to therapeutic treatment, in particular a treatment with an immune checkpoint inhibitor.
  • the term "immune checkpoint inhibitor” has its general meaning in the art and refers to any compound inhibiting the function of an immune inhibitory checkpoint protein. Inhibition includes reduction of function and full blockade.
  • Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins.
  • the immune checkpoint inhibitor is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-PD-L2 antibodies anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and anti-B7H6 antibodies.
  • FYN has its general meaning in the art and refers to FYN proto-oncogene, Src family tyrosine kinase encoded by the FYN gene (Gene ID: 2534) also known as SLK; SYN; or p59-FYN.
  • An exemplary human nucleic acid sequence is accessible in GenBank under the accessible number NM 002037.5 (isoform a), NM l 53047.3 (isoform b) or NM 153048.3 (isoform c).
  • Fyn inhibitor refers to any compound that is capable of inhibiting the activity or expression of Fyn.
  • Fyn activity includes any biological activity mediated by Fyn such as described in the EXAMPLE.
  • the Fyn inhibitor of the present invention is particular suitable for abrogating kinase activity.
  • Fyn inhibitors include but are not limited to polypeptides such as dominant- negative protein mutants, peptidomimetics, antibodies, ribozymes, antisense oligonucleotides, or other small molecules which specifically inhibit the activity or expression of Fyn.
  • the Fyn inhibitor is particularly suitable for inhibiting the activation downstream of Fc, B-cell and T-cell antigen receptors. More particularly, the Fyn inhibitor is particularly suitable for restoring and maintaining homeostasis of the immune system.
  • Small molecule inhibitors of Fyn activity include PP1 (4-Amino-5-(4-methylphenyl)-7- (t-butyl)pyrazo lo [3 ,4-d]pyrimidine) .
  • the inhibitor is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of Fyn.
  • shRNA short hairpin RNA
  • shRNA is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference.
  • shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited.
  • the shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • siRNA Small interfering RNA
  • silencing RNA are a class of 20-25 nucleotide- long double- stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene.
  • RNAi RNA interference
  • Anti-sense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos.
  • Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siR A, shR A or ribozyme nucleic acid to the cells and typically mast cells.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus
  • adenovirus adeno-associated virus
  • SV40-type viruses polyoma viruses
  • Epstein-Barr viruses Epstein-Barr viruses
  • papilloma viruses herpes virus
  • vaccinia virus
  • the inhibitor is an intrabody having specificity for Fyn.
  • the term "intrabody” generally refer to an intracellular antibody or antibody fragment.
  • Antibodies in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention.
  • the intrabody is a single domain antibody.
  • the antibody according to the invention is a single domain antibody.
  • single domain antibody sdAb or “VHH” refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.
  • a “therapeutically effective amount” of the inhibitor as above described is meant a sufficient amount to provide a therapeutic effect. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • an effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the inhibitor is administered to the subject in the form of a pharmaceutical composition.
  • the inhibitor may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • “Pharmaceutically” or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the active principle alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • saline solutions monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts
  • dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the inhibitor can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine,
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions the typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • the preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • a further object of the present invention relates to a method of screening a drug suitable for the treatment of an autoimmune inflammatory diseases comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression or activity of Fyn.
  • the assay fist comprises determining the ability of the test compound to bind to Fyn.
  • a population of immune cells (mastocytes, monocytes, B cells, T cells?) is then contacted and activated so as to determine the ability of the test compound to inhibit the activation downstream of Fc, B-cell and T-cell antigen receptors.
  • the SHP-1 activation is determined and in particular the identification of serine into tyrosine phosphorylation.
  • control substance refers a molecule that is inert or has no activity relating to an ability to modulate a biological activity or expression.
  • Assays for determining the test compound to dampen the immune signal response are well known in the art and are typically described in the EXAMPLE. It is to be understood that test compounds capable of inhibiting immune response, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo.
  • the test compound is selected from the group consisting of peptides, petptidomimetics, small organic molecules, antibodies (e.g. intraantibodies), aptamers or nucleic acids.
  • the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo.
  • the test compound may be selected form small organic molecules.
  • small organic molecule refers to a molecule of size comparable to those organic molecules generally sued in pharmaceuticals. The term excludes biological macromolecules (e.g.; proteins, nucleic acids, etc.); preferred small organic molecules range in size up to 2000da, and most preferably up to about 1000 Da.
  • FIGURES
  • FIG. 1 Fyn aggravates spontaneous autoimmune nephritis development in FcyRIIA T mice.
  • A Body weight.
  • B Serum BUN.
  • C Representative photographs of one- year-old mice demonstrating that the absence of Fyn prevents FcyRIIA-mediated glomerulopathy with marked reduction in IgG deposits and fibrosis (H&E and Masson's stain). Scale Bars: 200 ⁇ .
  • D Detection of CD1 lb + , F4/80 + and CD3 + cells in kidney sections of WT, hFcyRIIA Tg and Fyn _/" hFcyRIIA Tg mice by IHC. Quantification of positive cells is indicated on right panels.
  • LN human nephritis
  • the LN group was composed of 6 patients attending or referred to the Bichat' s Hospital specialist nephrology unit between July 2014 and January 2016 meeting at least four ACR systemic lupus erythematosus criteria (32) presenting with active disease with nephritis proven by kidney biopsy (2 at class IV and 4 at class V) and in whom peripheral blood by venepuncture was obtained immediately prior to immunosuppressive therapy administration. All patients were female with age varying between 25 and 42. Ethical approval for this study was obtained from the Bichat Hospital Local Research. Ethics Committee and informed consent was obtained from all subjects enrolled.
  • mice expressing the WT human FcyRIIA on CD 1 lb-positive cells were from Jackson Laboratory (JAX, Bar Harbor, ME, USA).
  • Fyn ' hFcyRIIA 18 , Lyn _/" hFcyRIIA Tg were obtained by the intercross of hFcyRIIA Tg mice with mice knockout for Fyn (JAX) or for Lyn (previously described in (15)). All mice carrying the hFcyRIIA transgene were used as heterozygous animals. Mice were bred and maintained at the mouse facilities of the Bichat Medical School campus. All experiments were performed in accordance with the French Council of Animal Care guidelines and national ethical guidelines of INSERM Animal Care Committee (Animal Use Protocol number 75-1596).
  • NTN Nephrotoxic nephritis
  • NTN was induced by i.p. injection (200 ⁇ 1/20 g body weight) of rabbit anti-mouse glomerular basement membrane (GBM) in C57BL/6 hFcyRIIA Tg , Lyn _/" , Fyn _/" , Fyn _/" hFcyRIIA Tg , Lyn ' hFcyRIIA ⁇ mice (7 to 9 wk old). Briefly, mice were preimmunized i.p. with normal rabbit IgG (0.5 mg/20 g body weight) in CFA 5 days prior to i.p. administration of NTN serum. Blood samples were collected and animals were sacrificed at day 7 following NTN injection. Renal function parameters (urinary proteins and BUN), histological and immunohistological parameters were studied.
  • GBM rabbit anti-mouse glomerular basement membrane
  • CAIA Collagen Antibody-Induced Arthritis
  • Arthritis was induced as described (9, 30) using the Arthrogen-CIA® Arthritogenic Monoclonal Antibody kit (Chondrex, Inc.). Mice were injected i.v. with anti-CII Ab cocktail (Day 0) followed by LPS (i.p.) 3 days later. Animals were injected i.p. with 10 mg/20 g body weight of 500 ⁇ g serum human IgA (purchased from Biomedicals)/20 g body weight or 100 ⁇ g AT-10 F(ab)' 2 or irrelevant mAb F(ab') 2 (clone 320) for 10 days at 2-day intervals. The first dose was administered 2 days prior to anti-CII Ab cocktail injection. Paw thickness was measured with a pocket thickness gauge. On day 10, animals were sacrificed and hind paws and knees were fixed in formalin or snap-frozen.
  • mice and Ramos human cell lines were maintained in RPMI-1640 supplemented with 10% FCS and antibiotics. FCS was removed from the culture medium immediately before stimulation as described (34).
  • Mouse mAb anti-hFcyRII (clone AT-10), anti-hCD3 (clone HIT-3a) or anti-hCD79a (clone ZL7-4) were purchased from Santa Cruz and used in their F(ab') 2 fragment forms.
  • Mouse mAb anti-hFcaRI (clone A77) and irrelevant control mAb (320) were purified in-house and were used as F(ab') 2 , as previously described (3, 9).
  • Jurkat cells were incubated with or without anti-CD3 F(ab') 2 fragment or with preformed complexes of anti-CD3 F(ab') 2 plus anti-kappa F(ab') 2 fragments. Cells were then stimulated or not with flagellin (1 ⁇ g) for 6 hours. PMA (40 nM) and ionomycin (InM) were used as positive stimuli for 6 hours. Brefeldin A was added after 2 hours stimulation and maintained for 4 hours. The stimulation was stopped by adding 1 ml cold PBS.
  • Intracellular cytokine staining was performed on fixed/permeabilized cells in residual permeabilization wash buffer (Biolegend, USA) using a conjugated antibody (anti-IL-2 PE or appropriate isotype control) for 20 min in the dark at room temperature as described (36). Data acquisition was performed using a BD Biosciences LSR Fortessa cytometer, and results were analyzed using Flow Jo analysis software (Tree Star).
  • Cells (5 x 10 6 to 10 7 ) were solubilized in RIPA lysis buffer containing 1% Nonidet P- 40/0.1% sodium dodecyl sulfate (SDS) as described 8 .
  • SDS sodium dodecyl sulfate
  • cell lysates were incubated with 2 ⁇ g/ml of AT- 10 anti-FcyRIIA, A77 anti-FcaRI, HIT-3a anti-CD3 or ZL7-4 anti-CD79a mAbs and immunoprecipitated overnight at 4°C with Protein G-Sepharose (GE Healthcare).
  • Enzyme-linked immunosorbent assay ELISA
  • IL-8 and IL-2 were measured in the supernatants of stimulated cells using ELISA kits (R&D Systems) according to the manufacturer's instructions.
  • kidney sections 4 ⁇ in thickness were stained with PAS for morphological analysis.
  • frozen kidney sections were incubated with biotinylated antibodies against rabbit IgG or mAb anti-mouse CD l ib, -mouse F4/80, - mouse CD3, and -mouse Ly6G (Becton Dickinson) for 1 hour at room temperature.
  • the primary antibody incubation was followed by incubation with anti-rabbit IgG or anti-goat IgG (Southern Biotech Associates).
  • Slides were mounted with the Eukitt mounting medium (Electron Microscopy Sciences) and read with an upright microscope (DM2000; Leica) using the IM50 software (Leica).
  • RNA purification from ho mogen i zed_k i d ney s was performed by using RNAble (Eurobio).
  • cDNA was obtained by reverse transcription using using Moloney murine leukaemia virus (Invitrogen). Samples were analyzed by real-time PCR with Taq Man® Gene Expression Master Mix (Applied Biosystem). Primers were purchased from Eurofms (Supplementary Table 1). Gene quantification was performed using a Chrom o4 Real-Time PCR Detection System (Bio-Rad Laboratories). Data were normalized to ⁇ -actin values.
  • F indicates a forward primer
  • R indicates a reverse primer
  • P indicates a FAM-TAMRA probe.
  • the immune system is controlled by a finely tuned network of regulatory mechanism (1).
  • certains immunoreceptors have been shown to exert inhibitory and activating signal through IT AM motif (Yxx[L/I]x6-sYxx[L/I]) (2) depending on the valency of their respective ligand.
  • Low valency interactions were shown to induce anergy and an inhibitory crosstalk with heterologous receptors thereby reducing the susceptibility to autoimmune and inflammatory diseases (3-10).
  • high valency ligand interactions promote an activating signal launching inflammatory and immune cascades to fight the inflammatory insult and restore homeostasis, but in case of ill-regulation or chronic stimulation can also result in autoimmune and inflammatory diseases (1, 10).
  • the IT AM motif is found in the cytoplasmic domain of several transmembrane adapter molecules, such as the common ⁇ subunit of FcR (FcRy), the Iga and Ig subunits of the BCR, and the ⁇ , ⁇ , ⁇ and ⁇ subunits of the TCR-associated CD3 complex (1, 2), and in the cytoplasmic tail of other receptors, such as the FcyRIIA (11).
  • FcRy FcR
  • FcRy FcR
  • Iga and Ig subunits of the BCR and the ⁇ , ⁇ , ⁇ and ⁇ subunits of the TCR-associated CD3 complex (1, 2)
  • FcyRIIA FcyRIIA
  • Src-family kinases phosphorylate the ITAM motifs upon stimulus- induced receptor clustering leading to downstream effector recruitments and cell activation.
  • ITAM-bearing immunoreceptors translate ligand valency into opposite signals remains elusive.
  • TCR and BCR were performed using anti-CD3 or anti-CD79a F(ab') 2 fragments or complexed with anti- ⁇ light chains in representative lymphocytic cell lines.
  • FcRs while divalent targeting of TCR or BCR resulted in ITAMi signatures, multivalent crosslinking led to expected ITAM signature.
  • Lck or Lyn was required for TCR- or BCR-mediated ITAMi signals, whereas Fyn was essential for ITAM configuration.
  • TCR-Fyn dissociation led to SHP-1 recruitment suggesting that Fyn could inhibit SHP-1 recruitment.
  • TCR- and BCR-divalent targeting resulted in inhibition of IL-2 and IL-8 secretion induced by flagellin or Pam3csk4 which were dependent on Lck or Lyn, respectively
  • Fyn was required for these cytokine production induced upon TCR and BCR multivalent engagement.
  • NTN nephrotoxic nephritis
  • Lyn "/_ FcyRIIA Tg recipients only whereas Lyn _/" , Fyn 1' or Fyn 1' FcyRIIA Tg mice did not develop significant disease despite glomerular Ab deposits. Renal disease development in Lyn _/ ⁇ FcyRIIA Tg mice was characterized by a marked increase in urinary protein and blood urea nitrogen concentration (BUN). Lyn "/_ FcyRIIA Tg mice also exhibited severe renal injury involving extensive mesangial and capillary (subendothelial or even intracapillary) deposits associated with mild mesangial and endocapillary plus extracapillary proliferation.
  • BUN blood urea nitrogen concentration
  • Glomerular lesions were characterized by an intense macrophage infiltrate and cytokine production. These effects involved activation of ITAM signaling, as demonstrated by in situ phosphorylation of the Y 525 residue in Syk.
  • the protective role of Lyn was confirmed in another autoimmune model, the collagen antibody-induced arthritis (CAIA) model.
  • CAIA collagen antibody-induced arthritis
  • targeting human FcyRIIA or FcaRI in Tg animals for ITAMi signaling by AT- 10 F(ab') 2 or monomeric IgA (9, 30) prevented disease development, and this protection required the presence of Lyn.
  • the aggravating role of Fyn was highlighted by the absence of lesions in both Fyn ⁇ FcyRIIA ⁇ and Fyn /' FcoU Tg mice.
  • tissue analysis of phosphorylated kinases and phosphatases in joints of arthritic mice revealed that Lyn may protect the host against autoimmunity by constitutive phosphorylation of SHP-1 Y536 residue, whereas Fyn favours autoimmunity development by inactivation of SHP-1 through the phosphorylation of the SHP-1 S591 residue associated with induction of Syk Y525 ⁇ 526 phosphorylation.
  • SFKs determine the ITAMi/ITAM balance that governs the outcome of inflammatory and autoimmune responses.
  • Lyn was strongly associated with FcyRIIA in healthy individuals but weakly associated in patients, whereas Fyn and Syk were exclusively associated with FcyRIIA in LN patients.
  • SHP-1 and pSHP-l Y536 were associated with FcyRIIA exclusively in cells from healthy individuals.
  • pSHP- 1 S591 and pPKCa were exclusively observed in LN patient cell lysate samples, thus emphasizing an ITAM configuration.
  • this inactive pSHP-l S591 and pPKCa were not associated with FcyRIIA (Fig. IF, right panel).
  • Lyn or Lck are crucial to maintain ITAMi-mediated homeostasis, whereas Fyn is essential for ITAM-mediated cell activation by inducing the PI3K- PKC signaling axis that inactivates SHP-1 during inflammatory responses for most immunoreceptors. Under chronic pathogenic immunoreceptor triggering, this activating loop may amplify inflammatory processes aggravating autoimmune or inflammatory diseases.

Abstract

La présente invention concerne des procédés et compositions pharmaceutiques pour le traitement de maladies inflammatoires auto-immunes. La kinase Fyn de la famille Src était l'effecteur crucial pour l'inhibition de SHP-1, tandis que Lyn/Lck était nécessaire pour son activation en aval des récepteurs du Fc, des cellules B et des cellules T. Les inventeurs ont démontré qu'alors que les souris déficientes en Lyn présentent une aggravation des maladies inflammatoires, les souris déficientes en Fyn ont été protégées. De même, ils ont montré que les patients atteints de glomérulonéphrite lupique présentaient une signature activatrice associée à Fyn, ce qui formait contraste avec la signature inhibitrice associée à Lyn chez les sujets sains. En particulier, la présente invention concerne une méthode de traitement de maladies inflammatoires auto-immunes chez le patient le nécessitant, comprenant l'administration au patient d'une quantité thérapeutiquement efficace d'un inhibiteur de Fyn.
PCT/EP2017/064085 2016-06-10 2017-06-09 Procédés et compositions pharmaceutiques pour le traitement de maladies inflammatoires auto-immunes WO2017212018A1 (fr)

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EP3587416A1 (fr) 2018-06-29 2020-01-01 Institut Univ. de Ciència i Tecnologia, S.A. Dérivés de 2-oxopiperidin-3-yl et leurs utilisations
EP4356909A1 (fr) 2022-10-17 2024-04-24 Selabtec Sciences, SLU 1derives de (sulfonyl)-n-phenylpyrrolidine-2-carboxamides et leur utilisation

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3587416A1 (fr) 2018-06-29 2020-01-01 Institut Univ. de Ciència i Tecnologia, S.A. Dérivés de 2-oxopiperidin-3-yl et leurs utilisations
EP4356909A1 (fr) 2022-10-17 2024-04-24 Selabtec Sciences, SLU 1derives de (sulfonyl)-n-phenylpyrrolidine-2-carboxamides et leur utilisation
WO2024083861A1 (fr) 2022-10-17 2024-04-25 Selabtec Sciences, Slu Dérivés 1-(sulfonyl)-n-phénylpyrrolidine-2-carboxamides et leur utilisation

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