BACKGROUND OF THE INVENTION

This invention is directed to the use of phosphotyrosine phosphatase inhibitors and phosphotyrosine kinase activators for controlling cellular proliferation, particularly proliferation of 5 lymphocytes.

Tyrosine phosphorylation is known to play an essential role in the control of lymphocyte function. This control is exerted by a network of tyrosine kinases 10 and phosphotyrosine phosphatases.

Apoptosis is a pattern of programmed cell death that involves a breakup of the cellular DNA and can be recognized by electrophoresis. Apoptosis is an

15 important lymphocyte response believed to play a prominent role in T and B lymphocyte maturation in the thymus (T cells) and in germinal centers (B cells) , an example being the process of negative selection of self- reactive cells (J.J. Cohen et al . , "Apoptosis and

20 Programmed Cell Death in Immunity, " Annu. Rev. Immunol . 10:267-293 (1992) ; D.R. Green et al. , "Activation- Induced Apoptosis in Lymphoid Systems," Sem. Immunol. 4:379-388 (1992) ) .

t* ^» 25 Three different means of inducing apoptosis in lymphocytes all appear to require tyrosine phosphorylation. First, stimulation of the antigen receptor can lead to lymphocyte apoptosis. In the case of B cells, treatment of immature B cell lymphomas, but

not mature B cells, with soluble anti-Ig antibodies often induces apoptosis (L.E. Benhamou et al . , "Anti- Immunoglobulins Induce Death by Apoptosis in EHI-231 B Lymphoma Cells," Eur. J. Immunol . 20:1405-1407 (1990) ; J. Hasbold & G.G.B. Klaus, "Anti-Immunoglobulin Antibodies Induce Apoptosis in Immature B Cell Lymphomas," Eur. J. Immunol . 20:1685-1690 (1990)) . Signaling by anti-Ig antibodies requires tyrosine phosphorylation as an early and essential step (A.L. DeFranco, "Structure and Function of the B Cell Antigen Receptor," Annu. Rev. Cell Biol . 9:377-410 (1993)) .

In immature B cells, stimulation of slgM (surface immunoglobulin M) by either antigen or anti- immunoglobulin antibodies activates the cells (G.J.V. Nossal, Annu. Rev. Immunol. 1:33-62 (1983)) . Stimulation of slg (surface immunoglobulin) in B cells induces tyrosine phosphorylation (M.R. Gold et al . , Nature 345:810-813 (1990) ; M.A. Campbell & B.M. Sefton, EMBO J. 9:2125-2131 (1990) , which is essential for productive slg signaling (P.J.L. Lane et al . , J. Immunol. 146:715-722 (1991)) .

Furthermore, specific tyrosine kinases appear to be involved in the B cell signaling that leads to apoptosis or growth arrest. For example, as a result of slg stimulation, Src family kinases are activated (A.L. Burkhardt et al. , Proc. Natl. Acad. Sci. USA 88:7410- 7414 (1991) ) . Additionally, expression of the Src family tyrosine kinase Blk was found to be essential in B cell lymphomas where slgM stimulation leads to growth arrest in apoptosis (X.R. Yao & D.W. Scott, "Expression of Protein Tyrosine Kinases in the Ig Complex of Anti-μ- Sensitive and Anti-μ-Resistant B Cell Lymphomas: Role of the p55 ^bl Kinase in Signaling Growth Arrest and Apoptosis," Immunol. Rev. 132:163-186 (1993)) . Similarly, the expression of the Blk tyrosine kinase has

been found to be necessary for antigen receptor induced apoptosis in CH31 lymphoma cells (X.R. Yao & D.W. Scott, "Antisense Oligodeoxynucleotides to the blk Tyrosine Kinase Prevent Anti-μ-Chain-Mediated Growth Inhibition and Apoptosis in a B Cell Lymphoma, " Proc. Natl . Acad. Sci. USA 90:7946-7950 (1993)) , wherein the Lyn tyrosine kinase has been shown to be necessary for antigen receptor induced growth arrest in both human and murine B cell lymphoma lines (R.H. Scheuermann et al . , "Lyn Tyrosine Kinase Signals Cell Cycle Arrest but Not Apoptosis in B-Lineage Lymphoma Cells," Proc. Natl. Acad. Sci. USA 91:4048-4052 (1994)) .

Thus, on slgM stimulation, tyrosine kinases such as Blk phosphorylate one or more proteins on tyrosine residues, and once phosphorylated, these proteins are then able to induce apoptosis. However, it has also been shown that the abundant phosphotyrosine phosphatase CD45 is required for slg signal transduction (L.B. Justement et al. , Science 252:1839-1842 (1991)) .

The correlation of the ability of monoclonal anti-idiotypic antibodies to induce tyrosine phosphorylation signaling and their ability to produce lymphoma regression in human patients also supports the role of tyrosine phosphorylation in these processes ( .M.J. Vuist et al., "Lymphoma Regression Induced by Monoclonal Anti-idiotypic Antibodies Correlates with Their Ability to Induce Ig Signal Transduction and Is Not Prevented by Tumor Expression of High Levels of Bcl- 2 Protein," Blood 83:899-906 (1994)) . In the case of T cells, the tyrosine kinase inhibitor herbimycin A prevented superantigen induced cell death that otherwise resulted from cross-linking multiple antigen receptors (N.K. Damle et al . , "Activation with Superantigens

Induces Programmed Death in Antigen-Primed CD4+ Class 11+ Major Histocompatibility Complex T Lymphocytes Via a

CDlla/CD18-Dependent Mechanism, " Eur. J. Immunol . 23:1513-1522 (1993) ) .

A second example of the role of tyrosine phosphorylation in lymphocyte apoptosis is the case of Fas induced cell death in T cells. Crosslinking of Fas antigen on Jurkat T cell leukemia cells with anti-Fas antibodies induces both apoptosis and activation of tyrosine kinases leading to cellular tyrosine phosphorylation (CM. Eischen et al . , "Tyrosine Kinase Activation Provides an Early and Requisite Signal for Fas-Induced Apoptosis," J. Immunol. 153:1947-1954 (1994) ) . Herbimycin A blocked both the Fas induced tyrosine phosphorylation and death in these cells, demonstrating the essential role of the tyrosine kinases.

Apoptosis induced by ionizing radiation provides a third example. Ionizing radiation causes tyrosine kinase activation in human B cell lymphocyte precursors at therapeutically relevant doses that lead to growth arrest in apoptotic cell death (F.M. Uckun et al. , "Ionizing Radiation Stimulates Unidentified Tyrosine-Specific Protein Kinases in Human B-Lymphocyte Precursors Triggering Apoptosis and Clonogenic Cell

Death," Proc. Natl. Acad. Sci. USA 89:9005-9009 (1992)) . Ionizing radiation is standard therapy for B cell malignancies such as leukemias and lymphomas. Tyrosine kinase inhibitors blocked the radiation-induced tyrosine phosphorylation and apoptosis. The phosphotyrosine phosphatase (PTP) inhibitor vanadate, which when used alone had little effect, greatly augmented the radiation-induced tyrosine phosphorylation and cell death (F.M. Uckun et al. (1992) , supra) . The activation of tyrosine kinases by ionizing radiation was essential for the induction of apoptosis because the tyrosine kinase inhibitors genistein and herbimycin A blocked the

effects of the radiation. These results indicate that not only is tyrosine kinase activation essential for radiation induced apoptosis in B cells, but also suggest that phosphatases can act to limit these responses. The 5 state of lymphocyte development also appears to be important in apoptotic responses since immature B cell lines frequently respond to Ig antibody treatment with apoptosis or growth arrest, whereas mature B cells respond by proliferation (R.H. Scheuermann et al. 10 (1994), supra; X.R. Yao & D. . Scott, Immunol. Rev.

(1993), supra; G.J.V. Nossal, "Cellular and Molecular Mechanisms of B Lymphocyte Tolerance, " Adv. Immunol. 52:283-331 (1992) ) .

15 In addition to blocking proliferation of malignant B cells or T cells in diseases such as leukemias and lymphomas, in a number of situations it may be desirable to slow the growth and/or differentiation of normal B cells or T cells. Such

20 occasions include organ transplantation, in which the immune response, at least in the short term, must be suppressed. Limited control of the proliferation of B cells may also be desirable in the treatment of autoimmune diseases such as rheumatoid arthritis and

25 lupus erythematosus.

Accordingly, there exists a need for improved methods of controlling proliferation of B cells or T cells in malignant and non-malignant conditions without

30 requiring the use of radiation. Such an approach

*- preferably involves the production of programmed cell i death (apoptosis) in susceptible cells. Preferably, such methods should be specific for lymphocytes and not ■ cause neutropenia. Also, such methods should be of

35 value in treating tumors overexpressing one or more members of the EGF receptor family, other receptor tyrosine kinases, or other tyrosine kinases.

SUMMARY

I have developed a method of inhibiting the proliferation of B cells or T cells by using inhibitors of phosphotyrosine phosphatase and/or stimulators of tyrosine kinase.

Several types of inhibitors can be used, including: (1) compounds in which at least one peroxo group is bound to the metal; and (2) compounds in which no peroxo group is bound to the metal, but at least one oxo group is bound to the metal. In general, these compounds comprise: (1) a metal ion selected from the group consisting of molybdenum (VI) , vanadium (V) , or tungsten (VI) ;

(2) at least one oxo group coordinate- covalently bound to the metal ion; (3) at least one organic moiety coordinate- covalently bound to the metal ion through at least one N-containing, O-containing or As-containing moiety capable of donating electrons to a coordinate-covalent bond; and (4) optionally, one or two peroxo groups coordinate-covalently bound to the metal ion and occupying two sites in the coordination sphere of the metal ion. The coordinate-covalent complex has an affinity for the active site of a phosphotyrosine phosphatase sufficient to detectably inhibit the activity of one or more phosphotyrosine phosphatases. Preferably, the coordinate-covalent complex has an affinity for the active site of phosphotyrosine phosphatase at least about equal to the affinity of bis(maltolato) oxovanadium (IV) for the active site of phosphotyrosine phosphatase.

One particularly useful class of coordinate- covalent complex comprises :

(1) a metal ion selected from the group consisting of molybdenum (VI) , tungsten (VI) , and vanadium (V) ;

(2) an oxo group coordinate-covalently bound to the metal ion;

(3) at least one peroxo group coordinate- covalently bound to the metal ion; and (4) at least one organic moiety coordinate- covalently bound to the metal ion through at least one N-containing or O-containing functional group capable of donating electrons to a coordinate-covalent bond.

Preferred coordinate-covalent complexes include (1, 10-phenanthroline) oxodiperoxovanadium (V) (known as pV(phen)) , oxalato-oxodiperoxovanadium (V) , (2, 2 ' -bipyridine) oxodiperoxovanadium (V) , (4,7- dimethyl-1, 10-phenanthroline) oxodiperoxovanadium (V) , (3,4,7, 8-tetramethyl-l, 10-phenanthroline) oxodiperoxovanadium (V) , (pyridine-2-carboxylic acid) oxodiperoxovanadium (V) , (5- hydroxypyridine-2- carboxylic acid) oxodiperoxovanadium (V) , (pyridine-2, 6- dicarboxylic acid) oxodiperoxovanadium (V) , and derivatives thereof possessing substantially equivalent affinity for the active site of phosphotyrosine phosphatase. Other preferred coordinate-covalent complexes include (pyridine-2, 6-dicarboxylato) (hydrato) oxoperoxovanadium (V) , bis (dimethylformamido) oxodiperoxomolybdenum (VI) , hydrogen (pyridine-2- carboxylato) oxodiperoxomolybdenum (VI) hydrate, [ (N- salicylidene) -2-hydroxybenzeneamine] [ethanol] oxoperoxomolybdenum (VI) , (pyridine-2 , 6-dicarboxylato) (hydrato) oxoperoxomolybdenum (VI) , and bis (N- phenylbenzohydroxamato) oxoperoxomolybdenum (VI) .

Another class of metal-organic coordinate- covalent complexes suitable for processes according to the present invention includes an oxo group coordinate- covalently bound to the metal ion, but no peroxo group. These complexes comprise:

(1) a metal ion selected from the group consisting of molybdenum (VI) , tungsten (VI) , and vanadium (V) ;

(2) an oxo group coordinate-covalently bound to the metal ion; and

(3) at least one organic moiety coordinate- covalently bound to the metal ion through at least one N-containing or O-containing moiety capable of donating electrons to a coordinate-covalent bond. The coordinate-covalent complex has an affinity for the active site of phosphotyrosine phosphatase at least about equal to bis (maltolato) oxovanadium (IV) for the active site of phosphotyrosine phosphatase.

Preferred coordinate-covalent complexes belonging to this class include bis [benzene-1, 2- di (dimethylarsino) ] [chloro] oxomolybdenum (VI) and meso-tartrato oxovanadium (V) .

These coordinate-covalent complexes can be used in the following methods:

(1) a method for inhibiting proliferation of a cell type selected from the group consisting of B cells, T cells, and cells derived from malignant transformation of B cells or T cells, comprising the step of contacting proliferating cells selected from the group consisting of B cells, T cells, and cells derived from malignant transformation of B cells or T cells with a coordinate-covalent complex as described above;

(2) a method of treating a subject suffering from a malignant proliferative disorder selected from

the group consisting of leukemias and lymphomas wherein proliferating malignant cells are selected from the group consisting of B cells, T cells, cells derived from malignant transformation of B cells or T cells, and myeloid cells, the method comprising the step of contacting the proliferating malignant cells with a coordinate-covalent complex as described above;

(3) a method of treating a subject suffering from a malignant proliferative disorder selected from the group consisting of leukemias and lymphomas wherein the proliferating malignant cells are selected from the group consisting of B cells, T cells, cells derived from malignant transformation of B cells or T cells, and myeloid cells, the method comprising: (i) contacting the proliferating malignant cells with a coordinate-covalent complex as described above; and

(ii) delivering ionizing radiation to the cells contacted with the coordinate-covalent complex, the ionizing radiation being delivered in a dose sufficient to induce a substantial degree of cell killing among the malignantly proliferating cells, the degree of cell killing induced being substantially greater than that induced by either the coordinate- covalent complex or the ionizing radiation alone;

(4) a method of preventing the class- switching of antibody-producing cells comprising administering to antibody-producing cells a quantity of a coordinate-covalent complex as described above; (5) a method of inducing tyrosine phosphorylation in a cell type selected from the group consisting of B cells, T cells, and cells derived from malignant transformation of B cells or T cells, comprising the step of contacting cells selected from the group consisting of B cells, T cells, and cells derived from malignant transformation of B cells or T

cells with a coordinate-covalent complex as described above;

(6) a method of suppressing growth of tumor cells overexpressing a tyrosine kinase, such as, but not necessarily limited to, a tyrosine kinase selected from the group consisting of HER1, HER2, HER3, HER4, and Src, the method comprising the step of contacting tumor cells overexpressing a tyrosine kinase with a coordinate- covalent complex as described above; (7) a method of suppressing growth of tumor cells requiring phosphotyrosine phosphatase for their growth and/or survival comprising the step of contacting tumor cells requiring phosphotyrosine phosphatase for their growth and/or survival with a coordinate-covalent complex as described above; and

(8) a method of activating a tyrosine kinase that is a member of the Syk family of tyrosine kinases comprising the step of contacting a cell containing a member of the Syk family of kinases with a coordinate- covalent complex as described above.

In particular, the compound pV(phen) demonstrates antitumor activity in vivo and against tumor cell lines in culture.

Another aspect of the present invention is novel coordinate-covalent metal-organic complexes. These coordinate-covalent metal-organic complexes include: (1, 10-phenanthroline) oxodiperoxomolybdenum (VI), (1,10- phenanthroline) oxodiperoxotungsten (VI) , oxolato oxodiperoxomolybdenum (VI) , (4,7-dimethyl-1,10- phenanthroline) oxodiperoxomolybdenum (VI), (4,7- dimethyl-1, 10-phenanthroline) oxodiperoxotungsten (VI) , (3,4,7, 8-tetramethyl-l,10 phenanthroline) oxodiperoxomolybdenum (VI), (3,4, 7, 8-tetramethyl-l, 10- phenanthroline) oxodiperoxotungsten (VI) , (pyridine-2-

carboxylic acid) oxodiperoxomolybdenum (VI) , (pyridine- 2-carboxylic acid) oxodiperoxotungsten (VI) , (5- hydroxypyridine-2-carboxylic acid) oxodiperoxomolybdenum (VI) , (5-hydroxypyridine-2-carboxylic acid) oxodiperoxotungsten (VI) , (pyridine-2, 6-dicarboxylic acid) oxodiperoxomolybdenum (VI) , and pyridine-2, 6- dicarboxylic acid) oxodiperoxotungsten (VI) .

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description, appended claims, and accompanying drawings where: Figure IA is a photograph of an anti- phosphotyrosine western blot following sodium dodecyl sulfate-polyacrylamide electrophoresis of cell lysates from Ramos cells, a human B cell lymphoma cell line, after treatment with bis (maltolato) oxovanadium (IV)

(BMLOV) , showing the dose dependence of the resulting phosphorylation after a one-hour exposure to BMLOV;

Figure IB is a similar photograph of an anti- phosphotyrosine western blot showing the effects of treating the cells with 100 μM BMLOV for varying times; Figure 1C is a similar photograph of an anti- phosphotyrosine western blot, showing the high levels of phosphorylation reached after exposure to 50 μM BMLOV for 16 hours; Figure 2A is a photograph of a stained electropherogram of an agarose gel of DNA from Ramos cells and human promyelocytic leukemia HL-60 cells after treatment of the cells with BMLOV, showing the breakdown of the DNA into fragments characteristic of apoptosis; Figure 2B is a similar photograph of a stained electropherogram of an agarose gel of DNA from the human T cell leukemia cell lines Jurkat and CEM, and the human

colon carcinoma cell line 3347, after treatment of the cells with BMLOV, showing that apoptosis did not occur, demonstrating the selectivity of BMLOV;

Figure 3 is a graph showing the effects of varying doses of BMLOV on thymidine incorporation in normal tonsillar cells stimulated with varying combinations of ligands;

Figure 4 is a similar graph showing the effects of varying doses of BMLOV on thymidine incorporation with tonsillar cells from a second donor;

Figure 5 is a graph showing the results of clonogenic assays after treatment of Ramos B cells with radiation alone, BMLOV alone, or radiation and BMLOV;

Figure 6 is a graph showing the inhibition by BMLOV of growth of peripheral blood lymphocytes (PBL) driven by anti-CD28 antibody plus interleukin-2;

Figure 7 is a graph showing the inhibition by BMLOV of growth of peripheral blood lymphocytes (PBL) driven by anti-CD28 antibody plus anti-CD3 antibody; Figure 8 is a graph showing the inhibition by

BMLOV of growth in mixed lymphocyte response cultures dependent on CD28 costimulation;

Figure 9 is a graph showing the inhibition of pp60° ^~Src kinase activity in two colon carcinoma cell lines expressing Src by BMLOV;

Figure 10 is a graph showing the results of fluorescence-activated cell sorting (FACS) analysis of the cell cycle stage of Ramos B cells after treatment with BMLOV; Figure 11 is a photograph of an anti- phosphotyrosine western blot similar to those in Figures 1A-1C showing the effects of treating the cells with vanadyl acetylacetonate and analogs of vanadyl acetylacetonate in which the vanadium is replaced by molybdenum, chromium, iron, manganese, or copper, showing the induction of phosphorylation by vanadyl acetylacetonate in a pattern similar to that of BMLOV;

Figure 12A is a photograph of anti- phosphotyrosine immunoblots showing the induction of cellular tyrosine phosphorylation in Ramos B cells by BMLOV; Figure 12B is a similar photograph of anti- phosphotyrosine immunoblots comparing the induction of cellular tyrosine phosphorylation in Ramos B cells by BMLOV and orthovanadate;

Figure 12C is a similar photograph of anti- phosphotyrosine immunoblots comparing the induction of cellular tyrosine phosphorylation in murine B cell lymphoma A20 cells and the human T cell leukemia lines CEM and Jurkat after 16-hr treatments with the indicated concentrations of BMLOV; Figure 13 is a photograph of a stained electropherogram on an agarose gel of DNA from Ramos, HL-60, CEM, Jurkat, and 3347 cells treated with the indicated concentrations of BMLOV for the indicated times, showing the apoptosis induced; Figure 14 is a series of graphs showing the effect of BMLOV on cell cycle progression for Ramos cells growing in log phase for various concentrations of BMLOV; DNA in the cells was stained with propidium iodine and quantitated by flow cytometry; the calculated percentage of cells at the stages of the cell cycle is shown ("A" marks apoptotic cells lacking normal amounts of DNA in the 5 μM dose panel) ;

Figure 15A is a graph showing the inhibition of cell proliferation by BMLOV; cells were grown for 5 days in the presence of 10 μM, 25 μM, or no BMLOV and proliferation was measured by [ ³ H] -thymidine incorporation; the percentage inhibition of proliferation by BMLOV is shown; assays were performed with 4 replicates and error bars show the standard error of the mean; asterisks mark conditions giving 99.9% or greater inhibition of proliferation;

Figure 15B shows graphs of flow cytometric results to determine the viability of cells after growth in the presence of BMLOV; viability was measured by staining with propidium iodine followed by cytometric analysis, with the percentage of cells in each quadrant being indicated; the lower right quadrant contains the viable cells;

Figure 16A is a graph showing the effects of BMLOV on thymidine incorporation in normal B cells; normal peripheral B cells were cultured in RPMI media containing 10% fetal bovine serum and with the addition of 1 μg/ml G28-5 plus 10 ng/ml IL-4 as indicated; cells were grown for 5 days in the indicated concentrations of BMLOV and proliferation was measured by [ ³ H] thymidine incorporation;

Figure 16B shows graphs of flow cytometric results to determine the viability of normal B cells after growth in the presence of BMLOV; normal peripheral B cells were grown for 4 days with the indicated concentrations of BMLOV; viability was measured by staining with propidium iodine followed by cytometric analysis as in Figure 15B;

Figure 17 is a graph of the effect of the phosphotyrosine phosphatase inhibitors BMLOV and (1,10- phenanthroline) oxodiperoxovanadium (V) (pV(phen)) on cell proliferation for REH, Ramos, CEM, Jurkat, HL-60, and THP-1 cells; cells were treated for 5 days with the concentration of inhibitor indicated and [ ³ H] thymidine incorporation was measured to determine proliferation; Figure 18 is a photograph of a stained electropherograms on agarose gels of DNA from L1210 murine T cell leukemia cells treated with the indicated concentrations of p(V)phen for the indicated times, showing the apoptosis induced as fragmentation of the DNA;

Figure 19 is a photograph of an anti- phosphotyrosine Western blot similar to those in Figures

1A-1C showing the induction of tyrosine phosphorylation by pV(phen) ; Ramos B cells and Jurkat T cells were treated with the indicated concentrations of pV(phen) for 16 hours or with anti-μ (to cross-link surface immunoglobulins) for 1 minute or with monoclonal antibody G19-4 (anti-CD3) for 1 minute; the cells were then lysed and cellular tyrosine phosphorylation was examined by an anti-phosphotyrosine Western blot of the cellular proteins; Figure 20 is a comparison of the tyrosine phosphorylation induced by pV(phen) and BMLOV; Ramos B cells were treated with the indicated concentrations of BMLOV or pV(phen) for 16 hours; the cells were then lysed and Western blotting performed as in Figure 19; and

Figure 21 shows Western blotting results indicating the response of Syk to pV(phen) ; Ramos B cells were treated with the indicated concentrations of BMLOV or pV(phen) for 16 hours; the cells were then lysed and the Syk tyrosine kinase was immunoprecipitated with anti-Syk antibodies; the tyrosine phosphorylation of Syk was examined by an anti-phosphotyrosine Western blot of the immunoprecipitated Syk protein following electrophoresis (shown at left) ; the amount of Syk protein recovered was determined by anti-Syk Western blot of identical samples (shown at right) .

DESCRIPTION

I have developed an effective means of inhibiting phosphotyrosine phosphatase, particularly in B cells as well as in T cells, as well as novel compounds, including vanadyl compounds, molybdate compounds, and tungstate compounds, possessing inhibitory activity for phosphotyrosine phosphatase, and in some cases, stimulatory activity for tyrosine kinase.

The inhibition of phosphotyrosine phosphatase, as well as the stimulation of tyrosine kinase, can be used to inhibit the proliferation of both malignant and normal B cells or T cells, as well as other physiological functions depending on the balance between phosphorylated and dephosphorylated tyrosine residues.

PHOSPHOTYROSINE PHOSPHATASE INHIBITORS

In general, two types of phosphotyrosine phosphatase inhibitors are useful in the methods of the present invention. These are metal-organic coordinate- covalent compounds and nonhydrolyzable phosphotyrosine analogs. Other phosphotyrosine phosphatase inhibitors are also useful in the methods of the present invention.

A. Metal-Organic Coordinate-Covalent Compounds

Metal-organic coordinate-covalent compounds useful in the methods of the present invention include metal-organic coordinate-covalent compounds lacking an oxo or peroxo ligand coordinate-covalently bound to the metal, and metal-organic coordinate-covalent compounds having at least one oxo or peroxo ligand bound to the metal ion.

1. Compounds Lacking an Oxo or Peroxo Ligand

Metal-organic coordinate-covalent compounds lacking at least one oxo or peroxo ligand bound to the metal ion and useful in the methods of the present invention comprise a metal selected from the group consisting of vanadium (IV) , copper (II) and gallium (II) coordinate-covalently bound to an organic moiety that can be either: (1) a keto-enol tautomer with keto and enol groups on adjacent carbon atoms and that forms

a 5-membered ring including the metal; (2) a beta diketone in which the two keto groups are separated by one carbon atom that forms a 6-membered ring including the metal; (3) complexes in which the metal is preferably vanadium and the organic moiety contains at least one carbon-oxygen or carbon-nitrogen double bond; or (4) complexes of vanadyl and cysteine or cysteine derivatives.

For those compounds where the metal can be selected from the group consisting of vanadium (IV) , copper (II) and gallium (II) , the metal is preferably vanadium (IV) . Other metals can give different patterns of inhibition in particular cell types.

Keto-Enol Tautomers

If the organic moiety is a keto-enol tautomer forming a 5-membered ring including the metal, it is preferably one of the following moieties: maltol, kojic acid, 2-hydroxy-2,4, 6-cycloheptatrien-l-one, 3-bromo-2- hydroxy-2, , 6-cycloheptatrien-l-one, 2-hydroxy-4- isopropyl-2,4,6-cycloheptatrien-l-one, 2-hydroxy-4- methyl-2,4, 6-cycloheptatrien-l-one, 3-hydroxy-l, 2- dimethyl-4 (1H) -pyridone, 3-ethyl-2-hydroxy-2- cyclopenten-1-one, 3,4-dihydroxy-3-cyclobuten-1, 2-dione, ethyl 2-hydroxy-4-oxo-2-pentenone, 2,3, 5, 6-tetrahydroxy- 1,4-benzoquinone, 2 ' ,4 ' -dihydroxy-2-methoxyacetophenone, 4-hydroxy-5-methyl-4-cyclopenten-1,3-dione, 2-chloro-3- hydroxy-1,4-naphthoquinone, 2- (4-bromophenyl) -3- hydroxymaleimide, 2-hydroxy-3-methyl-2-cyclopenten-1- one, 2 ' , 3 ' , ' -trihydroxyacetophenone, furoin, 2-hydroxy- 2-methylpropiophenone, maclurin, 6- (pyrrolidinomethyl)kojic acid, alpha-acetyl-4-hydroxy- beta- (hydroxymethyl) -3-methoxycinnamic acid gamma- lactone, 4-hydroxy-5-phenyl-4-cyclopenten-l, 3-dione, 6- (morpholinomethyl) kojic acid, 1- (4, 5-dimethoxy-2-

hydroxyphenyl) -3-methyl-2-buten-l-one, purpurogallin, 2, 3-dihydroxy-l,4-phenazinedione, alizarin orange, 1- hydroxy-l-methylnaphthalen-2 (1H) -one, alizarin, 6- (piperidinomethyl) kojic acid, 1,2,7- trihydroxyanthraquinone, 6- (4- methylpiperazinomethyl) kojic acid, fisetin, 3-oxo-4,5,6- trihydroxy-3 (H) -xanthene-9-propionic acid, benzoin, 4'- chlorobenzoin, quercetin, morin, myricetin, or 4,4'- dimethylbenzoin. More preferably, the organic moiety is maltol, and the resulting compound is bis (maltolato) oxovanadium (IV) ("BMLOV") .

b. Beta Diketones

If the organic moiety is a beta diketone, the organic moiety is preferably one of the following moieties: acetylacetone, 2-acetyl-l-tetralone, benzoylacetone, 1-benzoylacetylacetone, 1, 1, 1-trifluoro- 2,4-pentanedione, S-methyl-4,4,4-trifluoro-3- oxothiobutyrate, 2-acetyl-l, 3-cyclopentanedione, 3- chloro-2,4-pentanedione, 1,1,1,5,5, 5-hexafluoro-2,4- pentanedione, 3-ureidomethylene-2, -pentanedione, 2- acetylcyclopentanone, 2-acetylcyclohexanone, 3-methyl- 2,4-pentanedione, 2,4, 6-heptatrione, 3-ethyl-2,4- pentanedione, thenoyltrifluoroacetone, S-t _. -butyl- acetothioacetate, 3-acetyl-5-methylhexan-2-one, 3- acetyl-2-heptanone, 2,2-dimethyl-6, 6,7,7,8,8,8- heptafluoro-3, 5-octanedione, 4-hydroxy-5-phenyl-4- cyclopenten-1, 3-dione, 4,4,4-trifluoro-1-phenyl-1,3- butanedione, 3-acetyl-2-octanone, 1 (2-hydroxy-4- methylphenyl) -1, 3-butanedione, 1- (2-hydroxy-5- methylphenyl) -1, 3-butanedione, 3-benzylidene-2,4- pentanedione, 1- (2-hydroxy-5-methylphenyl) -1,3- pentanedione, 2, 2, 6, 6-tetramethyl-3, 5-heptanedione, 3- acetyl-5-hydroxy-2-methylchromone, (+)-3-

(trifluoroacetyl) camphor, 4, 9-dihydro-6-methyl-5H- furo (3,2-g) (1) benzopyran-4, 5, 9-trione, 3- (2-

nitrobenzylidene) -2,4-pentanedione, 1, 3-bis (4- chlorophenyl) -1, 3-propanedione, 1, 3-bis- (4- fluorophenyl) -1,3-propanedione, 4,4,4-trifluoro-1- (2- naphthyl) -1,3-butanedione, 1- (2- hydroxypheny1) -3- (4- methoxyphenyl) -1,3-propanedione, 2-bromo-l, 3-diphenyl- 1,3-propanedione, dibenzoyl ethane, 2- (4- chlorobenzylidene) -1-phenyl-1, 3-butanedione, 2- (2- nitrobenzylidene) -1-phenyl-1, 3-butanedione, bis (4- methoxybenzoyl) methane, and curcumin. Preferably, the organic moiety is acetylacetone, and the resulting compound is vanadyl acetylacetonate.

Among novel compounds containing vanadium coordinate-covalently bound to an organic moiety that are believed to have activity as phosphotyrosine phosphatase inhibitors are the compounds vanadyl 2- acetyl-1-tetralone, vanadyl 2-hydroxy-4-isopropyl-2,4, 6- cycloheptatrien-1-one, and vanadyl 2-hydroxy-4-methyl- 2,4, 6-cycloheptatrien-l-one. c. Complexes in which the Metal is

Preferably Vanadium and the Organic Moiety Contains at Least one Carbon- Oxygen or Carbon-Nitrogen Double Bond

Other metal-organic coordinate covalent compounds useful for the processes of the present invention include complexes in which the metal is preferably vanadium and the organic moiety is one of formulas I through IX, of which formula I is a general formula and formulas II through IX represent particular classes of compounds of formula I .

(I)

(ID

10

(III)

30

35

R ⁸ R ⁹ N 10

CHR

(V)

HO C 0

o: c- 13

R

(VII) _R 15

HO c: CR 14

In formula I, X ¹ and X ³ are independently oxygen, sulfur, or NX ⁶ , preferably oxygen or NX ⁶ . X ² is nitrogen or CX ⁷ . X ⁴ , X ⁵ , X ⁶ , and X ⁷ are independently non-labile protons or optionally substituted alkyl,

aryl, aralkyl or alkaryl . Alternatively, at least one pair of X ⁴ to X ⁷ , preferably X ⁴ and X ⁵ , together with the intervening atoms can represent an optionally substituted, saturated or unsaturated homocyclic or heterocyclic ring. Alternatively, where X ¹ is a NX ⁶ group, X ⁴ can represent a group X ⁸ H where X ⁸ is oxygen or sulfur, and one proton attached to X ¹ or X ⁸ , preferably a proton attached to X ¹ , is labile.

Typical organic moieties of formula I are α- amino acids (other than cysteine) , hydroxamates, thiohydroxamates, a-hydroxycarbonyls such as α- hydroxypyridinones or α-hydroxypyrones.

When the organic moiety comprises a homocyclic or heterocyclic ring, the ring is preferably a 5-, 6-, or 7-membered ring. If the ring is heterocyclic, it can contain 1, 2, or 3 heteroatoms, typically 1. The heteroatoms are selected from O, N, and S, and are preferably O or N. Each aryl group is preferably phenyl or naphthyl, typically phenyl. Each alkyl group or moiety contains 1 to 6 carbon atoms, typically 1 to 4. The optional substituents, which do not include thiol groups, are preferably selected from hydroxy, alkoxy, oxo, amide, and amine groups, as well as alkyl groups carrying such substituents. These groups can be selected for their ability to enhance the hydrophilicity or lipophilicity of the complex or to enable the complex to be conjugated to another molecule such as a protein, a polymer, or another biologically active molecule.

Particularly suitable organic moieties include the hydroxamates of formula II, the α-hydroxypyridinones of formula III, the α-hydroxypyrones of formula IV, the α-amino acids of formula V, the hydroxycarbonyls of formulas VI and VII, and the thiohydroxamates of formulas VIII and IX. In these formulas, R ¹ to R ¹⁹ are

hydrogen or optionally substituted, e.g., hydroxylated, C _x to C ₄ alkyl.

d. Complexes of Vanadyl and Cysteine or Cysteine Derivatives

Still other metal-organic coordinate covalent compounds are useful in processes according to the present invention. These coordinate covalent compounds are complexes of vanadyl and cysteine or cysteine derivatives and have the general structure shown in formula X:

C V0 < _x (X)

[ Y- -{ NH CH CO] _r C VO ² *) ,

- χ j _c

C I H,SZ

In formula X, either x is 1 and y is 0 or x is 0 and y is 1. The values of n, p, and m are 1 or 2. The cysteine moiety can be D-cysteine or L-cysteine.

When p is equal to 1, then Y is selected from the group consisting of a hydrogen atom and a R'-CO group.

When n is equal to 1 and m is equal to 2, X is selected from the group consisting of an OH group, an OR group, and an NHR group wherein R is selected from the group consisting of an alkyl group comprising from 2 to

9 carbon atoms, an aryl group, or an aralkyl group. In this structure, when X is an OH group, Y is a R'-CO group wherein R' is selected from the group consisting of an alkyl group comprising from 2 to 9 carbon atoms, and, when X is selected from the group consisting of an OR group and a NHR group, Y is H.

When n is equal to 2 and m is equal to 1, X is selected from the group consisting of a difunctional amine of formula WC[CH ₂ NH-] ₂ , a difunctional alcohol of formula WC[CH ₂ 0-] ₂ / and a difunctional amine-alcohol of formula WC(CH ₂ NH-) (CH ₂ 0-) , wherein W is an alkyl group of from 2 to 9 carbon atoms.

When p is equal to 2, then n is equal to 1, m is equal to 1, X is an OH group, and Y is selected from the group consisting of ZCH(CO-) ₂ , -CH ₂ -, or ZCH(CH ₂ -) ₂ in which Z is an alkyl, aryl, or aralkyl group.

One class of compounds of this type has x equal to 1, y equal to 0, and p equal to 1. In this class of compounds, Y is hydrogen and Z is a minus charge. This class of compounds has a free amine group that is bound to the vanadyl; the ligand bound to the vanadyl is bimolecular, in that two organic moieties are complexed to a single vanadyl ion. The ligand has a negative charge on a sulfur atom. This class of compounds is depicted in formula XI .

(XI)

In formula XI, X is an -OR group or a -NHR (amine) group, in which the R moiety is an aryl or aralkyl group or an alkyl group other than methyl. Where X is a -NHR group, the compound is a complex of vanadyl with an amide of cysteine such as a butylamide of cysteine or an octylamide of cysteine. The vanadyl- octylamide complex of L-cysteine is shown in formula XIa.

(XIa)

Where X is an -OR group, the compound is, for example, a complex of vanadyl with an ester of the cysteine, such as an octylester of cysteine or a butylester of cysteine.

Another class of compounds of this type has n equal to 2 and m equal to 1. In this class of compounds, the ligand is monomolecular. This class of compounds is depicted in formula XII.

In formula XII, X corresponds to a difunctional amine of formula WC[CH ₂ NH-] 2' a difunctional alcohol of formula WC[CH ₂ 0-] ₂ , or a difunctional amine- alcohol of formula WC(CH ₂ NH-) (CH ₂ 0-) . R is an alkyl group of from 2 to 9 carbon atoms.

Yet another class of compounds of this type has x equal to 0, y equal to 1, and n equal to l. In this class of compounds, X is a 0 ^" group and Z is hydrogen. This class of compounds includes a bimolecular ligand comprising a free carboxyl group and is depicted in formula XIII.

In molecules of formula XIII, Y corresponds to a R-CO (ketone) group in which the R moiety is an alkyl, aryl, or aralkyl group.

Yet another class of compounds of this type has p equal to 2 and m equal to 1. This class of compounds includes a monomolecular ligand comprising two free carboxyl groups and is depicted in formula XIV.

(XIV)

In molecules of formula XIV, Y corresponds to a group of formula ZCH(CO-) ₂ , -CH ₂ -, or ZCH(CH ₂ -) ₂ in which Z is an alkyl, aryl, or aralkyl group.

Still another class of compounds of this type has x equal to 0, y equal to 1, Y a CH ₂ group, and Z a minus charge. In this class of compounds, n and p are each 1 and m is 2. This class of compounds includes a monomolecular ligand with substituted amino and carboxyl groups and is depicted in formula XV.

CH, •NH- ^■ CH- •cox V02 ^"

CH ₂ S ^"

(XV)

In formula XV, X is an -OR group or a -NHR (amine) group, in which the R moiety is an aryl or aralkyl group or an alkyl group other than methyl.

B. Coordinate-Covalent Metal-Organic Compounds Containing at Least One Oxo or Peroxo Ligand Bound to the Metal

Another class of metal-organic coordinate- covalent compounds useful in the processes of the present invention has at least one oxo or peroxo ligand coordinate-covalently bound to the metal. These coordinate-covalent metal-organic compounds include: (1) compounds in which at least one peroxo group is bound to the metal; and (2) compounds in which no peroxo group is bound to the metal, but at least one oxo group is bound to the metal. In general, these compounds comprise: (1) a metal ion selected from the group consisting of molybdenum (VI) , vanadium (V) , or tungsten (VI) ;

(2) at least one oxo group coordinate- covalently bound to the metal ion; (3) at least one organic moiety coordinate- covalently bound to the metal ion through at least one N-containing, O-containing or As-containing moiety capable of donating electrons to a coordinate-covalent bond; and (4) optionally, one or two peroxo groups coordinate-covalently bound to the metal ion and occupying two sites in the coordination sphere of the metal ion. The coordinate-covalent complex has an affinity for the active site of a phosphotyrosine phosphatase sufficient to detectably inhibit the activity of one or more phosphotyrosine phosphatases. As used herein, the term "detectably inhibit" means a degree of inhibition that can be determined, within experimental error, by assays measuring the dephosphorylation of a suitable substrate for phosphatases, such as phosphorylated myelin basic protein. Typical phosphotyrosine phosphatases that can

be used to determine the activity of compounds include, but are not limited to, the phosphatases PTPIB and CD45 Preferably, the coordinate-covalent complex has an affinity for the active site of phosphotyrosine phosphatase at least about equal to the affinity of bis (maltolato) oxovanadium (IV) for the active site of phosphotyrosine phosphatase.

1. Coordinate-Covalent Metal-Organic Compounds With at Least One Peroxo Ligand

Bound to the Metal Ion

One particularly useful class of coordinate- covalent complex comprises: (1) a metal ion selected from the group consisting of molybdenum. (VI) , tungsten (VI) , and vanadium (V) ;

(2) an oxo group coordinate-covalently bound to the metal ion; (3) at least one peroxo group coordinate- covalently bound to the metal ion; and

(4) at least one organic moiety coordinate- covalently bound to the metal ion through at least one N-containing or O-containing functional group capable of donating electrons to a coordinate-covalent bond.

The coordinate-covalent complex has an affinity for the active site of a phosphotyrosine phosphatase sufficient to detectably inhibit the activity of one or more phosphotyrosine phosphatases. Preferably, the coordinate-covalent complex has an affinity for the active site of phosphotyrosine phosphatase at least about equal to the affinity of bis (maltolato) oxovanadium (IV) for the active site of phosphotyrosine phosphatase.

In one group of metal-organic compounds useful in the processes of the present invention, the metal ion is vanadium (V) .

In one group of metal-organic compounds useful for the processes of the present invention, the coordinate-covalent complex has two peroxo groups bound to the metal ion. Each of the peroxo groups occupies two sites in the coordination sphere of the metal ion.

When the metal is vanadium (V) , preferred coordinate-covalent complexes include (1,10- phenanthroline) oxodiperoxovanadium (V) , oxalato- oxodiperoxovanadium (V) , (2,2' -bipyridine) oxodiperoxovanadium (V) , (4, 7-dimethyl-l, 10- phenanthroline) oxodiperoxovanadium (V) , (3,4,7,8- tetramethyl-1, 10-phenanthroline) oxodiperoxovanadium (V) , (pyridine-2-carboxylic acid) oxodiperoxovanadium (V) , (5- hydroxypyridine-2-carboxylic acid) oxodiperoxovanadium (V) , (pyridine-2, 6-dicarboxylic acid) oxodiperoxovanadium (V) , and derivatives thereof possessing substantially equivalent affinity for the active site of phosphotyrosine phosphatase. In particular, the 1, 10-phenanthroline, 2, 2 ' -bipyridine, 4, 7-dimethyl-l, 10-phenanthroline, 3,4, 7, 8-tetramethyl- 1, 10-phenanthroline, pyridine-2-carboxylic acid, 5- hydroxypyridine-2-carboxylic acid, and pyridine-2, 6- dicarboxylic acid nuclei can be substituted with hydroxy, lower alkyl (C ₁ -C ₅ ) , or other substituents that do not significantly affect the binding to the metal ion.

One particularly preferable coordinate- covalent complex for use in methods according to the present invention is (1, 10-phenanthroline) oxodiperoxovanadium (V) , also referred to as pV(phen) .

When the metal is vanadium and the coordinate- covalent complex has one peroxo group bound to the metal ion, a preferred coordinate-covalent complex is (pyridine-2,6-dicarboxylato) (hydrato) oxoperoxovanadium (V) .

When the metal ion in the coordinate-covalent complex is molybdenum (VI) , and the coordinate-covalent complex has two peroxo groups bound to the metal ion, preferred coordinate-covalent complexes include bis(dimethylformamido) oxodiperoxomolybdenum (VI) and hydrogen (pyridine-2-carboxylato) oxodiperoxomolybdenum (VI) hydrate.

When the metal is molybdenum (VI) and the coordinate-covalent complex has one peroxo group bound to the metal ion, preferred coordinate-covalent complexes include [ (N-salicylidene) -2- hydroxybenzeneamine] [ethanol] oxoperoxomolybdenum (VI) , (pyridine-2,6-dicarboxylato) (hydrato) oxoperoxomolybdenum (VI), and bis(N- phenylbenzohydroxamato) oxoperoxomolybdenum (VI) .

2. Coordinate-Covalent Complexes Including an Oxo Group Coordinate-Covalentlv Bound to the Metal Ion but Without a Peroxo Group

Another class of metal-organic coordinate- covalent complexes suitable for processes according to the present invention includes an oxo group coordinate- covalently bound to the metal ion, but no peroxo group. These complexes comprise:

(1) a metal ion selected from the group consisting of molybdenum (VI) , tungsten (VI) , and vanadium (V) ;

phosphoramidates; (2) N-aryl phosphorothioates; or (3) N-aryl phosphonates. The N-aryl phosphoramidates have the structure shown in formula XVI, the N-aryl phosphorothioates have the structure shown in formula XVII, and the N-aryl phosphonates have the structure shown in formula XVIII.

(XVIII)

In either the N-aryl phosphoramidates, the N- aryl phosphorothioates, or the N-aryl phosphonates, the aryl moiety can be optionally substituted at any of the ortho, meta, and/or para positions. Similarly, one or two of the oxygen atoms bound to the phosphorus are optionally esterified.

In the N-aryl phosphoramidate of formula XVI, each of R _x through R ₇ can be selected from the group consisting of hydrogen and alkyl, which can be either straight-chain or branched-chain. Preferably, each of R ₁ through R _B is hydrogen. Preferably, when each of R _x through R ₅ is hydrogen, at least one of R ₆ and R ₇ is other than hydrogen.

When the nonhydrolyzable phosphotyrosine analog is a phosphorothioate of formula XVII, preferably each of R- _L through R ₇ is hydrogen or C- _L - _; alkyl, which can be either straight-chain or branched-chain. Preferably, each of R _x through R ₅ is hydrogen.

Preferably, each of R _λ through R ₅ is hydrogen, at least one of R ₆ and R ₇ is other than hydrogen.

Similarly, when the nonhydrolyzable phosphotyrosine analogue is a phosphonate of formula XVIII, preferably each of R ₁ through R ₇ is hydrogen or C- _L -Cg alkyl, which can be either straight-chain or branched-chain. Preferably, each of R ₁ through R ₅ is hydrogen. Preferably, when each of R _x through R ₅ is hydrogen, at least one of R ₆ and R ₇ is other than hydrogen.

D. Additional Phosphotyrosine Phosphatase Inhibitors

Additional phosphotyrosine phosphatase inhibitors exist that are useful in methods according to the present invention. These additional phosphotyrosine phosphatase inhibitors include dephostatin and 4- (fluoromethyl)phenyl phosphate and its esterified derivatives.

1. Dephostatin

Dephostatin is a phosphotyrosine phosphatase inhibitor isolated from Streptomvces sp. MJ742-NF5 (M. Imoto et al., "Dephostatin, a Novel Protein Tyrosine Phosphatase Inhibitor Produced by Streptomyces. I . Taxonomy, Isolation, and Characterization, " J. Antibiotics 46: 1342-1346 (1993)) . It has structure XIX shown below.

and is a competitive inhibitor of phosphotyrosine phosphatase, competing with the substrate for the enzyme. Dephostatin can be extracted from the broth

filtrate of a Streptomvces culture with ethyl acetate and purified by silica gel chromatography and high- pressure liquid chromatography (HPLC) .

2. 4- (Fluoromethyl) Phenyl Phosphate and Its

Esterified Derivatives

The inhibitor of human prostatic acid phosphatase 4- (fluoromethyl)phenyl phosphate (formula XX; R _λ and R ₂ each H) (J.K. Myers & T.S. Widlanski, "Mechanism-Based Inactivation of Prostatic Acid Phosphatase," Science 262: 1451-1453 (1993)), together with its esterified derivatives, are also phosphotyrosine phosphatase inhibitors that are useful in processes according to the present invention. In compounds of formula XX useful in processes according to the present invention, R and R ₂ are either hydrogen or C- _L -Cg alkyl, which can be either straight-chain or branched-chain. Preferably, at least one of R and R ₂ is Ci-Cg alkyl.

E. Synthesis of Compounds

1. Synthesis of Metal-Organic Coordinate- Covalent Compounds

Metal-organic coordinate-covalent compounds can be synthesized by the general method described for bis (maltolato) oxovanadium (IV) in.J.H. McNeill et al. ,

"Bis (maltolato) Oxovanadium (IV) Is a Potent Insulin Mimic," J. Med. Chem. 35:1489-1491 (1992) .

In general, the compounds are prepared by combining the organic ligand (maltol for BMLOV) and vanadyl sulfate in a 2:1 ratio, raising the pH of the solution to 8.5, refluxing overnight, and collecting the compound that precipitates on cooling.

In a generalization of this method, the organic moiety is dissolved in water at alkaline pH. Depending upon the compound, a higher pH may be required for solubilization. For some compounds, a water- miscible, less polar solvent may need to be added to dissolve the compound. Such solvents can include aprotic solvents such as acetonitrile, dimethylsulfoxide, or dimethylformamide, although other suitable solvents are also known in the art . Vanadyl sulfate, or another metal salt if desired, is then added in a sufficient quantity to achieve a 2:1 molar ratio between the organic moiety and the metal salt . The solution is then heated with a condenser attached to the reaction flask to permit refluxing. Following the reaction, the solution is then cooled and the product is recovered as a precipitated solid. In some cases, the product may not precipitate, and can then be recovered by rotary evaporation of the solvent or by other means, such as chromatography.

Vanadium chelates with ligands of Formula I could be prepared in a one-pot synthesis analogous to that described for gallium, aluminum, or indium complexes by Zhang et al. , Can. J. Chem. 67:1708-1710 (1989) .

In general, the compounds of Formula X are synthesized by the following process: (1) reacting a

with the aid of hydrochloric acid and dioxane; (3) adding vanadyl sulfate which is dissolved in water, under a nitrogen atmosphere, to the solution of the hydrochloride of the cysteine derivative in a dimethylformamide-borate buffer mixture at a pH of about 10 with a cysteine:vanadyl ratio of about 5:1; (4) stirring the mixture for 2 hours in a nitrogen atmosphere; (5) recovering the complex formed by precipitation by filtration; and (6) washing with water and drying the precipitate.

Another method for preparing the compound of Formula XI or XV comprises: (1) reacting with cysteine or a derivative thereof with vanadyl sulfate at a pH of about 7 in water; (2) recovering the complex after evaporation; (3) redissolving the complex formed in dimethylformamide as above; (4) coupling with a monofunctional amine or a monofunctional alcohol in the presence of dimethylaminopropylethylcarbodiimide; and (5) recovering the complex after vacuum evaporation of the solvent, washing with ether and with water, dissolving the product in methanol, evaporating, and reprecipitating by ethyl ether.

The preparation of the compound of XII comprises reacting a bifunctional amine or a bifunctional alcohol or an amine-alcohol with the cysteine or a derivative thereof. The remaining procedure is identical to that described above for the preparation of compounds XI and XV.

The preparation of the compound of Formula XIII where Y corresponds to a RCO- group comprises: (1) coupling an activated derivative (an ester or an acyl chloride) of the RCOOH acid with a cysteine previously protected on its thiol function by a t.-butyloxycarbonyl group; (2) deprotecting the thiol function by

acidolysis; and (3) complexing the resulting N-acylated derivative with the vanadyl sulfate in a DMF-water medium at a pH of 10.

A method for preparing the compound of Formula

XI where Y corresponds to RCH(CO-) ₂ comprises: (1) coupling an activated derivative (ester or acyl chloride) of the diacid RCH(COOH) ₂ with a cysteine previously protected on its thiol function with a t.- butyloxycarbonyl group; (2) deprotecting the thiol function by acidolysis; (3) reducing the N-acylated derivative thus obtained; and (4) complexing the reduced derivative in a DMF-water medium at a pH of 10 in the presence of vanadyl sulfate.

Other methods for producing coordinate- covalent complexes suitable for use in processes according to the present invention are also known in the art. For example, when the organic moiety is one of 1, 10-phenanthroline, 4, 7-dimethyl-l, 10-phenanthroline, 3,4,7, 8-tetramethyl-l, 10-phenanthroline, bipyridine, oxalic acid dianion, pyridine-2-carboxylate, 5- hydroxypyridine-2-carboxylate, and pyridine-2, 6- dicarboxylic acid dianion, a suitable method is dissolving V ₂ 0 ₅ in aqueous KOH, followed by the addition of 30% hydrogen peroxide, the organic ligand, and, in some cases, ethanol to facilitate precipitation of the product.

In a typical procedure, (1, 10-phenanthroline) oxodiperoxovanadium (V) (pV(phen)) can be prepared by adding H ₂ 0 (30 ml) to V ₂ 0 ₅ (2.75 g; 15 mmol) and KOH (1.95 g; 34 mmol) in a 125-ml Erlenmeyer flask followed by 1.5 ml of H ₂ 0 ₂ (30%, w/v, solution) . The mixture is then gently shaken until most of the solids dissolve

(about 2 min) . The brightly colored orange solution is then filtered through a sintered glass filter and is

allowed to stand for 15 to 30 min. More H ₂ 0 ₂ (30 ml) is then added to the filtrate followed almost immediately by phenanthroline (5.7 g; 32 mmol) in 25 ml of ethanol. The reaction is stirred for 30 min after which 100 ml of ethanol is added dropwise while stirring to effect precipitation of the product (4-5 g; 66-83% yield) as a fine yellow powder (B.I. Posner et al. , "Peroxovanadium Compounds," J. Biol. Chem. 269:4596-4604 (1994)).

Similar methods can be used to prepare complexes including these organic ligands in which the metal is another metal such as molybdenum (VI) or tungsten (VI) .

Other methods for preparing similar oxodiperoxovanadium (V) complexes with bidentate ligands such as oxalate or bipyridine, with various counterions, such as potassium or ammonium, are described in N. Vuletic & C. Djordjevic, "Oxodiperoxovanadate (V) Complexes with Bidentate Ligands," J. Chem. Soc. Dalton Trans. 1137-1141 (1973) . For example, for the preparation of ammonium oxalato-oxodiperoxovanadium (V) , ammonium trioxovanadate (V), NH ₄ V0 ₃ (0.59 g) , is dissolved with cooling in hydrogen peroxide (20%, 15 ml) and added to a solution (10 ml) of ammonium oxalate

(0.71 g) and hydrogen peroxide (20%) . Ethanol is then added gradually until a precipitate starts to appear. The precipitate is redissolved with a small amount of hydrogen peroxide and the reaction mixture is set aside to crystallize at 5°C. The orange crystals obtained are filtered off and dried on the filter paper.

For the preparation of potassium (oxalato) oxodiperoxovanadium (V), vanadium pentoxide (0.91 g) and potassium hydroxide (1.95 g) are dissolved in water (20 ml) and to the solution oxalic acid (1.26 g) dissolved in water (10 ml) and hydrogen peroxide (30%, 20 ml) are

added. Precipitation is initiated by ethanol as described above, the precipitate redissolved, and the reaction mixture set aside to crystallize at room temperature or in a cool place. The orange crystals obtained are filtered off and dried on the filter paper.

For ammonium (2,2' -bipyridine) oxodiperoxovanadium (V) , ammonium trioxovanadate (V) is dissolved under cooling in hydrogen peroxide (20%, 20 ml) , and 2,2 ' -bipyridine (0.8 g) , dissolved in ethanol (10 ml) , is added with stirring. After 2-3 minutes, more ethanol (20 ml) is added and the reaction mixture is set aside to crystallize at 5°C. Yellow crystals obtained are filtered off, washed once with ethanol, and dried in air.

For ammonium (1, 10-phenanthroline) oxodiperoxovanadium (V) , a similar preparation can be used, but the quantities of water and hydrogen peroxide are preferably reduced to half the above quantities because of the solubility properties of this complex.

For sodium (2,2 ' -bipyridine) oxodiperoxovanadium (V), and sodium (1,10- phenanthroline) vanadium (V) , the above procedures can generally be followed. However, crystallization typically takes place more slowly for complexes with these counterions. Crystals are dried on the filter paper in air.

For potassium (2,2' -bipyridine) oxodiperoxovanadium (V) , vanadium pentoxide (0.91 g) and potassium hydroxide (0.65 g) are dissolved in water (10 ml) , cooled, and, if necessary, the dissolution is completed with a small amount of hydrogen peroxide. To the clear, cooled solution, hydrogen peroxide (10 ml) and 2,2 ' -bipyridine (1.6 g) dissolved in ethanol (7-8

ml) are added. Pure crystals are obtained as described for the analogous oxalato-derivative of potassium. Potassium (1, 10-phenanthroline) vanadium (V) can be obtained similarly.

In general, other complexes can be prepared according to these methods. In some cases, the volumes or quantities of vanadium or other metal ions used can be adjusted depending on the solubility of the complex. In addition, the conditions used for the filtration and washing steps can also be varied. These variations would be known to one of ordinary skill in the art and need not be described further; they also depend on the solubility of the complexes involved.

2. Synthesis of Non-Hydrolyzable Phosphotyrosine Analogs

The nonhydrolyzable phosphotyrosine analogs, which are phosphoramidates, phosphorothioates, or phosphonates, can be synthesized by methods well understood in the art for synthesis of these compounds. Phosphorothioyltyrosine can be synthesized by sulfurization of the intermediate phosphite triester using phenylacetyl disulfide (D.B.A. Debont et al . , "Solid-Phase Synthesis of O-Phosphothioylserine- Containing and O-Phosphorothreonine-Containing Peptides as Well as of O-Phosphoserine-Containing and 0- Phosphothreonine-Containing Peptides," J. Org. Chem. 58: 1309-1317 (1993)) . Phosphonates of tyrosine can be synthesized with the use of the reagent 4- [ (di-t- butylphosphono)methyl] -N- (fluoren-9-ylmethoxycarbonyl) - D,L-phenylalanine (T.R. Burke et al . , "Preparation of Fluoro-4- (Phosphonomethyl) -D,L-Phenylalanine and Hydroxy-4- (Phosphonomethyl) -D,L-Phenylalanine Suitably Protected for Solid-Phase Synthesis of Peptides

Containing Hydrolytically Stable Analogues of O- Phosphotyrosine, " J. Or . Chem. 58: 1336-1340 (1993)) .

II. USE OF PHOSPHOTYROSINE PHOSPHATASE INHIBITORS AND PHOSPHOTYROSINE KINASE ACTIVATORS

Phosphotyrosine phosphatase inhibitors and phosphotyrosine kinase activators, including metal- organic coordinate covalent compounds such as vanadyl compounds and non-hydrolyzable phosphotyrosine analogs, can be used to block or alter a number of processes involving phosphotyrosine metabolism.

The exact physiological effects seen depend on whether the compound used is an inhibitor of phosphotyrosine phosphatase, an activator of phosphotyrosine kinase, or, as in some cases, both an inhibitor of phosphotyrosine phosphatase and an activator of phosphotyrosine kinase.

A. Use Of Phosphotyrosine Phosphatase Inhibitors

1. Inhibition Of Proliferation Of B Cells

In B cells, the level of tyrosine phosphorylation is used for metabolic regulation. Accumulation of an excessive level of tyrosine phosphorylation, such as by the continued activity of tyrosine kinases in the absence of significant phosphotyrosine phosphatase activity, leads to apoptosis, which is programmed cell death marked by fragmentation of cellular DNA. Therefore, the administration of inhibitors of phosphotyrosine phosphatase can be used to control the proliferation of B cells. This is particularly desirable for the treatment of malignancies of B cell origin, such as

leukemias and lymphomas. Such treatment also has the effect of sensitizing the cells to ionizing radiation, so that the effects of ionizing radiation and phosphotyrosine phosphatase inhibitors are not merely additive but synergistic. When both the coordinate- covalent metal-organic complex and ionizing radiation are used, the degree of cell killing induced is substantially greater than that induced by either the coordinate-covalent complex or the ionizing radiation alone.

However, phosphotyrosine phosphatase inhibitors can also be used to control proliferation of normal B cells, particularly in situations in which downregulation of the immune response is desired. Such situations include induction of immunosuppression to prevent transplant rejection, as well as in the treatment of autoimmune diseases such as rheumatoid arthritis, Hashimoto's thyroiditis, and systemic lupus erythematosus, as well as other autoimmune diseases.

Phosphotyrosine is also involved in proliferation of protozoans, such as amoebae and trypanosomes, a number of species of which are serious parasites. Accordingly, such phosphotyrosine phosphatase inhibitors can also be useful in treating protozoan-based diseases. It is predicted that the developmentally stage-specific tyrosine phosphorylation is disrupted in these organisms. This disruption is ■ predicted to lead to death of the protozoa (M. Parsons et al., "Distinct Patterns of Tyrosine Phosphorylation During the Life Cycle of Trypanosoma brucei. " Molec. Biochem. Parasitol. 45:241-248 (1990)).

The method of inhibiting B cell proliferation comprises the step of contacting proliferating B cells with a phosphotyrosine phosphatase inhibitor as

described above. The compound is administered in a quantity sufficient to detectably inhibit proliferation of the cells as measured by incorporation of nucleotides into DNA. The term "detectably inhibit proliferation, " as used herein, refers to a detectable decrease in either DNA synthesis or cell number, inasmuch as cell division follows DNA synthesis. Typically, the dose required is in the range of 1 μM to 100 μM, more typically in the range of 5 μM to 25 μM. The exact dose required can be readily determined from in vitro cultures of the cell and exposure of the cells to varying dosages of the phosphotyrosine phosphatase inhibitor. The effect of the phosphotyrosine phosphatase inhibitor, or other treatments such as radiation, can be judged by clonogenic assays, assays measuring the incorporation of radioactively labeled nucleotides into DNA, or other assays measuring cell proliferation.

For this and other treatment methods that are aspects of the present invention, one of ordinary skill in the art could determine effective dosages and other treatment modalities by animal studies. This approach is described in D.J. Brusick, "The Use of Short Term In Vitro and Submammalian Tests as Alternatives to Large Scale Animal Bioassays, " in The Role of Animals in Biomedical Research (J.A. Sechzer, ed. , New York Academy of Sciences, New York, New York, 1983 (Annals of the New York Academy of Sciences, vol. 406)), pp. 68-73, as well as in M.D. Rawlins, "What Is Expected from Repeated-Dose Studies by Clinical Pharmacologists," in Long-Term Animal Studies: Their Predictive Value for Man (S.R. Walker & A.D. Dayan, eds. , MTP Press Ltd., Lancaster, 1986) , pp. 13-22. Other approaches for determination of effective or optimal dosages and treatment modalities, such as the use of cell or tissue culture and

mathematical modeling, are known to those of ordinary skill in the art.

2. Treatment Of Lvmphoproliferative Disorders

Because of the effect of phosphotyrosine phosphatase inhibitors on proliferation of cells, particularly B cells, but also myeloid cells, such inhibitors can be used in a method of treating malignant proliferative disorders. The diseases that can be treated include leukemias and lymphomas, and the proliferating cells can be either B cells, or, alternatively, myeloid cells. The method comprises the step of contacting the proliferating malignant cells with a phosphotyrosine phosphatase inhibitor as described above. The compound is administered in a quantity sufficient to significantly inhibit proliferation of the malignantly proliferating cells, as that term is defined above. The dosage range in general is as described above. Further guidance for the dosage is given in the Examples below.

The compositions can be administered using conventional modes of administration including, but not limited to, intravenous, intraperitoneal, oral, or intralymphatic. Other routes of injection can alternatively be used. Oral or intraperitoneal administration is generally preferred. The composition can be administered in a variety of dosage forms which include, but are not limited to, liquid solutions or suspensions, tablets, pills, powders, suppositories, polymeric microcapsules or microvesicles, liposomes, and injectable or infusible solutions. The preferred form depends on the mode of administration and a quantity administered.

The compositions for administration including the phosphotyrosine phosphatase inhibitors preferably also include conventional pharmaceutically acceptable carriers and adjuvants known in the art such as human serum albumin, ion exchangers, alumina, lecithin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, and salts or electrolytes such as protamine sulfate. The most effective mode of administration and dosage regimen for the phosphotyrosine phosphatase inhibitors as used in the methods of the present invention depend on the severity and course of the disease, the patient's health, the response to treatment, the particular type of malignantly proliferating cells characteristic of the particular leukemia or lymphoma, pharmacokinetic considerations such as the condition of the patient's liver and/or kidneys that can affect the metabolism and/or excretion of the administered phosphotyrosine phosphatase inhibitors, and the judgment of the treating physician. Accordingly, the dosages should be titrated to the original patient. Nevertheless, an effective dose of the phosphotyrosine phosphatase inhibitors for use in the treatment methods of the present invention can be in the range from about 1 μM to about 100 μM in the blood and/or tissues.

Preferably, the dose of phosphotyrosine phosphatase inhibitor used is sufficient to induce apoptosis in the malignantly proliferating cells.

The treatment method can further comprise a step of delivering ionizing radiation to the cells contacted with the phosphotyrosine phosphatase inhibitor. The ionizing radiation is delivered in a dose sufficient to induce a substantial degree of cell killing among the malignantly proliferating cells, as judged by assays measuring viable malignant cells. The

degree of cell killing induced is substantially greater than that induced by either the phosphotyrosine phosphatase inhibitor alone or the ionizing radiation alone. Typical forms of ionizing radiation include beta rays, gamma rays, alpha particles, and X-rays. These can be delivered from an outside source, such as X-ray machine or a gamma camera, or delivered to the malignant tissue from radionuclides administered to the patient . The use of radionuclides is well understood in the art and need not be detailed further. The use of ionizing radiation in the treatment of malignancies is described, for example, in S. Hellman, "Principles of Radiation Therapy" jLn Cancer: Principles & Practice of Oncology (V.T. DeVita, Jr., S. Hellman, & S.A. Rosenberg, eds . , 4th ed., 1993, J.B. Lippincott Co., Philadelphia) , ch. 15, pp. 248-275.

A range of dosages that can be used is between about 1 and 500 cGy (i.e., from about 1 to about 500 rads) .

When phosphotyrosine phosphatase inhibitors according to the present invention are used along with ionizing radiation, typically, the dose of the phosphotyrosine phosphatase inhibitor used is equal to or less than the dose that would be used if the phosphotyrosine phosphatase inhibitor were used alone.

3. Inhibition of Phosphotyrosine Phosphatases

Methods according to the present invention can also be used to inhibit phosphotyrosine phosphatases for purposes other than that of treating malignant disease.

In particular, phosphotyrosine phosphatase inhibitors can be used to suppress the immune system in

order to prevent organ and tissue rejection during transplantation and also in the treatment of autoimmune diseases such as rheumatoid arthritis, Hashimoto's thyroiditis, systemic lupus erythematosus, Guillain- Barre Syndrome, and possibly multiple sclerosis.

Methods according to the present invention can also be used in the treatment of protozoan infections, such as amoebae and trypanosomes, whose proliferation also depends on phosphorylation.

Additionally, methods according to the present invention can be useful in the studies of B cells in culture, in order determine their susceptibility to ionizing radiation and other reagents, such as alkylating agents and intercalating agents that can interfere with DNA synthesis, and can be used to screen reagents for their activity to interfere with DNA synthesis. In particular, methods according to the present invention can be used to block B cell development in vitro in order to determine the effect of inhibition of proliferation of B cells on development of immune responses. For example, such methods could be used to augment signals and enhance calcium levels in cells (see Example 8) . Such methods can also be used to block the effects of CD28. Accordingly, such methods can therefore be used to screen B cell cultures for abnormal UV-induced signals. Such screening could be clinically useful in determining susceptibility to conditions related to ultraviolet exposure, such as skin cancer. This may be particularly useful in studying conditions linked to abnormal ultraviolet sensitivity. Such conditions include Bloom's Syndrome and xeroderma pigmentosum.

. Prevention Of Class-Switching In Antibodies

Methods according to the present invention can further be used to prevent class-switching in antibodies

from Ig or IgM to IgE. It is desirable to block IgE production because this type of antibody mediates many allergic responses, particularly immediate-type hypersensitivity reactions such as anaphylaxis, atopy, and urticaria. The CD40 ligand gp39 and the cytokine IL-4 act on B cells to induce the switching of the type of ^" antibody produced from IgM to IgE. CD40 and IL-4 mechanisms of action are known to involve tyrosine phosphorylation. Phosphotyrosine phosphatase inhibitors such as BMLOV disrupt the normal pattern of tyrosine phosphorylation, thus disrupting the class-switching process. The administration of BMLOV, in particular, can markedly inhibit the production of IgE antibody while much less markedly inhibiting the production of

IgG subclasses such as IgGl and IgG . This leads to the result that the ratio of IgG to IgE increases (Example 14) . These results lead to the conclusion that phosphotyrosine phosphatase inhibitors such as BMLOV have value in the treatment of allergy.

Accordingly, a method of preventing the class- switching of antibody-producing cells comprises administering to antibody-producing cells a quantity of a phosphotyrosine phosphatase inhibitor sufficient to detectably reduce the production of IgE antibody by the cells. Typically, the cells also produce IgG antibody, and the quantity of the phosphotyrosine phosphatase inhibitor is such that the ratio of the quantity of IgG antibody produced by the cells to the quantity of IgE antibody produced by the cells increases.

Any of the phosphotyrosine phosphatase inhibitors disclosed above can be used to prevent class- switching, including metal-organic coordinate-covalent compounds, non-hydrolyzable phosphotyrosine analogs, dephostatin, and 4- (fluoromethyl) phenyl phosphate and its esterified derivatives. However, a preferred phosphotyrosine phosphatase inhibitor for preventing class-switching is BMLOV.

5. Purgation of Bone Marrow for Autologous

Transplantation

Another use for phosphotyrosine phosphatase inhibitors according to the present invention is in purging bone marrow in preparation for autologous transplantation following whole body irradiation and/or chemotherapy. This type of therapy is often attempted for multiple myeloma, which is a B cell malignancy. Thus, reagents that inhibit replication of B cells, such as phosphotyrosine phosphatase inhibitors according to the present invention, would be expected to inhibit growth of the residual B cells present in bone marrow and assist in purging bone marrow prior to autologous transplantation.

B. Use of Reagents That Stimulate Tyrosine Kinases

Because the degree of phosphotyrosine phosphorylation in cells can be affected either by inhibition of phosphotyrosine phosphatase or the stimulation of tyrosine kinase, methods according to the present invention can also be practiced with reagents that either stimulate tyrosine kinases, or both inhibit phosphotyrosine phosphatase and stimulate tyrosine kinases.

Typically, reagents in this category are coordinate-covalent complexes comprising: (1) a metal ion selected from the group consisting of molybdenum (VI) , vanadium (V) , or tungsten (VI) ; (2) at least one oxo group coordinate-covalently bound to the metal ions; (3) at least one organic moiety coordinate-covalently bound to the metal ion through at least one N- containing, O-containing or As-containing moiety capable of donating electrons to a coordinate-covalent bond; and (4) optionally, one or two peroxo groups coordinate- covalently bound to the metal ion, each occupying two sites in the coordination sphere of the metal ion.

The coordinate-covalent complex has an affinity for the active site of a phosphotyrosine phosphatase sufficient to detectably inhibit the activity of one or more phosphotyrosine phosphatases. Preferably, the coordinate-covalent complex has an affinity for the active site of phosphotyrosine phosphatase at least about equal to the affinity of bis (maltolato) oxovanadium (IV) for the active site of phosphotyrosine phosphatase.

Preferably, a coordinate-covalent complex possessing both phosphotyrosine phosphatase inhibiting and tyrosine kinase stimulating activity comprises: (1) a metal ion selected from the group consisting of molybdenum (VI) , tungsten (VI) , and vanadium (V) ; (2) an oxo group coordinate-covalently bound to the metal ion; (3) at least one peroxo group coordinate-covalently bound to the metal ion; and (4) at least one organic moiety coordinate-covalently bound to the metal ion through at least one N-containing or O-containing functional group capable of donating electrons to a coordinate-covalent bond.

In one particularly preferred class of compounds that affect both tyrosine kinase and phosphotyrosine phosphatase activity, the metal ion is vanadium. When the metal ion is vanadium, the coordinate-covalent complex preferably has two peroxo groups bound to the metal ion, but can, alternatively, have one peroxo group bound to the metal ion. When the complex has two peroxo groups bound to the metal ion, particularly preferred coordinate-covalent complexes include, but are not limited to, (1, 10-phenanthroline) oxodiperoxovanadium (V) , oxalato-oxodiperoxovanadium (V), (2, 2 ' -bipyridine) oxodiperoxovanadium (V) , (4,7- dimethyl-1, 10-phenanthroline) oxodiperoxovanadium (V) , (3,4,7, 8-tetramethyl-l, 10-phenanthroline) oxodiperoxovanadium (V) , (pyridine-2-carboxylic acid) oxodiperoxovanadium (V) , (5-hydroxypyridine-2-carboxylic acid) oxodiperoxovanadium (V), (pyridine-2, 6- dicarboxylic acid) oxodiperoxovanadium (V) , and derivatives thereof possessing substantially equivalent affinity for the active site of phosphotyrosine phosphatase.

A particularly preferred vanadium-containing coordinate-covalent complex is (1, 10-phenanthroline) oxodiperoxovanadium (V) .

When the metal is vanadium and the coordinate- covalent complex has one peroxo group bound to the metal ion, a preferred coordinate-covalent complex is (pyridine-2, 6-dicarboxylato) (hydrato) oxoperoxovanadium (V) .

Alternatively, the metal ion in the coordinate-covalent complex can be molybdenum (VI) . The coordinate-covalent complex can have one or two peroxo groups bound to the metal ion when _. molybdenum is the metal ion. When the coordinate-covalent complex has two

peroxo groups bound to the metal ion, preferred coordinate-covalent complexes with molybdenum (VI) as the metal ion include, but are not necessarily limited to, bis (dimethylformamido) oxodiperoxomolybdenum (VI) and hydrogen (pyridine-2-carboxylato) oxodiperoxomolybdenum (VI) hydrate.

When the metal ion is molybdenum, and the coordinate-covalent complex has one peroxo group bound to the metal ion, preferred coordinate-covalent complexes include, but are not necessarily limited to, [ (N-salicylidene) -2-hydroxybenzeneamine] [ethanol] oxoperoxomolybdenum (VI) , (pyridine-2, 6-dicarboxylato) (hydrato) oxodiperoxomolybdenum (VI) , and bis (N- phenylbenzohydroxamato) oxodiperoxomolybdenum (VI) .

Alternatively, a coordinate-covalent complex suitable for the processes of the present invention can comprise: (1) a metal ion selected from the group consisting of molybdenum (VI) , tungsten (VI) , and vanadium (V) ; (2) an oxo group coordinate-covalently bound to the metal ion; and (3) at least one organic moiety coordinate-covalently bound to the metal ion through at least one N-containing or O-containing moiety capable of donating electrons to a coordinate-covalent bond. The coordinate-covalent complex has an affinity for the active site of a phosphotyrosine phosphatase sufficient to detectably inhibit the activity of one or more phosphotyrosine phosphatases. Preferably, the • coordinate-covalent complex has an affinity for the active site of phosphotyrosine phosphatase at least about equal to the affinity of bis (maltolato) oxovanadium (IV) for the active site of phosphotyrosine phosphatase.

When the metal ion is molybdenum, a particularly preferred coordinate-covalent complex is

bis (benzene-1,2-di [dimethylarsino) ] [chloro] oxomolybdenum (VI) . When the metal ion is vanadium, preferred coordinate-covalent complexes include, but are not necessarily limited to, meso-tartrato oxovanadium (V) .

In addition to the particular coordinate- covalent complexes disclosed above, other coordinate- covalent complexes can be used for the processes of the present invention. These include derivatives of these complexes substituted with hydroxy or lower alkyl (C^Cg) substituents that do not interfere with the formation of coordinate-covalent bonds.

Typically, the coordinate-covalent complexes are salts in which the coordinate-covalent complex is an anion; in these salts, the cation can be sodium, potassium, ammonium, a quaternary ammonium, or another cation with a +1 charge.

In particular, (1,10-phenanthroline) oxodiperoxovanadium (V) , known as pV(phen) , activates at least one type of tyrosine kinase, the Syk family kinases. Compounds described herein are believed to share this activation activity. Although Applicant is not bound by this theory, it is believed that pV(phen) causes relative lymphopenia or depletion of lymphocytes in the blood and also causes substantial neutrophilia, or increase in neutrophils. This is significant because most cytotoxic anti-cancer drugs cause neutropenia, a diminution of neutrophils which is a severe problem and a serious side effect of many cytotoxic drugs in patients administered such drugs.

In contrast to the compounds described above in Section A, the ability of pV(phen) to induce tyrosine phosphorylation in both T and B cells correlates with

its biological effects on both cell types. This is in contrast to BMLOV (Example 4) , which induces tyrosine phosphorylation in B cells much more than in T cells and which has much more potent biological effects on B cells than on T cells.

Accordingly, these compounds can inhibit proliferation of cell types including B cells, T cells, and cells derived from malignant transformation of B cells or T cells.

Methods of inhibiting proliferation of these cell types can be used to treat a subject suffering from a malignant proliferative disorder wherein the proliferating malignant cells are B cells, T cells, cells derived from malignant transformation of B cells or T cells, or myeloid cells. These methods can be combined with the use of ionizing radiation as described above. The degree of cell killing induced by the combination of the metal-containing coordinate-covalent complex and ionizing radiation is preferably substantially greater than that induced by either the coordinate-covalent complex or the ionizing radiation alone.

These coordinate-covalent compounds can also be used to prevent the class-switching of antibody- producing cells, as described above.

More generally, these metal-containing coordinate-covalent complexes can be used in a method of inducing tyrosine phosphorylation in a cell type selected from the group consisting of B cells, T cells, and cells derived from malignant transformation of B cells or T cells.

One particular application of these methods according to their present invention is in treating tumors, both tumors derived from B cells or T cells, such as leukemias or lymphomas, and solid tumors of other origins such as melanomas. Tumors susceptible to treatment by these methods include tumors comprised of tumor cells overexpressing one or more members of the EGF receptor family (HER1, HER2 or neu, HER3, and HER4) , other receptor tyrosine kinases (RPTK) or other tyrosine kinases (PTK) such as, but not limited to, Src. These phosphotyrosine phosphatase inhibitors and/or phosphotyrosine kinase activators, such as pV(phen) , are likely to selectively kill tumor cells that express RPTK such as HER2. Tumors susceptible to treatment by these methods also include tumors comprised of tumor cells requiring phosphotyrosine phosphatase for their growth and/or survival. An example of this latter type of tumor cells is melanoma cells, which, although not known to overexpress tyrosine kinases, are nevertheless sensitive in vivo to pV(phen) (Example 17, infra) .

Numerous published papers have reported that a desirability of using tyrosine kinase inhibitors to block signals from RPTK such as HER2 in tumor cells. The use of PTP inhibitors is the opposite of this widely held concept because it would instead greatly increase the signal . This concept is highly relevant to cancer treatment because RPTK such as HER2 are overexpressed in a variety of cancers, notably breast and ovarian cancers, as well as other cancers. Furthermore, the overexpression of HER2 has been shown to be an indicator of poor prognosis in patients, in that tumors frequently resist all known therapies. Therefore, treatments with • PTP inhibitors using processes according to the present invention would thus affect the tumors with the worst prognosis.

Overexpression of these kinases can be determined by assay of the kinases in culture and comparison to cells not of malignant origin.

The invention is illustrated by the following Examples. The Examples are for illustrative purposes only and are not intended to limit the invention.

EXAMPLES

Example 1

Inhibition of Phosphotyrosine Phosphatases by Bis (Maltolato) Oxovanadium (IV) (BMLOV)

Bis (maltolato) oxovanadium (IV) (BMLOV) was synthesized as described in J.H. McNeill et al . , "Bis (maltolato) oxovanadium (IV) is a Potent Insulin Mimic," J. Med. Chem. 35:1489-1491 (1992) . Briefly, the compound was synthesized by combining maltol (3-hydroxy-

2-methyl-4-pyrone) and vanadyl sulfate in a 2:1 ratio in water. The pH of the solution was raised to 8.5, and the solution was refluxed overnight. A deep purple- green birefringent compound precipitated on cooling and was collected. This compound is the BMLOV.

BMLOV was then assayed for its activity in inhibiting several phosphatases, including phosphotyrosine phosphatases PTPIB and CD45, as well as serine/threonine phosphatases PP1 and PP2A and calf intestinal alkaline phosphatase.

The tyrosine phosphatases PTPIB and CD45 were assayed with phosphorylated yelin basic protein as described in K. Guan et al . , Nature 350:359-362 (1991) except that 2-minute assays were performed. The PTPIB used was a GST fusion protein (Upstate Biotechnology,

Lake Placid, NY) . The CD45 was immunoprecipitated from Jurkat T cells with the monoclonal antibody 9.4 (ATCC No. HB 10270, deposited with the American Type Culture Collection, Rockville, MD, on October 20, 1989) . The phosphatases PPl and PP2A (Upstate Biotechnology) and calf intestinal alkaline phosphatase (Sigma, St. Louis, MO) were assayed with β-nitrophenyl phosphate as a substrate.

The results are shown in Table 1.

TABLE 1

Phosphatase Inhibition by BMLOV

Phosphatase IC ₅₀ ,NM SEM

PTPIB 26 7

CD45 25 1

PPl 6156 360

PP2A 3337 208

Alkaline Phosphatase >5xl0 ⁵

BMLOV was found to be a potent inhibitor of PTPIB and CD45, both of which are phosphotyrosine

phosphatases. The drug showed substantial selectivity for the phosphotyrosine phosphatases relative to other phosphatases. Much higher concentrations of the drug were required to inhibit the serine phosphatases PPl and PP2A. Intestinal alkaline phosphatase was highly resistant to inhibition by BMLOV.

Example 2

Induction of Tyrosine Phosphorylation in Transformed Lymphocytes

Ramos cells, a human B cell lymphoma cell line, were treated with varying doses of BMLOV. The cells were lysed and lysates were subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) . The resulting electropherograms were subjected to Western blotting with anti-phosphotyrosine antibody to detect the presence of phosphotyrosine in the cells.

The results are shown in Figures 1A-1C. Figure IA demonstrates that BMLOV induces tyrosine phosphorylation in a dose-dependent manner when the cells were treated with varying doses for a period of 1 hour. Figure IB shows the effects of treating cells with 100 μM BMLOV for varying times. The drug was found to begin to induce tyrosine phosphorylation in the cells by 8 minutes and the phosphorylation increased with further exposure.

Figure 1C demonstrates that treatment of the cells for 16 hours with 50 μM BMLOV leads to high levels of tyrosine phosphorylation.

Example 3

Induction of Apoptosis by BMLOV

BMLOV was demonstrated to kill malignant leukemia and lymphoma cells by inducing apoptosis. Apoptosis is the process of programmed cell death. The hallmark of apoptosis is the fragmentation of DNA into fragments whose size distribution is observed as a ladder of bands on agarose gels. Figure 2A demonstrates that BMLOV induced apoptosis in Ramos B cell lymphoma cells by 48 hours at a dose of 10 μM, with apoptosis apparent at 24 hours with higher doses. Apoptosis was also observed to be induced in the human acute promyelocytic leukemia cell line HL60 in a dose dependent manner (Figure 2A) . The induction of apoptosis was specific in that apoptosis was not observed in the human T-cell leukemia cell lines CEM or Jurkat, nor was it observed in the human colon carcinoma cell line 3347 (Figure 2B) .

For these experiments, cell samples (2xl0 ⁶ cells) were centrifuged at 200 x g, the media removed by aspiration and the cell pellets stored at -70°C until processing. Cell pellets were resuspended in 300 μl digestion buffer (100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 25 mM EDTA, 0.5% SDS, 0.1 mg/ml protease K) and incubated for 12 hours at 50°C. DNase-free ribonuclease (5 μg) in 200 μl TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) was added to each sample followed by incubation for 2 hours at 37°C. DNA was then extracted once with phenol :chloroform, once with chloroform and then precipitated with 0.5 volumes of ammonium acetate and 2 volumes of ethanol at -70°C for 12 hours. The precipitated DNA was resuspended in 200 μl TE buffer and quantitated by absorbance at 260 nanometers. The DNA (15 μg) was applied to a 1.3% agarose gel in TBE buffer

(89 mM Tris base, 89 mM boric acid, 2 mM EDTA) resolved at 20 mA constant current and the DNA was visualized by staining with ethidium bromide. Fragmented DNA due to apoptosis appeared as a ladder of bands on the gel.

Example 4

Selective Cvtotoxicity of BMLOV as Examined in Clonogenic Assays

The selective cytotoxicity of BMLOV was examined in clonogenic assays. Cells were grown in methylcellulose media and the number of colonies formed after seven days of treatment with various doses of the drug were determined. The clonogenic assays were performed with six replicates for each treatment as described [F.M. Uckun et al. , J. Exp. Med. 163:347-368 (1986) ; F. M. Uckun & J. A. Ledbetter, Proc. Natl. Acad. Sci. USA 85:8603-8607 (1988)) . The data is given plus/minus the standard error of the mean.

The data is shown in Table 2. The three B- cell lines, Ramos, Raji, and REH, were all highly sensitive to doses of 5 to 10 μM BMLOV. The myeloid cell line THP-1 and the promyelocytic cell line HL-60 also were highly sensitive to BMLOV at a dose of only lμM. BMLOV gave 99.8% clonogenic cell death for the Raji transformed B cell line at a dose of 10 μM, and 99.4% clonogenic cell death for HL-60 promyelocytic leukemia cells at 1 μM.

TABLE 2

Inhibition of Leukemia and Lvmphoma Cell Line Growth

by BMLOV as Measured in Clonogenic Assays

Dose,μM Cell Line

Ramos Raii REH THP-1 HL-60

0 4065±840 3252±672 5122±1296 6453±1563 5122±1241

1.0 179±33 1291±283 1810±385 1613±235 31±8 (95.6%) (60.3%) (64.7%) (75.0%) (99.4%)

5.0 7±2 4±0.6 71±8 127±35 31±8 (99.8%) (99.9%) (98.6%) (98.0%) (99.4%)

10.0 0 3±0.8 8±1 127+35 15+4

(99.9%) (99.8%) (98.0%) (99.7%)

Data is given in colonies per 10 ⁴ cells; the percentage of inhibition is shown in parentheses.

By contrast, the T cell line CEM or MDAMB-453 breast carcinoma cells were inhibited only moderately by a dose as high as 100 μM of BMLOV.

Example 5

The Effect of BMLOV on Incorporation of Nucleotides into DNA

In order to determine the effects of BMLOV on various cell types, including a number of cell types that did not grow well in the methylcellulose clonogenic assay, the cells were grown for seven days in the presence of BMLOV. For each cell type, the cells were passaged uniformly for all drug doses given during the seven-day period. The cells were then pulsed for six hours with [ ³ H] thymidine. The extent of DNA synthesis was then determined by counting the radioactivity incorporated into the cells. The data is shown in Table 3.

TABLE 3

Inhibition of Leukemia and Lvmphoma Cell Line Growth bv BMLOV as Measured in Thymidine Incorporation Assays

Dose, μM [ ³ H] cpm Incorporation for Cell Line;

Ramos BC ₁ A20 THP-1 HL-60 Jurkat CEM

0 292342 271144 389480 202301 249183 143917 395157

1 289936 265036 419947 201670 191637 149950 368721

5 65545 226714 289932 178290 276505 118720 403576

10 167 55396 66157 199993 222178 98057 371256

25 149 195 281 112209 11398 26511 313714

Ramos cells were highly sensitive to doses of 10 and 25 μm BMLOV. The murine cell line BC ^ is a highly tumorigenic and lethal leukemia considered to be a model of human chronic lymphocytic leukemia (CLL) . These cells were highly sensitive to the drug at a dose of 25 μM. THP-1 cells showed only partial sensitivity to the drug at a dose of 25 μM. In general, cells were more sensitive to the drug in the clonogenic assays than in the thymidine incorporation assay. These results suggest a greater requirement for phosphatase activity for cells to grow as colonies in methylcellulose relative to growth in free suspension in liquid media.

The murine B cell lymphoma line A20, which forms highly aggressive tumors in mice, was very sensitive to a dose of 25 μM of the drug. The human promyelocytic leukemia cell line HL60 and the human acute T cell leukemia cell line Jurkat were moderately sensitive to BMLOV at a dose of 25 μM, whereas the human T cell acute lymphocytic leukemia CEM was resistant . These results indicate that malignant cells of B cell origin, including lymphoma, acute lymphocytic leukemia, and chronic lymphocytic leukemia are sensitive to BMLOV. Some leukemias of myeloid and T cell origin also show sensitivity to BMLOV whereas others are resistant .

Example 6

Effect of BMLOV on Normal B Cell Proliferation

BMLOV can inhibit normal tonsillar B cell proliferation driven by stimulation of CD40 via either anti-CD40 antibody or gp39 ligand plus either anti-CD20 antibody or phorbol 12-myristate 13-acetate (PMA) . In one experiment, doses of 0.1 to 10 μM had little effect on proliferation (Figure 3) . In a second experiment, a dose of 5 μM gave substantial inhibition of proliferation and a dose of 50 μM completely blocked proliferation (Figure 4) . Variations between the individuals from which the tonsils were derived could account for the differences between these experiments.

Similarly, BMLOV can inhibit the proliferation of normal peripheral B cells . Normal B cell proliferation is mediated in part by the B cell surface molecule CD20, the B cell surface molecule CD40 in conjunction with its ligand gp39, and by the cytokine IL-4. The pharmacologic agent phorbol 12-myristate 13- acetate (PMA) can also be used in combination with these

biological stimulation agents to further increase proliferation.

In the experiment reported in Table 4, monoclonal antibodies to CD20 and CD40 were used to stimulate proliferation. Peripheral B cells were isolated from two healthy human volunteers. The cells were stimulated as listed in Table 4 and the effects of various doses of BMLOV on proliferation, as measured by [ ³ H] -thymidine incorporation, were determined. BMLOV was able to inhibit proliferation induced by CD20, CD40, IL- 4 and PMA in the various combinations tested. The cells from donor 2 were more sensitive, indicating some variation among individuals in their sensitivity to the drug.

It is important to note that the extent to which the proliferation of normal B cells was inhibited in this example (Table 4) was less than for the B cell leukemia and lymphoma cells examined in Examples 4 and 5. Phosphotyrosine phosphatase inhibitors such as BMLOV thus have the potential to act more selectively on malignant B cells than on normal B cells, offering an important advantage in the treatment of leukemia and lymphoma.

TABLE 4

INHIBITION OF NORMAL PERIPHERAL B CELL GROWTH BY BMLOV AS MEASURED IN Hl -THYMIDINE INCORPORATION ASSAYS

Standard error did not exceed 8% for donor 1 and 15% for donor 2. Stimulation of CD20 was via monoclonal antibody 1F5 and stimulation of CD40 was via monoclonal antibody G28-5.

Example 7

Sensitization of Ramos B Cells to Ionizing Radiation by BMLOV

Ionizing radiation in conjunction with bone marrow transplantation is a major therapy for leukemia. However, many leukemias are resistant to radiation, limiting the efficacy of this therapy.

It was previously demonstrated that ionizing radiation stimulates tyrosine kinases in human B lymphocyte precursors, triggering apoptosis and clonogenic cell death, an effect that was markedly enhanced by the phosphatase inhibitor orthovanadate.

In clonogenic assays, as shown in Figure 5, the combination of 1 μM BMLOV with 265 cGy radiation gave a 10-fold increase in cell death relative to radiation alone. The combination of 2μM BMLOV with 265 cGy radiation gave a 50-fold increase in clonogenic cell death relative to radiation alone. These effects were synergistic rather than additive in that an approximately 5-fold enhancement in sensitization for the combination treatment was observed relative to expected additive effects based on the use of either treatment alone. These results suggest that phosphatase inhibitors such as BMLOV can be of value for use in combination with radiation therapy for the treatment of B cell malignancies.

Example 8

Inhibition by BMLOV of CD28-Mediated Ca ²⁺ Signals and Cell Proliferation

BMLOV reproducibly caused marked inhibition of CD28 Ca ²⁺ signals in PHA T cell blasts. The inhibition was specific in that such inhibition was not observed for signals generated by CD3 alone or in combination with CD2 or CD4.

PBL growth driven by anti-CD28 antibody plus interleukin-2 (IL-2) was markedly inhibited by 1 μM BMLOV (Figure 6) and growth driven by anti-CD3 antibody plus anti-CD28 antibody was strongly inhibited by 10 μM BMLOV (Figure 7) .

In contrast, substantially less inhibition was observed for IL-2 alone, PMA in combination with anti- CD28, or the factor-independent growth of the T cell line CEM, indicating the specificity of these effects.

Mixed lymphocyte response cultures that are dependent on CD28 costimulation were inhibited by over 50% by 0.5 to 5 μM BMLOV (Figure 8) . These results suggest the phosphatase inhibitor may selectively block CD28 effects and therefore can be used to block the generation of antibodies to antigens in animals.

An ^' importa.it question has been how does the costi ulatory CD28 signal differ from the primary CD3 dependent signal in T cells, since both signals induce tyrosine phosphorylation and Ca ²⁺ flux. These results raise the possibility that the CD28 costimulatory or second signal requires a BMLOV-sensitive phosphatase activity that the primary signals do not.

Example 9

Augmentation of Signals in T and B Cells bv BMLOV

BMLOV was found to markedly enhance basal calcium levels in PHA blasts when the cells were treated with doses of 50 to 100 μM for fifteen hours. These results indicate that phosphatase activity is required to maintain normal calcium levels in the cell.

Treatment of the cells with 25-50 μM, BMLOV was found to prolong Ca ²⁺ signals generated by crosslinking CD3 alone or in combination with CD2 and CD4. BMLOV greatly increased and prolonged UV-induced Ca ²⁺ signals in Ramos B cells, indicating that phosphatase activity may limit UV-induced signals. In contrast, BMLOV did not alter Ca ²⁺ signals induced in Ramos B cells by crosslinking slg. However, BMLOV enhanced the tyrosine phosphorylation response of Ramos B cells to slg crosslinking, particularly at early time points. These results suggest a potential for BMLOV to augment some cell responses.

Example 10

BMLOV Inhibits Src-Family Kinase Activity in Lymphocytes and Colon Carcinoma Cells

The Src-family kinases are known to require cellular phosphotyrosine phosphatase activity in order for them to respond in biologically stimulated cells. This is because Src-family kinases require a C-terminal tyrosine phosphorylation site to be dephosphorylated for activation to occur. Phosphotyrosine phosphatase inhibitors may therefore be expected to inhibit Src kinases indirectly by preventing their activation.

Treatment of Ramos cells with BMLOV for 15 hours inhibited the activity of the Src kinases Lyn and Fyn from the cells by approximately 50%.

Many colon carcinomas are known to express the

Src oncogene product at high levels. In two colon carcinoma cell lines expressing Src, pp60 ^c"src activity was strongly inhibited by BMLOV in a dose-dependent fashion (Figure 9) , while pp60 ^c"src protein level remained constant. The treated cells showed morphological changes, including decreased adherence.

Example 11

BMLOV Alters the Cell Cycle Progression of Treated Cells

A variety of anti-cancer therapies are known to alter the cell cycle progression of tumor cells. For example, both cisplatin and radiation therapy result in accumulation of cells in G2/M phase.

Ramos B cells treated with various concentrations of BMLOV for 16 hours were examined for their DNA content by propidium iodide staining followed by FACScan flow cytometric analysis using the SFIT program.

The results are shown in Figure 10. The percentages of cells in Gl, S, and G2/M are listed for doses of 0 to 5 μM of BMLOV. Percentages could not be calculated for higher doses due to the ^' strong effects of the drug in causing apoptosis. Apoptotic cells lacking normal amounts of DNA are marked "A" in the 5μM dose panel. BMLOV was found to preferentially deplete the proportion of cells in Gl while increasing the proportion in S phase. Loss of DNA due to apoptotic

cell death was readily apparent in a dose of 5μM and was greatly increased at high doses.

Example 12

Effects of Analogs of BMLOV

Two analogs of BMLOV were prepared, vanadyl 1- benzoyl acetonate and vanadyl 2-acetyl 1-tetralonate.

The synthesis of vanadyl 2-acetyl 1- tetralonate was as follows. A quantity of 2-acetyl 1- tetralone (1.13 g; 0.006 moles) was dissolved in 74 ml of water with the pH adjusted to 13 with NaOH. A small amount of insoluble material was removed. Vanadyl sulfate (0.003 moles) was then added. The solution was then heated and allowed to reflux for 1 hour. The solution was then cooled on ice and 0.884 g product (a green solid) was collected and dried over P ₂ 0 ₅ . The yield was 33%. A similar procedure was used to synthesize vanadyl 1-benzoyl acetonate, starting with 1- benzoyl acetone. Vanadyl acetylacetonate, available commercially, was also studied.

Of these analogs, only vanadyl acetylacetonate and vanadyl 1-benzoyl acetonate strongly induced cellular tyrosine phosphorylation. As shown in Figure 11, anti-phosphotyrosine western blot analysis of whole cell lysates following SDS-PAGE revealed that vanadyl acetylacetonate strongly induced tyrosine phosphorylation in a pattern very similar to that of BMLOV.

Analogs of vanadyl acetylacetonate were prepared in which the vanadium was replaced with other metals, namely molybdenum, chromium, iron, manganese, or copper. Of these analogs with differing metal ions,

only cupric acetylacetonate induced tyrosine phosphorylation, but in a different pattern than for the vanadyl compounds (Figure 11) . Thus, only certain metals are active in this type of compound.

Clonogenic assays of Ramos Burkitt lymphoma cells treated with cupric acetylacetonate showed that the cells were sensitive to the compound at doses of 10 and 25 μM (Table 5) .

TABLE 5

Effect of Cupric Acetylacetonate on Ramos B Cells as Measured in Clonogenic Assays

Dose, μM Colonies SE

0 9126 1944

1 5749 1070

5 226 26

10 127 35

25 31 8

Example 13

BMLOV Was Tolerated by Animals

BMLOV is tolerated by animals when it was administered by oral or intraperitoneal routes. Mice were treated with 1.6 mM of BMLOV in their drinking water continuously for 10 days, and the level of the drug in their blood serum was determined by atomic absorption spectroscopy after 1 and 10 days of treatment, as shown in Table 6. The mice displayed no overt ill effects from the drug treatment. Subsequently, mice have received intraperitoneal doses of BMLOV of up to 1 mg and oral doses of up to 1.6 mg without ill effects except for some temporary lethargy.

TABLE 6

BMLOV Levels in Mouse Sera

Mouse Day BMLOV in Serum, μM

647 9.5

648 5.3

649 7.5

650 8.7

647 10 11.4

648 10 11.0

649 10 7.5

650 10 7.7

Example 14

Prevention of Class-Switching in Antibody-Producing

B Cells bv BMLOV

The effects of BMLOV were assayed on class- switching in antibody-producing B cells. Human B cells producing antibody were treated with anti-CD40 antibody plus IL-4, which increased production of IgE over 10- fold (Table 7) . However, in the presence of 5.6 or 17 μM BMLOV, the increased production of IgE was markedly inhibited. In contrast, the production of IgGl and IgG4 was much less affected, particularly at a dose of 5.6 μM BMLOV. This selective effect is important because the IgG antibody production is an important response to infectious disease. It would be of value to suppress IgE production for the treatment of allergies while maintaining IgG production, particularly in conditions in which an allergy coexists with an infectious disease. A common example is the exacerbation of allergic rhinitis (hay fever) as the result of a respiratory infection.

TABLE 7

EFFECT OF BMLOV ON CLASS-SWITCHING IN ANTIBODY-PRODUCING B CELLS

Anti-CD40+

IL-4+0.002 μM BMLOV 450 1.7 20.4 3.9

Anti-CD40+

IL-4+0.008 μM BMLOV 450 1.1 20.4 2.6

Anti-CD40+

IL-4+0.02 μM BMLOV 390 1.3 19.2 4.5

Anti-CD40+

IL-4+0.07 μM BMLOV 450 2.7 12.0 5.1

Anti-CD40+

IL-4+0.2 μM BMLOV 495 1.2 13.8 7.5

Anti-CD40+

IL-4+0.6 μM BMLOV 480 1.3 17.4 5.7

Anti-CD40+

IL-4+1.9 μM BMLOV 450 2.7 15.6 3.0

Anti-CD40+

IL-4+5.6 μM BMLOV 480 1.8 18.6 1.2

Anti-CD40+

IL-4+17 μM BMLOV 450 0.8 18.0 0.8

Anti-CD40+

IL-4+50 μM BMLOV 60 0.5 5.1 0.2

Example 15 Growth Inhibition and Selective Induction of Apoptosis in B Cell and Myeloid Leukemia Cells by

Phosphotyrosine Phosphatase Inhibition

This example synthesizes and correlates the results previously shown in Example 2, Example 3, Example 4, and Example 6, as well as other examples, using flow cytometry for cell cycle analysis.

Materials and Methods

Cells and reagents. The human Burkitt's lymphoma lines Ramos and Raji, the human T cell leukemia lines CEM and Jurkat, the human acute promyelocytic leukemia line HL-60 and the human acute monocytic leukemia line THP-1 were obtained from the American Type Culture Collection and were grown in RPMI 1640 medium (GIBCO, Gaithersburg, MD) with 10% fetal calf serum. The urine B cell lymphoma line A20 and the murine B cell leukemia line BCL1 were obtained from the American Type Culture Collection and were grown in RPMI 1640 media, 10% fetal bovine serum plus 50 μM β- mercaptoethanol . The human colon carcinoma cell line H3347 (J.S. Marken et al. , "Cloning and Expression of the Tumor-Associated Antigen L6," Proc. Natl. Acad. Sci. USA 89:3503-3507 (1992)) was grown in IMDM medium (GIBCO) with 10% fetal bovine serum. Anti-human CD3 e chain monoclonal antibody G19-4, anti-human CD45 monoclonal antibody 9.4, and anti-human CD40 monoclonal antibody G28-5 have been previously described (J.A. Hansen et al . , "Human T Lymphocyte Cell Surface Molecules Defined by the Workshop Monoclonal

Antibodies," in Leukocyte Typing I (A. Bernard et al. , eds., Springer-Verlag, New York 1984), pp. 195-212; B.F.

Haynes, "Summary of T Cell Studies Performed During the Second International Workshop and Conference on Human Leukocyte Differentiation Antigens," in Leukocyte Typing II (E.L. Reinherz, ed. , Springer-Verlag, New York, 1986) , pp. 3-30; E.A. Clark & J.A. Ledbetter,

"Activation of Human B Cells Mediated Through Two Distinct Cell Surface Antigens, Bp35 and Bp50," Proc. Natl. Acad. Sci. USA 83:4494-4498 (1986) ; J.A. Ledbetter et al . , "CD45 Regulates Signal Transduction and Lymphocyte Activation by Specific Association with

Receptor Molecules on T or B Cells, " Proc. Natl. Acad. Sci. USA 85:8628-8632 (1988)) . Rabbit anti-human IgM (Fab') ₂ was from Jackson Immunoresearch Labs (West Grove, PA) . Protein tyrosine phosphatase PTPIB and serine phosphatases PPl and PP2A were from Upstate

Biotechnology (Lake Placid, NY) . Calf intestinal alkaline phosphatase was from Sigma (St. Louis, MO) . Bis (maltolato) oxovanadium (IV) was synthesized as previously described (J.H. McNeill et al. , (1992), supra; C.P. Stewart & A.L. Porte, "Electron Paramagnetic Resonance Spectra of Some Oxovanadium (IV) Chelates, " J. Chem. Soc. Dalton Trans. 1661-1666 (1972)) . The product was characterized by infrared and mass spectroscopy.

Analysis of Tyrosine Phosphorylation and Phosphatase Activity

Anti-phosphotyrosine immunoblotting was performed as previously described (D. Dailey et al. , "Novel Yeast Protein Kinase (YPK1 Gene Product) is a 40- Kilodalton Phosphotyrosyl Protein Associated with Protein Tyrosine Kinase Activity, " Mol. Cell Biol. 10:6244-6256 (1990)) . Antibody binding was detected using [ ¹²⁵ I] protein A followed by autoradiography. CD45 and PTPIB were assayed using the substrate phosphorylated myelin basic protein as described (K. Guan et al. , "A Tyr/Ser Protein Phosphatase Encoded by

Vaccinia Virus," Nature 350:359-362 (1991)) , except that two-minute assays were performed. The bovine brain myelin basic protein was [ ³² P] labeled on tyrosine by phosphorylation with recombinant Lck tyrosine kinase expressed in a baculovirus-Sf9 insect cell system. Lck specifically phosphorylates bovine myelin basic protein on the tyrosine 67 residue (Q. Wang et al. , "Identification of Tyrosine 67 in Bovine Brain Myelin Basic Protein as a Specific Phosphorylation Site for Thymus ^~ p5β ^lck , " Biochem. Biophys. Res. Comm. 178:1393- 1399 (1991) ) . Baculovirus encoding the lck gene were provided by Dr. Joseph Bolen (Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ) . CD45 was immunoprecipitated from Jurkat T cells with mAb 9.4 and assayed as the immune complex. PPl, PP2A, and calf intestinal alkaline phosphatase were assayed with p- nitrophenyl phosphate as a substrate.

Cellular Proliferation and Clonogenic Assays. For assays of cellular proliferation, cells were grown five days in the presence of the indicated concentrations of BMLOV, pulsed 6 hr with [ ³ H] -thymidine (New England Nuclear, Boston, MA) , and counted in a liquid scintillation counter. Human peripheral blood lymphocytes (PBL) from normal volunteers were isolated from lymphocyte separation medium (Litton Bionetics, Kensington, MD) by density gradient centrifugation. Monocytes were depleted from PBL by two rounds of plastic absorption for 30 minutes at 37°C. B cells were enriched for by negative selection using complement plus monoclonal antibodies to CD2, CD3, CD4, and CD8 for complement depletion. Clonogenic assays of colony formation in methylcellulose medium were performed as previously described (F.M. Uckun et al. (1992), supra) .

DNA Fragmentation and Cell Death. Cellular DNA was extracted and then the state of DNA fragmentation

was analyzed by agarose gel electrophoresis as previously described (K.L. Donaldson et al. , "Cytotoxicity of the Anti-Cancer Agents Cisplatin and Taxol During Cell Proliferation and the Cell Cycle, " Int. J. Cancer 57:847-855 (1994)). Cell death was measured by staining cells with propidium iodine followed by flow cytometry (EPICS C, Coulter) as previously described (N.K. Damle et al. (1993), supra; Y. Liu & C.A.J. Janeway, "Interferon Gamma Plays a Critical Role in Induced Cell Death of Effector T Cells: A Possible Third Mechanism of Self-Tolerance, " J. Exp. Med. 172:1735-1739 (1990)) . Flow cytometric data on forward scatter and red fluorescence arising from propidium iodine binding to cellular DNA was collected for 5,000 cells and analyzed by the Quadstat Program (N.K. Damle, (1993) , supra) . The membranes of dead cells are permeable to propidium iodine (Y. Liu & C . Janeway, (1990) , supra) , but cells undergoing the process of apoptosis can shrink in size while temporarily maintaining plasma membrane integrity (J.J. Cohen et al. , "Apoptosis and Programmed Cell Death and Immunity," Annu. Rev. Immunol. 10:267-293 (1992)). Dead cells exhibiting both reduced size (lower forward scatter) and staining by propidium iodide (elevated red fluorescence) are found in the upper left quadrant, cells with damaged membranes but of normal size are found in the upper right quadrant and compromised cells of reduced size but which are impermeable to propidium iodide are found in the lower left quadrant. Only cells displaying normal size and resistance to staining by propidium iodide are considered viable cells and are found in the lower left quadrant (M.K. Damle et al. (1993), supra) . The percentage of cells in each quadrant was calculated by the Quadstat program.

Cell Cycle Analysis. Cells were fixed, permeabilized and stained for their DNA content with

propidium iodide as previously described (D.H. Pluznik et al . , "Colony Stimulating Factor (CSF) Controls Proliferation of CSF-Dependent Cells by Acting During the Gl Phase of the Cell Cycle," Proc. Natl. Acad. Sci. USA 81:7451-7455 (1984)) . The stained cells were analyzed by flow cytometry on a FACScan (Becton- Dickinson) instrument. The percentage of cells in various stages of the cell cycle was calculated using the CellFIT program.

Results of experiments in which the ability of BMLOV to inhibit various types of phosphatases was studied are shown in Table 8. BMLOV was a highly potent and selective inhibitor of phosphotyrosine phosphatases such as CD45 and PTPIB, displaying IC50 values of approximately 25 nM for these phosphotyrosine phosphatases while requiring over 100-fold greater concentration to give similar inhibition of the serine/ threonine phosphatases PPl and PP2A. The selective inhibition of phosphotyrosine phosphatases relative to serine/threonine phosphatases by BMLOV is similar to results in previous studies on sodium orthovanadate (J.A. Gordon, "Use of Vanadate as a Protein- Phosphotyrosine Phosphatase Inhibitor, " Meth. Enzvmol . 201B:477-482 (1991)) . Calf intestinal alkaline phosphatase was inhibited less than 10% by 500 μM BMLOV, whereas sodium orthovanadate has been reported to strongly inhibit calf intestinal alkaline phosphatase at concentrations of less than 20 μM (A.M. Cortizo et al. , "Vanadium Compounds: Their Action on Alkaline

Phosphatase Activity," Biol. Trace Elem. Res. 41:331-339 (1994)), indicating a greater degree of selectivity by BMLOV relative to orthovanadate.

TABLE 8

SELECTIVE INHIBITION OF PHOSPHOTYROSINE

PHOSPHATASES BY BMLOV

SEM = standard error of the mean n = number of experiments

The potent PTP inhibitor phenylarsine oxide has been reported to strongly induce tyrosine phosphorylation in T cells, but the high reactivity of this compound with protein sulfhydryl groups limits its utility for biological studies and for in ^' vivo use (P.G. Garcia-Morales et al., "Tyrosine Phosphorylation in T Cells Is Regulated by Phosphatase Activity: Studies with Phenylarsine Oxide," Proc. Natl. Acad. Sci. USA 87:9255- 9259 (1990) ; M.C. Fletcher et al . , "Complex Effects of Phenylarsine Oxide in T Cells, " J. Biol. Chem. 268:23697-23703 (1993)) . Accordingly, the effect of BMLOV on the induction of cellular tyrosine phosphorylation was examined. BMLOV displayed a dose- dependent induction of tyrosine phosphorylation in Ramos B cells (Fig. 12A) .

In the experiments shown in Figures 12A-12C, anti-phosphotyrosine immunoblotting of cellular proteins was performed using [ ¹²⁵ I] -protein A followed by autoradiography to detect antibody binding. In Figure 12A, Ramos B cells were treated with the indicated concentrations of BMLOV for 16 h, or were stimulated with goat anti-human IgM F(ab') ₂ (α-μ) for 1 min. The α- μ lane at right is a 6-fold shorter autoradiographic exposure than the left α-μ lane to show greater detail.

In Figure 12B, Ramos B cells were untreated (lane 1) or treated 30 min with either 100 μM sodium orthovanadate (lane 2) or 100 μM BMLOV (lane 3) .

In Figure 12C, murine B cell lymphoma A20 cells and the human T cell leukemia CEM and Jurkat were treated 16 hr with the indicated concentrations of BMLOV.

At a dose of 25 μM, the pattern of tyrosine phosphorylation was very similar to that induced by crosslinking surface IgM, but the level of phosphorylation was substantially less than that obtained following a maximal stimulation of the antigen receptor (anti-μ) . The phosphorylation was prolonged, however, as evidenced by the results of continuous exposure to the drug shown in Fig. 12A. These results indicate that BMLOV is active in cells, in addition to its activity against PTPs in direct enzyme assays.

The induction of cellular tyrosine phosphorylation by BMLOV was compared to that induced by the widely used PTP inhibitor sodium orthovanadate. As shown in Figure 12B, BMLOV was more potent than orthovanadate in the induction of tyrosine phosphorylation in Ramos B cells. The strong induction of cellular tyrosine phosphorylation at 30 min (Fig.

12B) indicates that BMLOV acts rapidly to induce cellular tyrosine phosphorylation. However, the ability of BMLOV to induce tyrosine phosphorylation was cell type specific. BMLOV induced only low levels of tyrosine phosphorylation in the human T cell leukemia lines CEM and Jurkat, whereas the murine B cell lymphoma line A20 was highly responsive (Fig. 12C) . These results indicate that BMLOV shows selectivity for B cells relative to T cells in the induction of tyrosine phosphorylation.

In addition, the induction of apoptosis and altered cell progression by BMLOV was examined. BMLOV induced apoptosis in Ramos B cell lymphoma lines within 48 hours at a dose of 10 μM, as demonstrated by the induction of extensive DNA fragmentation (Fig. 13) . In Figure 13, cells were treated with BMLOV for the times and at the concentrations indicated. DNA was extracted and the state of DNA fragmentation was examined by agarose gel electrophoresis. This pattern of DNA fragmentation is a hallmark of apoptosis in many cell types, including lymphocytes, as a result of cleavage of DNA between nucleosomes (J.J. Cohen (1992) , supra) . Higher doses induced DNA fragmentation within 24 hours of treatment. DNA fragmentation was also induced in the human acute promyelocytic leukemia cell line HL60 in a dose dependent manner (Fig. 13) . Induction of apoptosis was specific for myeloid and B cell lineages in that DNA fragmentation was not observed in the human T cell leukemia cell lines CEM or Jurkat, nor was it observed in the human colon carcinoma cell line 3347 (Fig. 13) .

Since crosslinking slg can induce growth arrest at the Gl stage of the cell cycle in B-lineage lymphoma cells in a process requiring the Lyn tyrosine kinase (R.H. Scheuermann et al. (1994), supra) . the effect of PTP inhibition on cell cycle progression was

investigated. The DNA content of Ramos B cells was examined by staining with propidium iodide followed by flow cytometry (Fig. 14) .

In Figure 14, Ramos cells growing in log phase were treated with BMLOV for 48 hr at the indicated concentrations. DNA in the cells was stained with propidium iodide and quantitated by flow cytometry as described previously. The calculated percentage of cells at the stages of the cell cycle is shown. "A" marks apoptotic cells lacking normal amounts of DNA in the 5 μM dose panel .

BMLOV preferentially depleted the proportion of cells in Gl in a dose dependent manner. The proportion of cells dropped from 34% of the control cells to only 14% in cells treated with 5 μM of the inhibitor. Simultaneously, the proportion of cells in S phase increased from 56% for control cells to 77% for cells treated with 5 μM BMLOV. The proportion of cells in G2/M did not change substantially. At a dose of 5 μM, apoptotic cells lacking normal amounts of DNA were readily apparent, and the proportion of cells undergoing apoptosis showed a clear increase with increasing doses of the inhibitor (Fig. 14) . At higher doses the percentages of cells at various stages of the cell cycle could not be calculated due to the strong induction of apoptosis. These findings confirm that BMLOV induces apoptosis in a dose dependent manner and indicate that the inhibitor also perturbs normal cell cycle progression in the cells, resulting in a decrease in the fraction in cells in Gl phase and an increase in the fraction of cells in S phase.

Therefore, the ability of the inhibitor to induce apoptosis and alter cell cycle progression sufficiently to block clonogenic cell growth was also

examined. In vitro methylcellulose colony assays of clonogenic cell growth revealed that the B cell Burkitt lymphoma lines Ramos and Raji were both highly sensitive to BMLOV treatment, displaying greater than 99.9% inhibition when treated with 10 μM of the inhibitor (Table 9) . These results are consistent with the induction of apoptosis previously observed for the Ramos cells (Example 11) . These results for BMLOV contrast strongly with previous studies of vanadate, which only gave slight effects on Ramos cells in clonogenic assays (F.M. Uckun et al. (1992), supra) . BMLOV is much more effective than orthovanadate in the induction of Ramos cellular tyrosine phosphorylation (Fig. 12B) , and this may account for the difference in clonogenic assay results. The acute monocytic leukemia line THP-1 was also strongly inhibited by BMLOV, displaying 98% inhibition of clonogenic cell growth.

TABLE 9

INHIBITION OF CLONOGENIC CELL GROWTH BY BMLOV

Data is given in terms of colonies formed per 10,000 cells + the standard error of the mean

Since other cell types of interest did not grow well in the clonogenic assay, their proliferation was examined utilizing [ ³ H] -thymidine incorporation assays (Fig. 15A) . In the experiments for which data is shown

in Figure 15A, cells were grown for 5 days in the presence of 10 μM, 25 μM, or no BMLOV and proliferation was measured by [ ³ H] -thymidine incorporation. The percentage inhibition of proliferation by the BMLOV treatment is shown. Assays were performed with 4 replicates and error bars show the standard error of the mean. Asterisks (*) mark conditions giving 99.9% or greater inhibition of proliferation.

Ramos cells were highly sensitive in this assay, consistent with the results in the previous assays. Murine B cell leukemia BCL _X cells and murine B cell lymphoma A20 cells were also highly sensitive to the inhibitor. Whereas all the B cell lines examined were highly sensitive to BMLOV, the myeloid and T cell lines examined were much more variable. At the lower dose of 10 μM, all the B cell lines were much more sensitive to BMLOV than were the other cell types. However, the myeloid lines HL-60 and THP-1 showed an inhibition of growth at the higher dose of 25 μM. Although only small effects were observed for CEM T cells, Jurkat T cell proliferation was substantially inhibited at 25 μM, even though apoptosis was not observed for these cells.

The extent to which the T cells were being killed by BMLOV treatment relative to the Ramos B cells was therefore examined. In Figure 15B, cells were grown for 4 days either in the absence (control) or continuous presence of 25 μM BMLOV. Viability was measured by staining with propidium iodide followed by flow cytometric analysis as described above. The percentage of cells in each quadrant is indicated. The lower right quadrant contains the viable cells. As shown in Figure 15B, both the CEM and Jurkat T cell leukemia lines were resistant to killing by BMLOV, with the CEM cells showing the greatest resistance. These results indicate

that the inhibition of Jurkat cell proliferation was not primarily due to cell death. By contrast, the Ramos B cells were substantially more sensitive to killing by BMLOV, indicating that the induction of cell death plays an important role in the virtually complete inhibition of proliferation observed for the Ramos cells.

The strong effects of BMLOV on B cell leukemia and lymphoma cells suggested that the effect of BMLOV on the growth of normal peripheral B cells be studied (Fig. 16A) . Normal peripheral B cells require a mitogenic stimulus to grow, so the combination of anti-CD40 monoclonal antibody plus IL-4 was employed to induce proliferation, a combination that is known to result in prolonged proliferation (J. Banchereau et al . , "Long- Term Human B Cell Lines Dependent on Interleukin-4 and Antibody to CD40," Science 251:70-72 (1991)) . In Figure 16A, normal peripheral B cells were cultured in RPMI media containing 10% fetal bovine serum and with the addition of 1 μg/ml G28-5 plus 10 ng/ml IL-4 as indicated. Cells were grown for 5 days in the indicated concentrations of BMLOV and proliferation was measured by [ ³ H] -thymidine incorporation. BMLOV treatment inhibited proliferation in a dose-dependent manner, but there was considerable variation in sensitivity between cells from two different donors (Fig. 16A) . Nonetheless, at a dose of 10 μM, the inhibition of proliferation of the normal B cells was markedly less than the strong inhibition observed for the transformed B cell lines (Fig. 15A) .

In addition to promoting B cell proliferation, anti-CD40 antibodies are known to protect B cells in germinal centers from undergoing apoptosis (Y. Liu et al., "Mechanism of Antigen-Driven Selection in Germinal Centres," Nature 342:929-931 (1989)) . The combination of IL-4 plus anti-CD40 has been reported to be more

effective than either treatment alone in protecting B cells from apoptosis induced by hypercross-linking of surface IgM or IgD receptors (S.L. Parry et al . , "Hypercross-linking Surface of IgM or IgD Receptors on Mature B Cells Induces Apoptosis that Is Reversed by Costimulation with IL-4 and Anti-CD40," J. Immunol . 152:2821-2829 (1994)) . Accordingly, it was examined whether BMLOV induced cell deaths in the normal B cells (Fig. 16B) . In Figure 16B, normal peripheral B cells were grown for 4 days with the indicated concentrations of BMLOV and then stained with propidium iodine and analyzed for viability by flow cytometry. The percentage of cells in each quadrant is indicated. The lower right quadrant contains the viable cells. In the absence of any treatment, 67% of the normal B cells remained viable after 4 days in culture, whereas only 27% of the cells were viable following treatment with BMLOV (Fig. 16B) . Treatment with G28-5, an anti-human CD40 monoclonal antibody, plus IL-4 protected the cells from the BMLOV induced death, with 58% of the cells remaining viable as opposed to 50% viability observed without BMLOV. In contrast to these results, treatment with combinations of anti-CD20 plus anti-CD40, phorbol ester plus IL-4, or phorbol ester plus anti-CD40 did not protect the cells from BMLOV induced death, even though these combinations are mitogenic for B cells (E.A. Clark & J.A. Ledbetter (1986) , supra) . Taken together, these results indicate that BMLOV can inhibit B cell growth in the absence of inducing cell death and suggest that BMLOV induced death can be blocked by appropriate biological stimulation that is known to protect against activation-induced cell death in vivo.

These results can be interpreted in view of the role of activation induced cell death in lymphocyte development. Activation induced cell death is extremely important to lymphocyte development, acting to remove

self-reactive immature lymphocytes. Immature lymphocytes whose antigen receptors bind self antigen strongly and give a strong activation signal are eliminated by apoptosis (D.R. Green et al . (1992) , supra) . When lymphocytes mature, productive activation requires not only the primary stimulation of the cells' antigen receptor, but also costimulatory signals. In T cells, the binding of CD28 by its ligands of the B7 family serves this costimulatory role, whereas in B cells, costimulation occurs via binding of CD40 to its ligand gp39 (E.A. Clark & J.A. Ledbetter, "How B and T Cells Talk to Each Other," Nature 367:425-428 (1994)) . In B cells, CD40 plays several important roles, promoting proliferation, preventing apoptosis of germinal center B cells, and promoting immunoglobulin class-switching. In the absence of costimulation, lymphocytes that receive only antigen receptor stimulation become non-responsive or anergic and their growth is inhibited (E.A. Clark & J.A. Ledbetter (1994), supra) .

Antigen receptor signal transduction in T cells by the T cell receptor or in B cells via the B cell receptor involves tyrosine kinase activation and phosphorylation of substrates as early and essential steps (A. Weiss & D.R. Littman, "Signal Transduction by Lymphocyte Antigen Receptors," Cell 76:263-274 (1994)) . In this study a PTP inhibitor was employed that induced tyrosine phosphorylation in B cell lines in the absence of receptor ligation. It was observed that BMLOV selectively induces apoptosis in B cell lines and in cells of myeloid linkage but not in T cell lines or in a colon carcinoma cell line. In addition, cellular proliferation was strongly inhibited in the B cell lines, but was less strongly inhibited in cells of T cell or myeloid linkage. Both the growth inhibition and selective induction of apoptosis may arise from the

effects of PTP inhibition on the tyrosine phosphorylation signal pathways that regulate lymphocyte responses.

These results suggest that the accumulation of tyrosine phosphorylated proteins in B cells treated with a phosphatase inhibitor such as BMLOV leads to apoptosis via a mechanism similar to activation-induced cell death. The pattern of cellular tyrosine phosphorylation induced by BMLOV treatment of the B cells was quite similar to that induced by anti-μ stimulation of the B cell receptor (Fig. 12A) . Thus, BMLOV treatment appears to result in the accumulation of many of the same tyrosine phosphorylated species as are found after antigen receptor stimulation.

The tyrosine phosphorylation response of the cell lines examined was well correlated with the apoptotic response of the cells. All the B cell lines examined displayed both a strong tyrosine phosphorylation response and induction of apoptosis, whereas the T cell line showed little tyrosine phosphorylation response or apoptosis. HL-60 cells, which underwent apoptosis in response to BMLOV, also showed a tyrosine phosphorylation response to that compound. The ability of anti-CD40 plus IL-4 stimulation to protect B cells from BMLOV induced cell death also supports the hypothesis of activation induced cell death, since that combination has been previously shown to be highly effective in blocking B cell receptor induced cell death even when it is triggered by a maximal stimulation such as hypercross-linking of surface Ig (S.L. Parry et al. (1994), supra) .

The reason for the selectivity of BMLOV effects on B cell versus T cells or other cell types remain to be identified. B cells might have higher levels of

basal tyrosine kinase activity and therefore require higher levels of PTP activity to maintain most substrates in a non-phosphorylated state, making the cells more susceptible to PTP inhibition. Alternatively, the PTPs regulating B cell tyrosine phosphorylation may be more sensitive to BMLOV. It is likely that CD45 plays an important role since immature B cells negative for CD45 are much more sensitive to apoptosis induced by anti-IgM stimulation (M. Ogimoto et al., "Negative Regulation of Apoptotic Death in Immature B Cells by CD45," Inter. Immunol. 6:647-654 (1994)). The induction of apoptosis in cells by accumulation of tyrosine phosphorylation requires that the cells have such a signal pathway in place capable of inducing apoptosis. Although such signal pathways are known in lymphocytes, they have not been reported for most other cell types, such as colon cells. For example, BMLOV can weakly induce tyrosine phosphorylation in H3347 carcinoma cells, but the cells do not undergo apoptosis, and there is no reported example of tyrosine phosphorylation dependent signals inducing apoptosis in colon cells. The expression of oncogenes and tumor suppressor genes might also be expected to influence the activity of cells to PTP inhibitor induced apoptosis. HL-60 cells lack an intact p53 gene, while Raji cells have a mutated p53 allele (M.B. Kastan et al. , "Participation of p53 Protein in the Cellular Response to DNA Damage," Cancer Res. 51:6304-6311 (1991), so wild type p53 does not appear to be essential for BMLOV induced apoptosis or inhibition of clonogenic cell growth.

Recent studies have shown that radiation and a wide variety of chemotherapy agents act to induce apoptosis in tumor cells with the sensitivity of the tumors to induction of apoptosis being well correlated with the prognosis for successful therapy (D.E. Fisher,

"Apoptosis in Cancer Therapy: Crossing the Threshold, " Cell 78:539-542 (1994)). The tumor suppressor p53 plays a major role in this sensitivity, particularly in cases where DNA damage due to radiation or chemotherapeutic drugs appears to trigger apoptosis, even when the level of such damage would not be lethal in itself (D.E. Fisher (1994) , supra) . The selective induction of apoptosis in tumors by means other than DNA damage would thus of potential value in cancer therapy. One approach would be to target those malignant cell types that retain the signal pathways that induce apoptosis in their normal precursor cells. These results suggest that phosphotyrosine phosphatases play an important role in the regulation of cellular proliferation and apoptosis in lymphocytes. Phosphotyrosine phosphatase inhibitors such as BMLOV may potentially provide a new approach in the treatment of leukemia or lymphoma by selectively inducing apoptosis and preventing clonogenic cell growth.

Most investigations of pharmacologic agents to control cellular tyrosine phosphorylation are focused on inhibitors of tyrosine kinases rather than phosphotyrosine phosphatases. These findings on BMLOV indicate that inhibition of PTP activity can be of value in a variety of biological systems. In addition, the properties of agents such as BMLOV would be expected to make them useful research tools for the study of receptor mediated tyrosine kinase signal transduction, kinase activation, and tyrosine phosphorylation dependent signal events such as calcium mobilization. These agents could also therefore be used to screen for therapeutic agents and have diagnostic potential, because the response of B cells is important to monitor in a variety of clinical conditions.

Example 16

Biological Activity of Sodium (1,10-Phenanthroline) Oxodiperoxovanadium (V) (" V(phen)" The biological activity of sodium (1,10- phenanthroline) oxodiperoxovanadium (V) ("pV(phen))" was examined. This compound has been reported to be a PTP inhibitor and to normalize glucose levels in diabetic animals. This compound is substantially more potent than BMLOV in biological assays and has activity towards additional cell types, particularly T cells, although it retains a high level of activity with respect to B cells.

The inhibition of murine leukemia and lymphoma cells is shown in Table 10. The cell lines A20, 70Z-3, L1210, and P388D were used. A20 is a B cell lymphoma cell line; 70Z/3 is a B cell leukemia cell line; L1210 is a T cell leukemia cell line; and P388D is a myeloid leukemia cell line. The cells were grown for 5 days in the presence of pV(phen) and pulsed with [ ³ H] -thymidine on day 5. The extent of DNA synthesis was then determined by counting the radioactivity incorporated into the cells, as in Example 5, supra. The data shown in Table 10 show that thymidine incorporation into these cells was almost completely inhibited by even the lowest dose of pV(phen) , 3 μM. The effects on L1210 cells and P388D cells are particularly noteworthy because these cell lines have been extensively used by the National Cancer Institute to identify anti-cancer drugs.

TABLE 10

INHIBITION OF MURINE LEUKEMIA AND LYMPHOMA

CELL PROLIFERATION BY pV(phen)

ND = not one

Similar results are shown in Figure 17 for the cell lines REH, Ramos, CEM, Jurkat, HL-60, and THP-1. Cells were treated 5 days with the amount -of inhibitor indicated and [ ³ H] -thymidine incorporation was then measured to determine proliferation. The percent inhibition was calculated relative to untreated controls. Results are shown for three concentrations of BMLOV (5 μM, 10 μM, and 25 μM and 5 μM of pV(phen)) . These results indicate that pV(phen) inhibits all cell types studied including cells of both B and T lineage.

The effect of the variation of treatment time with pV(phen) on the inhibition of proliferation in Ramos cells is shown in Table 11. These cells were grown in pV(phen) for the indicated times, washed, and then grown in fresh media for the balance of the 5-day assay. These cells were pulsed with [ ³ H] -thymidine on day 5.

TABLE 11

TREATMENT TIME DEPENDENCE OF INHIBITION OF

PROLIFERATION OF RAMOS CELLS BY pV(phen)

These results indicate that the response in Ramos cells depends on both the treatment time and the dosage. After 8 hours of treatment with pV(phen) , substantially complete inhibition was seen only at the highest dosage level, 25 μM; after 24 hours of treatment, substantially complete inhibition of proliferation was seen even at a 5μM dose. If the dosage is maintained for the full 5-day treatment, even 1 μM of pV(phen) was sufficient to induce at least a 99% inhibition of proliferation measured by thymidine incorporation.

The induction of apoptosis in L1210 cells by pV(phen) is shown in Figure 18. L1210 murine T cell leukemia cells were treated with the indicated concentrations of pV(phen) for the indicated times and the DNA was examined by gel electrophoresis as in Example 3. The fragmentation of DNA demonstrates apoptosis.

In Figure 19, the induction of phosphotyrosine by pV(phen) is shown in Ramos and Jurkat cells. Ramos B cells and Jurkat T cells were treated with the indicated concentrations of pV(phen) for 16 hours or with anti-μ (to crosslink surface immunoglobulin) for 1 minute or with mAb G19-4 (anti-CD3) for 1 minute. The cells were then lysed and cellular tyrosine phosphorylation was examined by anti-phosphotyrosine Western blotting of the cellular proteins following SDS-polyacrylamide gel electrophoresis. These results indicate a high level of tyrosine phosphorylation induced by pV(phen) .

Similarly, a comparison of the tyrosine phosphorylation induced by pV(phen) and BMLOV is shown in Figure 20. Ramos B cells were treated with the indicated concentrations of BMLOV or pV(phen) for 16 hours. The cells were then lysed and cellular tyrosine phosphorylation was examined by an anti-phosphotyrosine Western blot of the cellular proteins following SDS- polyacrylamide gel electrophoresis. These results indicate that pV(phen) causes the tyrosine phosphorylation of a significantly larger group of proteins than does BMLOV, particularly at 10 μM.

BMLOV is known to inhibit Src-family tyrosine kinases. In contrast, pV(phen) not only inhibits PTP but also activates at least one type of tyrosine kinase, the Syk-family kinases. The response of Syk kinase to pV(phen) is shown in Figure 21. Ramos B cells were treated with the indicated concentrations of BMLOV or pV(phen) for 16 hours. The cells were then lysed and the Syk tyrosine kinase was immunoprecipitated with anti-Syk antibodies. The tyrosine phosphorylation of Syk was examined by an anti-phosphotyrosine Western blot of the immunoprecipitated Syk protein following SDS- polyacrylamide gel electrophoresis (shown at left) . The amount of Syk protein recovered was determined by anti-

Syk Western blotting of identical samples (shown at right) . Figure 21 demonstrates that pV(phen) strongly induces tyrosine phosphorylation of the Syk tyrosine kinase, whereas only very slight tyrosine phosphorylation of Syk is observed following BMLOV treatment. The anti-Syk Western blot of identical samples at the right shows that the recovery of Syk as a protein is markedly reduced following the pV(phen) treatment, so the specific activity of Syk tyrosine phosphorylation is even greater than is apparent from the strong tyrosine phosphorylation shown at the left .

Analysis of the human T cell leukemia cell lines CEM and Jurkat by propidium iodide staining followed by flow cytometry demonstrates that 10 μM pV(phen) kills 98% of these leukemia cells in two days and all the cells by three days.

Animal studies with BALB/C mice have shown that the maximum tolerated dose is 1 ml of a 1 mM solution in saline by intraperitoneal (i.p.) injection, as tested in 18-20 g mice. This dose is well tolerated when administered every 2 days for 2 weeks. In contrast, a dose of 2 mM resulted in some deaths. However, this dose is substantially greater than that required to induce inhibition of proliferation of susceptible lymphoid and myeloid cells.

The animal studies have shown that pV(phen) specifically acts against lymphocytes in vivo. BALB/C mice received one dose of pV(phen) i.p. and their blood was examined 24 hr later. As shown in Table 12, the number of peripheral blood lymphocytes in the treated animals was markedly reduced. This effect on lymphocytes was specific in that the number of neutrophils was substantially increased, whereas no consistent effect was observed for monocytes. The

increase in neutrophils provides a significant advantage for the PTP inhibitors relative to many cytotoxic drugs that deplete neutrophils, a serious side effect of cytotoxic drugs in patients. The depletion of lymphocytes also suggest that compounds such as pV(phen) can be of use in the treatment of autoimmune diseases such as arthritis or inflammatory bowel disease.

TABLE 12

EFFECT OF pV(phen) ON LEUKOCYTE COUNTS IN MICE

Example 17 In Vivo Antitumor Activity of pV(phen) The compound pV(phen) was tested for its antitumor activity in vivo. A summary of the antitumor test results is shown below in Table 13. In the i.p. M109 murine lung carcinoma model (Experiment No. 330; Table 13), consecutive daily i.p. injections each day from day 1 to 5 were effective in achieving an active result. A dose level of 12 mg/kg per injection resulted in a maximum T/C value of 144%. Values of T/C greater than or equal to 125% are considered to be indicative of activity in this test format. In an attempt to confirm

this result, the pV(phen) was retested on the same treatment schedule but with slightly escalated doses (Experiment No. 333; Table 13) . A maximum T/C of 143%, consistent with the first result, was obtained.

Against i.p. B16 murine melanoma (Experiment No. 799; Table 13) consecutive daily i.p. injections administered for each day from day 1 to day 9 resulted in a maximum T/C of 152% at a maximum tolerated dose (MTD) of 6 mg/kg per injection. The cumulative dose MTD of 54 mg/kg was similar to the 60 mg/kg cumulative MTD observed in the last M109 experiment.

Against the i.p. implanted P388 murine leukemia (Experiment No. 8457; Table 13) , single i.p. doses of greater than or equal to 32 mg/kg of pV(phen) were excessively toxic, i.e. lethal, and the next lower dose tested, 8 mg/kg was inactive in this assay format (T/C <125%) . In another P388 trial (Experiment No. 8459; Table 13) consecutive daily i.p. injections each day from days 1 to 5 likewise proved ineffective at doses as great as 12 mg/kg per injection and considered to be a MTD.

Intermittent injections of pV(phen) , e.g., 8 mg/kg failed to achieve an active result in this test format against the i.p. implanted ras transformed rat-1 tumor model (Experiment No. 6; Table 13) .

Finally, pV(phen) was tested against subcutaneously implanted human ovarian carcinoma, A2780, in Experiment No. 54, using a treatment schedule of once daily for 9 days. Treatments (i.p.) were begun on day 10 post implant when the tumors were small but definite, e.g. 50-60 mg, at the MTD of 5 mg/kg per injection

(cumulative dose of 45 mg/kg) . A delay in primary tumor growth of 4.0 days was achieved, reflective of 0.4 log

cell kill (LCK) , a basically inactive result in this assay format.

These results are of major importance because activity considered significant by generally-applied standards was seen in two types of tumors in vivo. This indicates a significant potential for antitumor activity of p(V)phen.

TABLE 13

^a Or highest dose tested, or maximum tolerated dose, if inactive ^b MTD reached.

The inactive results seen in some tests may have been due to peculiarities of administration or metabolism in vivo. Such results do not necessarily mean that the drug was ineffective under these conditions, particularly because some activity was yet seen. It is possible that the treatment regimen or

route of administration could be optimized to yield significant activity in the trials that yielded inactive results in this format.

Example 18

In Vitro Cvtotoxicity of pV(phen) in a Panel of Human Tumor Cell Lines

The vanadate compound pV(phen) was tested for in vitro cytotoxicity in a panel of human tumor cell lines. The results are given in Table 14. The cell lines used were a human colon carcinoma cell line, HCT116, and two drug-resistant sublines. The HCT116(VM46) subline was selected for resistance to VM- 26 and expresses the multidrug resistant (MDR) phenotype including resistance to lipophilic anticancer drugs such as paclitaxel, VP-16, VM-26, doxorubicin, and vinblastine. This cell line overexpresses P- glycoprotein, a cell surface drug efflux pump, which limits accumulation of anticancer drugs such as the ones mentioned above. The other cell line, HCT116 (VP35) was selected for resistance to VP-16 and is resistant to topoisomerase II active drugs such as VP-16 and VM-26 due to reduced levels of the topoisomerase II enzyme. The compound pV(phen) was also evaluated in the ovarian carcinoma cell line A2780 and a paclitaxel resistant subline A2780-Tax22. The latter cell line, which is 18- fold resistant to paclitaxel, does not overexpress P- glycoprotein, but appears to have altered tubulin.

In the cytotoxicity assays, cells were plated and 24 hr later drugs were added and serially diluted. After 72 hr of continuous drug exposure, the tetrazolium dye XTT was added. A dehydrogenase enzyme in live cells reduces the XTT to a form that absorbs light at 450 nm which can be quantitated spectrophotometrically. The greater absorbance, the greater is the number of live cells. The results are expressed as a IC ₅₀ which is a

drug concentration required to inhibit cell proliferation (i.e., absorbance at 450 nm) to 50% of that of untreated control cells.

The compound pV(phen) had a IC value of approximately 8 μM in both the HCT116 and A2780 cell lines. Likewise, the HCT116 (VP)35 and the A2780-Tax22 resistant cell lines did not appear to be cross- resistant to the test compound. Interestingly, however, the MDR cell line HCT116(VM46) was collaterally sensitive to the vanadate compound, showing greater sensitivity than the parent . This result is contrasted with the effect of paclitaxel on these cell lines; the MDR subline was 75-fold more resistant than paclitaxel than was the parent line HCT116 (Table 14) .

In other results, it was shown that the MDR cell line MCF-7-AdrR had a similar level of increased sensitivity to pV(phen) relative to the parental cell line. In previous studies it was shown that cisplatin was also collaterally sensitive in MDR cells, although not to the degree shown for pV(phen) . The degree of collateral sensitivity for cisplatin MDR cells was lower than for pV(phen) . What if any connection these two drugs have is unclear. P-glycoprotein is known to be phosphorylated by protein kinase C, and modulation of protein kinase C activity can affect the level of MDR. However, it is unclear how the level of phosphorylation P-glycoprotein would affect the sensitivity to pV(phen) alone.

This showing of collateral sensitivity is an unexpected result which holds out considerable promise, particularly for treatment of malignancies that are resistant to other drugs. In any case, these results are highly significant in showing killing of tumor cells in tests using cell lines recognized in the art as

models for human tumors. This data on cell lines is recognized in the art as predictive of utility for the treatment of tumors.

TABLE 14

Results of Testing pV(phen) on Tumor Cell Lines

BMS# HCT116 HCT116(VM)46 HCT116(VP)35 A2780 A2780/Tax22

196365 8.4 2.0 (0.2) ² 7.2 (0.9) 8.2 7.5 (0.9)

paclitaxel 0.003 0.226 (75) 0.002 (0.7) 0.004 0.075 (18)

¹ Cytotoxicity determined after 72 hr drug exposure by XTT assay.

² Value in parenthesis is fold resistance relative to parental cell line .

ADVANTAGES OF THE INVENTION

The present invention provides methods for inhibiting phosphotyrosine phosphatase, particularly in B cells and T cells. This yields improved methods of inhibiting the proliferation of these cells by exploiting the occurrence of apoptosis (programmed cell death) . These methods can be exploited for treatment of disorders marked by malignant proliferation of B cells or T cells, such as leukemias and lymphomas, and can be combined with other methods of treatment, including radiation. Such a combination yields synergistic effects over either radiation alone or the use of phosphotyrosine phosphatase inhibitors alone .

Methods according to the present invention can also be used for controlling proliferation of non- malignant B cells for regulation of the immune response. This is desirable for the treatment of autoimmune disease and for controlling transplant rejection, as

well as for controlling class-switching in antibody- producing cells.

The methods of the present invention are further useful for studying signaling in B cells and for screening for abnormalities of B cell signaling.

In particular, the compound pV(phen) has demonstrated activity in vivo against tumors in animals that are considered to be models for the therapeutic evaluation of antitumor agents, as well as activity against tumor cell lines that are also considered to be models for the therapeutic evaluation of antitumor agents. Significantly, the activity against tumor cell lines persisted in cell sublines that exhibited resistance to other antitumor agents such as paclitaxel and topoisomerase active agents. This activity holds promise for treatment of tumors, including tumors resistant to other drugs in use. Other analogous compounds are expected to display a similar range of activities.

Although the present invention has been described with considerable detail, with reference to certain preferred versions thereof, other versions are possible. Therefore, the spirit and scope of the appended claims should not be limited to the description of the preferred versions contained herein.