EP1225891A2 - Utilisation d'un inhibiteur de la phosphotyrosylphosphatase dans le traitement de patients immunodeprimes - Google Patents

Utilisation d'un inhibiteur de la phosphotyrosylphosphatase dans le traitement de patients immunodeprimes

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Publication number
EP1225891A2
EP1225891A2 EP00971189A EP00971189A EP1225891A2 EP 1225891 A2 EP1225891 A2 EP 1225891A2 EP 00971189 A EP00971189 A EP 00971189A EP 00971189 A EP00971189 A EP 00971189A EP 1225891 A2 EP1225891 A2 EP 1225891A2
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EP
European Patent Office
Prior art keywords
bpv
compound
immune
nfat
virus
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP00971189A
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German (de)
English (en)
Inventor
Michel J. Tremblay
Benoit Barbeau
Jean-François FORTIN
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Virocell Inc
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Virocell Inc
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Publication of EP1225891A2 publication Critical patent/EP1225891A2/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/40—Peroxides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • A61P37/04—Immunostimulants

Definitions

  • the present invention relates to a method for restoring a functional immunity in immunosuppressed patients. More specifically, the invention relates to the use of phosphotyrosyl phosphatase inhibitors for restoring normal immune functions in individuals infected with viruses such as the human immunodeficiency virus (HIV) .
  • viruses such as the human immunodeficiency virus (HIV) .
  • T-cell activation results in the induction of several genes which play crucial roles for the subsequent functions that these cells accomplish in the immune system.
  • Different types of transcription factors are thus induced early on and directly affect transcription of immune response genes such as inflammatory cytokines.
  • This T-cell activation is of prime importance for the immune system to mount an adequate response.
  • One of these transcription factors is known as the NF- ⁇ B complex. Binding sites in the locus for several T-cell specific genes have been identified for this factor.
  • the NF- ⁇ B (Rel) family is made of several members which either form homo- or heterodimers . Some of these dimers actually are involved in higher order transcriptional complexes which are in return important for the regulation of specific T-cell genes.
  • NF- ⁇ B can act in concert with the transcription factor NFAT to achieve regulation of the IL-2 and ⁇ -interferon genes (Sica et al . , J. Biol . Chem. 272:30412-30420, 1997), as well as the human immunodeficiency virus type-1 (HIV-1) regulatory elements (LTR) (Kinoshita et al . , Immuni ty 6:235-244, 1997) .
  • NFAT is another family of Rel-related transcription factors which are activated at early times after T-cell activation.
  • this factor (at least one member of the family called NFATc or NFAT2) is sequestered in the cytoplasm and is translocated to the nucleus upon increase in intracellular calcium content.
  • This elevation in intracellular Ca 2+ triggers conformational changes in calmodulin and increases its binding to the calcineurin serine/threonine phosphatase, leading in turn to activation of this enzyme by displacement of an auto- inhibitory domain from the catalytic site.
  • the ensuing dephosphorylation of the NFAT factor by calcineurin then makes the nuclear localizing sequence accessible and hence allows it to freely migrate into the nucleus.
  • CsA and FK506 were both acting on an upstream component of the NFAT activation cascade which was resulting in inhibition of the calcineurin phosphatase activity (Liu et al . , Cell 66:807-815, 1991).
  • NFAT Upon reaching the nucleus, NFAT is usually found associated with the complex AP-1 which is newly synthesized and, after association, acquires high transactivating potential upon binding to its consensus sequence 5' - (T/A)GGAAA(A/N) (A/T/C) -3 ' .
  • NFAT binding sites suggested to be cooperative with AP-1 are known to resemble NF- ⁇ B binding sites and include the CD28RE element located in the locus of the IL-2 and GM- CSF genes .
  • NFAT2 NFAT2
  • NFAT4/x NFAT4/x
  • NFATp NFATp
  • Numerous T- cell expressed genes are regulated by the NFAT transcription factors.
  • NFAT binding sites have been identified m the promoter region of the IL-2, IL-3, IL-4 IL-5, GM-CSF, ⁇ - mterferon and IL-13 genes, all of which were found to be sensitive m their transc ⁇ ptional regulation to either CsA or FK506. The importance of NFAT members in T-cell function is thus very clearly demonstrated.
  • T-cell Activation of T-cells is a very complex process which involves cell-to-cell interactions of several cell surface molecules.
  • the most important polypeptide complex remains the T-cell receptor (TCR) .
  • TCR T-cell receptor
  • CD4 and CD28 co-receptors
  • Several second messengers are orchestrating the signals induced from the membrane to the nucleus but one common theme is the increase in the overall intracellular tyrosme phosphorylation level. Different studies have shown the importance of this increase in phosphorylation level which is believed to be initially controlled by two specific protein tyrosme kmases, p561ck and p59fyn.
  • TRP- initiated cascade Other important tyrosme kmases are involved m the TCR- initiated cascade and include the syk family member ZAP-70. It is also known that the protein tyrosme phosphatases (PTPs) , enzymes responsible for the dephosphorylation of proteins on their tyrosme residues, are very important modulators of T-cell activation cascade.
  • PTPs protein tyrosme phosphatases
  • One aim of the present invention is to provide a method for activating T-cells for restoring a functional immunity in immunosuppressed or immunodeficient patients.
  • a bis-peroxovanadium (bpV) compound for restoring immune functions and an overall immune response of a patient suffering from an immune disorder.
  • the bpV compound of the present invention is a phosphotyrosyl phosphatase inhibitor, and is preferable made of an oxo ligand, two peroxo anions, and an ancillary ligand located in an inner coordination sphere of vanadate .
  • the compound of the present invention may be used for treating immune disordes caused for example by an infection with a virus, preferably a human immunodeficiency virus .
  • a pharmaceutical composition for restoring immune functions and an overall immune response of a patient suffering from an immune disorder.
  • the pharmaceutical composition comprises a bis-peroxovanadium (bpV) compound and a pharmaceutically acceptable carrier.
  • the bpV compound may be used for the manufacture of a medicament for restoring immune functions and an overall immune response of a patient suffering from an immune disorder.
  • the medicament is preferably formulated for intravenous, subcutaneous, intradermal or topical application.
  • the medicament may be administered via a patch, an implant or by inhalation of a powder or an aerosol spray.
  • the medicament may be formulated in a liposomal composition suitable for administration or in a tablet form.
  • the bpV compound of the present invention may also be used in combination with an antiviral agent or with another immunomodulator .
  • a method for restoring immune functions and an overall immune response in a patient suffering from an immune disorder comprising administering to a patient in need of such a treatment a therapeutically effective amount of a bis- peroxovanadium (bpV) compound.
  • bpV bis- peroxovanadium
  • Fig. 1 illustrates a bar chart indicating that the bpV-mediated induction of HIV-1 LTR is sensitive to NaSal, FK506 and CsA;
  • Figs. 2A and 2B illustrate a bar chart indicating that the induction of wild-type and NF- ⁇ B- mutated HIV-1 LTR by bpV compounds is sensitive to FK506;
  • Figs. 3A and 3B show that NFAT-driven luciferase gene expression is induced by the bpV molecules and is FK506-sensitive;
  • Figs. 4A and 4B illustrate a radiographic film representing the bpV-mediated induction of nuclear translocation of NFAT;
  • Fig. 5 illustrates a bar chart showing that expression of a dominant negative mutant of p21ras blocks NFAT activation by bpV compounds
  • Figs. 6A and 6B illustrate bar charts illustrating that the presence of p56lck is required for bpV-mediated activation of NFAT;
  • Figs. 7A and 7B illustrate a scattergram and bar chart indicating that bpV compounds induce a FK- 506-sensitive activation of the human IL-2 promoter which synergizes with PMA;
  • Figs. 8A and 8B illustrate bar charts illustrating the correlation between IL-2 promoter activity and IL-2 secretion.
  • the present invention relates to the use of bpV compounds, a new class of potent phosphotyrosyl phosphatase inhibitors, in the treatment of humans suffering from a deficient immune system, a pathological condition which can lead to the development of opportunistic infections and cancers .
  • the present invention comprises a class of biologically compounds which are acting as potent protein tyrosine phosphatase inhibitors which are useful in treating various pathological conditions in humans accompanying immune suppression. These compounds are useful for enhancing the protective response of the immune system and to restore natural immunity.
  • bpV compounds are made of an oxo ligand, two peroxo anions, and an ancillary ligand located in the inner coordination sphere of vanadate .
  • Ancillary ligands located in the inner coordination sphere of the vanadate atom include bipyridine [bipy] ; picolinic acid (i.e.
  • Treatment with bpV compounds represent a new therapeutic avenue to reconstitute and/or enhance mucosal, humoral and cellular immune responses in patients suffering from immune response related syndromes.
  • the materials used and the analyses and assays carried out were as follows: Cell lines The lymphoid T-cell lines used include Jurkat
  • JCaMl .6 is a derivative of the Jurkat leukemic T-cell line that is deficient in p56lck expression.
  • JCaMl. TAg is a derivative of JCaMl .6 cells that stably express the SV40 large T antigen.
  • the 1G5 T-cell line is a Jurkat derivative that harbors two stably integrated constructs constituted of the luciferase gene under the control of the HIV-1SF2 LTR.
  • the Jurkat derivatives deficient in capacitative calcium entry (clones CJ1 through CJ5 , plus the parental line CJ) (Fanger et al . , J. Cell . Biol . 131:655-667, 1995) were also used. These cells were maintained in complete culture medium made of RPMI-1640 supplemented with 10% fetal bovine serum, glutamine (2 mM) , penicillin G (100 U/ml) , and streptomycin (100 ⁇ g/ml) . Plasmids and antibodies pLTR-LUC (HIV-1 LTR from strain HXB2) and pm ⁇ BLTR-LUC plasmids were used (Henderson et al . , J. Virol .
  • molecular constructs contain the luciferase reporter gene under the control of wild-type (GGGACTTTCC) or NF- ⁇ B-mutated (CTCACTTTCC) HIV-1 LTR.
  • GGGACTTTCC wild-type
  • CTCACTTTCC NF- ⁇ B-mutated HIV-1 LTR.
  • the p ⁇ B ⁇ TATA-LUC plasmid contains the minimal HIV-1 KB region (-105/-70) and a TATA box placed upstream of the luciferase reporter gene (Sun et al., Mol . Cell . Biol . 16:1058-1065, 1996).
  • pIL-2-LUC containing the complete 320-bp IL-2 promoter controlling the luciferase gene
  • pNFAT-LUC containing the IL-2 minimal promoter with 3 tandem copies of the NFAT-1 binding site
  • pEFneoLck T encodes for the wild type human p56lck protein and pEFneo is the control plasmid (Liu et al., Proc. Natl . Acad. Sci . USA 90:8957-8961, 1993).
  • Preparation of bpV compounds bpV molecules were prepared as described previously (Posner et al . , J. Biol . Chem . 269:4596- 4604, 1994). Briefly, V 2 0 5 was dissolved in an aqueous KOH solution and then mixed with 30% H 2 0 2 and the respective ancillary ligand in addition to the ethanol for optimal precipitation.
  • transfected cells were pooled 24 h after transfection and were next separated into various treatment groups. Stably transfected Jurkat cells were obtained by electroporation.
  • Stably or transiently transfected cells were seeded at a density of 10 5 cells per well (100 ⁇ l) in 96-well flat-bottom plates.
  • Cells were either left unstimulated or were stimulated with the different bpV molecules at 10 ⁇ M (bpV[HOpic], bpV [bipy] , and bpVtpic]), phytohemagglutinin (PHA-P at 3 ⁇ g/ml), phorbol 12-myristate 13-acetate (PMA at 20 ng/ml), Ionomycin (Iono at 1 ⁇ M) , anti-CD3 antibody (clone OKT3 at 3 ⁇ g/ml), anti-CD28 antibody (clone 9.3 at 1 ⁇ g/ml) in a final volume of 200 ⁇ l .
  • luciferase assay buffer (20 mM tricine, 1.07 mM (MgC0 3 ) 4 -Mg (OH) 2 .5 H 2 0, 2.67 mM MgS0 4 , 0.1 mM EDTA, 270 ⁇ M coenzyme A, 470 ⁇ M luciferin, 530 ⁇ M ATP, and 33.3 mM DTT).
  • Light output was measured for 20 s with a two second delay. Values are expressed as RLU (Relative Light Units) as measured by the apparatus .
  • IL-2 production 20 mM tricine, 1.07 mM (MgC0 3 ) 4 -Mg (OH) 2 .5 H 2 0, 2.67 mM MgS0 4 , 0.1 mM EDTA, 270 ⁇ M coenzyme A, 470 ⁇ M luciferin, 530 ⁇ M ATP, and 33.3 mM DTT.
  • Light output was measured for 20 s
  • PBST phosphate-buffered saline
  • BSA 1% BSA
  • 50 ⁇ l of a biotinylated anti-IL-2 antibody at a final concentration of 0.25 ⁇ g/ml in PBST-BSA along with 50 ⁇ l of tested cell supernatant were dispensed in the wells for another incubation period of 1 h at 37°C.
  • Jurkat cells were either left untreated or were incubated for 1 h at 37°C with the bpV compounds (10 ⁇ M) .
  • the incubation of Jurkat cells with the various stimulating agents was terminated by the addition of ice-cold PBS and nuclear extracts were prepared as follow.
  • Sedimented cells were resuspended in 400 ⁇ l of cold buffer A (10 mM N-2-hydroxyethylpiperazine-N' -2- ethanesulfonic acid [HEPES ; pH 7.9], 1.5 mM MgCl 2 , 10 mM KC1, 0.5 mM dithiothreitol , and 0.2 mM phenylmethylsulfonyl fluoride) .
  • cold buffer A 10 mM N-2-hydroxyethylpiperazine-N' -2- ethanesulfonic acid [HEPES ; pH 7.9]
  • 1.5 mM MgCl 2 10 mM KC
  • Protein concentrations were determined by the bicinchoninic assay with a commercial protein assay reagent (Pierce, Rockford, IL) . Nuclear extracts were incubated for 30 min at 23°C in 15 ⁇ l of buffer (100 mM HEPES [pH 7.9], 40% glycerol, 10% Ficoll, 250 mM KC1 , 10 mM dithiothreitol, 5 mM EDTA, 250 mM NaCl , 2 ⁇ g poly[dI- dC] , 10 ⁇ g nuclease-free bovine serum albumin fraction V) containing 0.8 ng of 32 P-5 ' -end-labeled double stranded DNA (dsDNA) oligonucleotide.
  • buffer 100 mM HEPES [pH 7.9], 40% glycerol, 10% Ficoll, 250 mM KC1 , 10 mM dithiothreitol, 5 mM EDTA
  • Double stranded DNA (100 ng) was labeled with [ ⁇ - 32 P] -ATP and T4 polynucleotide kinase in a kinase buffer (New England Biolabs, Beverly, MA) . This mixture was incubated for 30 min at 37°C and the reaction was stopped with 5 ⁇ l of 0.2 M EDTA. The labeled oligonucleotide was extracted with phenol/chloroform and passed through a G-50 spin column.
  • the dsDNA oligonucleotide which was used as a probe or as a competitor, contained the consensus NFAT-binding site from the murine IL-2 promoter (5 ' -TCGAGCCCAA AGAGGAAAAT TTGTTTCATG-3 ' ) .
  • a dsDNA oligonucleotide containing a mutated NFAT-binding site was also used in competition experiments (5 1 - TCGAGCCCAA AGACCTTAAT TTGTTTCATA CAG-3 ' ) .
  • Oligonucleo- tides were purchased from Gibco-BRL.
  • DNA-NFAT complexes were resolved from free labeled DNA by electrophoresis in native 4% (w/v) polyacrylamide gels containing 0.5X TBE buffer. The gels were subsequently dried and autoradiographed .
  • Cold competitor assays were carried out by adding a 100-fold molar excess of homologous unlabeled dsDNA NFAT oligonucleotide or of dsDNA NF- ⁇ B oligonucleotide (5 ⁇ -ATGTGAGGGG ACTTTCCCAG GC-3 ' ) simultaneously with the labeled probe.
  • Supershift assays were performed by pre-incubation of nuclear extracts with 1 ⁇ l of either preimmune serum (control) or specific antibody in the presence of all the components of the binding reaction described above for 15 minutes at room temperature prior to the addition of the probe.
  • the antibody used for the supershift assay is specific for the NFATc subunit of the NFAT complex. This antibody along with the NF- ⁇ B oligonucleotide were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) .
  • the therapeutically effective amount of the inhibitor of the present invention to be administered will vary with the particular inhibitor used, the type or mode of administration, the concurrent use of other active compounds, host age and size, type, response of individual patients, and the like.
  • it will be administered in sufficient doses for restoring immunity and to obtain an effective peak or steady-state concentration of about 100 nM to 25 ⁇ M, usually about 10 ⁇ M in plasma as suggested by the concentrations of bpV compounds tested and found to be effective in in vi tro experiments.
  • An effective dose amount of the bpV compounds is thus to be determined by the clinician after a consideration of all the above- mentioned criteria.
  • compositions Any suitable type or mode of administration may be employed for providing a mammal, especially a human with an effective dosage of a bpV compound of the present invention.
  • oral, parenteral and topical may be employed.
  • Dosage forms include tablets, capsules, powders, solutions, dispersions, suspensions, creams, ointments and aerosols.
  • compositions of the present invention comprise a bpV compound as a phosphotyrosyl phosphatase inhibitor and as the active ingredient, and a pharmaceutically acceptable carrier and optionally other therapeutic ingredients.
  • the bpV compounds can be used in a variety of ways in vivo . It can be formulated into pharmaceutical compositions according to any known methods of preparing pharmaceutically useful compositions. In this manner, the bpV compounds are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, including human proteins, e.g., human serum albumin, are described for instance in Remington's Pharmaceutical Sciences (16th ed. Osol , A., ed. , Mack, Easton, PA [1980] ) .
  • compositions suitable for effective administration will contain a therapeutically effective amount of the bpV compound or amount resulting in the restoration of functional immunity, together with a suitable amount of carrier vehicle.
  • suitable amount of carrier vehicle e.g., a suitable amount of a pharmaceutically acceptable carrier vehicle.
  • the bpV compound can be formulated as a sterile pharmaceutical composition for therapeutic use which is suitable for intravenous or intraarterial administration.
  • the product may be in a solvent -free form and ready to be reconstituted for use by the addition of a suitable carrier or diluent, or alternatively, it may be in the form of solution which may be aqueous or organic.
  • a suitable carrier or diluent or alternatively, it may be in the form of solution which may be aqueous or organic.
  • a sterile diluent which may contain materials generally recognized for approximating physiological conditions.
  • the sterile diluent may contain salts and/or buffering agents to achieve a physiologically acceptable tonicity and pH, such as sodium chloride, phosphate and/or other substances which are physiologically acceptable and/or safe for use.
  • the pharmaceutical composition When used as an aqueous solution, the pharmaceutical composition will for the most part contain many of the same substances described above for the reconstitution of a solvent-free product. When used in solution in an organic solvent, a small volume of the solution containing the bpV compound will be diluted with an aqueous solution that will contain many of the same substances described above for the reconstitution of a solvent-free product. The pharmaceutical composition, for the most part, will thus contain many of the same substances described above for the reconstitution of a solvent -free product.
  • the bpV compound useful in the methods of the present invention may be employed in such forms as, for example, sterile solutions for injection or encapsulated (for instance in liposomes) or embedded (for example in suppositories) for slower long- lasting release .
  • the bpV compound may be used in combination with other agents including, but not limited to, antiviral agents or other immunomodulator .
  • the subject bpV compound is to be administered to a host as an inhibitor of phosphotyrosyl phosphatase
  • the bpV compound may be administered, for example, intraarterially, intravenously, intraperito- neally, subcutaneously, intramuscularly, by injection, by suppository, by inhalation, or the like.
  • the mode of administration by injection includes continuous infusion as well as single or multiple boluses.
  • Useful administration type or mode also includes the use of implantable internal pumps for continuous infusion into a blood vessel or at different sites such as the peritoneal cavity or subcutaneously .
  • implantable internal pumps for continuous infusion into a blood vessel or at different sites such as the peritoneal cavity or subcutaneously .
  • Such techniques are disclosed in Cecil ' s Text Book of Medicine (chapter 164, 19th Edition, 1992) for the treatment of hepatic cancers.
  • Transdermal administration by means of a patch containing the bpV compound of the present invention may also be a useful administration mode.
  • controlled release preparations may be achieved through the use of macromolecules to complex or absorb the bpV compound.
  • the controlled delivery may be achieved by selecting appropriate macromolecules (for example, polyesters, polyamino acids, polyvinyl pyrrolidone, ethylene-vinyl acetate, methyl cellulose, car- boxymethyl cellulose, protamine sulfate or serum albumin, the appropriate concentration of macromolecules, as well as the methods of incorporation) . In this manner, release of the bpV compound can be controlled.
  • Another possible method useful in controlling the duration of action by controlled release preparations is the incorporation of the bpV compound into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid), or ethylene-vinyl acetate copolymers .
  • microcapsules prepared, for instance, by coacervation techniques or by interfacial polymerization (for example, hydroxymethyl cellulose or gelatin microcapsules and polymethyl methacrylate microcapsules, respectively) , in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions .
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • compositions include compositions suitable for oral or parenteral administration. Conveniently they are presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy.
  • the bpV compound can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration.
  • any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions; elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, capsules and tablets. If desired, tablets may be coated by standard aqueous or nonaqueous techniques.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the bpV compound, as a powder or granules or as a solution or suspension in an aqueous liquid, a non- aqueous liquid, an oil-in-water emulsion or a water-in- oil emulsion.
  • Such compositions may be prepared by any of the methods of pharmacy such methods including the step of bringing the bpV compound into association with the carrier which includes one or more necessary ingredients.
  • compositions are prepared by uniformly and intimately admixing the bpV compound with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
  • a tablet may be prepared by compression of molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent .
  • the bpV compound is to be administered in pharmacologically or physiologically acceptable amounts, by which is to be understood amounts not harmful to the patient, or amounts where any harmful side effects in individual patients are outweighed by the benefits.
  • the bpV compound is to administered in a therapeutically effective amount, which is to be understood is an amount meeting the intended therapeutic objectives, and providing the benefits available from administration of bpV compound.
  • the cell line 1G5 a Jurkat derivative containing stably integrated HIV-1 LTR-LUC constructs, was used.
  • 1G5 cells were either left untreated or were pretreated with NaSal (2.5 mM) , FK506 (10 ng/ml) , CsA (10 ng/ml) or a combination of NaSal with either FK506 or CsA for 1 h at 37°C.
  • cells were either stimulated or not for 8 h with the following agents: PHA (3 ⁇ g/ml), bpV[bipy] , bpV[HO-pic] or bpV [pic] (lO ⁇ M) .
  • Figs. 2A and 2B Jurkat cells were transiently transfected by DEAE-Dextran with 15 ⁇ g of either p ⁇ B-TATA-LUC (Fig. 2A) or pm ⁇ BLTR-LUC (Fig. 2B) . After 24 h, transfected cells were either left untreated (D) or were pretreated with FK506 (10 ng/ml) ( ⁇ ) for 1 h at 37°C.
  • NFAT-dependent luciferase expression is markedly stimulated by the bpV compounds
  • the enhancer region (-105/-70) of the HIV-1 LTR is a binding site for both NF- ⁇ B and NFAT transcription factors
  • the obvious candidate for the NF- ⁇ B-independent transcription factor induced by the bpV is NFAT.
  • transient transfections were performed in Jurkat cells with pNFAT-LUC, a vector made of three tandem repeats of the human IL-2 -derived NFAT binding site positioned in the context of the human IL-2 mini- mal promoter upstream of the luciferase reporter gene.
  • Luciferase activity was evaluated at different time points after initiation of the stimulation by either the combination of PHA/PMA or the bpV compound bpV[pic] .
  • Fig. 3A Jurkat cells were transiently transfected by DEAE-Dextran with 15 ⁇ g of pNFAT-LUC.
  • Fig. 3B Jurkat cells stably transfected with pNFAT-LUC were either left untreated (D) or were pretreated with FK506 (10 ng/ml) ( ⁇ ) .
  • J-NFAT-LUC stably pNFAT-LUC- transfected Jurkat cells
  • NFAT inducers with or without pre-treatment with FK506.
  • J-NFAT-LUC cells were highly responsive to the inducer PHA added alone or in the presence of PMA (48.9-and 72.1-fold increase, respectively), whereas PMA alone had no effect, a pattern consistent with the need of a calcium-inducing agent for the induction of NFAT.
  • the bpV compound bpV[pic] also led to a significant induction of luciferase activity in J-NFAT-LUC cells
  • Electrophoretic Mobility Shift Assays were next performed to demonstrate the bpV-mediated nuclear translocation of NFAT.
  • Fig. 4A Jurkat cells were either left untreated or were stimulated with PMA (20 ng/ml) /Iono (1 mM) (P/I) or bpV[pic] (10 ⁇ M) for 1 h.
  • Nuclear extract of bpV-stimulated Jurkat cells (10 ⁇ g) were incubated with 32 P-labeled murine IL-2 derived NFAT binding site for 20 min in the absence (lane 2) or presence of 10-, 100- , 200-fold excess of unlabeled NFAT oligonucleotide (lanes 4, 5, and 6, respectively) or 100-fold excess of unlabeled NF- ⁇ B oligonucleotide
  • NFAT oligonucleotides were incubated with nuclear extracts from unstimulated Jurkat cells as a negative control (lane 1) or from PMA/Iono- stimulated Jurkat cells as a positive control (lane 3) . In untreated Jurkat cells, no specific signal was seen
  • bpV compound bpV[pic] also led to the induction of a new signal which migrated at the same height as the one observed with P/I and which was competed by increasing concentrations of unlabelled NFAT oligonucleotide (compare lane 3 with lanes 4 to 6) but not by 100-fold excess of unlabelled NF- ⁇ B oligonucleotide (lane 7) .
  • Fig. 4B nuclear extracts from bpV-stimulated Jurkat cells were either untreated
  • NFAT activation by bpV compounds is dependent on p21ras and p561ck
  • NFAT activation requires the induction of the calcineurin pathway leading to dephosphorylation of the NFAT factor and its subsequent translocation and also necessitates the participation of p21ras. Indeed for
  • NFAT NFAT to become fully transcriptionally active
  • the transcription factor AP-1 is often required which becomes induced by a ras-dependent pathway.
  • bpV molecules activate the calcineurin-dependent pathway as bpV-mediated NFAT activation is blocked by FK506 (Figs. 3A and 3B) .
  • Jurkat cells were transiently transfected by DEAE-Dextran with 15 ⁇ g of the pNFAT-LUC plasmid plus 15 ⁇ g of either a control vector (D) or the RasN17 ( ⁇ ) encoding for the dominant negative mutant of p21ras.
  • the expression of the p21ras dominant negative protein in the Jurkat cells inhibited PHA- and PHA/PMA-mediated NFAT activation by approximately 60 to 70 percent, confirming p21ras implication in NFAT-based transcriptional regulation by these agents.
  • the p56lck-deficient T-cell line when transfected with the pNFAT-LUC plasmid was found to be unresponsive to all activators employed including the bpV compound bpV[pic] except for the Ionomycin (Iono) /PMA combination which acts more downstream than the other agents. Similar observations were made in parental lck-deficient JCaMl .6 cell line, which was again found to be unresponsive to NFAT activation by bpV molecules.
  • EXAMPLE 6 bpV molecules in combination with PMA can activate transcription from the complete IL-2 promoter in a FK506-sensitive manner
  • IL-2 expression is known to be highly regulated by NFAT binding sites and pervanadate has previously been shown to upregulate the expression of IL-2 mediated by agents such PMA, anti-CD3 and anti-CD28 antibodies. Since it is demonstrated that bpV molecules led to the activation of NFAT, it is next investigated as to whether the bpV compounds alone or with other activators would be able to also activate the IL-2 promoter.
  • Fig. 7A Jurkat cells were transiently transfected by DEAE-Dextran with 15 ⁇ g of the pIL-2-LUC plasmid. After a 24 h incubation, cells were either left untreated ( ⁇ ) or treated with PHA (3 ⁇ g/ml) /PMA
  • J-IL-2-LUC cells were either left untreated (D) or were pretreated ( ⁇ ) for 15 min with FK506 (10 nM) before being stimulated with PHA (3 ⁇ g/ml) /PMA (20 ng/ml) or bpV[pic] (10 ⁇ M)/PMA(20 ng/ml) .
  • Cells were lysed 8 h post -stimulation and luciferase was monitored. Results are the means ⁇ S.D. for triplicate samples and are representative of two independent experiments .
  • the kinetic of luciferase induction observed with the bpV[pic]/PMA combination is grossly the same as PHA/PMA, except that the maximal luciferase induction is higher and that the luciferase activity is reaching a plateau between 8 and 12 h returning to backgroung level after 24h.
  • J-IL-2-LUC stable IL-2-LUC transfectant Jurkat cells
  • J- IL-2 -LUC cells were treated with various activators (PHA, 3 ⁇ g/ml; bpV[pic], 10 ⁇ M; Iono, 1 ⁇ M; PMA, 20 ng/ml; anti-CD3 antibody, 3 ⁇ g/ml; anti-CD28 antibody, 1 ⁇ g/ml) .
  • PHA 3 ⁇ g/ml
  • bpV[pic] 10 ⁇ M
  • Iono 1 ⁇ M
  • PMA 20 ng/ml
  • anti-CD3 antibody 3 ⁇ g/ml
  • anti-CD28 antibody 1 ⁇ g/ml
  • Fig. 8B J- IL-2 -LUC cells were treated with the same activators as in Fig. 8A. Cell -free supernatants were collected after 24 h post-stimulation and IL-2 was quantified by an enzymatic assay. Results for both Figs. 8A and 8B are the means ⁇ S.D. for triplicate samples and are representative of two independent experiments. PHA, Iono or PMA alone did not result in any measurable activation of the IL-2 promoter, while bpV[pic] alone only resulted in a modest increase in promoter activity. However, when combining bpV[pic] with those activators, activation of the IL-2 promoter was observed. With the bpV[pic]/PHA combination, a fair increase in IL-2 promoter was obtained with 21-fold increase (Fig. 8A) .
  • the bpV [pic] /Iono combination was somewhat stronger, resulting in a 49-fold increase of luciferase activity.
  • a more stronger activation was achieved when combining bpV[pic] and PMA, as luciferase activity was increased from about 143 -fold compared to basal level.
  • the strongest increase was obtained when combining bpV[pic] with Iono and PMA, where the luciferase activity could be induced to a 220-fold, almost 7-times the value obtained with the Iono/PMA treatment.
  • the same potentiation effect of the bpV compound was observed when using more physiological stimuli. As previously determined in
  • anti-CD3 antibody and CD28 resulted in only a modest increase in IL-2 promoter activity, whereas stimulation through each of the receptor alone did not result in any detectable activation.
  • addition of bpVfpic] to either anti-CD3, anti-CD28 antibodies or both resulted in a strong stimulation of IL-2 promoter- driven luciferase activity (36-, 119- and 156-fold, respectively) .
  • IL-2 promoter stimulating agents were added for 24 h to the J- IL-2 -LUC and supernatants were tested for IL-2 production by enzymatic assays. Overall, the IL-2 production in the supernatant fairly reflected the IL-2 promoter activity as measured by luciferase activity
  • NFAT activation is known to be mediated by several different inducers which are also strong activators of T-cells.
  • inducers which are also strong activators of T-cells.
  • phosphotyrosyl phosphatase inhibitors have been shown to also induce T-cell activation and proliferation.
  • PTP inhibitors it is demonstrated for the first time that bpV compounds lead to the activation of the transcription factor NFAT and are demonstrated to be much stronger activators than the PHA/PMA combination.
  • Applicants were also interested in identifying the other transcription factor (s) which was positively affecting the regulatory sequences of HIV-1 in conjunction with NF- ⁇ B.
  • the first analysis was aimed at testing the sensitivity of the HIV-1 LTR expression induced by bpV to the immunosuppressors FK506 and CsA. It was found that those two molecules were greatly diminishing bpV- induced activation of HIV-1 LTR transcription. Similar inhibition was observed with the two immunosuppressors when the p ⁇ B-TATA-LUC construct, which contain only the tandem KB sites of the HIV-1 LTR (-105/-70), was instead used.
  • bpV-mediated activation of HIV-1 LTR results from a synergistic interaction between NFAT and NF- ⁇ B which accounts for the important induction observed for the HIV-1 LTR by bpV molecules (50-fold increase) .
  • p21ras in the activation of the pNFAT-LUC plasmid is thought to occur via the activation of the AP-1 factor which would act cooperatively with NFAT as demonstrated in several examples.
  • AP-1 activation by pervanadate, another PTP inhibitor has been described and, based on preliminary results from the inventors, it seems clear that AP-1 can be equally activated by bpV molecules.
  • the p561ck dependence of NFAT activation by bpV molecules is an attribute of a likely membrane-initiated signaling pathway.
  • bpV compounds are acting as complementing agents leading to the production of IL-2 induced by several agents such as ionomycin, PMA, PHA, anti-CD3 and anti-CD28 antibodies. This is most likely due to the fact that bpV induces NF- ⁇ B, NFAT and most probably AP-1, all of these considered as transcription factors known to be important in IL-2 regulation. More importantly, the powerful combination ionomycin/PMA was observed to be much more fully potentialised in the presence of bpV.
  • pervanadate PTP inhibitor was generating an increase in intracellular calcium levels in Jurkat T-cells (Imbert et al . , J. Inflammation 46:65-77, 1996; and O ' Shea et al., Proc . Natl . Acad. Sci . USA 89:10306-10310, 1992). It is hence demonstrated that the activation of IL-2 expression by bpV PTP inhibitors is likely to occur through concomitant activation of the NF- ⁇ B, NFAT and AP-1 transcription factors and has a dependency toward calcium flux.
  • bpV molecules have therapeutical values in inducing IL-2 expression in immunosuppressed individuals, including AIDS patients. It should be stressed out that bpVs by themselves do not induce IL-2 production. Hence, these compounds could help specifically prime the cells which have already been in contact with nominal antigen and partially alleviate the unresponsive cellular state which is a feature of the immune cells of these persons.

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Abstract

L'invention concerne un procédé de restauration des fonctions immunes et de la réponse globale immune, ce procédé consistant à administrer un composé bis-peroxovanadium (bpV) faisant partie d'une famille de puissants inhibiteurs de la phosphotyrosylphosphatase. On peut utiliser ce procédé pour traiter des individus présentant des pathologies d'ordre immunitaire ou dans le traitement de patients soufrant d'infections provoquées par des virus détruisant la réponse immune naturelle, tels que le virus du syndrome immunodéficitaire acquis. Ce composé bpV peut s'utiliser en combinaison avec divers immunomodulateurs et/ou des agents antiviraux. L'invention concerne également l'utilisation d'un tel composé bpV, dans la restauration des fonctions immunes et de la réponse immune globale, chez un patient nécessitant un tel traitement.
EP00971189A 1999-10-26 2000-10-23 Utilisation d'un inhibiteur de la phosphotyrosylphosphatase dans le traitement de patients immunodeprimes Withdrawn EP1225891A2 (fr)

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PCT/CA2000/001258 WO2001030325A2 (fr) 1999-10-26 2000-10-23 Utilisation d'un inhibiteur de la phosphotyrosylphosphatase dans le traitement de patients immunodeprimes

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WO2002009677A2 (fr) * 2000-08-02 2002-02-07 Virocell Inc. Procede servant a traiter des maladies infectieuses et/ou a augmenter l'efficacite antimicrobienne de medicaments
US7425537B2 (en) 2000-08-22 2008-09-16 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services SH2 domain binding inhibitors
EP1383792A2 (fr) 2000-08-22 2004-01-28 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES Inhibiteurs de liaison au domaine sh2

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WO1988000236A1 (fr) * 1986-07-03 1988-01-14 Tompkins Wayne A F Procedes et materiaux d'immunomodulation au vanadium
DE4336642C2 (de) * 1993-10-22 1995-08-10 Deutsches Rheuma Forschungszen Verwendung von Vanadiumverbindungen mit antiviraler Wirkung
US5565491A (en) * 1994-01-31 1996-10-15 Bristol-Myers Squibb Company Use of phosphotyrosine phospatase inhibitors for controlling cellular proliferation
CA2280249A1 (fr) * 1999-08-12 2001-02-12 Universite Laval Composes de vanadium utilises comme facteurs anti-angiogeniques et comme inhibiteurs de production d'endotheline

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