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Title:
COMPOSITIONS AND METHODS FOR CHARACTERIZATION AND AMELIORATION OF RHEUMATOID ARTHRITIS
Document Type and Number:
WIPO Patent Application WO/2015/028662
Kind Code:
A1
Abstract:
According to some embodiments herein, methods and compositions for amerlioration of rheumatoid arthritis are provided. According to some embodiments herein, methods and compositions for characterization of rheumatoid arthritis are provided. The methods and compositions of some embodiments include inhibitors of ChoKα.

Inventors:
FIRESTEIN, Gary S. (University Of California, 1111 Franklin St. 5th FloorOakland, CA, CA94607-5200, US)
GUMA, Mónica (University Of California, 1111 Franklin St. 5th FloorOakland, California, CA94607-5200, US)
KARIN, Michael (University Of California, 1111 Franklin St. 5th FloorOakland, California, CA94607-5200, US)
SÁNCHEZ LÓPEZ, Elsa (University Of California, 1111 Franklin St. 5th FloorOakland, California, CA94607-5200, US)
LACAL, Juan Carlos (Consejo Superior De Investigaciones Cientificas, C/ Serrano nº 117, Madrid, E-28060, ES)
TIZIANI, Stefano (The University of Texas at Austin, Office of Technology Commercialization3925 W. Braker Lane, Suite 1.9A,Campus Mail Code: R350, Austin Texas, 78759, US)
Application Number:
EP2014/068501
Publication Date:
March 05, 2015
Filing Date:
September 01, 2014
Export Citation:
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Assignee:
CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS (CSIC) (C/ Serrano, nº 117, Madrid, E-28006, ES)
UNIVERSITY OF CALIFORNIA (1111 Franklin St. 5th Floor, Oakland, California, CA94607-5200, US)
BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM (201 West 7th Street, Austin, TX, 78701, US)
International Classes:
A61K31/4425; A61P19/02
Domestic Patent References:
WO2007138143A22007-12-06
Foreign References:
US20100068302A12010-03-18
US20110212994A12011-09-01
US20040202658A12004-10-14
EP1889920A12008-02-20
US8481256B22013-07-09
US20070185170A12007-08-09
US20100068302A12010-03-18
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Attorney, Agent or Firm:
PONS ARIÑO, Angel (Glorieta Rubén Darío 4, Madrid, E-28010, ES)
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Claims:
CLAIMS

1. A method of ameliorating rheumatoid arthritis in a subject in need of such amelioration, the method comprising administering a therapeutically effective dose of a choline kinase a (ChoKa) inhibitor to the subject.

2. The method of claim 1 , wherein the ChoKa inhibitor comprises a small molecule.

3. The method of claim 1 , wherein the ChoKa inhibitor comprises 1 ,4- (4-4'-Bis-((4-(dimethylamine)pyridinium-1 -yl) methyl)diphenyl) butane dibromide (MN58b).

4. The method of claim 1 , wherein the ChoKa inhibitor consists of MN58b.

5. The method of claim 4 or claim 5, wherein the dose of MN58b comprises about 1 mg per kg to about 10mg per kg.

6. The method of claim 4 or claim 5, wherein the dose of MN58b comprises about 3mg per kg.

7. The method of any of claims 1 to 6, wherein administration comprises injecting the ChoKa inhibitor.

8. The method of any of claims 1 to 7, wherein administration of the ChoKa inhibitor inhibits proliferation of at least one resident synovial fibroblast

(FLS) of the subject.

9. The method of any of claims 1 to 8, wherein administration of the ChoKa inhibitor inhibits migration of at least one FLS of the subject.

10. The method of any of claims 1 to 9, wherein administration of the ChoKa inhibitor reduces at least one of IL-1 b expression or MMP3 expression by the subject

1 1. The method of any of claims 1 to 10, further comprising detecting an amount of proliferation or migration of at least one FLS of the subject.

12. The method of any of claims 1 to 1 1 , further comprising detecting a presence or absence of at least one of Akt activation or MAPK activation in at least one FLS of the subject.

13. The method of any of claims 1 to 12, further comprising detecting an amount of expression of at least one of IL-1 b expression or MMP3 expression by the subject.

14. A pharmaceutical composition comprising a dosage of a choline kinase a (ChoKa) inhibitor suitable for treatment of rheumatoid arthritis.

15. The pharmaceutical composition of claim 14, wherein the dosage comprises about 1 mg per kg to about 10mg per kg of body mass of a subject in need of treatment of rheumatoid arthritis.

16. The pharmaceutical composition of claim 14, further comprising an antibody for treatment of rheumatoid arthritis.

17. The pharmaceutical composition of claim 16, wherein the antibody comprises an anti-CD20 antibody.

18. The pharmaceutical composition of claim 16, wherein the antibody comprises rituximab.

19. A method of determining a response to at least one clinical treatment of rheumatoid arthritis administered to a subject, the method comprising measuring the amount of at least one of ChoKa mRNA, ChoKa protein, or a choline metabolite in the subject.

20. The method of claim 19, wherein an amount of ChoKa mRNA or ChoKa protein is measured in an FLS of the subject.

21. The method of claim 19 or 20, wherein the at least one clinical treatment of rheumatoid arthritis comprises treatment with an anti-CD20 antibody.

22. The method of claim 21 , wherein the anti-CD20 antibody comprises rituximab.

23. The method any of claims 19 to 22, wherein the at least one clinical treatment of rheumatoid arthritis comprises treatment with a ChoKa inhibitor.

24. The method of claim 23, wherein the ChoKa inhibitor comprises MN58b.

25. A method of identifying a metabolic profile indicative of osteoarthritis or rheumatoid arthritis comprising comparing the level of at least one of GPC, ChoKa mRNA, ChoKa protein, or a choline metabolite in individuals with osteoarthritis or rheumatoid arthritis to individuals who do not have osteoarthritis or rheumatoid arthritis.

26. A method of diagnosing osteoarthritis or rheumatoid arthritis comprising determining whether a subject has level of at least one of GPC, ChoKa mRNA, ChoKa protein, or a choline metabolite indicative of osteoarthritis or rheumatoid arthritis.

27. The method of Claim 26, further comprising administering a treatment for osteoarthritis or rheumatoid arthritis if said subject has level of at least one of GPC, ChoKa mRNA, ChoKa protein, or a choline metabolite indicative of osteoarthritis or rheumatoid arthritis.

Description:
COMPOSITIONS AND METHODS FOR CHARACTERIZATION AND AMELIORATION OF RHEUMATOID ARTHRITIS

Field

Embodiments herein relate generally to compositions and methods for treatment of and characterization of rheumatoid arthritis. More particularly, some embodiments relate to choline kinase a (ChoKa), inhibitors thereof, and uses of such inhibitors.

SUMMARY

In some embodiments, a method of ameliorating rheumatoid arthritis in a subject in need of such amelioration is provided, in which the method comprises administering a therapeutically effective dose of a choline kinase a (ChoKa) inhibitor to the subject. In some embodiments, the ChoKa inhibitor comprises a small molecule. In some embodiments, the ChoKa inhibitor comprises 1 ,4-(4-4'- Bis-((4-(dimethylamine)pyridinium-1 -yl) methyl)diphenyl) butane dibromide (MN58b). In some embodiments, the ChoKa inhibitor consists of MN58b. In some embodiments, the dose of MN58b comprises about 1 mg per kg to about 10mg per kg, for example about 1 mg per kg, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg per kg, including ranges between any two of the listed values. In some embodiments, the dose of MN58b comprises about 3mg per kg. In some embodiments, administration comprises injecting the ChoKa inhibitor. In some embodiments, administration of the ChoKa inhibitor inhibits proliferation of at least one resident synovial fibroblast (FLS) of the subject. In some embodiments, administration of the ChoKa inhibitor inhibits migration of at least one FLS of the subject. In some embodiments, administration of the ChoKa inhibitor reduces at least one of IL-1 b expression or MMP3 expression by the subject. In some embodiments, the method further comprises detecting an amount of proliferation or migration of at least one FLS of the subject. In some embodiments, the method further comprises detecting a presence or absence of at least one of Akt activation or MAPK activation in at least one FLS of the subject. In some embodiments, the method further comprises detecting an amount of expression of at least one of IL-1 b expression or MMP3 expression by the subject.ln some embodiments, a pharmaceutical composition comprising a dosage of a choline kinase a (ChoKa) inhibitor suitable for treatment of rheumatoid arthritis is provided. In some embodiments, the dosage comprises about 1 mg per kg to about 10mg per kg of body mass of a subject in need of treatment of rheumatoid arthritis. In some embodiments,the dosage further comprises an antibody for treatment of rheumatoid arthritis. In some embodiments, the antibody comprises an anti-CD20 antibody, for example rituximab.

In some embodiments, a method of determining a response to at least one clinical treatment of rheumatoid arthritis administered to a subject is provided, in which the method comprises measuring the amount of at least one of ChoKa mRNA, ChoKa protein, or a choline metabolite in the subject. In some embodiments, an amount of ChoKa mRNA or ChoKa protein is measured in an FLS of the subject. In some embodiments, at least one clinical treatment of rheumatoid arthritis comprises treatment with an anti-CD20 antibody, for example, rituximab. In some embodiments, at least one clinical treatment of rheumatoid arthritis comprises treatment with a ChoKa inhibitor, for example MN58b. The skilled artisan will recognize that a variety of ChoKa inhibitors can be used in conjunction with embodiments herein, for example small molecules {e.g. MN58b and 1 ,1 '-(biphenyl- 4,4'-diylmethylene)bis [4-(4-chloro-N-methylanilino-)quinolinium]dibromide (RSM- 932A); see, e.g. U.S. Pat. No. 8,481256 and U.S. Pub. Nos. 2007/0185170 and 2010/0068302, each of which is incorporated by reference in its entirety herein), antibodies {e.g monoclonal antibodies such as AD3, AD8 and AD1 1 , described in PCT Pub. No. WO2007/138143, hereby incorporated by reference in its entirety), RNAi (see U.S. Pub. No. 2010/0068302), antisense nucleic acids, ribozymes, aptamers, and the like. In some embodiments, a method of identifying a metabolic profile indicative of osteoarthritis or rheumatoid arthritis is provided, in which the method comprises comparing the level of at least one of GPC, ChoKa mRNA, ChoKa protein, or a choline metabolite in individuals with osteoarthritis or rheumatoid arthritis to individuals who do not have osteoarthritis or rheumatoid arthritis. In some embodiments, the method of diagnosing osteoarthritis or rheumatoid arthritis is provided, in which the method comprises determining whether a subject has level of at least one of GPC, ChoKa mRNA, ChoKa protein, or a choline metabolite indicative of osteoarthritis or rheumatoid arthritis. In some embodiments, the method further comprises administering a treatment for osteoarthritis or rheumatoid arthritis if the subject has level of at least one of GPC, ChoKa mRNA, ChoKa protein, or a choline metabolite indicative of osteoarthritis or rheumatoid arthritis.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a photograph illustrating a RA synovial that was immunostained with IgG (A) and cholinekinase a antibody (B).

Figure 2 is a photograph of a western blot. RA FLS were stimulated by several cytokines and ChoKa expression was confirmed by WB. C: control; T: TNF; P: PDGF; E: EGF; L: LPS.

Figures 3A and 3B are graphs depicting NMR spectra. Ή NMR spectra obtained from FLS RA (Figure 3A) grown in culture (blue) no stimulus (red) after 24hrs of PDGF (10ng/ml) stimulation. (Figure 3B) IMR-90 fibroblast cell line spectra.

Figure 4 is a photograph of a western blot. Lysates of RA FLS were prepared when indicated after PDGF stimulation and were analyzed for the expression of the indicated proteins. Figure 5 is a graph depicting cell growth. FLS were cultured in presence of PDGF with different MN58b concentrations. Growth was measured on day 4 using MTT. Figure 6 is a series of photographs depicting effects of ChoKa inhibitor on synoviocyte migration.

FIGURES 7A-7D ARE GRAPHS DEPICTING CLINICAL SCORES (FIGURE 7A), HISTOLOGICAL SCORES (FIGURE 7B), RELATIVE IL-1 B MRNA EXPRESSION (FIGURE 7C), AND RELATIVE MMP3 MRNA EXPRESSION (FIGURE 7D) IN PASSIVE K/BXN SERUM TRANSFER ARTHRITIS AFTER VEHICLE AND CHOKa INHIBITOR (3 MG/KG) TREATMENT. DETAILED DESCRIPTION

Choline kinase a (ChoKa) is an enzyme essential for phosphatidylcholine (PtdCho ) biosynthesis, and is involved in cell proliferation, growth and invasion. It has been recently recognized as both a prognostic marker and a therapeutic target in various types of human cancers. In Rheumatoid Arthritis (RA), synovial hyperplasia contributes to inflammation and joint destruction. Synovial fibroblast (FLS) in the intimal lining, especially in the pannus are the major effectors of cartilage damage through production of extracellular matrix degrading enzymes such as MMPs and cathepsins. RA FLS possess unique aggressive phenotype, such as cartilage invasion. It is appreciated herein that ChoKa expression is regulated by inflammatory cytokines and regulates key FLS functions that might contribute to cartilage destruction in RA. As shown herein, ChoKa inhibition in a model of inflammatory arthritis decrease paw swelling and histological score. ChoKa inhibition is a novel therapy for RA and will be a non-invasive biomarker of inflammation/joint damage and response to therapy.

The enzyme has also been implicated in cancer disease progression, metastasis, and invasiveness. The unique tumor-like behavior of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) led us evaluate whether this pathway could play a role in inflammation and joint damage due to synovitis. Therefore, we examined the expression and function of ChoKa in RA FLS and performed a targeted metabolomics assessment of this pathway. These studies suggest that ChoKa contributes to the rheumatoid aggressive phenotype. Pathogenesis of RA disease can involve a complex interaction between the innate and the adaptive arm of the immune system in concert with the resident synovial fibroblasts (FLS). FLS contribute to synovial inflammation by producing inflammatory mediators, extracellular matrix degrading enzymes such as MMPs and cathepsins and recruiting and activating immune cells. RA FLS possess unique aggressive phenotype, such as cartilage invasion. However, the molecular mechanisms that regulate FLS behavior in RA are poorly understood and represent a major obstacle for developing therapeutic interventions that modulate these functions. Small molecule inhibitors that inhibit key signaling molecules in RA have the potential to improve efficacy and to overcome limitations of antibody based approaches. Thus, new rationally designed disease modifying agents that directly target FLS are needed to complement current therapies.

To the best of Applicant's knowledge at the time of filing, there are no disease modifying agents that directly target FLS. There are no specific biomarker of inflammation/joint damage and response to therapy in rheumatoid arthritis.

ChoKa is essential for PtdCho biosynthesis, which is required for the increased cancer cell proliferation, tumor progression and invasiveness, making it a potential prognostic marker of some cancers. Selective inhibition of ChoKa also attenuates MAPK and P13K/Akt signaling, which can be associated with a decrease in the growth of cells. RA cultured FLS possess a unique tumor like behavior, and aggressive phenotype, such as cartilage invasiveness. Moreover, MAPK and P13K/Akt are signal pathways that regulate synoviocyte function in RA such as metalloproteinase (MMP) expression and synoviocyte growth and survival, which may explain its aggressive phenotype. As such, it was determined if this unique aggressive phenotype could be secondary to a higher ChoKa activity level, similar to what it is observed in tumor cells, and to determine suitability as therapeutic target and biomarker of inflammation/joint damage.The hallmark of rheumatoid arthritis (RA) is synovial inflammation, hyperplasia and joint destruction[1]. Pathogenesis of the disease involves a complex interaction between the innate and the adaptive arm of the immune system in concert with the resident synovial fibroblasts (FLS). FLS contribute to synovial inflammation by producing inflammatory mediators and recruiting and activating immune cells[2]. In addition, FLS in the intimal lining, especially in the pannus are the major effectors of cartilage damage through production of extracellular matrix degrading enzymes such as MMPs and cathepsins[2]. RA FLS possess unique aggressive phenotype, such as cartilage invasion[3]. However, the molecular mechanisms that regulate FLS behavior in RA are poorly understood and represent a major obstacle for developing therapeutic interventions that modulate these functions. Small molecule inhibitors that inhibit key signaling molecules in RA have the potential to improve efficacy and to overcome limitations of antibody based approaches[4-6]. Thus, new rationally designed disease modifying agents that directly target FLS can be useful for replacing or complementing current therapies.

Metabolomics allows for a global assessment of a cellular state within the context of the immediate environment, taking into account genetic regulation, altered kinetic activity of enzymes, and changes in metabolic reactions[7]. Compared with genomics or proteomics, metabolomics reflects changes in phenotype and therefore function[7]. Studying patients using a metabolomic strategy may reveal underlying biochemical phenomena associated with the disease, thus providing insights that help the development of a better understanding of mechanisms underlying disease, and to develop new strategies for treatment. Metabolic profiling has also been used to identify biomarkers for several diseases. Systemic diseases like RA are likely associated with changes in a complicated array of chemical reactions and metabolites that stem from a diverse set of metabolic pathways. However, few works have addressed metabolic changes in RA[8-12]. In other fields, like oncology, the tumor metabolome is beginning to be characterized[13-15]. Using standard metabolomic methods, tumors, in general, display elevated phospholipid levels characterized by increases in the levels of phophocholine (PCho) and total choline-containing metabolites together with decreases in the glycerophosphocholine (GPC)/PCho ratio (a phenomenon known as the "GPC-to-PCho switch"; Fig.1 )[16-18]. Because choline-containing compounds are detected by MRS, increased levels of these compounds provide a non-invasive biomarker of transformation, staging and response to therapy[19-23]. Elevated PCho levels can be partially attributed to an increased activity of choline kinase (ChoK) catalyzing the first step in the Kennedy pathway, an enzyme recently recognized as both a prognostic marker and a therapeutic target in various types of human cancers[18, 23, 24]. ChoK can also take on a rate-limiting, regulatory role in phosphatidylcholine (PtdCho) biosynthesis under some circumstances. ChoK is primarily located in the cytoplasm of cells from various tissues. At least three isoforms of ChoK exist in mammalian cells, and these are encoded by two genes: ChoKa and ChoKb. The two functional isoforms, ChoKa-1 and ChoKa-2, are derived from ChoKa by alternative splicing. Homodimeric or heterodimeric forms of ChoK are enzymatically active. The upregulation of ChoK activity in cancer probably results from an increase in ChoKa expression, which would lead to a higher proportion of ChoKa homodimers in cancer cells and in turn a higher ChoK activity level. ChoKa is essential for PtdCho biosynthesis, which is required for the increased cancer cell proliferation, tumor progression and invasiveness, making it a potential prognostic marker of some cancers. Selective inhibition of choline kinase also attenuates MAPK and PI3K/Akt signaling, which was associated with a decrease in the growth of cells[25].

As mentioned above, RA cultured FLS possess a unique tumor like behavior, and aggressive phenotype, such as cartilage invasiveness[2, 3]. Moreover, MAPK and PI3K/Akt are signal pathways that regulate synoviocyte function in RA such as metalloproteinase (MMP) expression and synoviocyte growth and survival, which may explains its aggressive phenotype[26, 27]. All together led us to do a targeted metabolomics assessment of choline metabolism in RA FLS to determine if its unique aggressive phenotype could be secondary to a higher ChoKa activity lever, similar to what it is observed in tumor cells, and to determine suitability as therapeutic target and biomarker of inflammation/joint damage. Our preliminary data demonstrates that ChoKa is highly expressed in RA synovial tissue, especially in the intimal lining and in cultured FLS, and its expression in FLS is regulated by inflammatory cytokines. MRS studies of choline-containing compounds in RA FLS showed an increase of PCho similar to what is observed in tumor cells. We used then a specific competitive choline kinase inhibitor, MN58b, to determine ChoK functions in vivo and in vitro. MN58b[28] (1 ,4-(4-4'-Bis-((4- (dimethylamine)pyridinium-l -yl) methyl}diphenyl) butane dibromide) exhibits selective inhibition of choline kinase, inhibits proliferation of cancer cells in vitro with an IC50 of 1 -1 OnM, and displays therapeutic activity against human tumor xenografts in vivo. We show here that ChoKa regulates key FLS functions that might contribute to cartilage destruction in RA. Initial studies also show a novel role for choline kinase in vivo. We hypothesized that ChoKaDis a key regulator ofD FLS functions that contribute to aggressive behavior and joint destruction in RA and that selective ChoKa inhibition in RA will be disease modifying by directly modulating synoviocyte mediated cartilage destruction and that choline-containing compounds detected by MRS might be a non-invasive biomarker of inflammation/joint damage and response to therapy.

Despite the successes in the last two decades, treatment of RA remains an unmet medical need. There is an ongoing effort to develop more effective, safer and less costly therapies to achieve free remission. Furthermore, to the best of Applicant's knowledge, the currently available disease modifying drugs do not directly target synoviocytes, cells that play a major role in RA pathogenesis. New rationally designed disease modifying agents that directly target key signaling proteins in FLS are needed to replace or complement current therapies. Metabolomics is an emerging field of biomedical research can offer a better understanding of mechanisms underlying disease, and help to develop new strategies for treatment. Variations in metabolite concentrations can also serve as diagnostic or prognostic biomarkers. Unique metabolomic profiles have been identified in the serum of patients with several diseases. Little is known about metabolomics changes in RA that could help in treatment decision-making. Of note, University of California, San Diego has been recently awarded by the NIH Common Fund and will play a central role in a new program from the National Institutes of Health (NIH) to accelerate metabolomics. Using a targeted metabolomics approach, we have identified a potential target in synoviocytes, ChoKa, that has been related to cancer cell proliferation, tumor progression and invasiveness. A highly selective choline kinase inhibitor could potentially block synovial hyperplasia and cartilage invasion by synoviocytes. We will explore the unexpected choline kinase function and signaling in synoviocytes biology and in RA pathogenesis. We will use selective small molecule inhibitor to test our hypothesis in vitro cell culture and in preclinical models of arthritis. We will also explore choline metabolites as biomarkers. If successful, these studies could pave the way for new treatments for patients with RA.

Our data demonstrates that ChoKa is highly expressed in RA synovial tissue, especially in the intimal lining and in cultured FLS, and its expression in FLS is regulated by inflammatory cytokines. MRS studies of choline-containing compounds in RA FLS showed an increase of PCho similar to what is observed in tumor cells. We used then a specific competitive choline kinase inhibitor, MN58b, to determine ChoKa functions in vivo and in vitro. MN58b (1.4-(4-4'-Bis-((4- (dimethylamine)pyridinium-l -yl) methyl}diphenyl) butane dibromide) exhibits selective inhibition of ChoKa, inhibits proliferation of cancer cells in vitro with an IC 50 of 1 -10 μΜ, and displays therapeutic activity against human tumor xenografts in vivo. Our data shows that ChoKa regulates key FLS functions that might contribute to cartilage destruction in RA. Initial studies also show that ChoKa inhibition in a model of inflammatory arthritis decrease paw swelling and histological score.

The data is using the specific competitive ChoKa inhibitor, MN58b. In some embodiments, any of a series of other compounds could also be included since findings herein imply cause of existing ChoKa inhibitorAccording to some embodiments herein, a ChoKa inhibitor, for example MN58b, also comprises a pharmaceutically acceptable excipient. Therefore, some embodiments are also directed to a pharmaceutical composition as disclosed above, wherein the pharmaceutical composition additionally comprises a pharmaceutically acceptable excipient.ln general all excipients known by a person skilled in the art are suitable within embodiments herein. Examples of such excipients are calcium carbonate, kaolin, sodium hydrogen carbonate, lactose, D-mannitol, starches, crystalline cellulose, talc, granulated sugar, porous substances, etc.A ChoKa inhibitor, for example MN58b, may be used as bulk itself but usually be formulated into pharmaceutical preparations together with a suitable amount of "carrier for pharmaceutical preparation" according to ordinary methods.

Thus, compositions and methods described herein may also contain additionally diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.

Further on, "carriers for pharmaceutical preparation" comprises, for example, excipients as defined herein, binders, e.g., dextrin, gums, a-starch, gelatin, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, pullulan, etc., thickening agents, e.g., natural gums, cellulose derivatives, acrylic acid derivatives, etc., disintegrators, e.g., carboxy- methyl cellulose, croscarmellose sodium, crospovidone, low-substitution hydroxypropyl cellulose, partial a-starch, etc., solvents, e.g., water for injections, alcohol, propylene glycol, macrogol, sesame oil, corn oil, etc., dispersants, e.g., Tween 80, HCO60, polyethylene glycol, carboxymethyl cellulose, sodium alginate, etc., solubilizers, e.g., poly- ethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisami- nomethane, triethanolamine, sodium carbonate, sodium citrate, etc., suspending agents, e.g., stearyl triethanolamine, sodium lauryl sulfate, benzalkonium chloride, polyvinyl alcohol, polyvinylpyrrolidone, hydroxyethyl cellulose, etc., pain-reducing agents, e.g., benzyl alcohol, etc., isotonizing agents, e.g., sodium chloride, glycerin, etc., buffers, e.g., phosphates, acetates, carbonates, citrates, etc., lubricants, e.g., magnesium stearate, calcium stearate, talc, starch, sodium benzoate, etc., colorants, e.g., tar pigments, caramel, iron sesquioxide, titanium oxide, riboflavins, etc., tasting agent, e.g., sweeteners, flavors, etc., stabilizers, e.g., sodium sulfite, ascorbic acid, etc., preservatives, e.g., parabens, sorbic acid, etc., and the like.

Preferred methods of administration of the pharmaceutical compositions described above include oral and parenteral, e.g., i.v. infusion, i.v. bolus and i.m. injection formulated so that a unit dosage comprises a therapeutically effective amount of each active component or some submultiples thereof. The compounds may be employed in powder or crystalline form, in liquid solution, or in suspension. Theses compounds may be formulated by any method well known in the art and may be prepared for administration by any route, including, without limitation, parenteral, oral, sublingual, by inhalation spray, transdermal, topical, intranasal, intra- tracheal, intrarectal via ophthalmic solution or ointment, rectally, nasally, buccally, vaginally or via implanted reservoir. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

Pharmaceutical compositions for injection, a preferred route of delivery according to some embodiments herein, may be prepared in unit dosage form in ampules, or in multidose containers. The composition will generally be sterile and pyrogen- free, when intended for delivery by injection into the subject. The injectable compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain various formulating agents. Alternatively, the active ingredient may be in powder (lyophilized or non-lyophilized) form for reconstitution at the time of delivery with a suitable vehicle, such as sterile water. Carriers suitable for an injectable pharmaceutical composition according to some embodiments are typically comprised sterile water, saline or another injectable liquid, e.g., peanut oil for intramuscular injections. Also, various buffering agents, preservatives and the like can be included. The pharmaceutical composition according to some embodiments may also be administered parenterally in a sterile medium. Depending on the vehicle and concentration used, the drug can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anaesthetic, preservative and buffering agents can be dissolved in the vehicle. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. It is also preferred to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatine. Intra-venous infusion is another possible route of administration for the compounds used according to some embodiments herein. Orally administrable pharmaceutical compositions according to some embodiments herein may be in the form of tablets, capsules, powders, granules, lozenges, liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions. The oral compositions may utilize carriers such as conventional formulating agents, and may include sustained release properties as well as rapid delivery forms. Such compositions and preparations should contain at least 0.1 % of active compounds. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

Tablets and capsules for oral administration may be in unit dose presentation form, and may also contain conventional excipients such as binding agents, for example syrup, acacia, gelatine, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers for example lac- tose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricant, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrates for example potato starch, or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known to a person skilled in the art. Oral liquid preparations may be in the form of, for example, aqueous or oily sus- pensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatine hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles which may include edible oils, for example almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents. Pharmaceutical compositions according to some embodiments herein may also be prepared in suitable forms for absorption through the mucous membranes of the nose and throat or bronchial tissues and may conveniently take the form of powder or liquid sprays or inhalants, lozenges, throat paints, etc. For medication of the eyes or ears, the preparations may be presented as individual capsules, in liquid or semi-solid form, or may be used as drops, etc.

Suitable subjects for the administration of the formulation of some embodiments herein include mammals, primates, humans, and other animals. Typically the animal subject is a mammal, generally a domesticated farm mammal, e.g. horse, pig, cow, sheep, goat etc., or a companion animal, e.g. cat, dog etc.. In vitro antibacterial activity is predictive of in vivo activity when the compositions are administered to a mammal infected with a susceptible bacterial organism. EXAMPLES

In the following examples, unless stated otherwise, the following methods were used: ChoKa expression in RA synovium and RA FLS was evaluated by immunohistochemistry (IHC) and Western blot (WB), respectively. Osteoarthritis (OA) samples were used as controls in some experiments. The metabolic profile of FLS cells was determined by 1 H-MRS under conditions of ChoKa inhibition. FLS function using the ChoKa inhibitor MN58b (IC50= 4.2 μΜ) in medium and platelet derived growth factor (PDGF) stimulated cells was evaluated by measuring 1 ) migration into a cleared area in cultured FLS monolayers (scratch assay); 2) proliferation using an MTT assay; and 3) protein expression by WB. Cell survival was determined in H2O2 treated cells by phase contrast light microscopy. For arthritis experiments, mice were injected with K/BxN sera on day 0. MN58b (3mg/kg) was injected daily i.p. beginning on day 0 or day 4 after serum administration. Clinical arthritis scores were serially assessed. Joints were evaluated for inflammation and joint damage using histology and a semiquantitative scoring system.

EXAMPLE 1

ChoKa mRNA and protein were highly expressed in RA synovial tissue and in cultured FLS. Its expression in FLS was increased 2-3-fold after tumor necrosis factor (TNF) and PDGF stimulation, respectively with peak expression within 48 hours. Metabolomic studies of choline-containing compounds in cultured FLS extracts showed increased levels of phosphocholine in RA FLS, confirming activation of this pathway. ChoKa regulates key FLS functions that might contribute to cartilage destruction in RA. For example, ChoKa inhibition with MN58b (5 μΜ) reduced proliferation by 79±3.2% and migration by 54±15% (p<0.05). ChoKa inhibition also markedly increased H 2 O 2 -induced apoptosis in FLS. Additionally, Akt phosphorylation in response to PDGF as determined by WB was blocked by ChoKa inhibition Finally, ChoKa inhibition significantly decreased arthritis in pre-treatment protocols (day 0) as well as in established disease (day 4). For example, day 8 scores were 12±1 .6 and 7±2.6 (P<0.05) for vehicle and MN58b-treated mice, respectively, when initiated on day 0; and were 6.6±0.9 and 1 .6±2.5 (PO.05) for PBS and MN58b-treated mice when initiated on day 4. Joint histology scores for vehicle and MN58b-treated mice for inflammation were 3.2±0.5 and 1 .25±1 (pO.05), bone erosion scores were 2.7±0.5 and 0.25±0.5 (p<0.05), and cartilage damage scores were 1 .5+1 and 1 .6±0.5 (p<0.05) respectively. Careful dissection of the metabolic profile in RA FLS suggests that choline metabolism is abnormal and is similar to transformed cells. Blocking this pathway with a selective ChoKa inhibitor suppressed inflammatory arthritis in mice as well as the aggressive behavior of cultured RA FLS, including cell migration and resistance to apoptosis. These data suggest that ChoKa inhibition could be an effective strategy for arthritis. EXAMPLE 2: CHOLINE KINASE-A EXPRESSION IN RA SYNOVIUM

Immunohistochemistry (IHC) was done to determine if ChoKa is expressed in RA synovium. As shown in the Fig. 1 , ChoKa is highly expressed in synovial tissue, it is primarily expressed in the intimal lining with scattered positive cells in the sublining, suggesting the expression of ChoKa in FLS. EXAMPLE 3: CHOLINE KINASE EXPRESSION AND REGULATION IN

CULTURED FLS

ChoKa protein is localized to the intimal lining cells, we evaluated its expression and regulation in cultured RA FLS derived from this region. Protein expression was confirmed with Western blot analysis. To determine whether ChoKa is regulated by inflammatory mediators implicated in RA, we stimulated RA FLS with TNF, IL-1 , PDGF, EGF or LPS for 48 hrs, and protein was assayed by WB. ChoKa increased following TNF, PDGF and EGF stimulation (Fig. 2) while LPS had no effect.

EXAMPLE 4: ACTIVATED CHOLINE METABOLITES ARE DETECTED IN RA

FLS

One-dimensional 1 H NMR spectra of aqueous extracts revealed and activated choline profile in RA FLS (Fig. 3A). The relative areas of signal components due to individual PC metabolites (GPC, PCho and Cho) are more similar to tumor than non-tumor cells (Fig. 1 ), PCho becoming the predominant metabolite, which is further increased after PDGF stimulation (GPC:PC ratio <0.1 ). Fig. 3 shows 1 H NMR spectra of normal IMR-90 fibroblast cell line, which shows a GPC/PC ratio around 1.

EXAMPLE 5: CHOLINE KINASE REGULATES P-AKT AND P-MAPK

ACTIVATION IN FLS

ChoKa in tumor cells can activate Akt and MAPK. To explore ChoKa function in FLS, we next studied activation of Akt and MAPK in response to growth factor PDGF, a potent activator of these pathways in various cell types. FLS were treated with ChoKa inhibitor or vehicle for 8h then stimulated with PDGF-BB 10 ng/ml or medium for 30 min. Western blot analysis was done for P-Akt (S473) and P-MAPK and total protein to quantify the inhibitory effects of the compound. As shown in Fig. 4, MN58b partially decreased P-Akt and P-ERK in FLS.

EXAMPLE 6: CHOLINE KINASE REGULATES RA FLS GROWTH

We also tested whether ChoKa inhibition interferes with cell growth in vitro in response to PDGF. FLS were cultured in presence of PDGF-BB (10 ng/ml) with MN58b (0.1 , 0.5, 1 and 5 uM) or vehicle for 7 days. Growth was measured on day 4 using MTT assay. As shown, the MN58b decreased cell proliferation dose dependency (Fig. 5). Data is presented as mean absorbance units+/-SEM normalized to Day 0. Treatment with MN58b alone did not affect viability when compared with media (not shown).

EXAMPLE 7: CHOKa REGULATES FLS MIGRATION

Because PDGF is a known chemotactic agent for mesenchymal cells, we evaluated whether ChoKa is required for FLS migration. FLS were grown to a confluent monolayer in a 6-well plates and wounded area was generated with 1 ml micropipette tip. Cells were cultured in low serum Med (1 % FBS) alone or with PDGF-BB 10 ng/ml +/- MN58b (1 μΜ) or vehicle (Fig. 6). Cell migration in response to PDGF was dramatically decreased in presence of CHoKa inhibitor MN58b at 1 μΜ. These results were also confirmed after siRNA knock down of ChoKa (not shown).

EXAMPLE 8: EFFECT OF CHOKa INHIBITOR IN KXB/N MODEL OF

ARTHRITIS

To determine whether choline kinase contributes to arthritis we tested efficacy, tolerability and safety of a choline kinase inhibitor, MN58b, in passive K/BxN animals. WT mice were injected with 150ul serum from adult K/BxN mice on day 0. MN58b significantly decreased paw swelling in this model (p<0.01 for vehicle compared with daily 3 mg/kg) (Fig. 7A). It also decreased histological score (Fig. 7B) and IL-1 b and MMP3 mRNA expression (Fig 7 C and D). These results demonstrate that MN58b is effective in a rodent model of arthritis.

To evaluate the safety of ChoKa inhibition, we treated WT mice with 1.5mg/kg and 3mg/kg of MN58b for 7 days, and tested liver and kidney histology that did not show any significant change (no shown). We also realized cleaved caspase-3 staining that did not show any apoptotic damage in the tissues studied (no shown). Biochemistry and hematologic studies did not show an increase of liver enzymes, worsening of kidney function or anemia. EXAMPLE 9: LACK OF EFFECT OF CHOKa INHIBITOR IN ADAPTIVE IMMUNE

RESPONSE

To test adaptive response, we performed preliminary studies in an antigen- induced arthritis model. Mice were immunized with an intradermal injection on day 0 with methylated BSA (mBSA) in complete Freund's adjuvant. 2 weeks later mice were bled and antibodies against mBSA were analyzed by ELISA. MN58b treatment (daily 3mg/kg) did not change the titer of antibodies against mBSA suggesting that the inhibitor was acting more in the innate than in the adaptive response compartment. lgG1 antibodies levels were 0.54±0.1 and 0.53±0.12 (p=ns) and lgG2b were 0.18±0.04 and 0.17±0.06 (p=ns) for PBS and ChoKa inhibitor-treated mice.

EXAMPLE 10: STATISTICAL ANALYSIS

In vivo studies can be analyzed by ANOVA. For in vitro studies, paired or unpaired Student's t-test will be used when the data have a normal distribution. In other cases, non-parametric tests such as the Wilcoxon signed rank test will be used. Pearson's coefficient will be used for correlation studies. A p value <0.05 will be considered significant. Power analysis shows that 8 mice per group gives a 90% chance of detecting 30% differences in mouse arthritis studies. Statistical support will be provided by the UCSD Biostatistics Core (ctri.ucsd.edu). EXAMPLE 11 : DETERMINATION OF PATTERNS OF CHOLINE-CONTAINING

COMPOUNDS IN RA FLS BY MRS

Preliminary MRS studies in RA FLS shows increased PCho ratios similar to tumor cells. We hypothesize that this switch in choline metabolites is secondary to increased ChoKa activity in RA, which could explain its aggressive phenotype. We will determine the choline kinase expression and functional state of choline kinase from RA, osteoarthritis (OA) and normal FLS by MRS. We will also use tumor fibroblast cell lines derived from pigmented villonodular synovitis and sarcoma that are currently in our repository. We will then evaluate how choline metabolites are modified by cytokines. Although, rheumatoid synoviocytes display certain unique features that are reminiscent of transformed cells, it is not clear whether these features are inherent in RA FLS (transformed aggressors) or are somehow imprinted due to exposure to cytokines in the rheumatoid milieu in vivo (passive responders). Studies using normal and OA FLS after cytokine stimulation will determine if cytokines mimic the change in choline metabolism in RA FLS.

Preparation of synovium and synoviocytes. Synovium and FLS will be obtained from patients undergoing total joint replacement or synovectomy who meet the 1987 revised American College of Rheumatology criteria for seropositive RA or patients with OA as previously described[29]. RA patients will discontinue methotrexate for at least 1 month prior to surgery to minimize the influence of methotrexate on folate metabolism and methyl donors[30]. For FLS lines, tissue is enzymatically dispersed and cells allowed to adhere overnight. Nonadherent cells are washed off, and the adherent FLS are grown in DMEM containing 10% FCS. FLS are used from passage 3 through 8 during which time they are a homogeneous population of cells (<1 % CD1 1 b positive, <1 % phagocytic, and <1 % FcR II and FcR III receptor positive)[31].

Choline metabolites profiling using 1 H-MRS: 1 H-MRS platform has become an established tool for the comprehensive analysis of the metabolome in biological samples for both polar and lipophilic metabolites. To confirm that RA FLS have a tumor-like metabolite pattern, cells from normal, RA and OA FLS (5 cell lines each), as well as tumor fibroblasts (e.g., PVNS) and dermal fibroblasts will be isolated and prepared for the 1 H-MRS analysis as described[21 , 32]. Acquisition of the 1 H-MRS metabolic profiles of both the polar and apolar fractions will be performed using a Bruker Avance 700 MHz NMR spectrometer equipped with high throughput robotics. One and two-dimensional NMR spectra will be acquired for improved metabolite identification and quantification. We will look at the 3.20-3.24 ppm region where the PCho, GPC and Cho have been reported. We will analyze the metabolite concentrations of cellular components of fibroblast cell lines. Metabolite quantification will be expressed as nmoles and normalized to the number of extracted cells. Ratio of choline phospholipid metabolites (PCho/total choline and GPC/PCho) will be analyzed.

Is the RA tumor-like choline metabolite profile secondary to ChoKa in RA FLS? Elevated PCho levels are usually attributed to an increased ChoK activity. We will evaluate the functional state of choline kinase by 1 H-MRS after incubation of RA, OA, and normal (NL) FLS cells with ChoKa inhibitor MN58b (1 nM-5 uM) to show that the profile is due to a ChoKa. The optimum concentrations of MN58b will be used in Aim 2.

Is choline kinase expression greater in RA FLS and synovium? Expression will be determined by qPCR and WB. We will also determine the expression of ChoKa in OA, RA and normal FLS (5 cell lines each) before and after stimulation with cytokines (IL-1 at 2ng/ml, TNF or PDGF BB at 10ng/ml) by Western blot (WB) and qPCR. RA, OA and normal synovium will be immunostained (5 tissues each) to localize ChoKa. Digital image analysis will determine 1 ) whether intimal lining expression is greater than sublining expression; and 2) whether these proteins are more abundant in RA compared with OA or normal tissues.

Is the RA choline metabolite profile modified by cytokines? To determine if cytokine stimulation further increases ChoKa expression in RA FLS and if cytokine stimulation changes choline metabolism in OA and normal FLS, we will incubate normal, RA and OA FLS with PDGF BB and TNF for 2 to 48 hr as we have shown that these cytokines increase ChoKa expression in RA FLS (see Preliminary Data). We will perform dose response and kinetics to determine optimum time point for ChoKa induction and use these culture conditions to determine choline metabolites by MRS.

Anticipated results: As RA cultured FLS possess a unique tumor like behavior that differentiates them to other fibroblast cell lines, and based on our preliminary results, we expect a choline metabolites profile characterized by high PCho/total choline and low GPC/PCho ratios in RA FLS, similar to the pattern from tumor fibroblasts. In contrast, normal and OA FLS will probably show a different profile characterized by low PCho/total choline and similar GPC/PCho as found in normal fibroblasts. In that case, analysis of other metabolites such as phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanomaline can be addressed to further characterize choline metabolism. As choline phosphorylation is mostly due to ChoKa activity, incubation of the RA FLS with the ChoKa inhibitor should reverse the activated choline metabolism profile. We will compare the expression of ChoKa in both OA and RA synovium and FLS to determine if the FLS profile is mimicked by tissue. Because the RA FLS phenotype is restored by cytokines in vitro, we expect that choline metabolism will also be modified.

EXAMPLE 12: DETERMINING THE EFFECT OF CHOLINE KINASE INHIBITION ON GENE EXPRESSION, SIGNALING AND FUNCTION

OF FLS

Based on the observation of the activated choline metabolism in FLS and its expression in these cells, and with the hypothesis that increased ChoKa activity might contribute to the unique tumor-like behavior or RA cultured FLS, we will explore how ChoKa modulates RA FLS growth, migration and invasion. Our preliminary data show that ChoKa regulates Akt/MAPK phosphorylation, growth and migration of RA FLS suggesting that ChoKa inhibition might be chondroprotective in RA. Cells will be pre-treated with the ChoKa inhibitor at the concentrations determined in Specific Aim 1. Key findings seen with chemical inhibition will be confirmed with siRNA knock down.

Does ChoKa regulate PDGF-mediated FLS activation? Our preliminary data show that ChoKa regulates P-Akt and P-ERK after PDGF stimulation. To examine the role of ChoKa in PDGF function, RA FLS will be stimulated with PDGF for 5 to 30 min in the presence or absence of the MN58b or after ChoKa knockdown. P-Akt, P-JNK, P-ERK and P-p38 will be determined by WB. If confirmed, we will also determine whether ChoKa blockade or deficiency affects other PDGF-mediated functions, such as MMP1 and MMP3 expression by qPCR.

Anticipated results and potential pitfalls: ChoKa will probably decrease P-protein and MMP expression based on our preliminary studies. Cytokines, especially TNF and IL-1 , enhance the destructive properties of RA FLS and their effects will probably be potentiated by growth factors like PDGF.

Does ChoKa blockade or deficiency alter synoviocyte growth through an effect on proliferation and apoptosis? Our data predict that ChoKa inhibition could have chondroprotective effects in RA. To explore this possibility, we will determine if ChoKa regulate proliferation and survival of cultured RA FLS. Human FLS will be cultured with MN58b compound of after ChoKa knockdown

• Apoptosis— Human and murine FLS will be treated with anti-Fas antibody or 100 uM hydrogen peroxide to induce apoptosis. Cells will be evaluated from 4 to 24 hr later using trypan blue dye exclusion. A histone release assay performed by ELISA will be performed to confirm the results.

• Proliferation— Cell proliferation will be evaluated using cultured FLS (human and murine) and stimulating with medium or PDGF (10ng/ml) in the presence of ChoKa inhibitor. Tritiated thymidine will be used to quantify DNA synthesis after 2-7 days.

Anticipated results: We expect that apoptosis will be increased and proliferation will be decreased in cells after blockade or deficiency of ChoKa activity because this kinase contributes to Akt phosphorylation in FLS that is known to play a role in these functions.

Does ChoKa regulate synoviocyte migration and invasion in vitro? Cell migration is an essential process for synoviocytes to reach, invade and destroy articular cartilage in RA. In addition, it was recently proposed that FLS migrating through the blood stream might be responsible for progression of arthritis and that this migratory behavior is unique for RA but not OA FLS. Interfering with key regulators of these functions of synoviocytes has the potential to reduce articular damage, progression of disease and improve outcome in RA. Based on our preliminary results that ChoKaDinhibition decreases synoviocytes migration and MMP expression, we hypothesized that ChoKa is a majorD regulator of FLS invasion. RA FLS will be cultured with MN58b compound of after ChoKa knockdown

• In vitro migration using a scratch assay— .FLS will be grown into a monolayer and an area will be cleared using micropipette. Cells will be pre- incubated with ChoKa inhibitor or ChoKa knockdown. Medium or PDGF (10ng/ml) will then be added to the cultures and migration of cells into the cleared area quantified after 12, 24, and 48 hr by image analysis.

• Invasion using Cytoselect assay— Because invasion is feature of RA synoviocytes, we will test the effect of ChoKa inhibition on FLS invasion using Matrigel coated transwell inserts (Cell Biolabs, San Diego, CA). FLS will be pre-treated with ChoKa inhibitor or vehicle for 8 hr. The FLS are added to the upper chamber and then medium with or without PDGF (25 ng/ml) is added to lower chamber. Invasion is quantified after 24 hr by counting cells on the lower surface of the membrane.

Anticipated results: Our preliminary data suggest that the ChoKa contributes to migration, although we still need to quantify the effects, determine the effect on invasion, and perhaps evaluate other chemoattractants. Given the role of ChoKa in cell movement, the ChoKa inhibitor will probably decrease cell invasion into matrix. While Matrigel, which is mainly comprised of laminin, is an imperfect matrix, it is commonly used as a substrate for invasion assays. If an effect is seen, then we will consider evaluating the effect of ChoKa inhibition on MMP and integrin gene expression.

Does ChoKa regulate migration and MMP expression through activation of Akt/MAPK pathway respectively? Because ChoKa regulates MAPK and Akt signaling after PDGF stimulation in RA FLS, and these signaling have been involved in MMP and migration respectively, we will determine the effect of Akt and MAPK siRNA on those functions together with ChoKa inhibition. We hypothesize that if the effect of ChoKa is through these two pathways, ChoKa inhibitor will not add any further effect after blockade of MAPK and Akt by siRNAs or chemical inhibitors. MAPK (either JNK, p38 or ERK siRNA) will be chosen according to the effect of ChoKa inhibition on those phospho-protein after PDGF stimulation) and Akt will be knocked-down by siRNA in RA FLS and at day 5, MMP expression and migration assays after PDGF stimulation will be realized and assessed as detailed above after chemical ChoKa inhibition.

Anticipated results: Based on our preliminary results that ChoKa inhibition decreases P-Akt and p-MAPK in FLS, we expect that addition of ChoKa inhibition will not add any additional effect in both migration and MMP expression after akt and MAPK siRNA downregulation respectively. We could also consider using chemical MAPK inhibitors, like SP600125, SB203580, and PD98059 if multiple isoform of each MAPK are involved.

EXAMPLE 13: DETERMING EFFECTS OF CHOLINEKINASE INHIBITION IN

K/BXN ARTHRITIS MODEL

The lack of effect of ChoKa inhibitor in adaptive immune response and its effect on FLS, led us evaluate the role of this kinase in the K/BxN arthritis model[33]. Although arthritis models do not represent RA in mouse, in vivo studies allow investigation of pathogenic mechanism in RA. The K/BxN passive serum transfer model is useful because it is FLS dependent and requires only innate immunity. We will use the choline kinase inhibitor MN58b to evaluate choline kinase blockade in vivo. We hypothesized that ChoKa inhibition will ameliorate inflammation and cartilage damage. Preliminary toxicology studies did not show any evident toxicity in major organs such as liver or kidney in mice on this specific choline kinase inhibitor. We will also determine if choline pathway correlates with disease activity in this animal model. What is the effect of chemical inhibition of ChoKa in K/BxN arthritis? Initial experiments will determine optimal doses and efficacy of MN58b compound in vivo. WT mice will be treated daily with several doses of ChoKa inhibitor (1 -3 mg/kg) or vehicle starting on day 0 parallel with K/BxN serum transfer. To minimize potential toxicity, we will also test other dosing options such as administering the drug every day during the first 5 days of arthritis, or giving the drug on alternate days. To examine efficacy in established disease, we will start therapy on day 5. To induce arthritis, WT mice will be injected with 150 ul serum from adult K/BxN mice on day 0. Clinical scoring will be done daily and experiments will be terminated on day 10. The endpoints for our analysis: 1 ) Clinical scores and ankle diameter; 2) Ankle histology, for synovial hyperplasia, cartilage erosion, cartilage proteoglycan, inflammation and bone destruction 3) IHC for infiltrating cell types 4) Inflammatory gene expression (mRNA and protein analysis) and 5) Alteration in synovial signaling in the arthritic joints.

· Synovial proliferation and cartilage erosion. Hind paws of mice are harvested for paraffin sections and are stained with H&E, safranin O-fast green for proteoglycan content. We will determine in situ FLS proliferation by performing IHC for PCNA and Ki67, nuclear antigens used as an index of cell proliferation in tissue. Results will be quantified by image analysis. · Synovial mediator expression— Ankle extracts will be assayed by tissue extract ELISA and qPCR of pro-inflammatory cytokines (TNF and IL-6), prototypical Th1 , Th2, and Th17 cytokines (IFNy, IL-4 and IL-17A), and proteases (MMP3, MMP13) as possible mediators regulated by the Akt/MAPK pathway.

· Synovial signaling— WB will be performed to determine P-Akt, P-JNK, P-p38 and P-ERK in the joints.

Anticipated results: As we shown in our preliminary results, the compound is tolerated by mice and can be used in the animal model of arthritis. We expect that chemical inhibition of ChoKa will ameliorate arthritis. Based on our in vitro studies in FLS, we expect to see decreased synovial lining hyperplasia and significant reduction in cartilage erosion in the mutant mice. There will be decreased synovial inflammation due to possible role of ChoKa on macrophages, and decreased inflammatory mediator production by resident synoviocytes. These benefits will correlate with decreased P-Akt and P-MAPK. These results would make us move forward and test the inhibitor in a antigen-induced arthritis and collagen-induced arthritis model. To confirm the results, we will target ChoKa using anti-sense oligonucleotides. We recently used this technique to demonstrate the role of the kinase MKK7 in passive K/BxN arthritis[34]. Unfortunately, CHKA-deficient mice is lethal and there is not CHKA F F mice available in order to study which cell type confers protection in arthritis. One option is to use the choline inhibitor on cadherin 1 1 -deficient mice[35] to determine if ChoKa inhibition is additive with cadherin deficiency.

Toxicology studies: Although the ChoKa inhibitor is well tolerated by mice, we would like to extend our preliminary toxicology studies. We will treat the mice with several doses of MN58b inhibitor (1 -3mg/kg) for one week (8 mice/group) and we will realize histology of major organs such as liver, kidney, spleen and brain. We will also get basic hematology and biochemistry studies from blood and serum respectively. Apoptotic cells will be determined by cleaved-caspase 3 staining by IHC.

Anticipated results: MN58b inhibitor is well tolerated by mice at the doses tested, and preliminary data already shows no changes in histology of liver and kidney. Do the choline metabolites correlate with disease activity in K/BxN arthritis model? We will analyze choline metabolites by 1 H-MRS in joint extracts, peripheral blood cells and serum from WT mice at different time points after K/BxN serum injection (day 2,4,6,8 and 10), to determine when we can better detect choline metabolites concentrations and ratio between levels of choline phospholipid metabolites (PCho/total choline and GPC/PCho). We will then inject K/BxN serum to 10 WT mice that will be sacrificed at that time point. We will correlate choline metabolites with clinical and histological score, IL-6, IL-1 , TNF, MMP3, and MMP13 expression by qPCR from joints, and cytokines assayed in serum by Luminex multiplex technology. PCho and GPC are typically intracellular metabolites but other metabolites such as phosphatidylcholines can be detected extracellularly. We will also determine choline metabolites in joint extracts at the same time point after MN58b administration to prove the effect of the compound.

Anticipated results: We expect to see decreased PCho/total choline ratio in joints after MN58b administration. As our preliminary results using the inhibitor in vivo decreases both inflammation and joint damage, we expect to see correlation of choline metabolites ratio with disease activity and mentioned mediators of inflammation and joint damage.

EXAMPLE 14: DETERMINING SUITABILITY OF CHOLINE METABOLITES AS

BIOMARKER OF INFLAMMATION

Biomarkers associated with clinical response might not be the same biomarkers that predict risk of further joint damage. MRS is a commonly used analytical method to analyze the metabolome of body fluids such as urine and blood serum[36]. Recent studies demonstrate the applicability of NMR-based metabolomics using serum samples for the diagnosis and prognosis[37]. In cancer, increased levels of these compounds provide a non-invasive biomarker of transformation, staging and response to therapy disease. However, few studies have addressed metabolic changes in RA and little information of this metabolic pathway has been addressed in inflammation. We will explore the hypothesis that choline metabolites can be detected in different kind of samples from RA patients, and that correlates with clinical response. We will take advantage of samples that we already have in our department to explore the relationship between choline metabolites and clinical response.

Are choline metabolites present in serum, peripheral blood and synovial tissue of RA patients? Our laboratory has developed a biological sample repository, which include thousands of blood, serum, and synovial tissue samples from patients with a variety of forms of arthritis. We will first determine choline metabolic profiles by 1 H MRS of peripheral blood, serum and synovial tissue from RA and OA patients and normal samples (10 samples each). We will determine which metabolites can be detected in those samples and which profile is normal (present in samples from healthy controls) and which one is disease specific (OA vs RA). PCho and GPC and other metabolites such as phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanomaline are typically intracellular metabolites. However, some of these metabolites such as phosphatidylcholines can be detected in serum.

To determine the relationship between the choline metabolites pattern and clinical response to treatment in RA patients. Our group recently conducted a study using the anti-CD20 monoclonal antibody rituximab in patients with active rheumatoid arthritis. In this study, serum, blood and synovial tissue samples were obtained before and following treatment, and detailed clinical information collected (i.e. to define whether or not patients responded to treatment) [38]. Choline- containing compounds will be assayed in serum and synovial tissue before and after treatment. The relationship between choline kinase metabolites in synovial tissue and blood and clinical responses as well as changes in histologic scores and gene expression will be determined. Statistical Analyses: Comparison of pre- treatment to post-treatment values was performed using Wilcoxon's signed rank test for paired data and corrected for multiple comparisons. Associations between changes in various clinical and biomarker results and choline metabolites will be performed using Spearman's rank correlation coefficient.

List of References

All references discussed herein, including the references below, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

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31 . Alvaro-Gracia, J.M., N.J. Zvaifler, and G.S. Firestein, Cytokines in chronic inflammatory arthritis. V. Mutual antagonism between interferon-gamma and tumor necrosis factor-alpha on HLA-DR expression, proliferation, coiiagenase production, and granulocyte macrophage colony-stimulating factor production by rheumatoid arthritis synoviocytes. The Journal of clinical investigation, 1990. 86(6): p. 1790-8.

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35. Lee, D.M., et al., Cadherin-11 in synovial lining formation and pathology in arthritis. Science, 2007. 315(5814): p. 1006-10.

36. Psychogios, N., et al., The Human Serum Metabolome. Plos One, 201 1 .

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38. Kavanaugh, A., et al., Assessment of rituximab's immunomodulatory synovial effects (ARISE trial). 1: clinical and synovial biomarker results. Annals of the rheumatic diseases, 2008. 67(3): p. 402-8. With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.

It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and/or "an" should be interpreted to mean "at least one" or "one or more"); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., " a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to "at least one of A, B, or C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., " a system having at least one of A, B, or C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as "up to," "at least," "greater than," "less than," and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1 -3 articles refers to groups having 1 , 2, or 3 articles. Similarly, a group having 1 -5 articles refers to groups having 1 , 2, 3, 4, or 5 articles, and so forth.

While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention.

The term "comprising" as used herein is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims in any application claiming priority to the present application, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention.

The foregoing description and Examples detail certain embodiments. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the invention may be practiced in many ways and the invention should be construed in accordance with the appended claims and any equivalents thereof.