WO2014018862A1 - Pharmaceutical compositions comprising a heat shock protein inhibitor and a|purine de novo synthesis inhibitor for treating rheumatoid arthritis or cancer - Google Patents

Abstract

A pharmaceutical composition including an effective amount of the combination of: an heat shock protein (HSP) inhibitor, and a purine de novo biosynthesis inhibitor, or a pharmaceutically acceptable salt thereof. Also disclosed is a method of treating rheumatoid arthritis or cancer including administering an effective amount of the above mentioned pharmaceutical composition to a patient in need of such treatment, as defined herein.

Description

PHARMACEUTICAL COMPOSITIONS COMPRISING A HEAT SHOCK PROTEIN INHIBITOR

AND A|PURINE DE NOVO SYNTHESIS INHIBITOR FOR TREATING RHEUMATOID

ARTHRITIS OR CANCER

[0001] The entire disclosure of any publication or patent document mentioned herein is incorporated by reference.

Cross Reference to Related Applications

[0002] This application claims the benefit of priority under 35 U.S.C. §1 19 of U.S.

Provisional Application Serial No. 61/676,390, filed July 27, 2012, the content of which is relied upon and incorporated herein by reference in its entirety.

[0003] This application is related to U.S. Provisional Application No. 61/368,802 (SP10- 207), to Benkovic, S., et al., entitled "Methods to identify targets and molecules regulating purinosomes and their uses", filed July 29, 2010; and US Patent Application Publication 201 10207789, filed February 19, 2010, USSN 12/ 708840, to Fang, Y., et al., entitled "Methods related to casein kinase II (CK2) inhibitors and the use of purinosome-disrupting CK2 inhibitors for anti-cancer therapy agents," the content of which is relied upon and incorporated herein by reference in their entirety, but does not claim priority thereto.

Background

[0004] The disclosure is related to pharmaceutical compositions and methods for the treatment of rheumatoid arthritis and cancers.

Summary

[0005] The present disclosure provides pharmaceutical compositions and methods of use thereof for the treatment of rheumatoid arthritis and cancers.

[0006] The present disclosure provides a combination of an HSP70 or HSP90 inhibitor and a purine de novo synthesis inhibitor for the treatment of rheumatoid arthritis and cancers.

[0007] The present disclosure provides a pharmaceutical composition comprising a combination of the purine de novo synthesis inhibitor, methotrexate, with an HSP90 inhibitor, 17-AAG or geldamycin, for treating rheumatoid arthritis and certain cancers.

[0008] The present disclosure provides a combination of the purine de novo synthesis inhibitor, methotrexate, with the HSP70 inhibitor, 2-phenylethynesulfonamide, for treating rheumatoid arthritis and certain cancers. [0009] The present disclosure provides a combination of a HSP inhibitor and a purine de novo synthesis inhibitor for the treatment of rheumatoid arthritis and cancers. Specifically, when compared with either agent alone, the combination of the purine de novo synthesis inhibitor, methotrexate, with the HSP90 inhibitor, 17-AAG or geldamycin, has been demonstrated to have synergistic effects on disrupting the purinosome, a multienzyme complex important for purine de novo biosynthesis, and thus provides evidence that the disclosed therapeutic composition and method can provide a method of treating rheumatoid arthritis and certain cancers.

Brief Description of the Figures

[0010] In embodiments of the disclosure:

[0011] Fig. 1 shows a Western blot of FGAMS-9xcMyc (162kDa) that was pulled down by anti-cMyc antibody in the co-immunoprecipitation (co-IP).

[0012] Fig. 2 shows immunofluorescence images that demonstrate that Hsp90 and Hsp70 co- localize with purinosomes.

[0013] Fig. 3 shows cellular co-localization of PPAT-GFP and Hsp90-OFP, PAICS-GFP and Hsp90-OFP in HeLa cells grown in purine-depleted conditions.

[0014] Fig. 4 shows cellular co-localization of Hsp70-OFP and PPAT-GFP or PAICS-GFP in HeLa cells grown in purine-depleted conditions.

[0015] Fig. 5 shows cellular co-localization of FGAMS-GFP and Hsp90-OFP in C3A and A431 cells.

[0016] Fig. 6 shows HeLa cells transfected with Hsp70 and Hsp90 alone.

[0017] Fig. 7 shows the results of a Luciferase reporter assay and the interaction between

FGAMS and Hsp70 or Hsp90.

[0018] Fig. 8 shows cellular co-localization of FGAMS-OFP and cochaperone BAG2-GFP or

DnaJAl-GFP in HeLa cells grown in purine-depleted conditions.

[0019] Fig. 9 shows several co-chaperones that are also associated with purinosomes.

[0020] Figs. 10 shows the effect of Hsp90 inhibitor, 17-DMAG, on purinosome formation in

HeLa cells transfected by FGAMS-GFP.

[0021] Fig. 1 1 shows Hsp90 and Hsp70 inhibitors disrupt purinosomes and are synergistic with methotrexate.

[0022] Fig. 12 shows the Hsp70 inducer, geranylgeranylacetone, has a protective effect on HeLa cells treated with methotrexate. Detailed Description

[0023] Various embodiments of the disclosure will be described in detail with reference to drawings, if any. Reference to various embodiments does not limit the scope of the invention, which is limited only by the scope of the claims attached hereto. Additionally, any examples set forth in this specification are not limiting and merely set forth some of the many possible embodiments of the claimed invention.

[0024] In embodiments, the disclosed composition and the disclosed method of using provide one or more advantageous features or aspects, including for example as discussed below. Features or aspects recited in any of the claims are generally applicable to all facets of the invention. Any recited single or multiple feature or aspect in any one claim can be combined or permuted with any other recited feature or aspect in any other claim or claims.

[0025] "Include," "includes," or like terms means encompassing but not limited to, that is, inclusive and not exclusive.

[0026] "About" modifying, for example, the quantity of an ingredient in a composition, concentrations, volumes, process temperature, process time, yields, flow rates, pressures, viscosities, and like values, and ranges thereof, employed in describing the embodiments of the disclosure, refers to variation in the numerical quantity that can occur, for example: through typical measuring and handling procedures used for preparing materials, compositions, composites, concentrates, or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods; and like considerations. The term "about" also encompasses amounts that differ due to aging of a composition or formulation with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a composition or formulation with a particular initial concentration or mixture.

[0027] The indefinite article "a" or "an" and its corresponding definite article "the" as used herein means at least one, or one or more, unless specified otherwise.

[0028] Abbreviations, which are well known to one of ordinary skill in the art, may be used (e.g., "h" or "hr" for hour or hours, "g" or "gm" for gram(s), "mL" for milliliters, and "rt" for room temperature, "nm" for nanometers, and like abbreviations).

[0029] Specific and preferred values disclosed for components, ingredients, additives, and like aspects, and ranges thereof, are for illustration only; they do not exclude other defined values or other values within defined ranges. The methods of the disclosure can include any value or any combination of the values, specific values, more specific values, and preferred values, including intermediate values and ranges, described herein. [0030] Rheumatoid arthritis is a common autoimmune disease that is associated with progressive disability, systemic complications, early death, and socioeconomic costs. The cause of rheumatoid arthritis is unknown, and the prognosis is guarded. However, advances in understanding the pathogenesis of the disease have fostered the development of new therapeutics, with improved outcomes. Introduction of biologic agents, specifically agents that target the inflammatory cytokine tumor necrosis factor (TNF), has transformed therapy for rheumatoid arthritis. These biologies include TNF -neutralizing antibody drugs etanercept, infliximab, adalimumab, certulizumab pegol and golimumab; anti T-cell (and/or dendritic cell) therapy abatacept; the B-cell-depleting antibody rituximab; and the inter leukin- 1 antagonist anakinra; and interleukin-6 receptor antagonist tocilizumab. The availability of these biologic agents, frequently used in combination with classic DMARDs (disease- modifying antirheumatic drugs), has given rheumatologists new and very potent means to treat patients with this chronic inflammatory condition. Despite the obvious impact of these drugs, there are still challenges for new generation drugs to treat this disease. Current conventional and biologic disease modifying therapies sometimes fail or produce only partial responses. Reliable predictive biomarkers of prognosis, therapeutic response, and toxicity are lacking. Sustained treatment-free remission is rarely achieved and requires ongoing pharmacologic therapy. Even the achievement of a low disease activity state with therapy is not uniform with the current biologic agents. The mortality rate is higher among patients with rheumatoid arthritis than among healthy persons; and cardiovascular and other systemic complications remain a major challenge. Molecular remission and the capacity to reestablish immunologic tolerance remain elusive. The current biologic agents have a number of adverse effects, for example, various infectious complications and increased risks of malignancies, that make their persistent administration a concern.

[0031] Past decades have also witnessed increasing numbers of anticancer agents entering the market. These agents were primarily screened for their cytotoxic effects against rapid growth cells. Not surprisingly, agents developed under this guiding principle often lacked selectivity of action, resulting in high degrees of toxicity in normal tissues. Furthermore, drug resistance is a common cause of treatment failure for cancer. Our understanding of cancer biology has fundamentally changed and led to the characterization of a large number of unique molecular targets for therapeutic intervention, including heat shock proteins (HSPs) such as HSP90. Inhibition of HSP90 function causes degradation of client proteins via the ubiquitin- proteasome pathway, which results in the combinatorial down-regulation of signals being propagated via numerous signaling pathways and modulation of all aspects of the malignant phenotype. HSP90 inhibitors have shown promise in the late stage of clinical trials to treat several types of cancers.

[0032] The disclosure provides a pharmaceutical composition useful for the treatment of human cancers, rheumatoid arthritis, or both, comprising:

a therapeutically effective amount of a combination of a HSP90 inhibitor, and a purine de novo biosynthesis inhibitor, or a pharmaceutically acceptable salt of either or both components of the combination.

[0033] In embodiments, the disclosure provides a pharmaceutical composition comprising an effective amount of the combination of:

an heat shock protein (HSP) inhibitor; and

a purine de novo biosynthesis inhibitor; or

a pharmaceutically acceptable salt of either or both inhibitors.

[0034] In embodiments, the HSP inhibitor is an inhibitor of HSP90. In embodiments, the HSP inhibitor is an inhibitor of HSP70.

[0035] In embodiments, the composition can be used to treat rheumatoid arthritis. In embodiments, the composition can be used to treat an oncological disease. The oncological disease can be selected from, for example, a solid tumor or a hematologic neoplasia.

[0036] In embodiments, the composition can be used to treat a solid tumor, for example, selected from a group: a bladder cancer, a lung cancer, a pancreatic cancer, a prostate cancer, a colorectal cancer, a gastrointestinal cancer, a head and neck cancer, a malignant mesotheliomas, a melanoma, a breast cancer, a malignant melanoma, an ovarian cancer, a soft tissue sarcoma, an osteosarcoma, a hepatocellular carcinoma, a non-small cell lung cancer (NSCLC), a renal cancer, a cervical cancer, or a tissue sarcoma.

[0037] In embodiments, the composition can be used to treat, for example, a refractory or relapsed multiple myeloma, an acute or chronic myelogenous leukaemia, a myelodysplastic syndrome, or an acute lymphoblastic leukaemia.

[0038] In embodiments, the purine de novo biosynthesis inhibitor is a compound selected from the group consisting essentially of: methotrexate; piritrexm; azaserine; azathioprine; diazomycin; dideazatetrahydrofolate; lometrexol; fluorosulfonylbenzoyl-adenosine;

nitroaminoimidazole ribonucleotide; and mixtures thereof.

[0039] In embodiments, the HSP90 inhibitor can be a compound selected from the group consisting essentially of:

geldamycin; 17-AAG (17-allyl-17-demethoxygeldanamycin); 17-DMAG (17- desmethoxy-17-N,N-dimethylaminoethylaminogel danamycin); ΓΡΙ-504 (17-allylamino-17- demethoxygeldanamycin hydroquinone hydrochloride); IPI-493 (17-desmethoxy-17-amino geldanamycin); ΒΙΓΒ021 ([6-Chloro-9-(4-methoxy-3,5-dimethylpyridin-2-ylmethyl)-9H- purin-2-yl]amine); MPC-3100 ((S)-l-(4-(2-(6-amino-8-((6-bromobenzo[d][l,3]dioxol-5- yl)thio)-9H-purin-9-yl)ethyl)piperidin-l -yl)-2-hydroxypropan-l-one); Debio 0932 (2-((6- (dimethylamino)benzo [d] [ 1 ,3 ]dioxol-5-yl)thio)- 1 -(2-(neopentylamino)ethyl)- 1 H- imidazo[4,5-c]pyridin-4-amine); PU-H71 (6-Amino-8-[(6-iodo-l,3-benzodioxol-5-yl)thio]- N-(l-methylethyl)-9H-purine-9-propanamine); STA-9090 (5-[2,4-dihydroxy-5-(l- methylethyl)phenyl]-4-(l -methyl- lH-indol-5-yl)-2,4-dihydro-3H- l,2,4-triazol-3-one);

VER52296 (5-(2,4-Dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4-

(morpholinomethyl)phenyl)isoxazole-3-carboxamide); KW-2478 (2-(2-ethyl-3,5-dihydroxy-

6-(3-methoxy-4-(2-morpholinoethoxy)benzoyl)phenyl)-N,N-bis(2-methoxyethyl)acetamide);

AT-13387 ((2,4-dihydroxy-5-isopropylphenyl)(5-((4-methylpiperazin- 1 - yl)methyl)isoindolin-2-yl)methanone); Radicicol ((1 aR,2Z,4E, 14R, 15aR)-8-Chloro-

1 a, 14, 15 , 15 a-tetrahydro-9, 1 1 -dihydroxy- 14-methyl-6H-oxireno [e] [2]benzoxacyclotetradecin-

6,12(7H)-dione);

and celastrol; a combination thereof, or a pharmaceutically acceptable salt thereof.

[0040] In embodiments, the HSP70 inhibitor can be a compound selected from of the group of:

2-phenylethynesulfonamide (Pifithrin-μ);

MKT-077 (l -Ethyl-2-[[3-ethyl-5-(3-methyl-2(3H)-benzothiazolylidene)-4-oxo-2- thiazolidinylidene]methyl]-pyridinium chloride); methylene blue;

VER155088 (5'-0-[(4-Cyanophenyl)methyl]-8-[[(3,4-dichlorophenyl)methyl]amino]- adenosine); or a pharmaceutically acceptable salt thereof.

[0041] In embodiments, the HSP inhibitor can be geldamycin, and the purine de novo biosynthesis inhibitor can be methotrexate.

[0042] In embodiments, the HSP inhibitor can be 17-AAG, and the purine de novo biosynthesis inhibitor is methotrexate.

[0043] In embodiments, the HSP inhibitor can be celastrol, and the purine de novo biosynthesis inhibitor is methotrexate.

[0044] In embodiments, the HSP inhibitor can be BIIB021, and the purine de novo biosynthesis inhibitor is methotrexate.

[0045] In embodiments, the HSP inhibitor can be Pifithrin-μ, and the purine de novo biosynthesis inhibitor can be methotrexate. [0046] In embodiments, the pharmaceutical composition can further comprise, for example, at least one therapeutic agent selected from the group of, for example, a chemotherapeutic agent and a targeted therapeutic agent when the composition is used to treat cancer.

[0047] In embodiments, the at least one therapeutic agent can be, for example, a

chemotherapeutic agent selected from Asparaginase, Bleomycin, Busulfan, Carmustine, Chlorambucil, Cladribine, Cyclophosphamide, Cytarabine, Dacarbazine, Daunorubicin, Doxorubicin, Etoposide, Fludarabine, Gemcitabine, Hydroxyurea, Idarubicin, Ifosfamide, Lomustine, Mechlorethamine, Melphalan, Mitomycin, Mitoxantrone, Pentostatin,

Procarbazine, Topotecan, Vinblastine, Vincristine, Dexamethasone, Retinoic acid,

Prednisone, and mixtures thereof.

[0048] In embodiments, the at least one therapeutic agent can be, for example, a targeted therapeutics agent selected from alemtuzumab, bevacizumab, cetuximab, gemtuzumab, ibritumomab, panitumumab, rituximab, tositumomab, trastuzumab, dasatinib, erlotinib, everolimus, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, sorafenib, sunitinib, temsirolimus, vandetanib, vemurafenib, crizotinib, vismodegib, axitinib, ruxolitinib, and mixtures thereof.

[0049] In embodiments, the pharmaceutical composition can further comprise at least one therapeutic agent selected from the group of, for example, classical DMARDs, NSAIDs, biologies DMARDs, immunomodulators, and mixtures thereof.

[0050] In embodiments, the at least one therapeutic agent can be selected from the group of , for example, chloroquine, hydroxychloroquine, cyclosporin A, D-penicillamine,

aurothiomalate, auranofin, leflunomide, minocycline, sulfasalazine, ibuprofen, naproxen, meloxicam, etodolac, nabumetone, sulindac, tolementin, choline magnesium salicylate, diclofenac, diflusinal, indomethicin, ketoprofen, oxaprozin, piroxicam, etanercept, infliximab, adalimumab, certolizumab pegol, golimumab, anakinra, rituximab, abatacept, and tocilizumab, and mixtures thereof.

[0051] In embodiments, the pharmaceutically acceptable salt can be, for example, a pharmaceutically acceptable inorganic and organic, acid or base.

[0052] In embodiments, the pharmaceutical composition can comprise, for example, a dosage form suitable for simultaneous, separate, or sequential use in the treatment of rheumatoid arthritis or cancer.

[0053] In embodiments, the disclosure provides a method of treating rheumatoid arthritis or cancer, comprising: administering an effective amount of the disclosed pharmaceutical composition of to a patient in need of such treatment.

[0054] Those skilled in the art can recognize that some of the disclosed compounds contain chiral centers. Thus, the composition contains an inhibitor that can exist in the form of two different isomers. All such enantiomers and mixtures thereof including racemic mixtures are included within the scope of the invention. The single optical isomer or enantiomer can be obtained by a method well known in the art, such as chiral HPLC (high performance liquid chromatography), enzymatic resolution, and the use of a chiral auxiliary.

[0055] In embodiments, the pharmaceutical compositions of the present disclosure are useful for treating an oncological disease. In embodiments, the oncological disease can be selected from a solid tumor or a malignant human neoplasia. In embodiments, the oncological disease can be a hematologic neoplasia. In embodiments, the pharmaceutical combinations of the present disclosure are useful for treating a refractory or relapsed multiple myeloma, an acute or chronic myelogenous leukaemia, a myelodysplasia; syndrome, or an acute lymphoblastic leukemia.

[0056] In embodiments, the pharmaceutical compositions of the present disclosure are useful in cancer therapy, in particular in the treatment of cancer selected from the group comprising lung cancer, prostate cancer, bladder cancer, colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, ovarian cancer, soft tissue sarcoma, osteosarcoma, hepatocellular carcinoma, leukemia, lymphomas, and like cancers in a patient.

[0057] In embodiment, the pharmaceutical compositions of the present disclosure are useful in cancer therapy, such as in the treatment of cancers selected from the group including, for example, colorectal cancer, melanoma, gastric cancer, islet cell cancer of the pancreas, non- small cell lung cancer (NSCLC), renal cancer, cervical cancer, breast cancer, ovarian cancer, squamous cell cancer of the pelvis, liver cancer, abdominal cancer, and penile cancer.

[0058] In embodiments, the pharmaceutical compositions useful for the treatment of cancer can be, for example, a therapeutically effective amount of the HSP90 inhibitor and the purine de novo biosynthesis inhibitor.

[0059] In embodiments, the disclosure provides a pharmaceutical composition useful for the treatment of cancer comprising a therapeutically effective amount of the HSP90 inhibitor and the purine de novo biosynthesis inhibitor, and at least one further therapeutic agent chosen from the group comprising a chemotherapeutic agent, a targeted therapeutics, or a combination thereof. [0060] In embodiments, the further therapeutic agent can be, for example, a chemotherapeutic agent selected from Asparaginase, Bleomycin, Busulfan, Carmustine, Chlorambucil, Cladribine, Cyclophosphamide, Cytarabine, Dacarbazine, Daunorubicin, Doxorubicin, Etoposide, Fludarabine, Gemcitabine, Hydroxyurea, Idarubicin, Ifosfamide, Lomustine, Mechlorethamine, Melphalan, Mitomycin, Mitoxantrone, Pentostatin,

Procarbazine, Topotecan, Vinblastine, Vincristine, Dexamethasone, Retinoic acid,

Prednisone, and like agents, or mixtures thereof.

[0061] In embodiment, the further therapeutic agent can be, for example, a targeted therapeutic agent selected from alemtuzumab, bevacizumab, cetuximab, gemtuzumab, ibritumomab, panitumumab, rituximab, tositumomab, trastuzumab, dasatinib, erlotinib, everolimus, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, sorafenib, sunitinib, temsirolimus, vandetanib, vemurafenib, crizotinib, vismodegib, axitinib, ruxolitinib, and like agents, or mixtures thereof.

[0062] In embodiments, the individual components of such combination compositions disclosed above can be administered, for example, sequentially or simultaneously, in separate or combined pharmaceutical formulations.

[0063] In embodiments, the compositions referred to above can be conveniently presented for use in the form of a pharmaceutical formulation including, for example, a pharmaceutically acceptable carrier.

[0064] In embodiments, the pharmaceutical compositions of the present disclosure are useful to treat rheumatoid arthritis.

[0065] In embodiments, the disclosure provides a pharmaceutical composition useful for the treatment of rheumatoid arthritis comprising a therapeutically effective amount of the HSP90 inhibitor and the purine de novo biosynthesis inhibitor.

[0066] In embodiments, the disclosure provides a pharmaceutical composition useful for the treatment of cancer comprising a therapeutically effective amount of the HSP90 inhibitor and the purine de novo biosynthesis inhibitor, and at least one further therapeutic agent selected from the group of, for example, classical DMARDs, NSAIDs, biologies DMARDs, immunomodulators, and like agents, or mixtures thereof.

[0067] In embodiments, the at least one further therapeutic agent can be a classical DMARD selected from, for example, chloroquine, hydroxychloroquine, cyclosporin A, T D- penicillamine, aurothiomalate, auranofin, leflunomide, minocycline, sulfasalazine, and like agents, or mixtures thereof. In embodiments, a further therapeutic agent can be a NSAID selected from, for example, ibuprofen, naproxen, meloxicam, etodolac, nabumetone, sulindac, tolementin, choline magnesium salicylate, diclofenac, diflusinal, indomethicin, ketoprofen, oxaprozin, piroxicam, and like agents, or mixtures thereof.

[0068] In embodiments, a further therapeutic agent can be a DMARDs biologic selected from, for example, etanercept, infliximab, adalimumab, certolizumab pegol, golimumab, anakinra, rituximab, abatacept, tocilizumab, and like agents, or mixtures thereof.

[0069] In embodiments, the individual components of such compositions as defined above can be administered, for example, either sequentially or simultaneously in separate or combined pharmaceutical formulations.

[0070] In embodiments, the disclosure provides pharmaceutical composition for therapeutic treatment including administering an effective amount of the pharmaceutical composition to a patient in need of such treatment. The amount of each compound in the compositions of the present disclosure for use in treatment can vary not only with the particular compound selected but also with the route of administration, the nature of the condition for which treatment is required and the age and condition of the patient, and will be ultimately at the discretion of the attendant physician or veterinarian. In general however a suitable dose can be from about 0.01 to about 500 mg kg of body weight per day, preferably from about 0.2 to about 50mg kg/day, and more preferably from 1 to about 20 mg/kg/day.

[0071] In embodiments, the disclosure provides pharmaceutical formulations that can include formulations suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), transdermal, vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation. The formulations can, where appropriate, be conveniently presented in discrete dosage units and can be prepared by any of the methods known in the pharmacy arts. Treatment methods in accord with the disclosure can include, for example, a step of bringing into association the active compound and a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.

[0072] In embodiments, a pharmaceutical formulation suitable for oral administration can be conveniently presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules. In embodiments, the formulation can be presented as a solution, a suspension, or as an emulsion. In embodiments, the active ingredient can be presented as a bolus, electuary or paste. Tablets and capsules for oral administration can contain, for example, conventional excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents. The tablets may be coated according to methods known in the art. Oral liquid preparations can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which can include, for example, edible oils), or preservatives.

[0073] In embodiments, the pharmaceutical compositions of the present invention are formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and can be presented in unit dose form in ampoules, pre- filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.

[0074] The present disclosure also provides an engineered cell line for screening compounds that disrupt the purinosome comprising:

an engineered cell line expressing a transcriptional reporter tagged to the C-terminus of FGAMS via a TEV protease recognition site (FGAMS-tTa), a HSP90-TEV protease fusion protein, and the transcriptional reporter controls the expression of firefly luciferase.

[0075] The present disclosure also provides an engineered cell line for screening compounds that disrupt the purinosome comprising:

an engineered cell line expressing a transcriptional reporter tagged to the C-terminus of FGAMS via a TEV protease recognition site (FGAMS-tTa), a HSP70-TEV protease fusion protein, and the transcriptional reporter controls the expression of firefly luciferase.

[0076] The present disclosure also provides a method to detect compounds that disrupt the purinosome complex comprising:

providing an engineered cell expressing a transcriptional reporter tagged to the C- terminus of FGAMS via a TEV protease recognition site (FGAMS-tTa), a HSP70-TEV protease fustion protein, and the transcriptional reporter controls the expression of firefly luciferase;

contacting the cell with a compound;

incubating the cell with a substrate of firefly luciferase; and

measuring the chemiluminescence signal of the substrate.

[0077] In embodiments, the substrate can be, for example, D-luciferin. In embodiments, the transcription factor can be, for example, tetracycline trans activator (tTA) transcription factor. [0078] If the two proteins (FGAMS-tTa and Hsp70 or 90-TEV) interact closely to form a purinosome complex, the protease tagged to the HSP will cleave the transcription factor from the FGAMS-tTa protein. This transcription factor is then translocated to the nucleus where it turns on the production of firefly luciferase. The interaction between FGAMS and HSP70 or HSP90 was measured by firefly luciferase activity (RLU) with D-luciferin and normalized by renilla luciferase activity.

[0079] The present disclosure is advantaged by, for example, providing a pharmaceutical composition and method for treating rheumatoid arthritis or certain cancers. The disclosure also identifies a novel molecular mechanism of action that can lead to successful treatment. The disclosure also demonstrates the involvement of heat shock proteins (HSP) in organizing the purinosome, a multienzyme complex involved in purine de novo biosynthesis and the combination of the purine de novo synthesis inhibitor, methotrexate, with the HSP90 inhibitor, 17-AAG or geldamycin, shows synergistic effects on disrupting the purinosome. Such a synergistic effect is desired for drug combination, since the affect offers a method to increase the therapeutic effects, but can also reduce the therapeutic dose of drugs and minimize unwanted side effects.

1. Purine de novo biosynthesis

[0080] Purines are essential molecules for all life, serving not only as the building blocks of DNA and R A, but also playing roles in energy storage and in signaling pathways.

Biosynthetically, adenosine and guanosine nucleotides are derived from inosine

monophosphate (IMP), which is synthesized from phosphoribosyl pyrophosphate (PRPP) in both the de novo and salvage biosynthetic pathways. The salvage pathway catalyzes the one- step conversion of hypoxanthine to IMP by hypoxanthine phosphoribosyl transferase (HPRT). Purine de novo biosynthesis starts with phosphoribosyl pyrophosphate (PRPP) and generates inosine 5 '-monophosphate (IMP), which is further converted to AMP and guanosine monophosphate. Purine de novo biosynthesis requires 10 enzymatic steps to generate IMP. This process is catalyzed by six enzymes in eukaryotes: PRPP

amidotransferase (PPAT) (EC 2.4.2.14), trifunctional phosphoribosylglycinamide formyltransferase (GAR Tfase, EC 2.1.2.2)/phosphoribosylglycinamide synthetase (GARS, EC 6.3.4.13 )/phosphoribosylamino imidazole synthetase (AIRS, EC 6.3.3.1) (GART or TrifGART), phosphoribosyl formylglycinamidine synthase (EC 6.3.5.3) (FGAMS), bifunctional phosphoribosyl aminoimidazole carboxylase (CAIRS, EC

4.1.1.21)/phosphoribosyl aminoimidazole succinocarboxamide synthetase (SAICARS, EC 6.3.2.6) (PAICS), adenylosuccinate lyase (EC 4.3.2.2) (ADSL) and bifunctional 5- aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICAR Tfase, EC

2.1.2.3)/IMP cyclohydrolase (IMPCH, EC 3.5.4.10) (ATIC).

2. Purinosome

[0081] Purinosome describes a multienzyme complex consisting of multiple enzymes involved in purine de novo biosynthesis. A recent study using fluorescence microscopy to HeLa cells indicates that all six of the enzymes in the purine de novo biosynthesis pathway colocalize into protein clusters in the cellular cytoplasm, especially under conditions of purine starvation (see An, S., et al., Reversible compartmentalization of de novo purine biosynthetic complexes in living cells, Science, 2008, 320: 103-106). These functional cytosolic multienzyme complexes are believed to produce efficient substrate channels that link the 10 catalytic active sites. Additionally, clustering of the 10 active sites can provide efficient means of globally regulating purine flux under varying environmental conditions. Thus, these multienzyme complexes observed in the de novo purine biosynthetic pathway can constitute a "purinosome." Considering that individual intermediates of purine de novo biosynthesis have potent regulatory and cytotoxic properties, these intermediates are either undetectable or present in very low (micromolar) concentrations in cellular extracts and/or body fluids under physiologic conditions. Therefore, the dynamic assembly and disassembly of the purinosome is important for the efficiency of purine de novo biosynthesis at such low concentrations of individual intermediates.

[0082] The association and dissociation of these enzyme clusters can be regulated dynamically by changing purine levels in the media or by adding exogenous small molecules, such as kinase inhibitors (An S., et al. Dynamic regulation of a metabolic multi-enzyme complex by protein kinase CK2. J. Biol. Chem., 2010, 285 : 11093-11099) or G protein- coupled receptor (GPCR) ligands (Verrier, F., et al., G protein-coupled receptor signaling regulates the dynamics of a metabolic multienzyme complex, Nature Chemical Biology, 201 1, 7:909-915). In light with these findings and considering the relevance of de novo purine biosynthesis to human diseases, the purinosome represents a new pharmacological target for therapeutic intervention.

3. Heat shock proteins

[0083] Heat shock proteins (HSPs) are a class of functionally related proteins involved in the folding and unfolding of other proteins. Their expression is increased when cells are exposed to elevated temperatures or other stress. This increase in expression is transcriptionally regulated. The dramatic up-regulation of the heat shock proteins is a key part of the heat shock response and is induced primarily by heat shock factor (HSF). HSPs are found in virtually all living organisms, from bacteria to humans. Heat-shock proteins are named according to their molecular weight. For example, HSP60, HSP70 and HSP90 refer to families of heat shock proteins on the order of 60, 70, and 90 kilo-Daltons in size, respectively. The small 8-kilodalton protein ubiquitin, which marks proteins for degradation, also has features of a heat shock protein. HSP70 and HSP90 belong to a family of ubiquitously expressed proteins that are up-regulated in response to stress and serve many functions including assisting in protein folding and the transport, degradation, and prevention of unspecific aggregation of proteins. In addition, these chaperones have been demonstrated to participate in the formation and stabilization of protein complexes.

[0084] HSP70 family constitutes the most conserved and best studied class of HSPs. It encompasses proteins ranging from 66 to 78 kDa that are encoded by a multigene family consisting in human of 11 genes. Some of them are mainly localized in the cytosol like the inducible HSP70 (termed HSP70 or HSP72 or HSPA1) or the constitutively expressed HSC70 (HSP73 or HSPA8), while others are located into the mitochondria (mtHSP70) or in the endoplasmic reticulum (GRP78/Bip). Eukaryotic HSP70s contain two functional domains: the NH2-terminal ATP -binding domain and the COOH-terminal peptide-binding domain. Under normal conditions, HSP70 protein function as an ATP-dependent molecular chaperone by assisting the folding of newly synthesized polypeptides, the assembly of multi- protein complexes and the transport of proteins across cellular membranes. Several proteins such as HSP40, HSP 110, CHIP, HOP, HIP, BAG-1 and BAG-3 have been identified as HSP70 co-chaperones.

[0085] HSP90s consist of four proteins, including HSP90a (HSPC2), HSP90p (HSPC3), GP96 (HSPC4) and TRAPl (HSPC5). HSP90a and HSP90p are essential for the viability of eukaryotic cells. They are constitutively abundant, make up 1-2% of cytosolic proteins, but their expression can be further stimulated by a stress. These isoforms are closely (86%) related proteins and the crystal structures of the two N-terminal domains are very similar. Most works do not differentiate between the two isoforms. HSP90 is a homodimeric protein composed of two identical and symmetrical subunits. Each monomer is divided into three domains, namely N-terminal, middle and C-terminal domains, which have different functions. HSP90 contains a highly conserved ATP binding domain near its N terminus, responsible for the protein's ATPase activity; and the chaperoning activity of HSP90 requires both the binding and hydrolysis of ATP at this site. The charged middle linker region has high affinity for co-chaperones and client proteins. The C terminal dimerization domain contains the tetratricopeptide repeat-binding motif, and is the main region for dimer interaction and the binding of p60HOP and immunophilins. Besides its role in dimerization, it was suggested that the C terminal domain contains a second ATP -binding site of HSP90. The contribution of this second site to the overall regulation of the chaperone is still unknown, but some molecules have been reported to bind at this site and destabilize HSP90 client proteins.

[0086] HSPs are molecular chaperone proteins, since HSPs can bind to and stabilize an otherwise unstable conformer of another protein, and by controlled binding and release of the substrate protein facilitates its correct fate in vivo, thus allowing folding, oligomeric assembly, transport to and between subcellular compartments, or controlled switching between active/inactive conformations. Molecular chaperones also bind to and prevent aggregation of denatured or partially folded proteins, assisting the correct folding of these proteins. HSPs function as molecular chaperones in regulating cellular homeostasis and promoting cell survival. In contrast to other chaperones, many substrate proteins of HSP90 are known because they form stable and long-lived complexes with HSP90 which has allowed their isolation, e.g., by immunoprecipitation. HSP90 modulates the stability and/or transport of a diverse set of critical cellular proteins, known as "client proteins". Client proteins are proteins which transiently non-covalently bind to HSP. This binding may be necessary for their function. HSP90 clients range from signaling protein kinases (e.g. MEK, kinase suppressor of Ras, Akt kinase, MAK-related kinase, epidermal growth factor receptor, insulin-like growth factor receptor) to steroid hormone receptors (glucocorticoid receptor, androgen receptor, progesterone receptor, estrogen receptor) and small G proteins to viral enzymes and components of the telomerase complex, and the list continues to grow. HSP90 client proteins play important roles in numerous cellular processes, including signal transduction, gene regulation, cell cycle control, and apoptosis. Elevated expression of some HSP90 client proteins has been implicated in the maintenance and progression of a number of cancers.

4. Purine de novo biosynthesis and rheumatoid arthritis

[0087] Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease. Its hallmark feature is persistent symmetric polyarthritis (synovitis) that affects the hands and feet, although any flexible joint lined by a synovial membrane may be involved. RA may also affect many other tissues and organs with extra-articular involvement, including the skin, heart, lungs, and eyes. The pathology of RA involves an inflammatory response of the capsule around the joints (synovium) secondary to swelling (hyperplasia) of synovial cells, excess synovial fluid, and the development of fibrous tissue (pannus) in the synovium. The pathology of the disease process often leads to the destruction of articular cartilage and ankylosis (fusion) of the joints. Rheumatoid arthritis can also produce diffuse inflammation in the lungs, membrane around the heart (pericardium), the membranes of the lung (pleura), and white of the eye (sclera), and also nodular lesions, most common in subcutaneous tissue. Although the cause of rheumatoid arthritis is unknown, autoimmunity plays a pivotal role in both its chronicity and progression, and RA is considered a systemic autoimmune disease.

[0088] There is no known cure for rheumatoid arthritis, but many different types of treatment can alleviate symptoms, modify the disease process, or both. Optimal care of patients with RA requires an integrated approach of pharmacologic and non-pharmacologic therapies, such as DMARDs (disease-modifying antirheumatic drugs), biologies, NSAIDs (non-steroid antiinflammatory drugs), analgesics, glucocorticoids, and immunomodulators. Cortisone therapy has offered relief in the past, but its long-term effects have been deemed undesirable.

Cortisone injections using low dosages of daily cortisone (e.g., prednisone or prednisolone) can be valuable adjuncts to a long-term treatment plan. DMARDs are commonly used as early therapy to efficiently inhibit or halt the underlying immune process, thus delaying disease progression, and to produce durable symptomatic remissions. Many of the newer DMARD therapies, however, are immunosuppressive in nature, leading to a higher risk for partially masked serious bacterial, and sometimes fungal, infections. Analgesia (painkillers) and anti-inflammatory drugs are used to suppress the symptoms, and to improve pain and stiffness, but do not prevent joint damage or slow the disease progression. NSAIDs used in the treatment of RA include ibuprofen, naproxen, meloxicam, etodolac, nabumetone, sulindac, tolementin, choline magnesium salicylate, diclofenac, diflusinal, indomethicin, Ketoprofen, Oxaprozin, and piroxicam. Biologies for treating RA include tumor necrosis factor alpha (TNFa) blockers such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), golimumab (Simponi); interleukin 1 (IL-1) blockers such as anakinra (Kineret); monoclonal antibodies against B cells such as rituximab (Rituxan); T cell co-stimulation blocker such as abatacept (Orencia); and interleukin 6 (IL-6) blockers such as tocilizumab (RoActemra, Actemra).

[0089] Methotrexate is an immunosuppressive agent that has been in clinical use for over 50 years. Although originally introduced for chemotherapy in cancer and leukemia, methotrexate was coincidentally found to have immunosuppressive properties and is now the drug of choice in treating rheumatoid arthritis. Early studies in malignant cells regarding the mode of action of methotrexate focused on its role as an antifolate. The major target demonstrated for methotrexate was the inhibition of dihydro folate reductase (DHFR). However, the antiinflammatory activity of low dose methotrexate was noted that involved both purine ribonucleotide and pyrimidine deoxyribonucleotide synthesis. These included inhibition at the level of the first committed step of purine biosynthesis and the ninth folate-dependent step of purine synthesis catalyzed by 5-amino-4-imidazolecarboxamide riboside 5'- monophosphate (AICAribotide) transformylase, as well as the pyrimidine enzyme thymidylate synthetase. Methotrexate enters cells either in its native form or after conversion to its active metabolite, 7 -hydroxy-methotrexate, through an active transport mechanism. Once inside the cell, both methotrexate and 7 -hydroxy-methotrexate are converted into a polyglutamate form by the enzyme folylpolyglutamyl synthetase. The polyglutamate form of methotrexate, which can have up to four new glutamic acid moieties, has several important consequences: 1) it maintains a low intracellular level of the monoglutamate form of methotrexate that never reaches steady-state, thus allowing cells to accumulate vast quantities of polyglutamated methotrexate; 2) it is retained within cells for long periods; 3) it has an increased potency for inhibiting thymidylate synthetase, which converts deoxyuridylate to deoxythymidylate in the de novo pyrimidine biosynthetic pathway; and 4) it has increased potency for directly inhibiting enzymes, such as glycinamide ribonucleotide transformylase (GART) and 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR), that are involved in de novo purine biosynthesis. Methotrexate's ability to inhibit these enzymes increases as the number of glutamate moieties increases. The pentaglutamate form of methotrexate is approximately 2,500 times more potent in inhibiting AICAR, 250 times more potent in inhibiting thymidylate synthetase, and 32 times more potent in inhibiting GART than is the native monoglutamate form. The observation that the relative affinity of the pentaglutamate form of methotrexate for AICAR is 10-fold greater than it is for thymidylate synthetase suggests that the inhibition of pyrimidine biosynthesis by low-dose methotrexate will be minimal compared with that of purine biosynthesis.

5. Heat shock proteins and rheumatoid arthritis

[0090] Rheumatoid arthritis (RA) is a chronic inflammatory disorder that involves mainly joint synovium. One of the major characteristics of RA synovium is the tumor-like growth of fibroblast-like synoviocytes (FLSs) that invade adjacent articular cartilage and bone. Although the mechanism of FLS hyperplasia in RA is not fully understood, it is explained in part by excessive survival, anti-apoptotic signals to FLSs transmitted by inflammatory cells and cytokines, or both. For example, it has been well established that tumor necrosis factor- alpha, a key cytokine in RA, activates genes that mediate proliferative and inflammatory responses. Other anti-apoptotic apparatuses expressed in RA FLSs include FLIP (Fas- associated death domain-like interleukin- 1 β-converting enzyme inhibitory protein), sentrin, mutated p53, and the activation of the nuclear factor-kappa-B or the Akt signaling pathways or both.

[0091] Heat shock protein 70 (HSP70) is a molecular chaperone that is rapidly induced by physical and chemical stresses. The anti-apoptotic function of HSP70 depends on its ability to interact with protein substrates that are not always associated with the chaperoning activity. The mechanisms by which HSP70 exerts its anti-apoptotic function encompass the inhibition of the c-Jun N-terminal kinase (J K) signaling pathway, caspase activation, mitochondrial cytochrome c release, and apoptosome formation. Although the anti-apoptotic role of HSP70 has been demonstrated in a number of studies in various cell types and under different conditions, several other studies have shown that the overexpression of HSP70 promotes cell death, which suggests that HSP70 has dual functionality depending on cell and stimulus type. It has been reported that the expression of HSP70 is higher in both tissue and cultured RA FLSs than in the FLSs of osteoarthritis and that inflammatory cytokines, such as TNF-a and IL-Ι β, that exist abundantly in RA joint fluid further increase HSP70 expression in cultured RA FLSs. HSP70 down-regulation has been found to protect RA FLSs from NO- induced apoptosis, suggesting that HSP70 may be a pro-apoptotic protein in RA FLSs.

[0092] Macrophages that are prominent in the lining and sublining of joints from patients with rheumatoid arthritis are important mediators of chronic inflammation. Synovial macrophages express high levels of cytokines and chemokines such as IL- 1, TNF-a, GM- CSF, IL-6, and IL-8, which mediate inflammation and cartilage and bone destruction. A number of potential endogenous stress response proteins such as HSP60, HSP70, and the extracellular matrix component biglycan have been implicated as potential endogenous tolllike receptor ligands in RA. However, their role in the perpetuation of the chronic inflammation observed in the RA joint is not clear.

6. Purine de novo biosynthesis and cancers [0093] Coordinated and highly regulated metabolic processes are essential to biological function and occur in all components of the human body. The human body extracts hydrocarbons from ingested food and transforms the potential chemical energy in these nutrients to ATP, which ultimately fuels all physiological processes. Glucose and glutamine metabolism is redirected by oncogenes in order to support de novo nucleotide biosynthesis during proliferation. Tumor cells increase the use of anabolic pathways to satisfy the metabolic requirements associated with a high growth rate. Transformed cells take up and metabolize nutrients such as glucose and glutamine at high levels that support anabolic growth. To achieve elevated rates of nucleotide biosynthesis, neoplastic cells must divert carbon from PI3K/Akt-induced glycolytic flux into the non-oxidative branch of the pentose phosphate pathway to generate rib ose-5 -phosphate. This redirection of glucose catabolism appears to be regulated by cytoplasmic tyrosine kinases. Myc-induced glutamine metabolism also increases the abundance and activity of different rate-limiting enzymes that produce the molecular precursors required for de novo nucleotide synthesis.

[0094] Inhibition of cellular replication is one characteristic of cancer cells that has been effectively exploited in the past for the development of anticancer agents. Most drugs that kill cancer cells inhibit the synthesis of DNA or interfere with its function in some way. For a cell to divide into two cells, it must replicate all components including its genome, and unlike the synthesis of other major macromolecules (protein, RNA, lipid, etc.), the synthesis of DNA does not occur to a great degree in quiescent cells. In an adult organism most cells are quiescent and are not in the process of duplicating their genome, therefore, drugs targeting DNA replication affords some level of selectivity. Of course, certain tissues (bone marrow, gastrointestinal, hair follicles, etc.) are in a replicative state, and all cells must continually repair their DNA. Therefore, inhibition of DNA replication in normal tissues results in considerable toxicity which limits the amount of drug that can be tolerated by the patient. Human cells have the capacity to salvage purines and pyrimidines for the synthesis of deoxyribonucleotides that are used for DNA synthesis, and analogues of these nucleotide precursors have proven to be an important class of anticancer agents. There are 14 purine and pyrimidine antimetabolites that are approved by the FDA for the treatment of cancers, which account for nearly 20% of all cancer drugs. Some of the first FDA approved cancer drugs were in this class of compounds. 6-Mercaptopurine was approved in 1953 for the treatment of childhood leukemia, where it is curative and is still the standard of treatment for this disease. Since 1991, nine nucleoside analogues were approved by the FDA for the treatment of various malignancies. Four of these new agents were approved since 2004, and there are numerous agents that are currently being evaluated in clinical trials. The recent FDA approvals indicate that the design and synthesis of new nucleoside analogues is still a productive area for discovering new drugs for the treatment of cancer. In general, these compounds have been most useful in the treatment of hematologic malignancies, and even though there is still room for significant improvements in the treatment of these diseases, some of the newer agents are finding use in the treatment of solid tumors.

[0095] The basic mechanism of action of purine and pyrimidine antimetabolites is similar. These compounds diffuse into cells (usually with the aid of a membrane transporter) and are converted to analogues of cellular nucleotides by enzymes of the purine or pyrimidine metabolic pathway. These metabolites then inhibit one or more enzymes that are critical for DNA synthesis, causing DNA damage and induction of apoptosis. Even though the compounds in this class are structurally similar and share many mechanistic details, it is clear that subtle quantitative and qualitative differences in the metabolism of these agents and their interactions with target enzymes can have a profound impact on their antitumor activity.

[0096] Potent inhibitors of purine (and of pyrimidine) nucleotide biosynthesis can be either synthetic or natural-product analogues of intermediates of the pathway, or inhibitors can also be designed based of the catalytic mechanism. These inhibitors are effective drugs against cancer, inflammatory disorders and various infections. For treatment of human cancer, targeting the purine pathway is more common than targeting the pyrimidine pathway, where more toxic side effects are apparent. Design of inhibitors based on the X-ray structure of the target enzyme can yield drugs with only one site of action in human cells. Such approach resulted in the discovery of drugs acting against PPAT (e. g. piritrexm), GART (e.g., azaserine, diazomycin, dideazatetrahydrofolate, lometrexol), AIRC (fluorosulfonylbenzoyl- adenosine), and SAICARS (e.g., nitroaminoimidazole ribonucleotide).

7. Heat shock proteins and cancers

[0097] Cancer is a collection of diseases that arise from the progressive accumulation of genetic alterations in somatic cells. Human cancer is considered to be a pathway dysregulated disease. The ability of tumor cells to outgrow their neighboring cells is often driven by constitutive activation of downstream proteins. Genetic studies over several decades have discovered a wide range of tumor-associated genes and their mutations, many of which preferentially occur in signaling proteins involved in a small number of pathways. Genetic mutations are often enriched in positive regulatory loops (gain of function), and methylated genes in negative regulatory loops (loss of function), leading to the disruption of the normal cooperative behavior of cells and thus promoting tumor phenotypes.

[0098] HSPs can block apoptosis by interacting with key proteins at three levels: 1) upstream of the mitochondria, thereby modulating signaling pathways (e.g., HSP70 modulates the activation of stress-activated kinases such as Akt); 2) at the mitochondrial level, controlling the release of cytochrome C by its interaction with actin and HSP70 with Bax; and 3) at the post-mitochondrial level, by blocking apoptosis by their interaction with cytochrome C (HSP27), Apaf-1 (HSP70 or HSP90), AIF (HSP70) or Smac (HSP27). HSP90 by chaperoning oncogenic proteins such us FLT-ITD, Bcr-abl, c-Kit, ZAP70, and their downstream signaling molecules (such as STAT5/Bcl-Xl, JAK/STAT) leads to cell survival. HSP70 prevents the cleavage of GATA-1 by caspase-3 allowing differentiation instead of apoptosis.

[0099] The basal expression level of stress-inducible HSP70 in normal, non-transformed, cells and tissues is rather low or absent. In contrast, these HSPs are abundantly expressed in most cancer cells, in particular in hematological malignancies, including lymphoid diseases and chronic or acute myeloid leukemia. Cancer cells need this strong content on HSPs for their survival which is the rational of their inhibition in cancer therapy. Furthermore, a high level of HSP27, HSP70, and HSP90 correlates with a poor prognosis in acute myeloid leukemia and myelodysplasia syndromes.

[00100] HSP90 is required to maintain the conformational stability and function of a broad range of oncogenic proteins like mutant c-Kit, FLT3 with internal tandem repeat mutation (FLT3-ITD), and Bcr-abl. For instance, HSP90 protects wild -type and mutant receptor tyrosine kinase c-Kit from degradation. Accordingly, treatment of malignant mast cells with the HSP90 inhibitor 17-AAG strongly reduces the constitutive activity of c-kit and the downstream signaling molecules AKT and STAT3. Furthermore, D816V mutant c-Kit AML cells, which are resistant to imatinib, are sensitive to growth inhibition by HSP90 inhibitors. Different types of mutant Kit kinase have been recurrently identified in AML and it has been shown that the potency of either the selective Kit inhibitor KI-328 or HSP90 inhibitors is dependent on the Kit kinase mutation type.

[00101] HSP 70 is overexpressed in malignant melanoma and underexpressed in renal cell cancer. Cancerous cells over express a number of proteins, including growth factor receptors, such as EGFR, or signal transduction proteins such as PI3K and AKT (inhibition of these proteins may trigger apoptosis). HSP90 stabilizes various growth factor receptors and some signaling molecules including PI3K and AKT proteins, hence inhibition of HSP90 may induce apoptosis through inhibition of the PI3K/AKT signaling pathway and growth factor signaling generally.

8. HSP inhibitors

[00102] HSP90 is an ATP dependent molecular chaperone protein which integrates multiple oncogenic pathways. As such, HSP90 inhibition is a promising anti-cancer strategy. Several inhibitors that act on HSP90 by binding to its N-terminal ATP pocket have entered clinical evaluation. Robust pre-clinical data suggested anti-tumor activity in multiple cancer types. Clinically, encouraging results have been demonstrated in melanoma, acute myeloid leukemia, castrate refractory prostate cancer, non-small cell lung carcinoma, and multiple myeloma. In breast cancer, proof-of-concept was demonstrated by first generation HSP90 inhibitors in combination with trastuzumab mainly in human epidermal growth factor receptor 2 (HER2) positive metastatic breast cancers. There are a multitude of second generation HSP90 inhibitors currently under investigation. To date, however, there is no FDA approved HSP90 inhibitor nor standardized assay to ascertain HSP90 inhibition.

[00103] HSP90 inhibitors can be classified into multiple chemical classes.

Geldanamycin derivative HSP90 inhibitors include 17-AAG (17-allyl-l 7- demethoxygeldanamycin), 17-DMAG (17-desmethoxy- 17-N,N-dimethylaminoethylaminogel danamycin), ΓΡΙ-504 (17-allylamino-17-demethoxygeldanamycin hydroquinone

hydrochloride) and IPI-493 (17-desmethoxy-17-amino geldanamycin). Purine and purine-like HSP90 inhibitors include CNF 2024/ΒΠΒ021, MPC-3100, Debio 0932 (CUDC-305), and PU-H71. Resorcinol derivative HSP90 inhibitors include STA-9090 (Ganetespib), NVP- AUY922/VER52296, KW-2478, and AT-13387.

[00104] HSP70 inhibitors include, for example, VER 155008 (5'-0-[(4- Cyanophenyl)methyl]-8-[[(3,4-dichlorophenyl)methyl]amino]-adenosine), 2- phenylethynesulfonamide (Pifithrin-μ), MKT-07, or methylene blue.

9. Purine de novo biosynthesis inhibitors

[00105] Purine de novo biosynthesis inhibitors can include at least one of a series of drugs approved by FDA, including, for example, thiopurines (mercaptopurine and thioguanine), fludarabine, nelarabine, cladribine, clofarabine, pentostatin, and methotrexate. Others are PPAT inhibitor piritrexm, GART inhibitors including azaserine, diazomycin, dideazatetrahydrofolate, lometrexol, AIRC inhibitor fluorosulfonylbenzoyl-adenosine, SAICARS inhibitor nitroaminoimidazole ribonucleotide, and mixtures thereof. 10. Combination therapy

[00106] Many DMARDs have adverse side effects. The most common adverse events relate to liver and bone marrow toxicity (methotrexate, sulfasalazine, leflunomide, azathioprine, gold compounds, D-penicillamine), renal toxicity (cyclosporine A, parenteral gold salts, D-penicillamine), pneumonitis (methotrexate), allergic skin reactions (gold compounds, sulfasalazine), autoimmunity (D-penicillamine, sulfasalazine, minocycline) and infections (azathioprine, cyclosporine A). Hydroxychloroquine is a less potent DMARD and may cause ocular toxicity in rare cases, but does not affect the bone marrow or liver.

Methotrexate as an effective DMARD is the most important and useful DMARD and is often part of the initial line of treatment. Methotrexate is often preferred by rheumatologists because if it does not control arthritis on its own then it works well in combination with many other drugs, especially the biological agents, in the control of arthritis. However, methotrexate does have adverse effects, and patients on this drug must be monitored regularly. Methotrexate has been shown to have organ toxicity, such as gastrointestinal, hematologic, pulmonary, and hepatic. Methotrexate is also considered a teratogenic and as such, it is recommended patients should use contraceptives to avoid pregnancy and to discontinue the use of methotrexate if pregnancy is planned.

[00107] Combination therapy of multiple disease modifying drugs is a common practice for the treatment of rheumatoid arthritis, particularly as it has become apparent that using a combination of these drugs does not increase their relative toxicity profiles. Common combinations of DMARDs include methotrexate and hydroxychloroquine, methotrexate and sulfasalazine, sulfasalazine and hydroxychloroquine, and methotrexate and

hydroxychloroquine and sulfasalazine. Small molecule DMARDs include azathioprine, ciclosporin (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide, methotrexate (MTX), minocycline, sulfasalazine. However, no combination of methotrexate with HSP inhibitors has been reported for the treatment of cancers and rheumatoid arthritis.

11. Salt(s) and pharmaceutically acceptable salt(s)

[00108] The pharmaceutical compositions of the present disclosure can be used in the form of salts of, for example, inorganic or organic acids. Depending on the particular compound, a salt of the compound may be advantageous due to one or more of the salt's physical properties, such as enhanced pharmaceutical stability in differing temperatures and humidity, or a desirable solubility in water or oil. In some instances, a salt of a compound can also be used as an aid, for example, in the isolation, purification, resolution, and like processing steps of the compound.

[00109] If a salt is intended to be administered to a patient (as opposed to, for example, being used in an in vitro context), the salt preferably is pharmaceutically acceptable. The term "pharmaceutically acceptable salt" refers to a salt prepared by combining one or both compounds of the combination with an acid whose anion, or a base whose cation, is generally considered suitable for human consumption. Pharmaceutically acceptable salts are particularly useful as products of the methods of the present disclosure because of their greater aqueous solubility relative to the parent compound. For use in medicine, the salts of the compounds of this invention are non-toxic "pharmaceutically acceptable salts." Salts encompassed within the term "pharmaceutically acceptable salts" refer to non-toxic salts of the compounds of the disclosure, which can be generally prepared by reacting the free base with a suitable organic or inorganic acid.

[00110] Suitable pharmaceutically acceptable acid addition salts of the compounds of the disclosure when possible include those derived from inorganic acids, for example, hydrochloric, hydrobromic, hydrofluoric, boric, fluoroboric, phosphoric, metaphosphoric, nitric, carbonic, sulfonic, and sulfuric acids, and organic acids such as acetic,

benzenesulfonic, benzoic, citric, ethanesulfonic, fumaric, gluconic, glycolic, isothionic, lactic, lactobionic, maleic, malic, methanesulfonic, trifluoromethanesulfonic, succinic, toluenesulfonic, tartaric, and trifluoroacetic acids. Suitable organic acids generally include, for example, aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids.

[00111] Specific examples of suitable organic acids include acetate, trifluoroacetate, formate, propionate, succinate, glycolate, gluconate, digluconate, lactate, malate, tartaric acid, citrate, ascorbate, glucuronate, maleate, fumarate, pyruvate, aspartate, glutamate, benzoate, anthranilic acid, mesylate, stearate, salicylate, p-hydroxybenzoate, phenylacetate, mandelate, embonate (pamoate), methanesulfonate, ethanesulfonate, benzenesulfonate, pantothenate, toluenesulfonate, 2-hydroxyethanesulfonate, sufanilate, cyclohexylaminosulfonate, algenic acid, β-hydroxybutyric acid, galactarate, galacturonate, adipate, alginate, butyrate, camphorate, camphorsulfonate, cyclopentanepropionate, dodecylsulfate, glycoheptanoate, glycerophosphate, heptanoate, hexanoate, nicotinate, 2-naphthalesulfonate, oxalate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, thiocyanate, tosylate, and undecanoate.

Furthermore, where the compounds of the disclosure carry an acidic moiety, suitable pharmaceutically acceptable salts thereof can include alkali metal salts, i.e., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts. In embodiments, base salts can be formed from bases which can form non-toxic salts, including aluminum, arginine, benzathine, choline, diethylamine, diolamine, glycine, lysine, meglumine, olamine, tromethamine, and zinc salts.

[00112] Organic salts can be made from secondary, tertiary, or quaternary amine salts, such as tromethamine, diethylamine, Ν,Ν'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. Basic nitrogen-containing groups can be quaternized with agents such as lower alkyl (Ci.Ce) halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (i.e., dimethyl, diethyl, dibuytl, and diamyl sulfates), long chain halides (i.e., decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides), arylalkyl halides (i.e., benzyl and phenethyl bromides), and others.

[00113] In embodiments, hemisalts of acids and bases can also be formed, for example, hemisulphate and hemicalcium salts.

[00114] The compounds of the disclosure and their salts can exist in both unsolvated and solvated forms.

Materials and Methods:

[00115] Chemicals. 4,5,6,7-tetrabromobenzimidazole (TBI), MKT-077,

geranylgeranylacetone, celastrol, methotrexate and methylene blue were purchased from Sigma- Aldrich, dithiobis(succinimidyl propionate) (DSP) was purchased from Thermo Scientific, 4,5,6,7-tetrabromobenzotriazole (TBB) from Calbiochem (EMD), 17-N- allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17- demethoxygeldanamycin(17-DMAG) from Selleck Chemicals LLC, Pifithrin-μ (2- phenylethynesulfonamide) from Tocris Bioscience.

[00116] Plasmids. The constructs of six human enzymes involved in the de novo purine biosynthetic pathway and the tetrahydro folate (H4F)-utilizing enzyme (hClTHF) in this disclosure were previously used (see Science, 2008, 320: 103-106 (supra.)); the plasmids are hFGAMS-EGFP, hFGAMS-OFP, hTrifGART-GFP, hPPAT-EGFP, hPAICS-EGFP, hASL-EGFP, GFP-hATIC and hClTHF-EGFP. The fluorescent protein fusion vectors: pmEGFP-Nl and pmOFP-Nl were modified from the pEGFP-Nl (Clontech) and pRSET mOrange. G3BP-GFP was a gift from the Jamal Tazi group of the Institut de Genetique Moleculaire de Montpellier. GFP170 and GFP250 were gifts from Elizabeth Sztul of the University of Alabama at Birmingham.

[00117] The SHMT1 (SHMT1) gene was a gift from Patrick Stover of Cornell University, the BAG5 (BCL2 -associated athanogene 5) gene was purchased from ATCC, and cDNAs of other enzymes were obtained from the Arizona State University Biodesign Institute plasmid repository (DNASU). All genes were amplified by PCR with primers containing two restriction sites. hel and BamHI were used for HSP90 (Swiss-Prot

Accession number P08107), Nhel and Kpnl for HSP70 (P07900), Xhol and Bamffl for DnaJC7 (Q99615), EcoRI and Xhol for BAG5 (Q9UL15), Nhel and EcoRI for p23

(Q15185), BAG2 (095816), Stipl(P31948) and SHMT1. The PCR product was introduced into the pmEGFP-Nl or pmOFP-Nl to obtain the GFP or OFP fused protein construct. The FGAMS-9xcMyc used for the IP experiment contains a -9xcMyc-6xHis tag (subcloned from a pYL436 vector from ABRC) and was constructed by inserting the FGAMS gene using the Nhel and EcoRI sites and the tag region using the EcoRI and Notl sites into the mEGFP vector. The HSP90G97D and HSP70K71E mutants were made by site-directed mutagenesis. All of the gene inserts were confirmed by DNA sequencing. All plasmids were isolated by the QIAprep Spin Miniprep Kit (Qiagen) after transformation of XL 1 -Blue or DH5a competent cells. 9xcMyc is a low c-Myc amplification factor. c-Myc is a regulator gene that codes for a transcription factor Myc. Myc protein is a transcription factor that activates expression of a great number of genes through binding on consensus sequences (Enhancer Box sequences (E -boxes)) and recruiting histone acetyltransferases (HATs). It can also act as a transcriptional repressor. By binding Miz-1 transcription factor and displacing the p300 co- activator, it inhibits expression of Miz-1 target genes. In addition, Myc has a direct role in the control of DNA replication. In the human genome, Myc is located on chromosome 8 and is believed to regulate expression of 15% of all genes through binding on Enhancer Box sequences (E-boxes) and recruiting histone acetyltransferases (HATs). This means that in addition to its role as a classical transcription factor, Myc also functions to regulate global chromatin structure by regulating histone acetylation both in gene-rich regions and at sites far from any known gene.

[00118] Mammalian Cell Culture. Three human cancer cell lines, HeLa, C3A and A431 were obtained from the American Type Culture Collection (ATCC). Cells were maintained in Minimum Essential Medium Eagle (MEM) with Earle's salts and L-glutamine (Mediatech, Inc.) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals). In the purine-depleted condition, cells were maintained in RPMI 1640 with L-glutamine (Mediatech, Inc.) supplemented with 5% dialyzed FBS. Purines in FBS were removed by membrane (MWCO: 25k) dialysis against 0.9% NaCl at 4°C for 2 days as previously described (see Science 2008, 320: 103-106 (supra.).

[00119] Plasmid DNA Transient Transfection. For microscopic imaging, cells were plated in 35 mm diameter glass bottom culture dishes (MatTek Corporation, P35G-1.5-14-C). One day before transfection, 1.5-2.5 x 10⁵ cells were plated in 1.5 mL of growth medium without antibiotics in order to reach 80-90% confluence at the time of transfection. Each well was transfected with 3-6 micrograms of DNA and 3-5 microL of Lipofectamine 2000 (Invitrogen) (for HeLa) or 4-6 microL of X-tremeGENE HP (Roche) (for A431 and C3A) following the manufacturer's protocol. To avoid the high cytotoxicity, transfection complexes were removed by replacing with growth medium after 5 hours. Cells were then incubated at 37°C in a 5% CO₂ incubator for 12-18 hours prior to imaging.

[00120] Co-Immunoprecipitation. HeLa cells were transiently transfected with

FGAMS-9xcMyc and grown in purine-rich and purine-depleted media. Formation of purinosomes in the transfected cells grown in purine-depleted medium was enriched by incubating the cells with 23 micro M (10 microg/mL) TBI at room temperature for 1 hour. To capture the cellular interaction network within the purinosome, DSP crosslinking was performed before harvesting the cells. The commercially available c-Myc monoclonal antibody crosslinked to protein A that is immobilized to agarose beads (Clontech) was applied to immunoprecipitate c-Myc-tagged FGAMS and its interacting partners. HeLa cells growing in the purine-depleted medium (samples A, B, and C) and purine-rich medium (samples D, E, and F) respectively were compared. Mock transfection controls (A, B, D, and E) and controls without TBI treatment (A) or without TBB treatment (D) were carried out to rule out any possibilities of non-specific interactions. The presence of FGAMS-9xcMyc (about 162 KDa) only in the final IP eluate samples C and F was confirmed by Western blot using anti-cMyc antibody (Abeam ab9132). A tandem mass spectrometric analysis followed to identify protein components of the purinosome.

[00121] Sample preparation: Liquid chromatography - mass spectrometry (MudPLT) analysis. Protein was TCA precipitated using standard procedures and resuspended in 8M Urea in 50 mM Tris pH 8.0. Each sample was reduced with 10 mM TCEP for 30 minutes and alkylated with 12.5 mM fresh IAA for 30 minutes in the dark. Samples were diluted to 2M Urea with 50 mM Tris pH 8.0 and digested overnight in the presence of 1 mM CaC^ and trypsin (1 microL of 0.5 microg/ microL). Digested samples were acidified to 5% final formic acid and centrifuged for 30 minutes. Peptides were loaded onto a biphasic column with SCX and CI 8 for analysis on a LTQ XL ion trap mass spectrometer (Thermo Scientific) using an 5 step standard procedure. The mass spectrometer was set in a data-dependent acquisition mode with dynamic exclusion enabled with a repeat count of 1 , a repeat duration of 20 s, exclusion duration of 60 s and an exclusion list size of 300. All tandem mass spectra were collected using normalized collision energy of 35 % and an isolation window of 2 Da. One micro scan was applied for all experiments in the LTQ. Spray voltage was set to 2.50 kV. Each full MS survey scan was followed by 7 MS/MS scans.

[00122] Analysis of Mass Spectroscopic (MS) Data. RAW files were generated from mass spectra using XCalibur version 1.4, and MS/MS spectra data extracted using RAW Xtractor (version 1.9.1) which is publicly available (see for example,

fields.scripps.edu). MS/MS spectral data were searched using the Prolucid algorithm

(Version 3.0) against a custom made database containing 22,935 human sequences (longest entry for the IPI database for each protein) that were concatenated to a decoy database in which the sequences for each entry in the original database were reversed. In total the search database contained 45,870 protein sequence entries (22,935 real sequences and 22,935 decoy sequences). SEQUEST searches allowed for oxidation of methionine residues (16.0 Da), DSP modification (145.0 Da K,R and M), static modification of cysteine residues (57.0 Da - due to alkylation), no enzyme specificity, and a mass tolerance set to ±1.5 Da for precursor mass and ± 0.5 Da for product ion masses. The resulting MS/MS spectra matches were assembled and filtered using DTASelect2 (version 2.0.27). The validity of peptide/spectrum matches was assessed using DTASelect2 (version 2.0.27) and two SEQUEST-defined parameters, the cross-correlation score (XCorr), normalized difference in cross-correlation scores (DeltaCN). The search results were grouped by charge state (+1, +2, +3), tryptic status, and modification status (modified and unmodified peptides), resulting in 18 distinct subgroups. In each of these subgroups, the distribution of Xcorr and DeltaCN values for the direct and decoy database hits was obtained, then the direct and decoy subsets were separated by discriminant analysis. Outlier points in the two distributions were discarded. Full separation of the direct and decoy subsets is not generally possible so the discriminant score was set such that a false discovery rate of less than 1 % was determined based on the number of accepted decoy database peptides (number of decoy database hits/number of filtered peptides identifiedx 100). In addition, a minimum peptide length of seven amino acids residues was imposed and protein identification required the matching of at least two peptides per protein. Such criteria resulted in the elimination of most decoy database hits. [00123] Fluorescence Live Cell Imaging. All samples were washed 3 times for 5 min incubations with buffered saline solution (BSS: 20 mM HEPES (pH 7.4), 135 mM NaCl, 5 mM KC1, 1 mM MgCl₂, 1.8 mM CaCl₂ and 5.6 mM glucose) before imaging. Cells were imaged at ambient temperature (about 25°C) under a Nikon TE-2000E inverted microscope equipped with a 60X 1.49 numerical aperture objective and a photometries CoolSnap ES2 CCD detector. GFP detection was accomplished by using an S484/15x excitation filter, S517/30m emission filter, and Q505LP/HQ510LP dichroic (Chroma Technology). The OFP signal was obtained by using an S555/25x excitation filter, S605/40m emission filter, and Q575LP/HQ585LP dichroic (Chroma Technology). Nikon NIS-Elements (3.0) was used for collecting images samples and viewed using a mercury fiber illuminator. In the reversibility imaging experiment, cells were incubated in the 37°C, 5% CO2 incubator between images captured at different time points. All images were created using the ImageJ program and were in some cases cropped, inverted or shown in color for clarity, but were otherwise unmodified. Colocalization analyses were performed with the JACoP plugin in ImageJ. The threshold for each image was created using the Auto Local Threshold tool using the Sauvola method. The resulting thresholds were used for co-localization analyses. Values for Pearson's correlation coefficient and overlap coefficient are reported and range from 0 (no correlation/overlap) to 1 (complete correlation/overlap). To investigate the effect of heat shock protein

inhibitors/activators on the purinosome formation, the small molecule was added into the growth medium and the cells were incubated for an additional 1-2 hours inside the incubator; images were acquired before and after the incubation.

[00124] Luciferase Reporter Assay. For the transfection for this protocol, HeLa cells suspended in purine-rich or purine-depleted medium without antibiotics were inoculated into 24-well cell culture plate (BD) at 30,000 or 40,000 cells per well densities. The following day, the cells were transfected with XtremeGene transfection reagent (Roche), according to the manufacturer's protocol. Briefly, 100 microL cell culture medium containing no serum was mixed with plasmids and XtremeGene reagent and incubated at room temperature for 15 minutes before being added into a well on the 24-well cell culture plate. For each well, 100 ng of TRE-TIGHT firefly luciferase construct, 100 ng of TEV cleavage site-tTA fusion construct and 100 ng TEV only or TEV fusion construct, and 3 XtremeGene reagent were used. When measuring the luciferase signal induced by a tTA fusion protein in the absence of TEV or TEV fusion protein, 100 ng pEGFP-Nl plasmid was used in place of TEV or TEV fusion plasmid. When measuring normalized luciferase expression, an additional 10 ng of renillar luciferase expression plasmid (Promega) was added to each well. After adding the transfection reagent, the plate was returned to the cell culture incubator and incubated for 2 hours before being rinsed and replaced with fresh medium. The plates of transfected cells were then incubated for two days before being using for the luciferase assay.

[00125] For the assay, cells were harvested and luciferase expression was measured with a luciferase assay kit (Biotium), according to the manufacturer's protocol. Briefly, after removing cell culture media, cells in 24-well plates were rinsed with PBS and kept on ice. 100 microL cell lysis buffer was added to each well. The plates, on ice, were placed on a rocking platform with gentle rocking for 15 minutes before beginning the luciferase assay. 100 microL of luciferase assay buffer was mixed with 20 microL cell lysate from each well, and luminescence was measured on a Lumat LB 9501 luminometer (Berthold).

[00126] Cell death assay (MTT assay). A cell survival assay was used to monitor the cytotoxic effect of the various inhibitors. HeLa cells were plated in 96-well plates at approximately 1 x 10⁵ cells per mL and grown at 37°C in 5% CO₂ for 16 hours. Varying concentrations of the drug or vehicle control were added to the wells and the plate was returned to the incubator for an additional 72 hours. The media was removed and 5 mg/mL 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to the wells and further incubated for 3 hours. The media was removed and replaced with DMSO to solubilize the formazan product. After one hour incubation the plate was read at 550 nm. The data were plotted as factor affected vs. dose and fit with the median-effect equation fa = l/[l+(Dm/D)m , where fa is the factor affected, Dm is the median-effect dose (EC50), m is the slope of the curve and D is the dose.

[00127] For combinations of drugs with methotrexate, the drug was combined with methotrexate at a 1 : 1 ratio and the assay was carried out as described above. To determine if the drug combination had a synergistic effect, a Chou plot of factor affected vs. combination index was constructed. The combination index is defined as CI = (D)l/(Dx)l + (D)2/(Dx)2 , where D is the dose at some concentration and Dx = Dm[fa/(l-fa)] l/m. A combination index of less than one indicates synergism, while a value above one indicates antagonism. All samples were carried out in at least triplicate and are reported as average plus/minus standard error.

Results and Discussion

[00128] To investigate the composition of the purinosome and the factors that drive its assembly and disassembly, we carried out an immunoprecipitation (IP) of myc -tagged formylglycinamidine synthetase (FGAMS), from transiently transfected cells that had been treated with a chemical cross-linking reagent. FGAMS, the enzyme that catalyzes the fourth step in purine biosynthesis, is an established marker for purinosomes and has been speculated to act as a scaffold for the formation of purinosome. The purified samples, confirmed by Western blot to be enriched in FGAMS (Fig.1) were analyzed by mass spectrometry to identify co-precipitated proteins (Table 1). Amongst the proteins pulled down with FGAMS, of particular interest was a group of co-chaperone proteins including Bcl-2-associated anthogene (BAG) domain proteins, J-domain proteins (DnaJ or HSP40) and heat shock organizing protein (Stipl) that led us to investigate the possible role of the HSP70/HSP90 chaperone machinery in purinosome formation.

[00129] Referring to the Figures, Fig. 1 shows a Western blot of FGAMS-9xcMyc (162kDa) that was pulled down by anti-cMyc antibody in the co-immunoprecipitation (co- IP). Lanes A, B, C, D, E and F are co-IP eluates from HeLa cells growing under different conditions: only C (purine-depleted medium) and F (purine-rich medium) are transfected with FGAMS-9xcMyc. Lane A, B, D and E are mock transfection controls. Lane A does not contain TBI (4,5,6,7-tetrabromobenzimidazole), lane B contains TBI, lane D does not contain TBB (4,5,6,7-tetrabromobenzotriazole), and lane E contains TBB. Lane L is a protein standard.

[00130] Fig. 2 shows immunofluorescence images that demonstrate that Hsp90 and Hsp70 co-localize with purinosomes. Hsp90-GFP (A) and Hsp70-GFP (D) colocalize with the purinosome marker FGAMS-OFP (B and E, Pearson's coefficient of 0.92 and overlap coefficient of 0.93 for Hsp90, Pearson's coefficient of 0.90 and overlap coefficient of 0.91 for Hsp70) in HeLa cells. (C) and (F) show the merged images. The merged images of Hsp90- GFP and FGAMS-OFP serve to demonstrate that purinosomes appear under purine depleted conditions (G), disappear when conditions are changed to purine rich (H, 120 minutes) and reappear when purine depleted conditions are restored (I, 90 additional minutes after media change). Hsp90-OFP (K and M) does not co-cluster with control proteins Cl -THF-GFP (J) or SHMT1-GFP (L). The scale bar represents 10 micrometers (μιη).

[00131] Fig. 3 shows cellular co-localization of PPAT-GFP and Hsp90-OFP, PAICS- GFP and Hsp90-OFP in HeLa cells grown in purine-depleted conditions. PPAT (A, B, and C) is the enzyme that catalyzes the first step in the purine de novo biosynthetic pathway, while PAICS (D, E and F) catalyzes the sixth and seventh steps. Both PPAT-GFP (A and green in merged image C) and PAICS (D and green in merged image F) co-localize with Hsp90-OFP (B, E and red in C, F) in HeLa cells. The scale bar represents 10 micrometers (μιη). [00132] Fig. 4 shows cellular co-localization of Hsp70-OFP and PPAT-GFP or PAICS-GFP in HeLa cells grown in purine-depleted conditions. Both PPAT-GFP (B and green in merged image C) and PAICS (E and green in merged image F) are co-localized with Hsp70-OFP (A, D and red in C, F) in HeLa cells.

[00133] Fig. 5 shows cellular co-localization of FGAMS-GFP and Hsp90-OFP in C3A and A431 cells. Human liver cancer cell line, C3A (A, B and C) and human skin cancer cell line, A431 (D, E and F) were grown in purine-depleted conditions. FGAMS-GFP (A, D and green in C, F) co-localized with Hsp90-OFP (B, E and red in C, F) in both cells. (C) and (F) show merged images of (A) and (B), and (D) and (E), respectively. The scale bar represents 10 micrometers.

[00134] Fig. 6 shows HeLa cells transfected with Hsp70 and Hsp90 alone. Hsp90-GFP (A and B) and Hsp70-GFP (C and D) show a diffuse staining pattern when expressed in HeLa cells grown in purine depleted media.

[00135] Fig. 7 shows the results of a Luciferase reporter assay and the interaction between FGAMS and Hsp70 or Hsp90. In this method, a transcriptional reporter is tagged to the C-terminus of FGAMS while the sequence linking the protein and the tag contains a TEV protease recognition site. The Hsp70 or Hsp90 is fused to a modified form of TEV protease which, if the two proteins (FGAMS-tTa and Hsp70 or 90-TEV) interact closely, will cleave the transcription factor. This factor is targeted to the nucleus where it turns on the production of firefly luciferase. The interaction between FGAMS and Hsp70 or Hsp90 was measured by firefly luciferase activity (RLU) and normalized by renilla luciferase activity. When the FGAMS-tTA and the reporter constructs were introduced into HeLa cells in the absence of Hsp70-TEV or Hsp90-TEV construct, the expression of firefly reporter gene was at a very low level. When either Hsp70-TEV or Hsp90-TEV was introduced with FGAMS-tTA and the reporter constructs, the expression of firefly luciferase was greatly increased, indicating that FGAMS and Hsp70/Hsp90 associate closely in vivo. Note that for the FGAMS-tTA, Hsp70-TEV and Hsp90-TEV controls, the normalized RLU values are 1382/2182,

4977/5525, and 2404/31 15 for purine rich/purine depleted media respectively and thus do not show up.

[00136] Fig. 8 shows cellular co-localization of FGAMS-OFP and cochaperone BAG2-GFP or DnaJAl -GFP in HeLa cells grown in purine-depleted conditions. BAG2-GFP (A and green in C), DnaJAl-GFP (D and green color in F), FGAMS-OFP (B, E and red color in C, F). (C) and (F) show merged images of (A) and (B), and (D) and (E), respectively. The scale bar represents 10 micrometers. [00137] Fig. 9 shows several co-chaperones that are also associated with purinosomes. BAG5-GFP (A), Stipl -GFP (D), DnaJ-C7-GFP (G) and p23-GFP (J) co-localize with FGAMS-OFP (B, E, H, and K). Merged images are shown in (C), (F), (I), and (L), respectively. Overlap coefficients with FGAMS-OFP for BAG5, Stipl, DnaJ-C7, and p23 are 0.89, 0.86, 0.90 and 0.84 respectively. The scale bar represents 10 micrometers.

[00138] Figs. 10 shows the effect of Hsp90 inhibitor, 17-DMAG, on purinosome formation in HeLa cells transfected by FGAMS-GFP. Image (A) was taken prior to the 17- DMAG addition, showing the purinosomes formed by the FGAMS-GFP. Image (B) was taken after the cell was incubated with 500 micromolar (μΜ) of 17-DMAG for 1.5 hours. Image (C) was taken after 2 hours. Approximately 80% of clusters (as determined by ImageJ analyze particles tool) were disrupted after incubation with 17-DMAG for 2 hrs. The scale bar represents 10 micrometers.

[00139] Fig. 11 shows Hsp90 and Hsp70 inhibitors disrupt purinosomes and are synergistic with methotrexate. Images (A) and (C) show HeLa cells transfected with the purinosome marker FGAMS-GFP. The Hsp90 inhibitor celastrol disrupts purinosomes at 1.8 μΜ after 10 minutes (B), and the Hsp70 inhibitor MKT -077 disrupts purinosomes at 15 μΜ after 60 minutes (D). Disruption of purinosomes by inhibitors is reversible, as shown by the treatment of cells with the Hsp90 inhibitor 17-AAG (E, before treatment and F, after treatment with 200 μΜ for 90 minutes) followed by a media change to remove the inhibitor (G, 30 minutes after media change). Treatment of HeLa cells for 72 hours with 17-AAG alone (H, filled squares and dotted line) kills cells with an EC50 of 0.14 ± 0.4 μΜ, while treatment with methotrexate alone (H, filled diamonds and dashed line) gives an EC50 of 10.1 ± 0.4 nM. The combination of 17-AAG and methotrexate (H, filled circles and solid line) leads to a decrease in the EC50 to 4.7 ± 0.3 nM. A Chou plot of the results (J) verifies that the combination is synergistic (CI < 1). Similarly, the combination of the Hsp70 inhibitor Pifithrin-μ and methotrexate (I, filled circles and solid line) gives a decrease in EC50 (5.0 ± 0.3 nM) over either Pifithrin-μ (I, filled squares and dotted line, EC50 of 9.4 ± 0.8 microM) or methotrexate (I, filled diamonds and dashed line, EC50 of 10.1 ± 0.4 nM) alone. A Chou plot of the results (K) verifies that the combination is synergistic (CI < 1). The scale bar represents 10 micrometers.

[00140] Fig. 12 shows the Hsp70 inducer, geranylgeranylacetone, has a protective effect on HeLa cells treated with methotrexate. Image (A) shows treatment of HeLa cells with methotrexate alone for 72 hours kills cells with an IC5₀ of 10.1 ± 0.4 nM (filled circles and solid line), while treatment of cells with a 1 : 1 combination of methotrexate and geranylgeranylacetone (filled squares and dashed line) yields an IC₅₀ of 24.6 ± 3 nM.

Geranylgeranylacetone treatment alone did not effectively kill cells at the concentrations employed in our assay (up to 1 mM). Treatment of He la cells with geranylgeranylacetone stimulates purinosome formation. Hela cells, transfected with FGAMS-GFP, and grown in purine depleted conditions are shown before treatment (image B) and after treatment (image C) with geranylgeranylacetone (1 mM) for 85 minutes.

[00141] HSP70 and HSP90 belong to a family of ubiquitously expressed proteins that are up-regulated in response to stress and serve many functions including assisting in protein folding as well as the transport, degradation and prevention of unspecific aggregation of protein. In addition, these chaperones have been demonstrated to participate in the formation and stabilization of protein complexes. To investigate whether the HSP70/HSP90 machinery plays a role in the assembly of the purinosome, we first determined whether these proteins co-localize with FGAMS under conditions of purine starvation. Live cell imaging of fluorescently labeled constructs of HSP70 or HSP90 confirmed that these proteins co-localize with purinosome proteins in HeLa cells (Figs. 2, 3, and 4) and in skin and liver cancer cell lines (Fig. 5). Cells transfected with HSP90 or HSP70 alone yielded a diffuse staining pattern (Fig. 6). This co-localization was not observed for control proteins, Cl-tetrahydro folate synthase (Cl-THF) or serinehydroxymethyl transferase 1 (SHMT1) (Fig. 2J to Fig. 2M). In addition, both HSP70 and HSP90 were shown to be dynamically associated with the purinosomes as purine levels changed in the media (Fig. 2G to Fig. 21). As a further validation that HSP70 and HSP90 associate with purinosome proteins, we employed an in vivo protein proximity reporter assay to measure the interaction of HSP70 or HSP90 with FGAMS. These results indicate that both HSP70 and HSP90 strongly interact with FGAMS in vivo and that the level of interaction is increased in conditions that favor purinosome formation (Fig. 7). Note that a lower level of interaction was also observed in purine rich media, in accord with the hypothesis that the purinosome is present under normal conditions and upregulated in response to purine starvation.

[00142] The IP results obtained did not show a difference in the level of ubiquitin or polyubiquitin between samples with overexpressed FGAMS or untransfected controls (Table 1). We conclude from these data that purinosomes are not stress granules or aggresomes and that the function of HSP70 and HSP90 in these structures is the trafficking of protein members to, or the assembly or stabilization of, this protein complex. [00143] The functions of HSP90 and HSP70 are facilitated and regulated in vivo by a large number of co-chaperones. Accessory proteins such as J-domain proteins, Bcl-2- associated anthogene (BAG) domain proteins, heat shock organizing protein (HOP) and p23 assist in client binding and nucleotide exchange. Investigation of our IP results revealed that several of these cochaperone proteins were precipitated with FGAMS. We identified two J- domain proteins (DnaJ-Al and DnaJ-C7), two BAG proteins (BAG2 and BAG5), and the heat shock organizing protein (Stipl) in our IP data (Table 1). Co-localization experiments revealed that these proteins associate with FGAMS in purinosomes (Figs. 8 and 9). As controls, two additional J-domain proteins (DnaJ-Bl and -CI 4, not identified in our IP experiment) were examined and did not co-localize with FGAMS (data not shown). The presence of these proteins confirms that a complete complement of chaperones and cochaperones are associated with purinosomes and implicates the HSP90/HSP70-based chaperone machinery in the assembly of the purinosome. Table 1 lists the results of mass spectrometry analysis for the immunoprecipitation of proteins cross-linked to FGAMS. These proteins were found to be enriched for those cells that had been transfected with FGAMS- 9xcMyc in purine depleted medium and these proteins level exceeded those observed in controls.

Table 1.

1. where OS = Organism Name; GN = Gene Name; PE = Protein Existence; SV = Sequence Version; which

provides a unique identifier (or barcode) for the protein.

[00144] To further examine the role of HSP70 and HSP90 in the assembly of purinosomes, we tested known inhibitors of the chaperones for their ability to disrupt these protein complexes. Cells that were treated with the HSP90 inhibitors, 17-AAG, 17-DMAG or celastrol, lost the punctate staining pattern that is characteristic of purinosomes (Figs. 10 and 1 1). The effect was observed to be concentration and time dependent. In addition, a media change to remove the presence of the inhibitors reversed the effect and resulted in a reappearance of purinosomes (Fig. 1 IE to G). Similarly, treatment of cells with the HSP70 inhibitors 2-phenylethynesulfonamide (Pifithrin-μ), MKT-077 or methylene blue produced a similar reversible disruption of the purinosomes (Figs. 1 1 C, 1 ID, and 12). These results are consistent with the hypothesis that HSP70 and HSP90 are necessary to assemble or stabilize the purinosome, and indicate that these proteins may represent a novel target for the inhibition of purinosome formation and function.

[00145] Considering the importance of the purine biosynthetic pathway as a target for cancer chemotherapeutics and the role that purinosomes play in increasing flux through the pathway, we considered the possibility that drugs that target HSP70 or HSP90 may work cooperatively with current cancer treatments that disrupt purine biosynthesis. To test this hypothesis we investigated the effect of combining HSP70 or HSP90 inhibitors with the known cancer drug, methotrexate. This compound, which is currently used to treat several forms of cancer, inhibits purine biosynthesis by preventing the production of a cofactor that is essential at two separate steps of the pathway. The combination of methotrexate and the HSP90 inhibitor, 17-AAG, led to an increased cytotoxic effect on cervical cancer cells with a greater than twofold decrease in the EC5₀ (Fig. 1 1H). Analysis of the results using the Chou- Talalay method (36) verified that the drug combination was synergistic (Fig. 1 1 J). A similar synergistic effect was observed for the HSP70 inhibitor Pifithrin-μ (Figs. I l l and K).

Conversely, when the HSP70 activator geranylgeranylacetone was combined with methotrexate it had an antagonistic effect, increasing the EC50 and stimulating the production of purinosomes (Fig. 12). These data confirm a role for HSP70 and HSP90 in the assembly of the purine biosynthetic protein complex and validate the purinosome as a viable target for improved cancer chemotherapeutics.

[00146] The recent discovery of the purinosome has led to a paradigm shift regarding how metabolic enzymes associate intracellularly and has raised questions about the advantage of such multiprotein structures to the cell. Our findings reveal that together HSP90, HSP70, and several co-chaperones play a role in the assembly or stabilization of the purinosome. The involvement of this chaperone machinery begins to explain why the in vitro reconstitution of a purine biosynthetic complex has eluded researchers for so long. While these findings add another level of complexity, they help to elucidate the dynamic nature of the purinosome and may be the key to understanding the signals that drive the assembly and disassembly of this protein complex. The recent enthusiasm over the development of inhibitors to chaperones as anticancer drugs provides a rich resource that can be used to further our understanding of purinosome structure and function. In addition, the role of these proteins in assembling the purinosome can be exploited in the development of novel pharmaceuticals that target the purinosome alone or that combine disruption of the purinosome with some other complementary effect such as inhibition of purine biosynthesis.

[00147] The disclosure has been described with reference to various specific embodiments and techniques. However, it should be understood that many variations and modifications are possible while remaining within the scope of the disclosure.

Claims

What is claimed is:

1. A pharmaceutical composition comprising an effective amount of a combination of: an heat shock protein (HSP) inhibitor; and

a purine de novo biosynthesis inhibitor; or

a pharmaceutically acceptable salt of either or both inhibitors.

2. The pharmaceutical composition of claim 1, wherein the HSP inhibitor is an inhibitor of HSP90.

3. The pharmaceutical composition of any of claims 1 - 2, wherein the HSP inhibitor is an inhibitor of HSP70.

4. The pharmaceutical composition of any of claims 1 - 3, wherein the composition is used to treat rheumatoid arthritis.

5. The pharmaceutical composition in accordance with any of claims 1 - 4, wherein the composition is used to treat an oncological disease.

6. The pharmaceutical composition of any of claims 1 - 5, wherein the oncological disease is selected from a solid tumor or a hematologic neoplasia.

7. The pharmaceutical composition of any of claims 1 - 6, wherein the composition is used to treat a solid tumor selected from a group consisting essentially of: a bladder cancer, a lung cancer, a pancreatic cancer, a prostate cancer, a colorectal cancer, a gastrointestinal cancer, a head and neck cancer, a malignant mesotheliomas, a melanoma, a breast cancer, a malignant melanoma, an ovarian cancer, a soft tissue sarcoma, an osteosarcoma, a hepatocellular carcinoma, a non-small cell lung cancer (NSCLC), a renal cancer, a cervical cancer, or a tissue sarcoma.

8. The pharmaceutical composition of any of claims 1 - 6, wherein the composition is used to treat a refractory or relapsed multiple myeloma, an acute or chronic myelogenous leukaemia, a myelodysplasia syndrome, or an acute lymphoblastic leukaemia.

9. The pharmaceutical composition of any of claims 1 - 8, wherein the purine de novo biosynthesis inhibitor is a compound selected from the group consisting essentially of: methotrexate;

piritrexm;

azaserine;

azathioprine;

diazomycin;

dideazatetrahydrofolate;

lometrexol;

fluorosulfonylbenzoyl-adenosine;

nitroaminoimidazole ribonucleotide; and mixtures thereof.

10. The pharmaceutical composition of any of claims 1 - 2, wherein the HSP90 inhibitor is a compound selected from the group consisting essentially of:

geldamycin;

17-AAG (17-allyl- 17-demethoxygeldanamycin);

17-DMAG (17-desmethoxy- 17-N,N-dimethylaminoethylaminogel danamycin); IPI-504 (17-allylamino-l 7-demethoxygeldanamycin hydroquinone hydrochloride); IP 1-493 (17-desmethoxy- 17-amino geldanamycin);

ΒΠΒ021 ([6-Chloro-9-(4-methoxy-3,5-dimethylpyridin-2-ylmethyl)-9H-purin-2- yl] amine);

MPC-3100 ((S)-l -(4-(2-(6-amino-8-((6-bromobenzo[d][l ,3]dioxol-5-yl)thio)-9H- purin-9-yl)ethyl)piperidin- 1 -yl)-2 -hydro xypropan- 1 -one);

Debio 0932 (2-((6-(dimethylamino)benzo [d] [ 1 ,3 ]dioxol-5-yl)thio)- 1 -(2- (neopentylamino)ethyl)-lH-imidazo[4,5-c]pyridin-4-amine);

PU-H71 (6-Amino-8-[(6-iodo-l ,3-benzodioxol-5-yl)thio]-N-(l-methylethyl)-9H- purine-9-propanamine);

STA-9090 (5-[2,4-dihydroxy-5-(l-methylethyl)phenyl]-4-(l-methyl-lH-indol-5-yl)- 2,4-dihydro-3H- l,2,4-triazol-3-one);

VER52296 (5-(2,4-Dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4- (morpholinomethyl)phenyl)isoxazole-3-carboxamide);

KW-2478 (2-(2-ethyl-3,5-dihydroxy-6-(3-methoxy-4-(2- morpholinoethoxy)benzoyl)phenyl)-N,N-bis(2-methoxyethyl)acetamide); AT-13387 ((2,4-dihydroxy-5-isopropylphenyl)(5-((4-methylpiperazin- 1 - yl)methyl) iso indolin-2 -yl)methanone);

Radicicol ((1 aR,2Z,4E, 14R, 15aR)-8-Chloro-l a, 14, 15, 15a-tetrahydro-9, 1 1 -dihydroxy- 14-methyl-6H-oxireno[e][2]benzoxacyclotetradecin-6, 12(7H)-dione);

and

Celastrol ((9 β, 13 a, 14β,20α)-3 -Hydroxy-9 , 13 -dimethyl-2-oxo-24,25 ,26-trinoroleana- l(10),3,5,7-tetraen-29-oic acid);

a combination thereof, or a pharmaceutically acceptable salt thereof.

1 1. The pharmaceutical composition of any of claims 1 - 3, wherein the HSP70 inhibitor is a compound selected from of the group consisting essentially of:

2-phenylethynesulfonamide (Pifithrin-μ);

MKT-077 (l -Ethyl-2-[[3-ethyl-5-(3-methyl-2(3H)-benzothiazolylidene)-4-oxo-2- thiazolidinylidene]methyl]-pyridinium chloride);

methylene blue;

VER155088 (5'-0-[(4-Cyanophenyl)methyl]-8-[[(3,4-dichlorophenyl)methyl]amino]- adenosine);

or a pharmaceutically acceptable salt thereof.

12. The pharmaceutical composition of any of claims 1 - 11, wherein the HSP inhibitor is geldamycin, and the purine de novo biosynthesis inhibitor is methotrexate.

13. The pharmaceutical composition of any of claims 1 - 12, further comprising at least one therapeutic agent selected from the group consisting essentially of a chemotherapeutic agent and a targeted therapeutic agent when the composition is used to treat cancer.

14. The pharmaceutical composition of any of claims 1 - 13, wherein the at least one therapeutic agent is a chemotherapeutic agent selected from Asparaginase, Bleomycin, Busulfan, Carmustine, Chlorambucil, Cladribine, Cyclophosphamide, Cytarabine,

Dacarbazine, Daunorubicin, Doxorubicin, Etoposide, Fludarabine, Gemcitabine,

Hydroxyurea, Idarubicin, Ifosfamide, Lomustine, Mechlorethamine, Melphalan, Mitomycin, Mitoxantrone, Pentostatin, Procarbazine, Topotecan, Vinblastine, Vincristine,

Dexamethasone, Retinoic acid, Prednisone, and mixtures thereof.

15. The pharmaceutical composition of any of claims 1 - 13, wherein the at least one therapeutic agent is a targeted therapeutics agent selected from alemtuzumab, bevacizumab, cetuximab, gemtuzumab, ibritumomab, panitumumab, rituximab, tositumomab, trastuzumab, dasatinib, erlotinib, everolimus, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, sorafenib, sunitinib, temsirolimus, vandetanib, vemurafenib, crizotinib, vismodegib, axitinib, ruxolitinib, and mixtures thereof.

16. The pharmaceutical composition of any of claims 1 - 15, further comprising at least one therapeutic agent selected from the group consisting essentially of a classical DMARDs, NSAIDs, biologies DMARDs, immunomodulators, and mixtures thereof.

17. The pharmaceutical composition of any of claims 1 - 16, wherein the at least one therapeutic agent is selected from the group consisting essentially of chloroquine, hydroxychloroquine, cyclosporin A, T D-penicillamine, aurothiomalate, auranofin, leflunomide, minocycline, sulfasalazine, ibuprofen, naproxen, meloxicam, etodolac, nabumetone, sulindac, tolementin, choline magnesium salicylate, diclofenac, diflusinal, indomethicin, ketoprofen, oxaprozin, piroxicam, etanercept, infliximab, adalimumab, certolizumab pegol, golimumab, anakinra, rituximab, abatacept, and tocilizumab, and mixtures thereof.

18. The pharmaceutical composition of any of claims 1 - 17, wherein the

pharmaceutically acceptable salt is an inorganic or organic, acid or base.

19. The pharmaceutical composition of any of claims 1 - 18, wherein the composition comprises a dosage form suitable for simultaneous, separate, or sequential use in the treatment of rheumatoid arthritis or cancer.

20. A method of treating rheumatoid arthritis or cancer, comprising:

administering an effective amount of the pharmaceutical composition of claim 1 to a patient in need of such treatment.