Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/655—Azo (—N=N—), diazo (=N2), azoxy (>N—O—N< or N(=O)—N<), azido (—N3) or diazoamino (—N=N—N<) compounds
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates to a method of inhibiting in vitro the production of cytokines by cells, in particular animal or human, and their secretion.
• the present invention aims to develop a method of inhibiting in vitro production of cytokines by cells which does not endanger these cells.
• this process will make it possible to inhibit in these cells the production and the secretion of substances promoting the appearance of immunoallergic phenomena.
• R 1 and R 2 can be linked together to form a heterocyclic ring with their adjacent nitrogen atom
• R 3 and R 4 can be linked together to form a heterocyclic ring with their adjacent nitrogen atom
• X 1 and X 2 are identical or different and each independently represent an oxygen atom or a group NR 5 , in which R 5 is a hydrogen or halogen atom, an optionally substituted aliphatic or aromatic hydrocarbon radical, or a nitro group, and in which, when two NR 5 groups are simultaneously present, each R 5 may be identical to or different from l other, as well as their salts, esters and isomers.
• azo derivatives are compounds known, in particular for their antiviral activity, in particular against viruses of the retro-virus group, in particular the AIDS virus (see WO-A-9116054 and WO-A-9107876, as well as US-A-5585367).
• -azobisdimethylformamide also called diamide
• many researchers have studied the phenomenon of activation of the intracellular transcription factor NF kappa B and have shown that the diamide blocks the role of certain enzymes. in the activation cascade of this factor.
• the method comprises an in vitro inhibition of production and secretion of interleukins by cells.
• interleukins there may be mentioned in particular the interleukins IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 and IL-15, and consideration is particularly given to the inhibition of IL-2 and IL-5.
• the method comprises an inhibition of production and secretion of interferon- ⁇ (IFN- ⁇ ) by the cells.
• the method comprises an inhibition of production and secretion of a tumor necrosis factor ⁇ (TNF- ⁇ ).
• R 1 to R 5 each represent in the general formula given above an aliphatic or aromatic hydrocarbon radical containing from 1 to 6 carbon atoms.
• the heterocycles optionally formed in the general formula may contain, in addition to a nitrogen atom, at least one other heteroatom, for example oxygen.
• the heterocycle is for example pentagonal to octagonal, preferably it is hexagonal.
• the azo derivative according to the invention can be chosen from the group comprising azobisformamidine derivatives, such as 1,1'- azobisformamidine, 1, 1-azobisnitroformamidine, 2, 2 '-azobismethylformamidine, 1,1'- azobisfluoroformamidine, 1-monochloroazobisformamidine, and azobis- [chloroformamidine], azobisformamide derivatives , such as 1,1'-azobisformamide and dimethyl-azobisformamide, 1, l'- (azodicarbonyl) -dipiperidine, 1,1'- (azodicarbonyl) -dimorpholine, azodihydroxamic acid and its salts , and azodicarboxylic acid and its salts.
• azobisformamidine derivatives such as 1,1'- azobisformamidine, 1, 1-azobisnitroformamidine, 2, 2 '-azobismethylformamidine, 1,1'- azobisfluoroformamidine, 1-
• the method comprises an application of the azo derivative to the cells at a dose which does not induce apoptosis of the latter. It is preferably possible to envisage a micromolar concentration of approximately 0.4 to approximately 200, preferably of 2 to 20, advantageously of 2 to 10.
• 1,1'-azobisdimethylformamide also called diamide
• diamide can have the disadvantage in certain circumstances of causing cell apoptosis at certain too high concentrations, while offering a relatively short half-life of only a few minutes.
• the application of at least one of the azo derivatives indicated is carried out on cells isolated from macroorganisms or cells of microorganisms, which for example come from cell cultures.
• the cells treated can also be those of a multicellular organ or tissue extracted from a human or animal body, such as, for example, cells from a blood or lymph sample.
• the application of at least one of the azo derivatives indicated is for example carried out on cells isolated from human blood producing cytokines. These cells can be extracted globally and therefore we will observe global effects on the different lymphocyte types, antigen presenting cells as well as on macrophages in cocultures. A further stage of the investigation may consist in measuring the production of cytokines by highly purified cell lines, for example CD4 lymphocytes.
• the treatment of the cell lines concerned can also be done by the administration of azo derivatives to living organisms including humans and the measurement of the different groups of cytokines in biological fluids extracted from the human or animal body, before , during and / or after treatment, as well as by measuring the possibility of production of the lymphokines concerned after extraction of the cells contained in these liquids.
• Cytokines are produced in the body by different cell reservoirs.
• lymphocytes specialize in the specific production of cytokines and that, depending on the type of cytokines produced, CD4 Thl and Th2 lymphocytes are differentiated.
• Cytokines are also produced by CD8 lymphocytes (IL-4 and IL-5) but also by mastocytes and eosinophils and at the stage of implantation in the uterus by ectodermal cells of the tropho- blast.
• cytokine productions from these different cellular reservoirs are involved in the appearance of various inflammatory and allergic processes in the broad sense, as well as in the phenomenon of transplant rejection.
• the active substance according to the invention can be applied to the cells, alone or in admixture with other substances according to the invention, or alternatively in admixture with other substances having another effect on the cells. It is possible to provide for the application of the active substance (s) as such or in the form of a composition comprising at least one of the azo derivatives corresponding to the formula given above and an appropriate support or vehicle.
• Certain compounds according to the invention have at least one asymmetric C atom and consequently all the isomers, including the diastereoisomers and the rotary or enantiomeric isomers, are included as part of the invention.
• the invention includes the D and L isomers in pure form or as a mixture, including racemic mixtures.
• Certain acid-type compounds for example carboxylic acids, can form salts by example with metals, or with acceptable amines, or esters with compatible alcohols.
• the present invention also relates to the use of at least one of the derivatives corresponding to the formula given above, as well as their salts or isomers, for the manufacture of medicaments to be used in the treatment or prophylaxis of conditions human or animal resulting from pathological cellular production of said cytokines.
• Special provision may be made for the manufacture of medicaments for the treatment and / or prevention of autoimmune and / or inflammatory diseases involving T lymphocytes, of allergic inflammatory diseases or of rejection of allograft and / or xenograft of organs and graft versus host disease after cell allograft.
• said use will therefore be provided for manufacturing medicaments intended to be used in the treatment or prophylaxis of conditions such as those indicated above.
• the medicament thus prepared according to the invention can be administered by any suitable route, inter alia by oral, sublingual, rectal or vaginal route, by injection or infusion, by local, transcutaneous or transmucosal route.
• the drug contains a therapeutically effective amount of the azo derivative (s) indicated above.
• the dosage will vary from individual to individual according to their own immunological characteristics which are partly genetically determined.
• the drug can be in any suitable dosage form, for example in the form of capsules, pills, tablets, dragees, powders, injectable forms, creams, ointments, systems known transdermal distribution, products to inhale.
• the pharmaceutical formulations and compositions may contain the usual pharmaceutically acceptable excipients, as well as optionally common additives in pharmacy.
• excipients and additives include in particular compatible inert fillers, binders, disintegrating agents, buffers, preservatives, antioxidants, lubricants, sapid agents, thickeners, dyes, emulsifiers, etc.
• an object of the invention also relates to azo derivatives corresponding to the general formula given above, in which at least one of the groups A and B represents a carboxyl group, as well as their salts, esters and pharmaceutically acceptable isomers, for their application as therapeutically active substances.
• azodicarboxylic acids of the formula below are envisaged
• Another subject of the invention relates to azo derivatives corresponding to the general formula given above, in which the group A is one of the radicals R and R representing a hydroxy group and the other a hydrogen atom, and in which group B can simultaneously represent the group ⁇ N- ⁇ 4.
• R radicals R 3 and R 4 which may represent a hydroxy group and the other a hydrogen atom, as well as their pharmaceutically acceptable salts, esters and isomers, for their application as therapeutically active substances.
• the invention also relates to products containing at least one of the azo derivatives corresponding to the formula indicated above and their isomers and at least one cytokine, as a combination product for simultaneous, separate or spread over time use, in therapy or prophylaxis of human or animal diseases resulting from pathological cellular production of at least one cytokine, different from said at least one cytokine contained in the combination product, or in the therapy or prophylaxis of human affections having as side effect a pathological cellular production of at least one cytokine, different from said at least one cytokine contained in the combination product.
• PMBC Peripheral blood mononuclear cells
• the cells (5.10 6 cells / ml) are activated with 0.3 ⁇ g / ml of phytohemagglutinin (PHA) and cultured for 72 hours, at 37 ° C., in the presence of various concentrations of ADA (20, 10, 5, 1, 0.2 and 0.04 ⁇ g / ml).
• the culture medium consists of RPMI 1640 supplemented with 10% fetal calf serum and glutamine, and it contains mercaptoethanol.
• ADA solutions are prepared in diethyl sulfoxide (DMSO), the working concentration of DMSO during the experiments being 0.1%.
• DMSO diethyl sulfoxide
• the latter are carried out on 96-well plates (200 ⁇ l / well) and after centrifugation of the plates at 1500 revolutions per minute for 10 minutes, the supernatants are harvested and analyzed for their IL-5 content by an enzyme-linked immunosorbent assay ELISA.
• the decrease in cytokine production depends on the amount of ADA used (dose-dependent effect) and it is observed for ADA concentrations which do not induce cytotoxic effects.
• PBMCs from 3 healthy donors are purified by density gradient centrifugation (LymphoPrep).
• T lymphocytes are obtained from 15.10 6 PMBC incubated for 20 to 30 minutes in a water bath of 37 ° C with 0.8 ml of a LymphoKwik® mixture (One Lambda, Inc., CA, USA). This is a mixture of complement and specific antibodies for antigenic reasons of the membrane of the cells which lyse the unwanted cells (B lymphocytes, monocytes, ). The cells are centrifuged and then washed twice. By FACS analysis we see that the crops T cells produced in this way contain more than 85% of CD3 + cells.
• the results are expressed in% by considering the medium without ADA as 100%.
• the concentrations corresponding to 100% are respectively 90,000 pg / ml of IL-2, 1100 pg / ml of IL-5, 1900 pg / ml of IFN ⁇ and 2870 pg / ml of TNF- ⁇ .
• the results are the average of independent ELISA measurements.
• a single ip injection of azodicarbonamide (ADA) in a dose of 100 mg / kg of body weight or of the corresponding support is carried out on BALB / C mice (Harlan, Zeist, Netherlands), one day before inoculation 25 ⁇ g ip of 145-2C11 hamb mAb, a CD 3 mAb anti-mouse which induces massive activation of polyclonal T cells in vivo leading to a systemic release of cytokines.
• Sera are collected 2, 4, 8 and 24 hours after the anti-CD 3 treatment and the levels of cytokines (IL-2, IL-4) are determined by an ELISA test (Genzyme).
• FIGS. 1 and 2 graphs are shown with the quantities of IL-2 and
• IL-4 in serum as an average of 5 animals tested.
• the abscissa indicates the times of the blood samples.
• the empty bars correspond to the results obtained with the support and the gray bars to those obtained with a preliminary treatment of ADA.
• Skin grafts well known for their dependence on T cells are prepared from the tails of female C57BL / 6.CH-2 bm12 mice.
• mice (Jackson Laboratory, Bar Harbor, ME, USA). They are grafted onto the flanks of C57BL / 6 mice (Harlan, Zeist, Netherlands). A bandage is applied around the hole. This bandage is removed 10 days later and the transplants are checked every day. The test mice receive a daily ip injection of 50 mg / kg body weight of ADA and the controls receive an equivalent amount of the corresponding support, until an acute rejection is observed.
• the survival of the skin graft is indicated on the ordinate, in% and the number of days after the transplant on the abscissa.
• Mononuclear cells are isolated by Ficoll-Hypaque centrifugation and resuspended in a medium consisting of RPMI 1640 supplemented with 5% autologous serum, 1 mN of glutamine, 1001 IU / ml of penicillin, 10 ⁇ g / ml of streptomycin , 10 ⁇ g / ml phytohemagglutinin (pha), 10 ⁇ g / ml phorbol myristate acetate (pma) and test compounds, as indicated, by individual experience, at a cell density of 2,000,000 per ml .
• the cells are seeded in 96-well microtiter plates at 250 ⁇ l per well and they are incubated for 48 hours at 37 ° C. Next, the supernatants are harvested and the IL-2 and IL-5 concentrations are quantified by a standard ELISA, using a biotin-streptavidin technology. Alternatively, the pha was replaced by 0.1 ⁇ g / ml of monoclonal antibodies directed against human CD3, so as to obtain a more specific activation of T cells.
• Brown-Norway rats are actively sensitized to ovalbumin 21 days later, the rats are treated by aerosol with a solution of 5% ovalbumin in 0.9% NaCl. 24 hours later, the bronchial reactivity to metacholine is measured on the anesthetized rats. At the end of the bronchial reactivity measurement, splenocytes are removed from these rats and placed in culture to determine the production of IFN- ⁇ by these cells.
• ADA is administered 24 hours before the start of the ovalbumin sensitization phase and daily for the duration of the test, except the day of the measurements.
• ADA is administered orally, suspended in 1% methylcellulose, in a dosage of 500 mg / kg and 1000 mg / kg body weight, twice a day.
• the results are collated in FIG. 4.
• On the ordinate the amounts of IFN- ⁇ in pg / mg of protein are indicated.
• the empty bars A to F correspond to the following experiments: A - no stimulation with ovalbumin, no active substance B - no stimulation with ovalbumin, ADA at 500 mg / kg po C - no stimulation with ovalbumin, ADA at 1000 mg / kg po
• ADA has an effect of inhibiting the production of INF- ⁇ by the splenocytes of treated rats to show a strong bronchial reactivity.
• Example 9 Capsules to be administered orally.
• Composition of a capsule 500 mg of ADA
• Tablets containing 250 mg of azobisformamidine are commonly produced using the following excipients: hydroxypropylmethylcellulose, hydroxypropylcellulose, titanium dioxide, polyethylene glycol 400, black iron oxide.
• These tablets can be administered to adequately reduce the production of IgE by reducing IL-5 in allergic phenomena (seasonal allergic rhinitis for example). This treatment will be initiated in order to obtain a remission and its maintenance.
• Example 11 Form to be injected.
• An injectable form is produced based on 1 g of dimethylazobisformamide and pyrogen-free distilled water added with NaCl.
• Example 12 Cream or ointment.
• a cream or ointment is produced with 1, 1 '-azobis- [chloroformamidine] (choroazodine) and as an excipient, in particular glycerin, paraffin oil, petrolatum.
• This cream can be applied locally as an immunomodulator in scleroderma.
• Transdermal delivery systems for dimethylazobisformamide can also be provided.
• Example 14 Tablets for Oral Administration Composition of one tablet: 100 mg of ADA
• This composition is routinely introduced into a tableting machine.
• an administration of IL-2 can be provided to the patient.
• the abundant content of IL-2 in the body of the patient thus treated has the effect of stimulating an overproduction by the cells of the latter of other cytokines, and in particular of IL-5, which leads to side effects, such as pemphigus, thyroiditis, rheumatoid arthritis, Crohn's disease, scleroderma, hypereosinophilia, etc. It is therefore possible, in conjunction with the administration of IL-2, to take regular ADA tablets to counteract these side effects.
• IL-2 and ADA can be administered simultaneously, separately or in a spread in time .

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• 1997
  • 1997-11-26 BE BE9700949A patent/BE1011571A3/fr not_active IP Right Cessation
• 1998
  • 1998-11-25 AT AT98956724T patent/ATE290870T1/de not_active IP Right Cessation
  • 1998-11-25 CA CA002312023A patent/CA2312023A1/fr not_active Abandoned
  • 1998-11-25 JP JP2000522922A patent/JP2001524526A/ja active Pending
  • 1998-11-25 DE DE69829406T patent/DE69829406T2/de not_active Expired - Lifetime
  • 1998-11-25 US US09/555,258 patent/US6407081B1/en not_active Expired - Fee Related
  • 1998-11-25 AU AU13276/99A patent/AU757489B2/en not_active Ceased
  • 1998-11-25 WO PCT/BE1998/000183 patent/WO1999027937A2/fr not_active Ceased
  • 1998-11-25 ES ES98956724T patent/ES2241182T3/es not_active Expired - Lifetime
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