USE OF A PHOSPHOTYROSYL PHOSPHATASE INHIBITOR FOR THE TREATMENT OF IMMUNOSUPPRESSED PATIENTS

Abstract: The present invention provides a method for restoring the immune functions and the overall immune response by administration of a bis-peroxovanadium (bpV) compound, a potent class of phosphotyrosyl phosphatase inhibitors. The method can be utilized in treating individuals afflicted with immune disorders or for the treatment of patients suffering from infections caused by viruses that destroy the natural immune response, such as the human immunodeficiency virus (HIV). The bpV compound may be used in combination with various immunomodulators and/or antiviral agents. The present invention also relates to the use of such bpV compound for restoring the immune functions and the overall immune response in a patient in need of such a treatment.

Description: USE OF A PHOSPHOTYROSYL PHOSPHATASE INHIBITOR FOR THE TREATMENT OF IM UNOSUPPRESSED PATIENTS BACKGROUND OF THE INVENTION (a) Field of the Invention 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) . (b) Description of Prior Art 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-κB complex. Binding sites in the locus for several T-cell specific genes have been identified for this factor. The NF-κB (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-κB can act in concert with the transcription factor NFAT to achieve regulation of the IL-2 and γ-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. Like NF-κB, 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. This mechanistic description of the activation steps of NFAT has been also well established by the demonstration that the immunosuppressors cyclosporin A (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). 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 ' . Certain types of NFAT binding sites suggested to be cooperative with AP-1 are known to resemble NF-κB binding sites and include the CD28RE element located in the locus of the IL-2 and GM- CSF genes . Several members of the NFAT family of transcription factors are present in human T-cells such as NFATc (NFAT2) , NFAT4/x and NFATp (NFAT1) (Lyakh et al., Mol . Cell . Biol . 17:2475-2484, 1997). Numerous T- cell expressed genes are regulated by the NFAT transcription factors. Indeed, potentially functional NFAT binding sites have been identified m the promoter region of the IL-2, IL-3, IL-4 IL-5, GM-CSF, γ- mterferon and IL-13 genes, all of which were found to be sensitive m their transcπptional regulation to either CsA or FK506. The importance of NFAT members in T-cell function is thus very clearly demonstrated. 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) . Although by itself, the TCR/CD3 complex can induce a cascade of intracellular events, it also needs co-receptors such as CD4 and CD28 for optimal T-cell activation to proceed. 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. 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. Severe immunological abnormalities have been reported to precede the quantitative decline of CD4+ T- cell numbers seen in HIV-1-mfected persons. The exact mechanism (s) responsible for this unresponsiveness (anergy) is (are) still incompletely defined although in vi tro studies have demonstrated that signal transduction of the T-cell activation pathway was severely impaired in HIV-1-infected T-cells. It was first proposed, among several possibilities, that the reduced proliferative responses were resulting from the interaction between the CD4 surface molecule and the external viral envelope glycoprotein gpl20 because inhibition of T-cell receptor-dependent proliferative response has been observed following gpl20 treatment. The anergic state caused by gpl20 treatment was attributed to inhibition of mRNA IL-2 expression, as well as to IL-2 secretion, in CD4+ T lymphocytes since addition of exogenous IL-2 was able to restore proliferative responses (Oyaizu et al . , Proc . Natl . Acad . Sci . USA 87:2379-2383, 1990). The molecular mechanism responsible for the functional impairment of CD4+ T lymphocytes before CD4+ T-cell depletion seen in HIV-1-infected persons is still incompletely understood. However, for specific antiviral therapies to be effective in the improvement of seropositive individuals, immunomodulators should be considered as important additions. It would be highly desirable to be provided with a method for activating T-cells, such activation restoring a functional immunity in immunosuppressed or immunodeficient patients. SUMMARY OF THE INVENTION 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. In accordance with the present invention there is provided 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 . In accordance with the present invention there is also provided 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. In accordance with the present invention, 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. Alternatively, 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 . Also in accordance with the present invention, there is provided 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. BRIEF DESCRIPTION OF THE DRAWINGS 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-κB- 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; and Figs. 8A and 8B illustrate bar charts illustrating the correlation between IL-2 promoter activity and IL-2 secretion. DETAILED DESCRIPTION OF THE INVENTION In accordance with a preferred embodiment, 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. pyridine-2- carboxylic acid) anion [pic] ; 5-hydroxypyridine-2- carboxylic acid anion [HO-pic] ; 1 , 10-phenanthroline [phen] ; 4 , 7-dimethyl-l, 10-phenanthroline [Me2phen] ; 3 , 4, 7, 8-tetramethyl-l, 10-phenanthroline [Me4phen] ; oxalic acid dianion [ox] . Formulas and abbreviations of a number of structurally defined bpV compounds are listed herein to illustrate the invention rather than to limit it: K[VO(0 2 ) 2 bipy] -5H 2 0, bpV [bipy]; K 2 [VO (0 2 ) 2 pic] H 2 0, bpV [pic] ; K 2 [VO (0 2 ) 2 (EOpic) ] H 2 0, bpV [Hopic] ; K[VO(0 2 ) 2 phen] -3H 2 0, bpV [phen]; K [VO (0 2 ) 2 (4 , 7-Ne 2 phen) ] ; bpV [Me 2 phen] ; K[VO (0 2 ) 2 (Me 4 phen) ] , bpV [Me 4 phen] ; and K 3 [VO(0 2 ) 2 OX] -2H 2 0, bpV[ox] . 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. In relation to Examples 1 to 7 given hereinafter, the materials used and the analyses and assays carried out were as follows: Cell lines The lymphoid T-cell lines used include Jurkat (clone E6-1) , Sup-Tl, JCaMl .6 and JCaMl. Tag (Straus et al., J. Biol . Chem . 271:9976-9981, 1996). 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 μg/ml) . Plasmids and antibodies pLTR-LUC (HIV-1 LTR from strain HXB2) and pmκBLTR-LUC plasmids were used (Henderson et al . , J. Virol . 69:5337-5344, 1995). These molecular constructs contain the luciferase reporter gene under the control of wild-type (GGGACTTTCC) or NF-κB-mutated (CTCACTTTCC) HIV-1 LTR. The pκB~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, and pNFAT-LUC, containing the IL-2 minimal promoter with 3 tandem copies of the NFAT-1 binding site were also used (Timmerman et al . , Nature 383:837-840, 1996). The pLTR/N17 plasmid codes for a dominant negative of p21ras under the control of murine leukemia virus LTR (rasN17) (Baldari et al . , J " . Biol . Chem . 268:2693-2698, 1993) . 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. Characterization of the bpV molecules were carried out by infrared X H-NMR and Vanadium-51 ( 51 V) NMR spectroscopy. Stock solutions of bpV molecules (1 mM in phosphate buffered saline pH 7.4) were kept at -85°C until use. Transfeetions Transient transfections were done using the DEAE-Dextran method, as follow. Cells (5 x 10 6 ) were first washed once in TS buffer (25 mM Tris-HCl [pH 7.4], 5 mM KC1, 0.6 mM Na 2 HP0 4 , 0.5 mM MgCl 2 , and 0.7 mM CaCl 2 ) and resuspended in 0.5 ml of TS containing 15 to 30 μg of the indicated plasmid (s) and 500 μg/ml of DEAE-dextran (final concentration) . The cells/TS/plasmid/DEAE-dextran mix was incubated for 25 min at room temperature. Thereafter, cells were diluted at a concentration of 1 x 10 6 /ml using complete culture medium supplemented with 100 μM of chloroquine and transferred into 6 -well plates. After 45 minutes of incubation at 37°C, cells were centrifuged, resuspended in complete culture medium and incubated at 37 °C for 24 h. To minimize variations in plasmid transfection efficiencies, transfected cells were pooled 24 h after transfection and were next separated into various treatment groups. Stably transfected Jurkat cells were obtained by electroporation. Briefly, 10 x 10 6 cells in mid-log phase were washed once and resuspended in 400 μl of complete RPMI medium plus 20 μg of either pNFAT- LUC or pIL-2-LUC plasmid. This mixture was transferred to a 0.4 cm-gap electroporation cuvette. Cells were transfected in Bio-Rad apparatus using standard voltage and capacitance conditions (250 V and 960 μF) . After a 10 min incubation period, transfected cells were resuspended at a density of 1 x 10 6 /ml in complete RPMI medium for 24 h. Cells were then diluted at 5 x 10 4 cells/ml and 1 mg/ml Geneticin was added. After two weeks of selection, G418-resistant T-cells were pooled and identified as either J-NFAT-LUC or J- IL-2 -LUC. Stimulations and luciferase assays Stably or transiently transfected cells were seeded at a density of 10 5 cells per well (100 μl) in 96-well flat-bottom plates. Cells were either left unstimulated or were stimulated with the different bpV molecules at 10 μM (bpV[HOpic], bpV [bipy] , and bpVtpic]), phytohemagglutinin (PHA-P at 3 μg/ml), phorbol 12-myristate 13-acetate (PMA at 20 ng/ml), Ionomycin (Iono at 1 μM) , anti-CD3 antibody (clone OKT3 at 3 μg/ml), anti-CD28 antibody (clone 9.3 at 1 μg/ml) in a final volume of 200 μl . Next, cells were incubated at 37°C for 8 h unless otherwise specified. For some experiments, prior to the addition of activators, cells were either left untreated or pretreated with FK506 (10 ng/ml) or cyclosporin A (CsA at 100 ng/ml) for 15 min at 37°C or sodium salicylate (NaSal at 2.5 mM) for 1 h at 37°C. Luciferase activity was determined as follow. Briefly, following the incubation period, 100 μl of cell -free supernatant were withdrawn from each well and 25 μl of cell culture lysis buffer (25 mM Tris phosphate [pH 7.8], 2 mM DTT, 1% Triton X-100™, and 10% glycerol) were added for incubation at room temperature for 30 min. The extracts (20 μl) were analyzed in 96- well plates using a Dynex MLX luminometer. Each well was injected with 100 μl of 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 μM coenzyme A, 470 μM luciferin, 530 μM 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 Following stimulation for 24 h, cell -free supernatants from either Jurkat or J- IL-2 -LUC cells were removed and quantification of secreted IL-2 was carried out using a commercial cytokine matched antibody pair (Endogen) . Briefly, polyclonal anti-IL-2 antibody was first diluted to a concentration of 2 μg/ml in phosphate-buffered saline (PBS) and 100 μl/well were transferred in the wells of a 96 -well plate and left overnight at room temperature. After three washes with phosphate-buffered saline (PBS) containing 0.05% (v/v) Tween-20™ (PBST) , 200 μl of PBST plus 1% BSA (w/v) was added per well and incubated for 30 min at 37°C. Following three further washes with PBST-BSA, 50 μl of a biotinylated anti-IL-2 antibody at a final concentration of 0.25 μg/ml in PBST-BSA along with 50 μl of tested cell supernatant were dispensed in the wells for another incubation period of 1 h at 37°C. Three washes with PBST-BSA were then performed and 100 μl of a 1:4000 dilution of the streptavidin-tagged horseradish peroxidase enzyme (Streptavidin-HRP40 , Research Diagnostics Inc., Flanders, NJ) was added to the wells and left at room temperature for 30 min. After another three washes with PBST-BSA, 100 μl of TMB-S (Research Diagnostic Inc., Flanders, NJ) were finally dispensed in each well. After 15 min, the reaction was stopped by adding 50 μl of 1 M H 3 P0 4 and absorbance was read using a microtiter plate reader (450 nm filter, reference wavelength 690 nm) . Appropriate dilutions of recombinant human IL-2 were used to produce standard curves (3.9 to 250 U/ml) . Preparation of nuclear extracts Jurkat cells were either left untreated or were incubated for 1 h at 37°C with the bpV compounds (10 μM) . 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 μl 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) . After 10 min on ice, the lysate was vortexed for 10 s, and samples were centrifuged for 10 s at 12,000 x g. The supernatant fraction was discarded and the cell pellet was resuspended in 100 μl of cold buffer C (20 mM HEPES-KOH [pH 7.9], 25% glycerol, 420 mM NaCl , 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride) and incubated on ice for 20 min. Cellular debris were removed by centrifugation at 12,000 x g for 2 min at 4°C and the supernatant fraction was stored at -70°C until use. EMSA Electrophoretic mobility shift assay (EMSA) was performed with 10 μg of nuclear extracts. 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 μl 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 μg poly[dI- dC] , 10 μg nuclease-free bovine serum albumin fraction V) containing 0.8 ng of 32 P-5 ' -end-labeled double stranded DNA (dsDNA) oligonucleotide. Double stranded DNA (100 ng) was labeled with [γ- 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 μl 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-κB oligonucleotide (5 ■ -ATGTGAGGGG ACTTTCCCAG GC-3 ' ) simultaneously with the labeled probe. Supershift assays were performed by pre-incubation of nuclear extracts with 1 μl 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-κB oligonucleotide were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) . Dose Ranges 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. In the case of bpV compounds, 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 μM, usually about 10 μM 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. The dosage amount of agent necessary to obtain the desired concentrations in blood can be determined by pharmacokinetic studies, as described in Marleau et al . , J. Immunol . 150: 206, 1993, and Marleau et al, Br. J. Pharmacol . 112: 654, 1994. Pharmaceutical 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. For example, oral, parenteral and topical may be employed. Dosage forms include tablets, capsules, powders, solutions, dispersions, suspensions, creams, ointments and aerosols. The pharmaceutical 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. It should be recognized that 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] ) . In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions 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. The amounts required for restoring functional immunity can be determined by in vivo pharmacological studies. 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. For reconstitution of a solvent -free product in accordance with the present invention, one may employ a sterile diluent, which may contain materials generally recognized for approximating physiological conditions. In this manner, 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. 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 . Where 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 . 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. Additional pharmaceutical methods may be employed to control the duration of action. For exam- pie, 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 . Instead of incorporating the subject bpV compound into polymeric particles, it is also possible to entrap them in 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 . Such techniques are disclosed in Remington's Pharmaceutical Sciences (16th ed. Osol, A., ed. , Mack, Easton, PA [1980]). The 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. In practical use, 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. In preparing the compositions for oral dosage form, 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. Pharmaceutical 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. In general, the 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. For example, 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 . It will be understood that 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. Similarly, 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 present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope. EXAMPLE 1 bpV induction of HIV-1 LTR is FK-506- and CsA-sensitive Since NFAT has recently been shown to be of importance for the regulation of the HIV-1 LTR, it was first tested whether FK506 and CsA, two known inhibitors of calcineurin and thus of NFAT activation, could block the bpV-mediated activation of the HIV-1 LTR. To achieve this goal, the cell line 1G5, a Jurkat derivative containing stably integrated HIV-1 LTR-LUC constructs, was used. In Fig. 1, 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. Following treatment, cells were either stimulated or not for 8 h with the following agents: PHA (3 μg/ml), bpV[bipy] , bpV[HO-pic] or bpV [pic] (lOμM) . Cells were then lysed and evaluated for luciferase activity. Values are the mean of three different measured samples ± S.D. This is representative of two independent experiments. The addition of three different bpV molecules (i.e. bpVfbipy], bpV[HO-pic] and bpV[pic]) resulted in a strong induction of HIV-1 LTR-driven luciferase activity in the cell extract (49.8-, 49.4-, and 48.5-fold increase), which was stronger than with the mitogenic agent PHA (4.5-fold) (Fig. 1). In order to further corroborate our initial observation that a factor other than NF-κB was being activated by bpV compounds, we initially incubated 1G5 cells with sodium salicylate (NaSal), a known inhibitor of NF-KB translocation. As depicted in Fig. 1, NaSal led to a significant but incomplete loss of the induction of HIV-1 LTR-driven luciferase activity by the various bpV molecules whereas no effect was discernible with PHA stimulation. FK506 and CsA were both found to inhibit PHA-mediated HIV-1 activation as expected since it is mainly acting through NFAT. Surprisingly, both NFAT inhibitors also blocked bpV- mediated induction of luciferase activity. Furthermore, when either FK506 or CsA were added along with NaSal, a greater inhibitory action on measured HIV-1 LTR- dependent luciferase activity was apparent in cells treated with the three tested bpV molecules. It should be noted that no detectable toxicity was measured upon cell incubation with any combination of the inhibitors used as determined by the MTS assay. Inability to completely inhibit bpV-mediated HIV-1 LTR activation by the FK506/NaSal or CsA/NaSal combination is probably due to residual NF-κB activity, since FK506 completely inhibited NFAT activation. Higher concentrations of NaSal could not be used to achieve a more complete inhibition of NF-κB due to its intrinsic toxicity at these doses. The bpV molecule bpV[pic] was used for our subsequent experiments as it is representative of the other bpV compounds . EXAMPLE 2 Induction of wild-type and NF-κB-mutated HIV-1 LTR by bpV compounds is sensitive to FK506 and CsA Since several transcription factors binding in the -105/ -70 enhancer region of the HIV-1 LTR have been found to be of importance in virus gene transcription (including the classical NF-κB factor) , it was next examined if the bpV- induced signaling component which is sensitive to FK506 and CsA could equally be detected in the context of the isolated HIV-1 enhancer region or a NF-KB-mutated HIV-1 LTR. Jurkat cells were hence transiently transfected with two different constructs, one containing the -105/-70 region of the HIV-1 LTR (pKB-TATA-LUC) and the other harboring the HIV-1 LTR mutated at the NF-κB binding sites (pmκBLTR-LUC) , both of which were placed upstream of the luciferase reporter gene. In Figs. 2A and 2B, Jurkat cells were transiently transfected by DEAE-Dextran with 15 μg of either pκB-TATA-LUC (Fig. 2A) or pmκBLTR-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. Following treatment, cells were stimulated for 8 h with the following agents: PHA (3μg/ml) , PHA (3μg/ml)/PMA (20 ng/ml) or bpV[pic] (lOμM) . Cells were then lysed and evaluated for luciferase activity. Values are the mean of three different measured samples ± S.D. This is representative of two independent experiments . When pκB~ TATA-LUC was transfected in the Jurkat cells, strong induction of luciferase activity was seen upon the addition of the bpV compound bpVfpic] which was severely affected upon pretreatment of the transfected cells with FK506 (Fig. 2A) . As controls, inhibition of PHA and PHA/PMA induction of luciferase activity was also evaluated in cells pretreated with FK506, PHA/PMA being less inhibited most likely because of the concomitant strong activation of NF-κB by PMA (Fig. 2A) . Similar inhibitions for all three tested activators were observed when CsA was instead used. As depicted in Fig. 2B, the transfection of the pmκBLTR-LUC plasmid in Jurkat cells led to an induction of luciferase activity upon the addition of either PHA, PHA/PMA or bpV[pic]. In this case, a lesser difference in terms of HIV-1 LTR activation was observed between PHA and PHA/PMA because of the mutation of the two NF-κB binding sites on the HIV-1 LTR. When cells were pretreated with subcytotoxic concentrations of FK506, a strong down-modulation of HIV-1 LTR-mediated luciferase activity was again measured for all types of activators including bpV[pic]. Similar decrease of luciferase activity was apparent following treatment with CsA. These results further suggested that the NF-κB-independent signaling pathway which is activated by the bpV molecules was FK506- and CsA-sensitive and that it was acting through the -105/-70 HIV-1 enhancer region. EXAMPLE 3 NFAT-dependent luciferase expression is markedly stimulated by the bpV compounds Based on our previous results and since the enhancer region (-105/-70) of the HIV-1 LTR is a binding site for both NF-κB and NFAT transcription factors, the obvious candidate for the NF-κB-independent transcription factor induced by the bpV is NFAT. In order to test whether bpV compounds act directly on the NFAT factor, 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] . In Fig. 3A, Jurkat cells were transiently transfected by DEAE-Dextran with 15 μg of pNFAT-LUC. Following a 24 h incubation period, cells were left untreated (■) or were stimulated with either PHA (3μg/ml)/PMA (20 ng/ml) (D) or bpV[pic] (lOμM) (•) and lysed after 4, 6, 8, 12, and 24 h of stimulation. In Fig. 3B, Jurkat cells stably transfected with pNFAT-LUC were either left untreated (D) or were pretreated with FK506 (10 ng/ml) (■) . After 1 h, cells were stimulated for 8 h with PHA (3 μg/ml) , PMA (20 ng/ml) , PHA (3 μg/ml) /PMA(20 ng/ml), bpV[pic] (10 μM) or bpV[pic] (10 μM)/PMA(20 ng/ml) . Values are the mean of three different measured samples ± S.D. This is repre- sentative of two independent experiments. As expected, a strong induction of NFAT-dependent luciferase activity was mediated by the combined action of PHA and PMA (Fig. 3A) . Interestingly, a stronger induction of this same construct was observed upon stimulation of transfected Jurkat cells with the compound bpV[pic]. Time kinetic analysis showed a transient increase in luciferase activity which peaked around 6 to 8 hours for PHA/PMA (53.5-fold and 61.1-fold increase), while maximal activation was reached after 8 to 12 hours for bpV[pic] (92.2-fold and 73.2-fold increase). For the subsequent experiments, the 8 hour time point was hence chosen for time of treatment of pNFAT-LUC-transiently transfected cells. To further demonstrate the ability of bpV compounds to activate NFAT, stably pNFAT-LUC- transfected Jurkat cells (J-NFAT-LUC) were derived and pooled resistant clones were then treated with various NFAT inducers with or without pre-treatment with FK506. As illustrated in Fig. 3B, 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. Similarly, however, the bpV compound bpV[pic] also led to a significant induction of luciferase activity in J-NFAT-LUC cells (113.3-fold increase) which was further enhanced in the presence of PMA (208.1-fold increase). After pre- treatment with FK506, NFAT-driven luciferase induction by all tested activators including bpVfpic] was totally abrogated without any measurable effect on cell viability. These results hence represented the first indication that PTP inhibitors such as bpV compounds were inducing activation of the NFAT transcription factor. EXAMPLE 4 Nuclear translocation of NFAT by the bpV compounds Electrophoretic Mobility Shift Assays (EMSA) were next performed to demonstrate the bpV-mediated nuclear translocation of NFAT. In 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 μM) for 1 h. Nuclear extract of bpV-stimulated Jurkat cells (10 μg) 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-κB oligonucleotide (lane 7) . Labeled 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 (Fig. 4A, lane 1), while PMA/Ionomycin (P/I) , a strong NFAT-inducing combination, gave a specific signal (lane 2, arrow). Similarly, the 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-κB oligonucleotide (lane 7) . In Fig. 4B, nuclear extracts from bpV-stimulated Jurkat cells were either untreated (lane 2) or preincubated with an anti-NFATc polyclonal antibody (lane 3) or pre- immune serum (lane 4) for 15 min at room temperature. Extracts from unstimulated Jurkat cells were used as a negative control (lane 1) . Free probe and the specific signal observed are indicated at the left of Figs. 4A and 4B. To identify the composition of this shifted signal, antibodies were incubated in the presence of bpV-treated Jurkat nuclear extracts. As presented in Fig. 4B, the signal induced by bpVfpic] was completely abrogated by the addition of anti-NFATc antibodies to the nuclear extract prior to incubation with the labeled NFAT oligonucleotide (compare lanes 2 and 3) while no such decrease in band intensity was observed upon pre-treatment of these same extracts with pre- immune serum (lane 4) . The fast migrating band in these EMSA experiments was not clearly identified but most likely represents degradation of the NFAT signal. These experiments hence showed that bpV molecules indeed led to the activation (i.e. the translocation to the nucleus) of the NFAT transcription factor and that the major if not only isoform activated in Jurkat T-cells was the NFATc isoform. EXAMPLE 5 NFAT activation by bpV compounds is dependent on p21ras and p561ck Cooperativity between two different signaling pathways is known to be essential to achieve optimal transcriptional activation through the NFAT motifs of the human IL-2 gene. Previous reports have shown that 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 to become fully transcriptionally active, the transcription factor AP-1 is often required which becomes induced by a ras-dependent pathway. It is shown that 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 μg of the pNFAT-LUC plasmid plus 15 μg of either a control vector (D) or the RasN17 (■) encoding for the dominant negative mutant of p21ras. After 24 h incubation period, cells were treated or not with PHA (3 μg/ml) , PHA (3 μg/ml)/PMA (20 ng/ml), bpV[pic] (10 μM) or bpV[pic] (10 μM) /PMA (20 ng/ml). Cells were next lysed 8 h post- stimulation and luciferase activity was monitored. Results are the means ± S.D. for triplicate samples and are representative of two independent experiments. As shown in Fig. 5, 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. Involvement of p21ras in bpV [pic] -mediated activation of NFAT was also clearly demonstrated as expression of the rasN17 dominant negative mutant reduced NFAT activation by bpV with or without PMA down to 20 to 30 percent of the level observed in the absence of the dominant negative mutant. The implication of the tyrosyl kinase p561ck in the bpV- induced NFAT activation was also examined since this src family member has been previously demonstrated to act on the activation of NFAT. The p561ck-deficient Jurkat -derivative JCaM-TAg was initially tested to see whether bpV compounds could induce NFAT activation in this cell setting. In Figs. 6A and 6B, JCaM-TAg cells (p56lck- negative) were transiently transfected by DEAE-Dextran with 15 μg of the pNFAT-LUC plasmid (Fig. 6A) plus 30 μg of either the control vector pEFneo (D) or the p56lck-encoding vector pEFneo-Lck (■) (Fig. 6B) . After a 24 h incubation, cells were treated or not with PHA (3 μg/ml) , PMA (20 ng/ml) , Iono (lμM) , PHA (3 μg/ml)/PMA (20 ng/ml), bpV[pic] (lOμM) or Iono (lμM)/PMA (20 ng/ml). Cells were lysed after 8 h post- stimulation and luciferase activity monitored. Luciferase activity is presented on a log scale in Fig. 6A. Results are the means + S.D. for triplicate samples and are representative of two independent experiments. As presented in Fig. 6A, 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. To demonstrate that this lack of bpV-mediated NFAT induction was due to the absence of p561ck, the lck-negative JCaM-TAg cell line were cotransfected with the pNFAT-LUC plasmid plus either the p561ck-encoding vector pEFneo-lck or, as a negative control, the empty vector pEFneo. As depicted in Fig. 6B, no NFAT activation was observed with PHA/PMA when cotransfecting pNFAT-Luc with pEFneo . However, response to these stimulating agents was restored upon cotransfection of both pNFAT-Luc and the p561ck- encoding vector, consistent with the need for the presence of p561ck for PHA to mediate a signal through the T-cell receptor. The same p561ck dependency was observed with the bpV compound bpV[pic], confirming that p561ck is a mediator of the signal generated by the bpV PTP inhibitors. No strong differences in induction were apparent in cells similarly stimulated with Iono/PMA whether the p561ck expression vector was co-transfected or not, due to the downstream action of this combination. 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. In Fig. 7A, Jurkat cells were transiently transfected by DEAE-Dextran with 15 μg of the pIL-2-LUC plasmid. After a 24 h incubation, cells were either left untreated (■) or treated with PHA (3 μg/ml) /PMA (20 ng/ml) (D) or bpV[pic] (lOμM) /PMA (20 ng/ml) (•) . At fixed time after stimulation (0, 4, 6, 8, 12, and 24 h) , cells were lysed and luciferase activity was monitored. In Fig. 7B, 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 μg/ml) /PMA (20 ng/ml) or bpV[pic] (10 μM)/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 . As demonstrated in Fig. 7A, PHA/PMA treatment of Jurkat cells transiently transfected with the pIL-2- LUC reporter plasmid revealed a maximal induction of luciferase activity at 8 h followed by a gradual decrease and a return to basal level at 24 h. More importantly, the bpV compound bpV[pic] was found to be a potent activator of IL-2 -dependent luciferase activity in the presence of PMA. Neither PMA nor bpV[pic] alone results in an increase in IL-2 promoter activation in this transient transfection system. 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. With the use of stable IL-2-LUC transfectant Jurkat cells (J-IL-2-LUC) , the sensitivity of bpV [pic] /PMA-mediated activation of the IL-2 promoter to the immunosuppressive drug FK506 was next tested. Pretreatment of J-IL-2-LUC cells with 10 ng/ml of FK506 resulted in a 80% inhibition of the IL-2 promoter-driven luciferase activity induced by the bpV[pic]/PMA combination (Fig. 7B) . Similarly, PHA/PMA stimulation was reduced by more than 75% with FK506. EXAMPLE 7 Correlation between IL-2 promoter-driven luciferase expression and IL-2 secretion In order to look for potential synergy between bpV compounds and other stimuli, various combinations of activators were tested on J-IL-2-Luc cells and looked at luciferase activity and IL-2 secretion. In Fig. 8A, J- IL-2 -LUC cells were treated with various activators (PHA, 3 μg/ml; bpV[pic], 10 μM; Iono, 1 μM; PMA, 20 ng/ml; anti-CD3 antibody, 3 μg/ml; anti-CD28 antibody, 1 μg/ml) . Cells were lysed after 8 h post- stimulation and luciferase activity was monitored. In 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. However 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. Importantly, the same potentiation effect of the bpV compound was observed when using more physiological stimuli. As previously determined in Jurkat cells, simultaneous stimulation through TCR (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. However, 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) . To confirm these results at the protein level, these 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 (Fig. 8B) . Indeed, a strong correlation between IL-2 promoter activity and IL-2 secretion was noticed when IL-2 production was plotted against luciferase activity (r = 0.968) . The only major difference between these two types of measurements of IL-2 expression is the treatment PMA/Ionomycin where IL-2 secretion seems to be more pronounced. Therefore the induction of IL-2 expression and secretion by bpV molecules when used in combination with agents mimicking signal transduction mediated upon antigen recognition (e.g. anti-CD3 and anti-CD28 antibodies) is demonstrated. NFAT activation is known to be mediated by several different inducers which are also strong activators of T-cells. In the present application, phosphotyrosyl phosphatase inhibitors have been shown to also induce T-cell activation and proliferation. Using a new set of 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-κB. 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 pκB-TATA-LUC construct, which contain only the tandem KB sites of the HIV-1 LTR (-105/-70), was instead used. Since these calcineurin inhibitors have been shown to also affect NF-κB activation in certain cascade, the inhibitory potential of these same immunosuppressors on NF-κB-mutated LTR-LUC construct have been tested and it has been demonstrated that inhibition by FK506 and CsA of the bpV- induced HIV-1 LTR-based transcription could also be observed to the same extent. Two lines of evidence suggest that NFAT was most likely the other transcription factor activated by bpV molecules. First, NFAT is greatly affected by these two immunosuppressors and, second, it was recently demonstrated that NFAT is playing an important role in the regulation of HIV-1 replication. In the present application, the use of Jurkat T-cells either transiently or stably transfected with pNFAT- LUC-vector clearly showed a FK506/CsA-sensitive induction of reporter gene activity by bpV molecules. It was also determined by shift assays that bpV compounds led to the accumulation of the NFATc isoform in the nucleus. Although it seemed that this isoform is most likely the only isoform being activated in Jurkat T-cells, other NFAT members could also be similarly activated by bpV, especially in different T-cell types and also in peripheral blood leukocytes. Based on these findings, bpV-mediated activation of HIV-1 LTR results from a synergistic interaction between NFAT and NF-κB which accounts for the important induction observed for the HIV-1 LTR by bpV molecules (50-fold increase) . The role of 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. However, the importance of the TCR in the bpV- mediated induction of NFAT is not clear as the TCR- negative T-cell line Sup-Tl demonstrated reproducible, albeit lower, bpV-induced gene expression of NFAT- regulated plasmids. The results in the present application have also demonstrated that bpV led to the activation of the IL-2 promoter when added along with PMA. Similar observations have already been noticed when using the PTP inhibitor pervanadate along with PMA, anti-CD3 or anti-CD28 antibodies (Imbert et al . , J. Inflammation 46:65-77, 1996; 0 ' Shea et al . , Proc . Natl . Acad. Sci . USA 89:10306-10310, 1992; and Secrist et al . , J. Biol . Chem. 268:5886-5893, 1993). The results suggest that the multifactorial requirement for IL-2 expression is in part greatly helped by the bpV PTP inhibitor. In fact, in a correlative fashion, when measuring both luciferase-based IL-2 promoter activity and secreted IL-2, it is found that 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-κB, 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. This bpV-dependent potentiating effect was also illustrated in a more physiological setting for T-cell stimulation involving anti-CD3 and anti-CD28 antibodies where superinduetion of IL-2 -regulated luciferase expression and IL-2 secretion was noticed. It is believed that bpV molecules are acting primarily via calcium-dependent signaling pathway (s) since they are strongly potentiating agents known to initiate calcium-independent cascade such the one initiated by PMA or anti-CD28 antibodies. In fact, it is observed that activation of NFAT by bpV was severely hampered in calcium-deficient Jurkat-derived cell lines, which thus gives an importance of the calcium flux in the mechanistic action of bpV leading to NFAT activation. Other groups have previously demonstrated that 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-κB, NFAT and AP-1 transcription factors and has a dependency toward calcium flux. The results presented in the present application show that bpV PTP inhibitors activate NFAT. It is hereby demonstrated for the first time that blocking of PTP activity is resulting in an activation of NFAT. These results suggest that PTP activity acts by attenuating a constitutive kinase activity that otherwise would lead to a constant NFAT activation. The strong induction of IL-2 expression by bpV in conjunction with PMA or the more physiological stimuli such as anti-CD3/anti-CD28 antibodies suggest that bpV compounds, which are more stable PTP inhibitors than pervanadate, might be acting through an alternative complementary pathway to TCR-mediated T-cell signaling. In conclusion, 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. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia- tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims .

Claims: WHAT IS CLAIMED IS: 1. Use of a bis-peroxovanadium (bpV) compound for restoring immune functions and an overall immune response of a patient suffering from an immune disorder. 2. The use of claim 1, wherein the bpV compound is made of an oxo ligand, two peroxo anions, and an ancillary ligand located in an inner coordination sphere of vanadate . 3. The use of claim 1, wherein the immune disorder is caused by an infection with a virus. 4. The use of claim 3 , wherein said virus is a human immunodeficiency virus. 5. Use of a bpV compound for the manufacture of a medicament for restoring immune functions and an overall immune response of a patient suffering from an immune disorder. 6. The use of claim 5, wherein the bpV compound is made of an oxo ligand, two peroxo anions, and an ancillary ligand located in an inner coordination sphere of vanadate . 7. The use of claim 5, wherein the immune disorder is caused by an infection with a virus . 8. The use of claim 7, wherein said virus is a human immunodeficiency virus. 9. The use of claim 5, wherein said medicament is suitably formulated for intravenous, subcutaneous, intradermal or topical application. 10. The use of claim 5, wherein said medicament is suitably formulated in a patch or an implant . 11. The use of claim 5, wherein said medicament is suitably formulated for inhalation. 12. The use of claim 11, wherein said medicament is formulated in an aerosol spray. 13. The use of claim 11, wherein said medicament is in a powder form. 14. The use of claim 5, wherein said medicament is formulated in a liposomal composition suitable for administration. 15. The use of claim 5, wherein the medicament is in a tablet form. 16. The use of claim 1, wherein the bpV compound is used in combination with an antiviral agent. 17. The use of claim 1, wherein the bpV compound is used in combination with another immunomodulator . 18. A bis-peroxovanadium (bpV) compound for restoring immune functions and an overall immune response of a patient suffering from an immune disorder, whereby said bis-peroxovanadium compound is a phosphotyrosyl phosphatase inhibitor. 19. The compound of claim 18, wherein the bpV compound is made of an oxo ligand, two peroxo anions, and an ancillary ligand located in an inner coordination sphere of vanadate. 20. The compound of claim 18, wherein the immune disorder is caused by an infection with a virus. 21. The compound of claim 20, wherein said virus is a human immunodeficiency virus. 22. A pharmaceutical composition for restoring immune functions and an overall immune response of a patient suffering from an immune disorder, said composition comprising a bis-peroxovanadium (bpV) compound and a pharmaceutically acceptable carrier, whereby said bis-peroxovanadium compound is a phosphotyrosyl phosphatase inhibitor. 23. 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. 24. The method of claim 23, wherein the bpV compound is made of an oxo ligand, two peroxo anions, and an ancillary ligand located in an inner coordination sphere of vanadate. 25. The method of claim 23, wherein the immune disorder is caused by an infection with a virus. 26. The method of claim 25, wherein said virus is a human immunodeficiency virus. 27. The method of claim 23, wherein the bpV compound is administered intravenously, subcutaneously, intradermally, or by topical application. 28. The method of claim 23, wherein the bpV compound is administered using a patch or an implant . 29. The method of claim 23, wherein the bpV compound is administered by inhalation. 30. The method of claim 29, wherein the bpV compound is administered by aerosol spray. 31. The method of claim 29, wherein the bpV compound is inhalated in a powder form. 32. The method of claim 23, wherein the bpV compound is formulated in a liposomal composition suitable for administration. 33. The method of claim 23, wherein the bpV compound is administered as a tablet . 34. The method of claim 23, wherein the bpV compound is administered in combination with an antiviral agent. 35. The method of claim 23, wherein the bpV compound is administered in combination with another immunomodulator .

Inventors: BARBEAU BENOIT, FORTIN JEAN-FRANCOIS, TREMBLAY MICHEL J, BARBEAU, BENOIT, FORTIN, JEAN-FRANCOIS, TREMBLAY, MICHEL, J.
Applicants: BARBEAU BENOIT, FORTIN JEAN FRANCOIS, TREMBLAY MICHEL J, VIROCELL INC, BARBEAU, BENOIT, FORTIN, JEAN-FRANCOIS, TREMBLAY, MICHEL, J., VIROCELL INC.
Application year: 2000
Main classification: A61K31
IPC codes: A61K33