WO2010009403A2 - Methods for treating arthritis and other inflammatory or autoimmune diseases - Google Patents

Abstract

Methods and compositions for treating arthritis and other disorders are provided herein. Methods can include administering secretory phospholipase A2 Group V (sPLA2-V) protein or a variant thereof.

Description

METHODS FOR TREATING ARTHRITIS AND OTHER INFLAMMATORY OR AUTOIMMUNE DISEASES

CROSS REFERENCE TO RELATED APPLICATIONS

This application is copending with, shares at least one common inventor with, and claims the benefit of U.S. Provisional Application No. 61/082,159, filed July 18, 2008, the entire contents which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Inflammation is a series of defensive response process caused in the tissues, induced by the applied injurious events (inflammatory stimuli) on any parts of a human body. When the tissues are damaged by inflammatory stimuli that could be caused by bacterial infections, immunological responses or physical injuries, the tissues respond (acute inflammation) to the stimuli, followed by excluding the stimuli to repair the damages. Alternatively, if the exclusion is difficult, the damages are progressed to induce continuously tissue swelling (chronic inflammation). Inflammation is well known to be associated with several diseases such as rheumatoid arthritis, sepsis, psoriasis, pancreatitis.

Phospholipase A2 (PLA2) are a diverse family whose members have been implicated in multiple disease states including rheumatoid arthritis, sepsis, psoriasis, pancreatisis and cancer ^{!" 1}. PLA2 proteins share capacity to enzymatically hydrolyze membrane glycerophospholipids to release fatty acids and lysophospholipids. There are at least 15 mammalian PLA2 isoenzymes that have historically been grouped into three major classes, namely the calcium dependent and independent intracellular PLA2, and secreted PLA2 (sPLA2) ⁸'⁹. Of the intracellular PLA2 enzymes, the calcium independent intracellular PLA2 (group VI) demonstrates a complex transcriptional and translational regulation that results in numerous splice variants . The calcium dependent cytosolic PLA2 (group IV) is constitutively expressed in most tissues and is known to contribute significantly to eicosanoid synthesis .

The secreted PLA2 (sPLA2) family members are strikingly diverse. They are typically — 14-19 kDa heavily disulfide bridged proteins found not only in mammals, but also in insects, snake venoms, plants, bacteria, fungi and viruses ^12~16. Indeed, substantial insights concerning the molecular properties of sPLA2 were revealed by study of sPLA2 found in venoms, where sPLA2 not only hydrolyze lipids, but also have neurotoxic activity via direct binding to receptors¹⁷. A transmembrane receptor, known as M-receptor, was cloned and shown to bind a subset of mammalian sPLA2 ¹⁷. Sequence homology analysis led to the identification often mammalian enzymatically active sPLA2 (nine of which are expressed in humans where HC is a pseudogene) and two sPLA2-like proteins devoid of catalytic activity. These sPLA2 enzymes cluster into three subfamilies according to their sequence homology and number and position of disulfide bonds. The first large subfamily is comprised of the most catalytically active enzymes and includes sPLA2-IB, -HA, -IIC, -HD, -HE, -HF, -V, -X and otoconin-90 ¹⁸. The second subfamily is characterized by its homology to bee venom sPLA2 and contains the group III sPLA2. The 3^rd subfamily contains sPLA2-XIIA and -XIIB, the latter anticipated to be enzymatically inactive thanks to a mutation in its active site.

Functionally, sPLA2 are implicated in numerous physiologic processes in addition to generating substrate arachidonic acid (AA) for subsequent synthesis of eicosanoids. sPLA2-IIA has been implicated in host defense against negatively charged Gram-positive bacteria ¹⁹'²⁰. This is due to its strong predilection for negatively charged membranes such as those present in the cell wall of these bacteria. sPLA2-IB, also known as pancreatic sPLA2, was first identified in pancreatic juice and exhibits a role in dietary phospholipids digestion ²¹'²². Recently, sPLA2-V has been shown to play a role in host defense by promoting phagocytic uptake of zymosan- containing yeast by peritoneal macrophages (Balestrieri, JBC 281(10):6691-8, 2006) and regulating TLR2-dependent eicosanoids synthesis (Kikawada, Blood 110(2):561-7, 2007).

No drug has been developed which shows remedial effects in the clinical therapies by modulating specific sPLA2 activities.

SUMMARY OF THE INVENTION

The present invention provides novel and inventive therapeutic approaches for treating arthritis and other inflammatory or autoimmune diseases, disorders or conditions. Specifically, the present invention encompasses the surprising findings of distinct and opposing functions of secretory phospholipases A2 group V (sPLA2-V) as an anti-inflammatory agent and group HA (sPLA2-IIA) as a pro-inflammatory mediator. In one aspect, the present invention provides methods of treating arthritis and other inflammatory or autoimmune diseases, disorders or conditions by increasing activity of secretory phospho lipase A2 Group V (sPLA2-V). In some embodiments, the present invention provides methods of treating arthritis including a step of administering to a subject in need of treatment an effective amount of sPLA2-V protein or a variant thereof, such that at least one symptom or feature of the arthritis is reduced in intensity, severity, or frequency, or has delayed onset of, disorders or conditions associated with arthritis. In some embodiments, the arthritis is inflammatory arthritis. In some embodiments, the arthritis is selected from the group consisting of rheumatoid arthritis, spondylitic arthritis, psoriatic arthritis, juvenile idiopathic arthritis, lupus, reactive arthritis, lyme arthritis, osteoarthritis and combinations thereof.

In some embodiments, inventive methods of the invention include administering to the subject the sPLA2-V protein or a variant containing at least 85% amino acid sequence identity to human sPLA2-V. In some embodiments, inventive methods of the invention include administering to the subject human sPLA2-V. In some embodiments, sPLA2-V variants suitable for the invention contain a tryptophan residue in the sPLA2 interfacial binding site. In some embodiments, sPLA2-V variants suitable for the invention are fragments of human sPLA2-V. In some embodiments, suitable fragments of human sPLA2-V includes the sPLA2 interfacial binding site.

In some embodiments, sPLA2-V variants suitable for the invention lack catalytic activity. In some embodiments, sPLA2-V variants suitable for the invention contain an amino acid substitution at a position corresponding to position 48 in the human sPLA2-V protein. In some embodiments, suitable sPLA2-V variants contain a H48Q substitution.

In some embodiments, sPLA2-V variants suitable for the invention include mutant sPLA2-IIA proteins, wherein the mutant sPLA2-IIA proteins contain tryptophan substitutions at positions corresponding to positions 3 and/or 31 in the human sPLA2-IIA protein.

In some embodiments, inventive methods of the invention can be used to treat at least one symptom or feature of arthritis selected from the group consisting of joint effusion, leukocytic infiltration of synovial fluid, tissue leukocytic infiltration, synovial hyperplasia, synovial pannus, bone erosion, cartilage depletion, pain, swelling, morning stiffness, fatigue, loss of joint range of motion, and combinations thereof. In some embodiments, inventive methods further include a step of inhibiting sPLA2-IIA. In some embodiments, the step of inhibiting sPLA2-IIA does not inhibit sPLA2-V. In some embodiments, the step of inhibiting sPLA2-IIA includes administering an interfering RNA. In some embodiments, suitable interfering RNAs are selected from siRNA, shRNA or miRNA. In some embodiments, the step of inhibiting sPLA2-IIA includes administering an antibody, or a fragment thereof, that specifically binds sPLA2-IIA. In some embodiments, suitable antibodies, or fragments thereof, are selected from the group consisting of intact IgG, F(ab')2, F(ab)2, Fab', Fab, ScFvs, monoclonal antibodies, diabodies, triabodies, tetrabodies, single-domain antibodies and combinations thereof. In some embodiments, the step of inhibiting sPLA2-IIA includes administering a small molecule.

In some embodiments, sPLA2-V or variants thereof are administered parenterally . In some embodiments, sPLA2-V or variants thereof are administered intraveneously. In some embodiments, the effective amount administered ranges from 0.5 mg/kg to 10 mg/kg.

In another aspect, the present invention provides methods of treating inflammatory diseases, disorders or conditions including a step of administering to a subject in need of treatment an amount of sPLA2-V, or a variant thereof, effective to stimulate phagocytic clearance of immune-complexes. In some embodiments, inventive methods of the invention can be used to treat inflammatory diseases, disorders or conditions including, but not limited to, rheumatoid arthritis, spondylitic arthritis, psoriatic arthritis, and osteoarthritis.

In another aspect, the present invention provides methods of treating autoimmune diseases, disorders or conditions including administering to a subject in need of treatment an effective amount of sPLA2-V or a variant thereof, such that at least one symptom or feature of the autoimmune diseases, disorders or conditions is reduced in intensity, severity, or frequency, or has delayed onset. In some embodiments, inventive methods of the invention can be used to treat autoimmune diseases, disorders or conditions including, but not limited to, lupus, vasculitis, nephritis.

In another aspect, the present invention provides methods of treating arthritis comprising a step of specifically inhibiting sPLA2-IIA but not sPLA2-V. In some embodiments, the step of specifically inhibiting sPLA2-IIA includes inhibiting the catalytic activity of sPLA2-IIA. In some embodiments, the step of specifically inhibiting sPLA2-IIA includes inhibiting the binding activity of sPLA2-IIA to M-type receptor or other interacting proteins (e.g vimentin and coagulation components). In some embodiments, inventive methods of the present invention further include administering sPLA2-V or a functional variant thereof.

In some embodiments, the step of inhibiting sPLA2-IIA includes administering an interfering RNA specific against sPLA2-IIA. In some embodiments, the interfering RNA is selected from siRNA, shRNA or miRNA. In some embodiments, the interfering RNA is a siRNA molecule.

In some embodiments, the step of inhibiting sPLA2-IIA comprises administering an antibody, or a fragment thereof, that specifically binds the sPLA2-IIA protein, but not the sPLA2-V protein. In some embodiments, the antibody, or a fragment thereof, specifically binds to the catalytic or receptor binding domain of sPLA2-IIA. In some embodiments, the antibody, or a fragment thereof, is selected from the group consisting of intact IgG, F(ab')2, F(ab)2, Fab', Fab, ScFvs, monoclonal antibodies, diabodies, triabodies, tetrabodies, single-domain antibodies and combinations thereof. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized monoclonal antibody.

In some embodiments, the step of inhibiting sPLA2-IIA includes administering an aptamer that specifically binds the sPLA2-IIA protein, but not the sPLA2-V protein.

In some embodiments, the step of inhibiting sPLA2-IIA comprises administering a small molecule that specifically inhibits sPLA2-IIA activity.

In some embodiments, small molecules suitable for the present invention have a structure represented by formula I

wherein

Ri is hydrogen, halogen, Ci-C₆ alkyl, C₃-C₆ cycloalkyl, -0-(Ci-C₆ alkyl), -S-(Ci-C₆ alkyl);

R₂ is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; substituted or unsubstituted, branched or unbranched arylalkyl; substituted or unsubstituted, branched or unbranched heteroarylalkyl; -ORA; -C(=O)RA; -CO₂RA; -CN; -SCN; - SR_A; -SOR_A; -SO₂RA; -NO₂; -N(RA)₂; -NHC(O)RA; -OC(O)RA; -0C(0)0R_A; or -C(RA)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, a Ci-C₆ alkyl moiety, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or hetero arylthio moiety;

Xi is O, S, or NR₃;

R₃ is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; substituted or unsubstituted, branched or unbranched arylalkyl; substituted or unsubstituted, branched or unbranched heteroarylalkyl; C(=0)R_A; or -C(R_A)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, a Ci-C₆ alkyl moiety, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

Each occurrence of R₄ is independently hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; substituted or unsubstituted, branched or unbranched arylalkyl; substituted or unsubstituted, branched or unbranched heteroarylalkyl; - ORA; -C(=0)R_A; -C0₂R_A; -CN; -SCN; -SR_A; -SOR_A; -SO₂RA; -NO₂; -N(RA)₂; -NHC(0)R_A; - OC(O)R_A; -OC(O)OR_A; or -C(R_A)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, a Ci-C₆ alkyl moiety, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. In some embodiments, small molecules suitable for the present invention have a structure represented by formula II

wherein

R₅ is hydrogen, halogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, -0-(Ci-C₆ alkyl), -S-(Ci-C₆ alkyl);

R₆ is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; substituted or unsubstituted, branched or unbranched arylalkyl; substituted or unsubstituted, branched or unbranched heteroarylalkyl; or -C(R_A)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, a Ci-C₆ alkyl moiety, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

X₂ is O, S, or NR₃;

R₇ is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; substituted or unsubstituted, branched or unbranched arylalkyl; substituted or unsubstituted, branched or unbranched heteroarylalkyl; C(=0)R_A; or -C(R_A)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, a Ci-C₆ alkyl moiety, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; and Each occurrence of Rg is independently hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; substituted or unsubstituted, branched or unbranched arylalkyl; substituted or unsubstituted, branched or unbranched heteroarylalkyl; - ORA; -C(=O)R_A; -CO₂RA; -CN; -SCN; -SR_A; -SOR_A; -SO₂R_A; -NO₂; -N(RA)₂; -NHC(O)RA; - OC(O)RA; -OC(O)ORA; or -C(RA)₃; wherein each occurrence of RA is independently a hydrogen, a protecting group, a Ci-C₆ alkyl moiety, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety.

In some embodiments, small molecules suitable for the present invention have a structure as shown in Table 5, or a derivative thereof.

In some embodiments, inventive methods further includes administering an antiinflammatory agent. In some embodiments, suitable anti-inflammatory agents are selected from the group consisting ofNSAID's (Cox-2 selective or non-selective), Methotrexate, Sulfasalazine, Hydroxychloroquine, Leflunomide, Minocycline, Azathioprine, Etanercept, Infliximab, Adalimumab, Rituximab, Abatacept, Anakinra and combinations thereof. In some embodiments, suitable anti-inflammatory agents include anti-TNF agents (e.g., pegylated anti-TNF agents). In some embodiments, suitable anti-inflammatory agents include anti-IL-6 receptor antibodies or fragments thereof.

In another aspect, the present invention provides pharmaceutical compositions for treating arthritis and other inflammatory or autoimmune diseases, disorders or conditions. In some embodiments, pharmaceutical compositions of the present invention contain an effective amount of sPLA2-V, or a variant thereof. In some embodiments, pharmaceutical compositions of the present invention contain effective amount of sPLA2-IIA inhibitors as described herein.

In some embodiments, pharmaceutical compositions of the present invention further include an anti-inflammatory agent. In some embodiments, suitable anti-inflammatory agents include, but not limited to, NSAID's (Cox-2 selective or non-selective), Methotrexate, Sulfasalazine, Hydroxychloroquine, Leflunomide, Minocycline, Azathioprine, Etanercept, Infliximab, Adalimumab, Rituximab, Abatacept, Anakinra, anti-TNF agents (e.g., pegylated anti- TNF agents), anti-IL-6 receptor antibodies or fragments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Exemplary quantitation of sPLA2-IIA (Fig. IA), sPLA2-V (Fig. IB), and several other (Fig. 1C) sPLA2 in synovial fluid from RA patients showing that all sPLA2 isoforms are present. sPLA2-IIA, V, and XIIA are the most abundantly expressed. sPLA2-IIA is induced in RA as demonstrated by the comparison of protein levels detected in synovial fluid of patients with RA versus healthy controls. Synovial fluids from patients with RA (n=45) and healthy volunteers (n=l2) were analyzed. Levels of sPLA2-IIA in RA synovial fluid versus normal is induced P< 0.001.

Figure 2. Exemplary data illustrating that sPLA2-IIA contributes to arthritis. (Fig. 2A) Arthritis response in IIA-/V+ and control sufficient mice. n=5 mice/group/experiment, pooled data from three independent experiments. IIA+/V+ versus IIA-/V+, clinical index and Δ ankle thickness P < 0.001. (Fig. 2B) Histomorphometric arthritis quantification and histologic sections from IIA-/V+ and IIA+/V+ mice at experimental day 13. (Fig. 2C) Mice in C57BL/6J background over-expressing or not human sPLA2-IIA were transferred with K/BxN serum and arthritis monitored. n=5 mice/group/experiment, representative of three independent experiments. Control mice versus human sPLA2-IIA expressing mice. Clinical index and Δ ankle thickness have P < 0.001.

Figure 3. Exemplary data illustrating that sPLA2-V protects from the disease. (Fig. 3A) Arthritis response in IIA-/V+ and II A- V-. n=5 mice/group/experiment, pooled data from three independent experiments. IIA-/V+ versus IIA-/V- clinical index and Δ ankle thickness P < 0.001. (Fig. 3B) Histomorphometric arthritis quantification and histologic sections from IIA-/V+ and IIA-/V- mice at experimental day 13.

Figure 4. Exemplary data illustrating that intravenous injection of recombinant sPLA2-V protects mice from developing arthritis. IIA-/V- (Fig. 4A,B) and IIA+/V+ (Fig. 4C,D) mice were injected with diluent (PBS) or 50μg recombinant mouse sPLA2-V diluted in PBS 2 hours prior K/BxN serum injection and daily during 5 days and arthritis monitored. Clinical index and Δ ankle thickness have P < 0.001. Figure 5. Exemplary data illustrating that sPLA2-V is involved in clearance of immune- complexes. (Fig. 5A) Phagocytosis of IgG-opsonized sheep red blood cells by wild type and Pla2g5-null mouse macrophages. (Fig. 5B) Phagocytosis of complement-opsonized sheep red blood cells by wild type and Pla2g5-null mouse macrophages. (Fig. 5C) Cryostat sections of IIA-/V+ and IIA-/V- ankle joints stained with anti-IgG and anti-C3 fragments showing deposition of immune complexes. Mice, V+ (left panel) and V- (right panel) were injected with K/BxN serum and ankles harvested at day 7.

DEFINITIONS

Amino acid: As used herein, term "amino acid," in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H₂N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. "Standard amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. "Nonstandard amino acid" refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions. Amino acids, including carboxy- and/or amino-terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, and/or substitution with other chemical groups that can change the peptide's circulating half-life without adversely affecting their activity. Amino acids may participate in a disulfide bond. The term "amino acid" is used interchangeably with "amino acid residue," and may refer to a free amino acid and/or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.

Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans, at any stage of development. In some embodiments, "animal" refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.

Antibody: As used herein, the term "antibody" refers to any immunoglobulin, whether natural or wholly or partially synthetically produced. All derivatives thereof which maintain specific binding ability are also included in the term. The term also covers any protein having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. Such proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. An antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. As used herein, the terms "antibody fragment" or "characteristic portion of an antibody" are used interchangeably and refer to any derivative of an antibody which is less than full-length. In general, an antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, and Fd fragments. An antibody fragment may be produced by any means. For example, an antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody and/or it may be recombinantly produced from a gene encoding the partial antibody sequence. Alternatively or additionally, an antibody fragment may be wholly or partially synthetically produced. An antibody fragment may optionally comprise a single chain antibody fragment. Alternatively or additionally, an antibody fragment may comprise multiple chains which are linked together, for example, by disulfide linkages. An antibody fragment may optionally comprise a multimolecular complex. A functional antibody fragment typically comprises at least about 50 amino acids and more typically comprises at least about 200 amino acids.

Approximately: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

Combination therapy: The term "combination therapy", as used herein, refers to those situations in which two or more different pharmaceutical agents are administered in overlapping regimens so that the subject is simultaneously exposed to both agents.

Control: As used herein, the term "control" has its art-understood meaning of being a standard against which results are compared. Typically, controls are used to augment integrity in experiments by isolating variables in order to make a conclusion about such variables. In some embodiments, a control is a reaction or assay that is performed simultaneously with a test reaction or assay to provide a comparator. In one experiment, the "test" {i.e., the variable being tested) is applied. In the second experiment, the "control," the variable being tested is not applied. In some embodiments, a control is a historical control (i.e., of a test or assay performed previously, or an amount or result that is previously known). In some embodiments, a control is or comprises a printed or otherwise saved record. A control may be a positive control or a negative control.

Dosing regimen: A "dosing regimen", as that term is used herein, refers to a set of unit doses (at least one and often more than one) that are administered individually separated by periods of time. The recommended set of doses (i.e., amounts, timing, route of administration, etc.) for a particular therapeutic agent constitutes its dosing regimen.

Dysfunction: As used herein, the term "dysfunction" refers to an abnormal function. Dysfunction of a molecule (e.g., a protein) can be caused by an increase or decrease of an activity associated with such molecule. Dysfunction of a molecule can be caused by defects associated with the molecule itself or other molecules that directly or indirectly interacting with or regulating the molecule.

Functional: As used herein, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.

Inhibition: As used herein, the terms "inhibition," "inhibit" and "inhibiting" refer to processes or methods of decreasing or reducing activity and/or expression of a protein or a gene of interest. Typically, inhibiting a protein or a gene refers to reducing expression or a relevant activity of of the protein or gene by at least 10% or more, for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or more, or a decrease in expression or the relevant activity of greater than 1-fold, 2-fold, 3 -fold, 4-fold, 5 -fold, 10-fold, 50-fold, 100-fold or more as measured by one or more methods described herein or recognized in the art.

In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multicellular organism.

In vivo: As used herein, the term "in vivo" refers to events that occur within a multicellular organism such as a non-human animal.

Isolated: As used herein, the term "isolated" refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated. In some embodiments, isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, the term "isolated cell" refers to a cell not contained in a multicellular organism.

Modulator: As used herein, the term "modulator" refers to a compound that alters or elicits an activity. For example, the presence of a modulator may result in an increase or decrease in the magnitude of a certain activity compared to the magnitude of the activity in the absence of the modulator. In certain embodiments, a modulator is an inhibitor, which decreases the magnitude of one or more activities. In certain embodiments, an inhibitor completely prevents one or more biological activities. In certain embodiments, a modulator is an activator, which increases the magnitude of at least one activity. In certain embodiments the presence of a modulator results in a activity that does not occur in the absence of the modulator.

Nucleic acid: As used herein, the term "nucleic acid," in its broadest sense, refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g. nucleotides and/or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably. In some embodiments, "nucleic acid" encompasses RNA as well as single and/or double-stranded DNA and/or cDNA. Furthermore, the terms "nucleic acid," "DNA," "RNA," and/or similar terms include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. For example, the so-called "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. The term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and/or encode the same amino acid sequence. Nucleotide sequences that encode proteins and/or RNA may include introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated. The term "nucleic acid segment" is used herein to refer to a nucleic acid sequence that is a portion of a longer nucleic acid sequence. In many embodiments, a nucleic acid segment comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more residues. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., T- fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages). In some embodiments, the present invention may be specifically directed to "unmodified nucleic acids," meaning nucleic acids (e.g. polynucleotides and residues, including nucleotides and/or nucleosides) that have not been chemically modified in order to facilitate or achieve delivery.

Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a "protein" can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids.

Small molecule: In general, a "small molecule" is understood in the art to be an organic molecule that is less than about 5 kilodaltons (Kd) in size. In some embodiments, the small molecule is less than about 4 Kd, about 3 Kd, about 2 Kd, or about 1 Kd. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g/mol, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol. In some embodiments, small molecules are non-polymeric. In some embodiments, small molecules are not proteins, peptides, or amino acids. In some embodiments, small molecules are not nucleic acids or nucleotides. In some embodiments, small molecules are not saccharides or polysaccharides.

Subject: As used herein, the term "subject" or "patient" refers to any organism to which compositions in accordance with the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.). Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

Suffering from: An individual who is "suffering from" a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of the disease, disorder, and/or condition.

Susceptible to: An individual who is "susceptible to" a disease, disorder, and/or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition.

Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to any agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect.

Treating: As used herein, the term "treat," "treatment," or "treating" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease. Definitions of specific functional groups and chemical terms in connection with chemical compounds are described in more detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, the entire contents of which are incorporated herein by reference.

Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans -isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

Isomeric mixtures containing any of a variety of isomer ratios may be utilized in accordance with the present invention. For example, where only two isomers are combined, mixtures containing 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios are all contemplated by the present invention. Those of ordinary skill in the art will readily appreciate that analogous ratios are contemplated for more complex isomer mixtures.

If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.

One of ordinary skill in the art will appreciate that the synthetic methods, as described herein, utilize a variety of protecting groups. By the term "protecting group", as used herein, it is meant that a particular functional moiety, e.g., O, S, or N, is temporarily blocked so that a reaction can be carried out selectively at another reactive site in a multifunctional compound. In preferred embodiments, a protecting group reacts selectively in good yield to give a protected substrate that is stable to the projected reactions; the protecting group should be selectively removable in good yield by readily available, preferably non-toxic reagents that do not attack the other functional groups; the protecting group forms an easily separable derivative (more preferably without the generation of new stereogenic centers); and the protecting group has a minimum of additional functionality to avoid further sites of reaction. As detailed herein, oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized. Hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (/?-A0M), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4- methoxytetrahydrothiopyranyl S,S-dioxide, 1 -[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin- 4-yl (CTMP), l,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a- octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1 -ethoxyethyl, 1 -(2-chloroethoxy)ethyl, 1 -methyl- 1 -methoxy ethyl, 1 -methyl- 1 -benzyloxy ethyl, 1 -methyl- 1 -benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, /?-chlorophenyl, /?-methoxyphenyl, 2,4-dinitrophenyl, benzyl, /?-methoxybenzyl, 3,4-dimethoxybenzyl, o- nitrobenzyl, /?-nitrobenzyl, /?-halobenzyl, 2,6-dichlorobenzyl, /?-cyanobenzyl, /?-phenylbenzyl, 2- picolyl, 4-picolyl, 3-methyl-2-picolyl iV-oxido, diphenylmethyl, p,p '-dinitrobenzhydryl, 5- dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, /?-methoxyphenyldiphenylmethyl, di(/?-methoxyphenyl)phenylmethyl, tri(/?-methoxyphenyl)methyl, 4-(4 ' - bromophenacyloxyphenyl)diphenylmethyl, 4,4',4"-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4 ' ,4 " -tris(levulinoyloxyphenyl)methyl, 4,4 ' ,4 " -tris(benzoyloxyphenyl)methyl, 3-(imidazol- 1 - yl)bis(4 ' ,4 ' ' -dimethoxyphenyl)methyl, 1 , 1 -bis(4-methoxyphenyl)- 1 ' -pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, l,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t- butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-/?-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate,/?-chlorophenoxyacetate, 3- phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p- phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2- (trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2- (triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl /?-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p- methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl /?-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-l-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o- (dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4- (methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4- methylphenoxyacetate, 2,6-dichloro-4-( 1 , 1 ,3 , 3 -tetramethylbutyl)phenoxy acetate, 2,4-bis( 1,1- dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2- methyl-2-butenoate, o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'- tetramethylphosphorodiamidate, alkyl JV-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). For protecting 1,2- or 1,3-diols, the protecting groups include methylene acetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylidene ketal, (4- methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, /?-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1- methoxyethylidene ortho ester, 1-ethoxyethylidine ortho ester, 1 ,2-dimethoxyethylidene ortho ester, α-methoxybenzylidene ortho ester, l-(Λ/,Λ/-dimethylamino)ethylidene derivative, a-(N,N'- dimethylamino)benzylidene derivative, 2-oxacyclopentylidene ortho ester, di-t-butylsilylene group (DTBS), l,3-(l,l,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS), tetra-t- butoxydisiloxane-l,3-diylidene derivative (TBDS), cyclic carbonates, cyclic boronates, ethyl boronate, and phenyl boronate. Amino-protecting groups include methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7- dibromo)fluoroenylmethyl carbamate, 2,7-di-£-butyl-[9-( 10,10-dioxo- 10,10,10,10- tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2- phenylethyl carbamate (hZ), l-(l-adamantyl)-l-methylethyl carbamate (Adpoc), l,l-dimethyl-2- haloethyl carbamate, l,l-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), l,l-dimethyl-2,2,2- trichloroethyl carbamate (TCBOC), 1 -methyl- l-(4-biphenylyl)ethyl carbamate (Bpoc), l-(3,5-di- £-butylphenyl)-l-methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(/V,iV-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1- adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, TV-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p- methoxybenzyl carbamate (Moz), /?-nitobenzyl carbamate, /?-bromobenzyl carbamate, p- chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfϊnylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2- methylsulfonylethyl carbamate, 2-(/?-toluenesulfonyl)ethyl carbamate, [2-(l,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), l,l-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p- (dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6- chromonylmethyl carbamate (T croc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivative, N'-p-toluenesulfonylaminocarbonyl derivative, JV'-phenylaminothiocarbonyl derivative, t-amyl carbamate, 5^*-benzyl thiocarbamate, /?-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, /?-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl carbamate, o-(Λ/,Λ/-dimethylcarboxamido)benzyl carbamate, l,l-dimethyl-3-(/V,iV- dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p '-methoxyphenylazo)benzyl carbamate, 1- methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1 -methyl- 1 -eye lopropylmethyl carbamate, l-methyl-l-(3,5-dimethoxyphenyl)ethyl carbamate, 1 -methyl- \-(p- phenylazophenyl)ethyl carbamate, 1 -methyl- 1-phenylethyl carbamate, 1 -methyl- 1 -(4- pyridyl)ethyl carbamate, phenyl carbamate, /?-(phenylazo)benzyl carbamate, 2,4,6-tri-t- butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N- benzoylphenylalanyl derivative, benzamide,/?-phenylbenzamide, o-nitophenylacetamide, o- nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxycarbonylamino)acetamide, 3-(p- hydroxyphenyl)propanamide, 3 -(o-nitrophenyl)propanamide, 2-methyl-2-(o- nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4- chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N- 1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted l,3-dimethyl-l,3,5- triazacyclohexan-2-one, 5-substituted l,3-dibenzyl-l,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(I -isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5- dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4- methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7- dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (F cm), N-2-picolylamino N'- oxide, N-l,l-dimethylthiomethyleneamine, N-benzylideneamine, N-p- methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, NN'-isopropylidenediamine, N-/?- nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2- hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5 ,5 -dimethyl-3 -oxo- 1 - cyclohexenyl)amine, N-borane derivative, N-diphenylborinic acid derivative, N- [phenyl(pentacarbonylchromium- or tungsten)carbonyl] amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o- nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys), /?-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6- trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3 ,5 ,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β- trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'- dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.. Exemplary protecting groups are detailed herein, however, it will be appreciated that the present invention is not intended to be limited to these protecting groups; rather, a variety of additional equivalent protecting groups can be readily identified using the above criteria and utilized in the method of the present invention. Additionally, a variety of protecting groups are described in Protective Groups in Organic Synthesis, Third Ed. Greene, T.W. and Wuts, P.G., Eds., John Wiley & Sons, New York: 1999, the entire contents of which are hereby incorporated by reference.

It will be appreciated that the compounds, as described herein, may be substituted with any number of substituents or functional moieties. In general, the term "substituted" whether preceded by the term "optionally" or not, and substituents contained in formulas of this invention, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. Furthermore, this invention is not intended to be limited in any manner by the permissible substituents of organic compounds. Combinations of substituents and variables envisioned by this invention are preferably those that result in the formation of stable compounds useful in the treatment, for example, of infectious diseases or proliferative disorders. The term "stable", as used herein, preferably refers to compounds which possess stability sufficient to allow manufacture and which maintain the integrity of the compound for a sufficient period of time to be detected and preferably for a sufficient period of time to be useful for the purposes detailed herein.

The term "aliphatic", as used herein, includes both saturated and unsaturated, straight chain (i.e., unbranched), branched, acyclic, cyclic, or polycyclic aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, "aliphatic" is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term "alkyl" includes straight, branched and cyclic alkyl groups. An analogous convention applies to other generic terms such as "alkenyl", "alkynyl", and the like. Furthermore, as used herein, the terms "alkyl", "alkenyl", "alkynyl", and the like encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, "lower alkyl" is used to indicate those alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1- 6 carbon atoms.

In certain embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-4 carbon atoms. Illustrative aliphatic groups thus include, but are not limited to, for example, methyl, ethyl, n- propyl, isopropyl, cyclopropyl, -CH₂-cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl, tert- butyl, cyclobutyl, -CH₂-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl, - CH2-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, -CH2-cyclohexyl moieties and the like, which again, may bear one or more substituents. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

The term "alkoxy", or "thioalkyl" as used herein refers to an alkyl group, as previously defined, attached to the parent molecule through an oxygen atom or through a sulfur atom. In certain embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-20 alipahtic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-4 aliphatic carbon atoms. Examples of alkoxy, include but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy, and n- hexoxy. Examples of thioalkyl include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

The term "alkylamino" refers to a group having the structure -NHR', wherein R' is aliphatic, as defined herein. In certain embodiments, the aliphatic group contains 1-20 aliphatic carbon atoms. In certain other embodiments, the aliphatic group contains 1-10 aliphatic carbon atoms. In yet other embodiments, the aliphatic group employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the aliphatic group contains 1-6 aliphatic carbon atoms. In yet other embodiments, the aliphatic group contains 1-4 aliphatic carbon atoms. Examples of alkylamino groups include, but are not limited to, methylamino, ethylamino, n-propylamino, iso-propylamino, cyclopropylamino, n-butylamino, tert-butylamino, neopentylamino, n-pentylamino, hexylamino, cyclohexylamino, and the like.

The term "dialkylamino" refers to a group having the structure -NRR', wherein R and R are each an aliphatic group, as defined herein. R and R' may be the same or different in an dialkyamino moiety. In certain embodiments, the aliphatic groups contains 1-20 aliphatic carbon atoms. In certain other embodiments, the aliphatic groups contains 1-10 aliphatic carbon atoms. In yet other embodiments, the aliphatic groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the aliphatic groups contains 1-6 aliphatic carbon atoms. In yet other embodiments, the aliphatic groups contains 1-4 aliphatic carbon atoms. Examples of dialkylamino groups include, but are not limited to, dimethylamino, methyl ethylamino, diethylamino, methylpropylamino, di(n-propyl)amino, di(iso-propyl)amino, di(cyclopropyl)amino, di(n-butyl)amino, di(tert-butyl)amino, di(neopentyl)amino, di(n- pentyl)amino, di(hexyl)amino, di(cyclohexyl)amino, and the like. In certain embodiments, R and R' are linked to form a cyclic structure. The resulting cyclic structure may be aromatic or non-aromatic. Examples of cyclic diaminoalkyl groups include, but are not limited to, aziridinyl, pyrrolidinyl, piperidinyl, morpholinyl, pyrrolyl, imidazolyl, 1,3,4-trianolyl, and tetrazolyl.

Some examples of substituents of the above-described aliphatic (and other) moieties of compounds of the invention include, but are not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; -OH; -NO₂; -CN; -CF₃; -CH₂CF₃; -CHCl₂; - CH₂OH; -CH₂CH₂OH; -CH₂NH₂; -CH₂SO₂CH₃; -C(O)R_x; -CO₂(R_x); -CON(R_X)₂; -OC(O)R_x; - OCO₂R_x; -0C0N(R_x)₂; -N(R_X)₂; -S(O)₂R_x; -NR_x(CO)R_x wherein each occurrence of R_x independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein.

In general, the terms "aryl" and "heteroaryl", as used herein, refer to stable mono- or polycyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated moieties having preferably 3-14 carbon atoms, each of which may be substituted or unsubstituted. Substituents include, but are not limited to, any of the previously mentioned substitutents, i.e., the substituents recited for aliphatic moieties, or for other moieties as disclosed herein, resulting in the formation of a stable compound. In certain embodiments of the present invention, "aryl" refers to a mono- or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. In certain embodiments of the present invention, the term "heteroaryl", as used herein, refers to a cyclic aromatic radical having from five to ten ring atoms of which one ring atom is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.

It will be appreciated that aryl and heteroaryl groups can be unsubstituted or substituted, wherein substitution includes replacement of one, two, three, or more of the hydrogen atoms thereon independently with any one or more of the following moieties including, but not limited to: aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; -Cl; -Br; -I; - OH; -NO₂; -CN; -CF₃; -CH₂CF₃; -CHCl₂; -CH₂OH; -CH₂CH₂OH; -CH₂NH₂; -CH₂SO₂CH₃; - C(O)R_x; -CO₂(R_x); -CON(R_X)₂; -OC(O)R_x; -OCO₂R_x; -0C0N(R_x)₂; -N(R_X)₂; -S(O)₂R_x; - NR_x(CO)R_x, wherein each occurrence of R_x independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples that are described herein.

The term "cycloalkyl", as used herein, refers specifically to groups having three to seven, preferably three to ten carbon atoms. Suitable cycloalkyls include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the case of other aliphatic, heteroaliphatic, or hetercyclic moieties, may optionally be substituted with substituents including, but not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; -Cl; -Br; -I; -OH; -NO₂; -CN; -CF₃; -CH₂CF₃; -CHCl₂; - CH₂OH; -CH₂CH₂OH; -CH₂NH₂; -CH₂SO₂CH₃; -C(O)R_x; -CO₂(R_x); -C0N(R_x)₂; -OC(O)R_x; - OCO₂R_x; -0C0N(R_x)₂; -N(R_X)₂; -S(O)₂R_x; -NR_x(CO)R_x, wherein each occurrence of R_x independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples that are described herein.

The term "heteroaliphatic", as used herein, refers to aliphatic moieties that contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moieties may be branched, unbranched, cyclic or acyclic and include saturated and unsaturated heterocycles such as morpholino, pyrrolidinyl, etc. In certain embodiments, heteroaliphatic moieties are substituted by independent replacement of one or more of the hydrogen atoms thereon with one or more moieties including, but not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; -Cl; -Br; -I; -OH; -NO₂; - CN; -CF₃; -CH₂CF₃; -CHCl₂; -CH₂OH; -CH₂CH₂OH; -CH₂NH₂; -CH₂SO₂CH₃; -C(O)R_x; - CO₂(R_x); -CON(R_X)₂; -OC(O)R_x; -OCO₂R_x; -0C0N(R_x)₂; -N(R_X)₂; -S(O)₂R_x; -NR_x(CO)R_x, wherein each occurrence of R_x independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples that are described herein.

The terms "halo" and "halogen" as used herein refer to an atom selected from fluorine, chlorine, bromine, and iodine.

The term "haloalkyl" denotes an alkyl group, as defined above, having one, two, or three halogen atoms attached thereto and is exemplified by such groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.

The term "heterocycloalkyl" or "heterocycle", as used herein, refers to a non-aromatic 5-, 6-, or 7- membered ring or a polycyclic group, including, but not limited to a bi- or tri-cyclic group comprising fused six-membered rings having between one and three heteroatoms independently selected from oxygen, sulfur and nitrogen, wherein (i) each 5 -membered ring has O to 1 double bonds and each 6-membered ring has O to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally be oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocyclic rings may be fused to a benzene ring. Representative heterocycles include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl. In certain embodiments, a "substituted heterocycloalkyl or heterocycle" group is utilized and as used herein, refers to a heterocycloalkyl or heterocycle group, as defined above, substituted by the independent replacement of one, two or three of the hydrogen atoms thereon with but are not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; -Cl; -Br; -I; - OH; -NO₂; -CN; -CF₃; -CH₂CF₃; -CHCl₂; -CH₂OH; -CH₂CH₂OH; -CH₂NH₂; -CH₂SO₂CH₃; - C(O)R_x; -CO₂(R_x); -CON(R_X)₂; -OC(O)R_x; -OCO₂R_x; -0C0N(R_x)₂; -N(R_X)₂; -S(O)₂R_x; - NR_x(CO)R_x, wherein each occurrence of R_x independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples which are described herein.

The term "carbocycle," as used herein, refers to an aromatic or non-aromatic ring in which each atom of the ring is a carbon atom.

The term "independently selected" is used herein to indicate that the R groups can be identical or different.

As used herein, the term "labeled" is intended to mean that a compound has at least one element, isotope, or chemical compound attached to enable the detection of the compound. In general, labels typically fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes, including, but not limited to, ²H, ³H, ³²P, ³⁵S, ⁶⁷Ga, ^99mTc (Tc-99m), ¹¹¹In, ¹²³1, ¹²⁵ I, ¹⁶⁹Yb and ¹⁸⁶Re; b) immune labels, which may be antibodies or antigens, which may be bound to enzymes (such as horseradish peroxidase) that produce detectable agents; and c) colored, luminescent, phosphorescent, or fluorescent dyes. It will be appreciated that the labels may be incorporated into the compound at any position that does not interfere with the biological activity or characteristic of the compound that is being detected. In certain embodiments, hydrogen atoms in the compound are replaced with deuterium atoms (²H) to slow the degradation of a compound in vivo. In certain embodiments of the invention, photoaffϊnity labeling is utilized for the direct elucidation of intermolecular interactions in biological systems. A variety of known photophores can be employed, most relying on photoconversion of diazo compounds, azides, or diazirines to nitrenes or carbenes (See, Bayley, H., Photo generated Reagents in Biochemistry and Molecular Biology (1983), Elsevier, Amsterdam.), the entire contents of which are hereby incorporated by reference. In certain embodiments of the invention, the photoaffinity labels employed are o-, m- and p-azidobenzoyls, substituted with one or more halogen moieties, including, but not limited to 4-azido-2,3,5,6-tetrafluorobenzoic acid.

The term "unsaturated," as used herein, means that a moiety has one or more units of unsaturation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, among other things, methods and compositions for treating arthritis and other disorders, diseases and/or conditions, in particular, those associated with inflammation and/or autoimmunity. In some embodiments, the present invention provides methods and compositions for treating arthritis and other inflammatory or autoimmune diseases, disorders or conditions by administering secretory phospholipase A2 Group V (sPLA2-V) protein, or a variant thereof. In some embodiments, the present invention provides methods and compositions for treating arthritis and other inflammatory or autoimmune diseases, disorders or conditions by specifically inhibiting secretory phospholipase A2 Group HA (sPLA2-IIA). In some embodiments, inventive methods and compositions in accordance with the present invention do not inhibit sPLA2-V.

The present invention is, in part, based on the surprising discovery that sPLA2 isoform IIA and sPLA2 group V are distinct inflammatory mediators with opposing functions. As described in the Examples section below, the present inventors found that numerous sPLA2 iso forms, including sPLA2-V and sPLA2-IIA, are present in the joint fluid of patients with rheumatoid arthritis (RA). Surprisingly, despite the high level of homology between these two isoforms (i.e., about 40%), the inventors found that sPLA2-V and sPLA2-IIA have opposing activities in the pathogenesis of arthritis. The inventors found that sPLA2-IIA contributes to the pathophysiology of inflammatory arthritis. For example, the inventors observed that mice lacking sPLA2-IIA display substantial amelioration of clinical signs of arthritis compared to sPLA2-IIa sufficient wild-type mice (Example 2). Conversely, as described in Example 3, mice lacking sPLA2-V demonstrated substantially more severe autoantibody driven arthritic responses than mice expressing sPLA2-V. The inventors established that sPLA2-V-defϊcient mice treated with highly purified functional recombinant sPLA2-V were protected from developing symptoms of arthritis (see Example 4). The inventors further contemplate that sPLA2-V may modulate arthritis by stimulating phagocytic clearance of immune-complexes (see Example 5).

The apparent down modulating activity of GV sPLA2 in synovitis, evidenced by increased joint inflammation in GV null mice and confirmed by an ameliorating effect of systemically administered recombinant GV sPLA2, is a particularly unexpected and novel function for this isoform. Previous studies, focused predominantly on the enzymatic capacity for sPLA-GV to generate substrate arachidonic acid (AA) for pro-inflammatory eicosanoid synthesis have demonstrated that sPLA2-V is potent at releasing AA from the outer leaflet of healthy cells and can drive leukotriene synthesis when added to neutrophils ²³. Furthermore, bone marrow derived mast cells from sPLA2-V deficient mice have impaired eicosanoid synthesis ²⁴ and sPLA2-V deficient mice are less sensitive to airways hyperresponsiveness in an ovalbumin sentisation model ²⁵. In addition, LDL-receptor deficient mice lacking bone marrow-derived sPLA2-V have reduced atherosclerosis when compared to the sPLA2-V expressing counterparts when fed with high fat diet ²⁶. Thus, eicosanoid substrate generation was the predominant anticipated activity for sPLA-GV in inflammatory arthritis.

Based on these surprising discoveries, the present invention provides novel and inventive therapeutic methods for treating arthritis and other inflammatory and/or autoimmune diseases, disorders and conditions by administering anti-inflammatory sPLA2 isoenzyme (e.g., sPLA2-V or functional variants thereof), and/or by specifically inhibiting pro-inflammatory sPLA2 isoforms (e.g., sPLA2-IIA) and concurrently avoiding inhibition of anti-inflammatory sPLA2 isoforms or groups such as sPLA2-V.

Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of "or" means "and/or" unless stated otherwise. Structures ofsPLA2 isoenzymes

Typically, sPLA2s are disulfide rich, contain an N-terminal signal peptide and are secreted from their native tissues or from transfected mammalian cells. Several sPLA2s, including HA and V share a common three dimensional structure based on structural data and sequence alignment ⁹'²⁷'²⁸. Human sPLA2-IIA and sPLA-V share about 40% amino acid sequence identity ²¹.

It was observed that all sPLA2s share the common structural feature of a ~15 Angstrom deep active slot where a single phospholipid molecule binds to position the enzyme-susceptible ester next to the catalytic residues. See Lambeau et al. "Biochemistry and Physiology of Mammalian Secreted Phospholipases A2," Annu. Rev. Biochem. (2008) 77:495-520. The region of the protein's surface that surrounds the opening to the active site slot constitutes the interfacial binding surface that is in direct contact with the membrane when the enzyme transfers from the aqueous phase to the membrane surface.

Typically, the interfacial binding site and the enzymatically active site are distinct from one another. The sPLA2 protein could be bound via the interfacial binding surface to the membrane while maintaining an active site devoid of bound substrate ²⁹ .

The M type receptor is a well-known protein target of sPLA2 binding. The M type receptor has been shown to play a role in promoting inflammation in a mouse model of endotoxic shock. The M type receptor can counteract the enzymatic action of sPLA2s by inhibiting catalytic activity upon binding to the sPLA2 and internalizing and degrading sPLA2 ³⁰. In addition, inhibitors known to block sPLA2 activity are also found to be potent at inhibiting the interaction between sPLA2 and the M-type receptor ³¹. sPLA2-IIA is also involved in binding to vimentin, a protein which is partially exposed on the surface of apoptotic T cells. Specific motifs in the interfacial binding site are involved in the interaction with vimentin ³². These same motifs are also important for the sPLA2 binding to coagulation factor Xa ³³.

sPLA2-V

As used herein, the terms "sPLA2-V polypeptide," "sPLA2-V protein" and "sPLA2-V"

(used inter-changeably) encompass both naturally-occurring sPLA2-V sequences and sPLA2-V variants (which are further defined herein). An sPLA2-V polypeptide suitable for the invention may be isolated from a variety of sources, such as from human or non-human (e.g. , mouse) tissues, or prepared by recombinant or synthetic methods.

As used herein, a "naturally-occurring sPLA2-V" includes a polypeptide having the same amino acid sequence as a sPLA2-V polypeptide derived from nature sources. Such naturally- occurring sPLA2-V can be isolated from nature or can be produced by recombinant or synthetic means. The term "naturally-occurring sPLA2-V" also encompasses naturally-occurring truncated forms of the sPLA2-V proteins, naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants.

As non- limiting examples, the nucleotide sequence of human sPLA2 Group V is shown in Table 1. The amino acid sequence of human sPLA2 Group V is shown in Table 2.

TABLE 1

Human sPLA2 Group V (hPLA2-V) nucleotide sequence (GenBank Accession # NM 000929) (SEQ ID NO:!)

1 cagggttcta cccggctggg tccaggcaga agtttttcct ccccacctcc gggtttgtcc

61 tcatcatcgg tcactcccat tcacagcttt aagattctgg aggccaagaa tttgactccc

121 cccggatcca tggtctgtgg ataccaatgt tccgactgga gacggggagc ccgcgagacc

181 cgggtctcca gggtctgccc aaggaagttg ctcatgggag cagacctcta gagcaggatt

241 tgaggccagg ccaaagagaa ccccagagat gaaaggcctc ctcccactgg cttggttcct

301 ggcttgtagt gtgcctgctg tgcaaggagg cttgctggac ctaaaatcaa tgatcgagaa

361 ggtgacaggg aagaacgccc tgacaaacta cggcttctac ggctgttact gcggctgggg

421 cggccgagga acccccaagg atggcaccga ttggtgctgt tgggcgcatg accactgcta

481 tgggcggctg gaggagaagg gctgcaacat tcgcacacag tcctacaaat acagattcgc

541 gtggggcgtg gtcacctgcg agcccgggcc cttctgccat gtgaacctct gtgcctgtga

601 ccggaagctc gtctactgcc tcaagagaaa cctacggagc tacaacccac agtaccaata

661 ctttcccaac atcctctgct cctaggcctc cccagcgagc tcctcccaga ccaagacttt

721 tgttctgttt ttctacaaca cagagtactg actctgcctg gttcctgaga gaggctccta

781 agtcacagac ctcagtcttt ctcgaagctt ggcggacccc cagggccaca ctgtaccctc

841 cagcgagtcc caggagagtg actctggtca taggacttgg tagggtccca gggtccctag

901 gcctccactt ctgagggcag cccctctggt gccaagagct ctcctccaac tcagggttgg

961 ctgtgtctct tttcttctct gaagacagcg tcctggctcc agttggaaca ctttcctgag

1021 atgcacttac ttctcagctt ctgcgatcag attatcatca ccaccaccct ccagagaatt

1081 tttacgcaag aagagccaaa ttgactctct aaatctggtg tatgggtatt aaataaaatt

1141 cattctcaag gctaataaaa accacattgg cattttcctc tgctgtgggg gatcgctggt

1201 gcctctttct ctgccactgg ggcaataaac ccaaagatgt ctacattatc tccgaaacag

1261 aagggaagat tagtaaatgc agggttttct gggatgagct tcaggctttc tcttgggcta

1321 attttcttac accttggggt cctctccagt attgggtctc attcttcctc gatggggtca

1381 gggaaagata actggtgatt atgccagctt cagcttccag gccagagagg gtggcattca

1441 aatcccagtg ctggcttctt cagctgtgtg gtcttggacc cgttactgaa cctctttgac

1501 tttcagtctc tttgagaaat aaactgtctt gttccttgca atgtaaaatg agacttctaa

1561 agcccacctt gatgctgata tggagaatgc tgaggttcta ggatttcaca cagcaggaat

1621 ttttttttaa taggtgtcag ctgtggggtt tattttttac aaagtaagga cattaaaaaa 1681 accaacccgt ctatcaattc ataaaagaaa ggatgttctg ataccaagac tgaaagaaga

1741 aaggatgtat tccaaaacaa aggaacatcc ttccaagaaa ggacctatgg cttctttatt

1801 ccgacatacc ccaaaataac tgcatgataa ataggtctat atttaaaaag ctctagtgtc

1861 gaatgttttc aaaataaaat ttaattttat gagaaaaaaa aaaaaaaaaa a

TABLE 2

hPLA2-V polypeptide sequence (GenBank Accession # NP 000920)

MKGLLPLAWFLACSVP AVQGGLLDLKSMIEKVTGKNALTNYGFYGCYCGWGGRGTP KDGTDWCCWAHDHCYGRLEEKGCNIRTQSYKYRFAWGVVTCEPGPFCHVNLCACDR KLVYCLKRNLRSYNPQYQYFPNILCS (SEQ ID NO:2)

Human group V sPLA2, like other sPLA2s, has an interfacial binding site (IBS) that is structurally distinct from the catalytic site. The catalytic site is a slot on the protein where a single phospholipid substrate binds to undergo hydrolysis. Since naturally occurring phospholipids have virtually no solubility in water, sPLA2s need to bind to the membrane interface in order to gain access to their substrate. The surface of the protein that surrounds the opening to the catalytic site slot is the IBS. The IBS is composed of several amino acid residues, each of which makes relatively small contributions to the overall binding of the enzyme to the membrane surface. The catalytic site and IBS are independent sites in a functional sense. For example, the sPLA2 can be bound to the membrane interface via its IBS with or without a phospholipid molecule in its catalytic site.

The residues that may constitute the IBS of group V sPLA2 are shown above in the sequence of the protein as boldface residues.

An sPLA2-V polynucleotide sequence suitable for the invention includes a polynucleotide sequence provided in Table 1 , or a fragment thereof. The invention can also use mutant or variant sPLA2-V sequences whose bases may be changed from the corresponding base shown in Tables 1 while still encoding proteins that maintain the anti-inflammatory activities of sPLA2-V protein, or fragment of such nucleic acids. In some embodiments, up to 30% or more of the bases may be so changed (e.g., up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, or more bases may be changed). Thus, an sPLA2-V polynucleotide sequence suitable for the invention includes an sPLA2-V polynucleotide variant having 70-100%, including 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%, sequence identity to the polynucleotide sequence shown in Tables 1 (SEQ ID NO: 1). sPLA2-V polypeptides suitable for the invention includes a polypeptide sequence provided in Table 2 (SEQ ID NO:2), or fragments thereof. sPLA2-V polypeptides suitable for the invention also includes sPLA2-V mutant or variant proteins. Suitable sPLA2-V mutants or variants may contain residues that differ from the corresponding residues shown in Table 2, while still encoding a protein that maintains its anti-inflammatory activity, or fragments thereof. In some embodiments, up to 30% or more of the residues may be so changed (e.g., up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or more residues may be changed). Thus, sPLA2-V polypeptides suitable for the invention include polypeptides having an amino acid sequence at least 70%, including at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, identical to SEQ ID NO:2. In some embodiments, sPLA2-V variants suitable for the invention include a substantailly intact IBS (e.g., with no amino acid substitutions or with conservative substitutions of amino acids that costitute the IBS).

In some embodiments, sPLA2-V variants suitable for the invention are catalytically inactive while maintaining anti-inflammatory activity. Such catalytically inactive variants may contain mutations within the catalytically active site (e.g., residues 39 to 57) or the calcium- binding loop (e.g., residues 24 to 37) ¹⁷. For example, suitable catalytically inactive variants may contain an amino acid substitution, deletion or insert at a position corresponding to position 48, 30 or 49 in the human sPLA2-V protein. Exemplary substitutions at position 48 may include H48Q, D49K or G30S. These catalytically inactive variants are desirable because they may be safer drugs with reduced toxicity and/or side effects.

"Percent (%) nucleic acid sequence identity" with respect to the sPLA2-V sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the sPLA2-V sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Preferably, the WU-BLAST-2 software is used to determine amino acid sequence identity (Altschul et al., Methods in Enzymology. 266, 460-480 (1996); http://blast.wustl/edu/blast/README.html). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=l, overlap fraction=0.125, world threshold (T)=I 1. HSP score (S) and HSP S2 parameters are dynamic values and are established by the program itself, depending upon the composition of the particular sequence, however, the minimum values may be adjusted and are set as indicated above.

"Percent (%) amino acid sequence identity" with respect to the sPLA2-V sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the sPLA2-V sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Preferably, the WU- BLAST-2 software is used to determine amino acid sequence identity (Altschul et al., Methods in Enzvmology 266, 460-480 (1996); http://blast.wustl/edu/blast/README.html). WU-BLAST- 2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=l, overlap fraction=0.125, world threshold (T)=I 1. HSP score (S) and HSP S2 parameters are dynamic values and are established by the program itself, depending upon the composition of the particular sequence, however, the minimum values may be adjusted and are set as indicated above. sPLA2-V mutants or variants can be prepared by introducing appropriate nucleotide changes into the sPLA2-V DNA, or by synthesis of the desired sPLA2-V polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the sPLA2-V, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.

Variations in the sPLA2-V sequence or in various domains of the sPLA2-V polypeptides described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Pat. No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the sPLA2-V that results in a change in the amino acid sequence of the sPLA2-V as compared with a naturally-occurring sequence of sPLA2-V. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the sPLA2-V protein. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity in the in vitro assays known in the art or as described in the Examples below.

The variations can be made using methods known in the art such as oligonucleotide- mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al, Nucl. Acids Res., 13:4331 (1986); Zoller et al, Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the sPLA2-V variant DNA.

Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins, (W. H. Freeman & Co., N.Y.); Chothia, J. MoI. Biol, 150:1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used. As used herein, the terms "sPLA2-IIA polypeptide," "sPLA2-IIA protein" and "sPLA2- HA" (used inter-changeably) encompass both naturally-occurring sPLA2-IIA sequences and sPLA2-IIA variants (which are further defined herein).

As non- limiting examples, the nucleotide sequence of human sPLA2-IIA is shown in Table 3. The amino acid sequence of human sPL A-IIA is shown in Table 4.

TABLE 3

Human sPLA2 Isoform HA (hPLA2-IIA) nucleotide sequence (GenBank Accession # NM 000300) (SEQ ID NO:3)

1 gaaggaaaaa gagcaacaga tccagggagc attcacctgc cctgtctcca aacagccttg

61 tgcctcacct acccccaacc tcccagaggg agcagctatt taaggggagc aggagtgcag

121 aacaaacaag acggcctggg gatacaactc tggagtcctc tgagagagcc accaaggagg

181 agcaggggag cgacggccgg ggcagaagtt gagaccaccc agcagaggag ctaggccagt

241 ccatctgcat ttgtcaccca agaactctta ccatgaagac cctcctactg ttggcagtga

301 tcatgatctt tggcctactg caggcccatg ggaatttggt gaatttccac agaatgatca

361 agttgacgac aggaaaggaa gccgcactca gttatggctt ctacggctgc cactgtggcg

421 tgggtggcag aggatccccc aaggatgcaa cggatcgctg ctgtgtcact catgactgtt

481 gctacaaacg tctggagaaa cgtggatgtg gcaccaaatt tctgagctac aagtttagca

541 actcggggag cagaatcacc tgtgcaaaac aggactcctg cagaagtcaa ctgtgtgagt

601 gtgataaggc tgctgccacc tgttttgcta gaaacaagac gacctacaat aaaaagtacc

661 agtactattc caataaacac tgcagaggga gcacccctcg ttgctgagtc ccctcttccc

721 tggaaacctt ccacccagtg ctgaatttcc ctctctcata ccctccctcc ctaccctaac

781 caagttcctt ggccatgcag aaagcatccc tcacccatcc tagaggccag gcaggagccc

841 ttctataccc acccagaatg agacatccag cagatttcca gccttctact gctctcctcc

901 acctcaactc cgtgcttaac caaagaagct gtactccggg gggtctcttc tgaataaagc 961 aattagcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa

TABLE 4

hPLA2-IIA polypeptide sequence (GenBank Accession # NP 000291)

MKTLLLLAVIMIFGLLOAHGNLVNFHRMIKLTTGKEAALSYGFYGCHCGVGGRGSPK DATDRCCVTHDCCYKRLEKRGCGTKFLSYKFSNSGSRITCAKQDSCRSQLCECDKAAA TCFARNKTTYNKKYQYYSNKHCRGSTPRC (SEQ ID NO:4)

The first 20 amino acids may constitute the prepeptide (bold). H47 (bold) may be an important amino acid from the catalytic site. G29 and D48 (bold italic) may bind the calcium ion which is central for catalytic activity. Certain basic patches that may be involved in interacting with lipid vesicles are underlined.

An sPLA2-IIA polynucleotide sequence suitable for the invention includes a polynucleotide sequence provided in Table 3, or a fragment thereof. The invention can also use a mutant or variant sPLA2-IIA sequence whose bases may be changed from the corresponding base shown in Table 3 while still encoding a protein that maintains the activities and physiological functions of sPLA2-IIA proteins, or a fragment of such a nucleic acid. A sPLA2- IIA polynucleotide further includes a nucleic acid molecule whose sequences are complementary to the above-described sequences, including complementary nucleic acid fragments. The polynucleotides or nucleic acids suitable for the invention can have chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In some embodiments, up to 30% or more of the residues may be so changed (e.g., up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or more bases may be changed).

An sPLA2-IIA polynucleotide sequence suitable for the invention also includes an SPLA2-IIA polynucleotide variant having 70-100%, including 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%, sequence identity to the polynucleotide sequences shown in Tables 1 and 3 (SEQ ID NOs: 1 and 3, respectively). In particular, an sPLA2-IIA polynucleotide variant encodes a functional or active sPLA2-IIA protein as defined herein.

An sPLA2-IIA polypeptide suitable for the invention includes a polypeptide sequence provided in Table 4 (SEQ ID NO:4), or fragments thereof. An sPLA2-IIA polypeptide suitable for the invention also includes an sPLA2-IIA mutant or variant protein. A suitable sPLA2-IIA mutant or variant may contain residues that differ from the corresponding residues shown in Table 4, while still encoding a protein that maintains its biological activities and physiological functions, or a functional fragment thereof. In some embodiments, up to 30% or more of the residues may be so changed (e.g., up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or more residues may be changed). Thus, an sPLA2-IIA polypeptide suitable for the invention includes a polypeptide having an amino acid sequence at least 70%, including at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, identical to SEQ ID NO: 4.

Percent (%) nucleic acid sequence identity and Percent (%) amino acid sequence identity are determined as described in the sPLA2-V section.

Specific inhibitors ofsPLA2-IIA

Inhibitors of sPLA2-IIA suitable for the invention can be chemical compounds {e.g., small molecules), proteins or peptides, antibodies, co-crystals, nano-crystals, nucleic acids {e.g., DNAs, RNAs, DNA/RNA hybrids, siRNAs, shRNAs, miRNAs, ribozymes, aptamers, etc.), carbohydrates {e.g. mono-, di-, or poly-saccharides), lipids {e.g., phospholipids, triglycerides, steroids, etc.), natural products, any combination thereof. In some embodiments, inhibitors of sPLA2-IIA suitable for the invention are selective inhibitors of sPLA2-IIA. For example, Candidate agents can also be designed using computer-based rational drug design methods. Typically, a plurality of test agents {e.g., libraries of candidate agents) are tested in screening assays for potential modulators. In some embodiments, test agents are biodegradable and/or biocompatible.

Small molecules

Small molecule compounds suitable for the invention include those that can bind or inhibit sPLA2-IIA, in particular, those can selectively or specifically inhibit sPLA2-IIA. For example, small molecules particularly useful for the invention include those compounds that specifically inhibit sPLA2-IIA but not sPLA-V. In some embodiments, suitable small molecule compounds are indole-based compounds including substituted indoles, 6,7-benzoindoles, indolizines derived from LY315920 (the structure of LY315920 is described in Oslund et al. J. Med. Chem. Published on Web July 8, 2008, which is incorporated herein by reference) including, but not limited to, indolizine-1-glyoxylamides and 6,7-benzoindole-3-glyoxylamides, indolizine-1-acetamides, indolizine-1 -acetic acid hydrazides, indolizine-1-glyoxylamides, indolizine-3-acetamides, indolizine-3 -acetic acid hydraxides, and indolizine-3-glyoxylasides. In some embodiments, suitable small molecule compounds have a structure represented by the formula (I),

wherein

Ri is hydrogen, halogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, -0-(C₁-C₆ alkyl), -S-(Ci-C₆ alkyl);

R₂ is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; substituted or unsubstituted, branched or unbranched arylalkyl; substituted or unsubstituted, branched or unbranched heteroarylalkyl; -ORA; -C(=O)RA; -CO₂RA; -CN; -SCN; - SR_A; -SOR_A; -SO₂R_A; -NO₂; -N(RA)₂; -NHC(0)R_A; -OC(O)R_A; -OC(O)OR_A; or -C(RA)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, a Ci-C₆ alkyl moiety, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or hetero arylthio moiety;

Xi is O, S, or NR₃;

R₃ is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; substituted or unsubstituted, branched or unbranched arylalkyl; substituted or unsubstituted, branched or unbranched heteroarylalkyl; C(=0)R_A; or -C(R_A)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, a Ci-C₆ alkyl moiety, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

Each occurrence OfR₄ is independently hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; substituted or unsubstituted, branched or unbranched arylalkyl; substituted or unsubstituted, branched or unbranched heteroarylalkyl; - ORA; -C(=O)R_A; -CO₂RA; -CN; -SCN; -SR_A; -SOR_A; -SO₂R_A; -NO₂; -N(RA)₂; -NHC(O)RA; - OC(O)R_A; -OC(O)OR_A; or -C(R_A)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, a Ci-C₆ alkyl moiety, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety.

In some embodiments, suitable small molecule compounds are represented by the formula (II),

wherein

R₅ is hydrogen, halogen, Ci-C₆ alkyl, C₃-C₆ cycloalkyl, -0-(Ci-C₆ alkyl), -S-(Ci-C₆ alkyl);

R₆ is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; substituted or unsubstituted, branched or unbranched arylalkyl; substituted or unsubstituted, branched or unbranched heteroarylalkyl; or -C(R_A)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, a Ci-C₆ alkyl moiety, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

X₂ is O, S, or NR₃;

R₇ is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; substituted or unsubstituted, branched or unbranched arylalkyl; substituted or unsubstituted, branched or unbranched heteroarylalkyl; C(=O)R_A; or -C(R_A)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, a Ci-C₆ alkyl moiety, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; and

Each occurrence of Rg is independently hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; substituted or unsubstituted, branched or unbranched arylalkyl; substituted or unsubstituted, branched or unbranched heteroarylalkyl; - ORA; -C(=O)R_A; -CO₂RA; -CN; -SCN; -SR_A; -SOR_A; -SO₂RA; -NO₂; -N(RA)₂; -NHC(O)RA; - OC(O)RA; -OC(O)ORA; or -C(RA)₃; wherein each occurrence of RA is independently a hydrogen, a protecting group, a Ci-C₆ alkyl moiety, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety.

Exemplary small molecules suitable for the present invention are shown in Table 5. TABLE 5

In some embodiments, suitable sPLA2-IIA inhibitors also include derivatives or analogs of the small molecules described herein. In some embodiments, sPLA2-IIA inhibitors of the present invention include analogues or derivatives of the compounds as shown in Table 5. In some embodiments, derivatives or analogues of the compounds described herein are developed based on molecule modeling using methods described herein and known in the art, such as, pharmacophore modeling techniques. In some embodiments, sPLA2-IIA inhibitors of the present invention include derivatives or analogues that have a chemical structure substantially similar to any of the compounds described herein.

As used herein, two chemical structures are considered "substantially similar" if they share at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical linkage bonds (e.g., rotatable linkage bonds). In some embodiments, two chemical structures are considered "substantially similar" if they share at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical atom coordinates defining the structures, or equivalent structures having a root mean square of deviation less than about 5.0 A (e.g., less than about 4.5 A, less than about 4.0 A, less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, or less than about 1.0 A). In some embodiments, two chemical structures are considered "substantially similar" if they share at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical atom coordinates defining surface- accessible features (e.g., hydrogen bond donors and acceptors, charged/ionizable groups, and/or hydrophobic patches), or equivalent features having a root mean square of deviation less than about 5.0 A (e.g., less than about 4.5 A, less than about 4.0 A, less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, or less than about 1.0 A). As used herein, a derivative or an analog of a compound is not the compound itself.

Various non-specific sPLA2-IIA inhibitors have been reported. See, U.S. Patent Nos. 5654326, 5733923, 5919810, 6175021, 6433001, 6451839, 5919943, 6645976, the contents of all of which are hereby incorporated by references in their entireties. Based on the discovery by the inventors, these compounds can be screened for their specificity in inhibiting sPLA2-IIA. For example, experiments can be designed to determine if each of these known compounds selectively inhibit sPLA2-IIA but not sPLA2-V. Methods such as those described in Oslund et al. J. Med. Chem. Published on Web July 8, 2008, can be used to identify specific sPLA2-IIA inhibitors.

In addition, compound libraries synthesized de novo can be screened to identify novel compounds that have specific sPLA2-IIA inhibitory activity. In some embodiments, compounds can be synthesized using rational drug design techniques based on, for example, crystal structures of sPLA2 isoenzymes (e.g., sPLA2-IIA, sPLA2-V). The structure and physical properties of human sPLA2 are well know. See, Seilhamer et al. "Cloning and Recombinant Expression of Phospho lipase A2 Present in Rheumatoid Arthritic Synovial Fluid," J. Biol. Chem., Vol. 264, No. 10, Issue of April 5, pp. 5335-5338, 1989; and Kramer et al. "Structure and Properties of a Human Non-pancreatic Phospholipase A2 ," J. Biol. Chem., Vol. 264, No. 10, Issue of April 5, pp. 5768-5775, 1989; the disclosures of which are incorporated herein by reference.

In some embodiments, public libraries containing drugs (including FDA approved drugs) can be screened to identify existing compounds whose sPLA2-IIA specific inhibitory activities are previously unknown. In some embodiments, modified libraries containing derivatives or analogues of existing compounds can be synthesized using methods well known in the art and screened to identify novel or improved specific sPLA2-IIA inhibitors. Suitable small molecule compound libraries can be obtained from commercial vendors such as ChemBridge Libraries (www.chembridge.com), BIOMOL International, ASINEX, ChemDiv, ChemDB, ICCB- Longwood.

Antibody Therapy

Anti-sPLA2-IIA antibodies suitable for the invention include antibodies or fragments of antibodies that bind immunospecifically to any sPLA2-IIA epitopes. As used herein, the term "antibodies" is intended to include immunoglobulins and fragments thereof which are specifically reactive to the designated protein or peptide, or fragments thereof. Suitable antibodies include, but are not limited to, human antibodies, primatized antibodies, chimeric antibodies, bi-specific antibodies, humanized antibodies, conjugated antibodies {i.e., antibodies conjugated or fused to other proteins, radiolabels, cytotoxins), Simall Modular ImmunoPharmaceuticals ("SMIPs™ ), and antibody fragments. As used herein, the term "antibodies" also includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies {e.g. bi-specific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

As used herein, an "antibody fragment" includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; single domain antibodies; diabodies; triabodies; tetrabodies; linear antibodies; single-chain antibody molecules; and multi specific antibodies formed from antibody fragments. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. For example, antibody fragments include isolated fragments, "Fv" fragments, consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker ("ScFv proteins"), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.

Antibodies can be generated using methods well known in the art. For example, protocols for antibody production are described by Harlow and Lane, Antibodies: A Laboratory Manual, (1988). Typically, antibodies can be generated in mouse, rat, guinea pig, hamster, camel, llama, shark, or other appropriate host. Alternatively, antibodies may be made in chickens, producing IgY molecules (Schade et al, (1996) ALTEX 13(5):80-85). In some embodiments, antibodies suitable for the present invention are subhuman primate antibodies. For example, general techniques for raising therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al, international patent publication No. WO 91/11465 (1991), and in Losman et al, Int. J. Cancer 46: 310 (1990). In some embodiments, monoclonal antibodies may be prepared using hybridoma methods (Milstein and Cuello, (1983) Nature 305(5934):537-40.). In some embodiments, monoclonal antibodies may also be made by recombinant methods (U.S. Pat. No. 4,166,452, 1979).

In some embodiments, antibodies suitable for the invention may include humanized or human antibodies. Humanized forms of non-human antibodies are chimeric Igs, Ig chains or fragments (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of Abs) that contain minimal sequence derived from non-human Ig. Generally, a humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization is accomplished by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody (Riechmann et al , Nature 332(6162):323-7, 1988; Verhoeyen et al, Science. 239(4847): 1534-6, 1988.). Such "humanized" antibodies are chimeric Abs (U.S. Pat. No. 4,816,567, 1989), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In some embodiments, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent Abs. Humanized antibodies include human Igs (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit, having the desired specificity, affinity and capacity. In some instances, corresponding non-human residues replace Fv framework residues of the human Ig. Humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which most if not all of the CDR regions correspond to those of a non-human Ig and most if not all of the FR regions are those of a human Ig consensus sequence. The humanized antibody optimally also comprises at least a portion of an Ig constant region (Fc), typically that of a human Ig (Riechmann et ah, Nature 332(6162):323-7, 1988; Verhoeyen et al, Science. 239(4847): 1534-6, 1988.).

Human antibodies can also be produced using various techniques, including phage display libraries (Hoogenboom et al., Mol Immunol. (1991) 28(9):1027-37; Marks et al., J MoI Biol. (1991) 222(3):581-97) and the preparation of human monoclonal antibodies (Reisfeld and Sell, 1985, Cancer Surv. 4(l):271-90). Similarly, introducing human Ig genes into transgenic animals in which the endogenous Ig genes have been partially or completely inactivated can be exploited to synthesize human antibodies. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire (Fishwild et ah, High-avidity human IgG kappa monoclonal antibodies from a novel strain of minilocus transgenic mice, Nat Biotechnol. 1996 July; 14(7):845-51; Lonberg et ah, Antigen-specific human antibodies from mice comprising four distinct genetic modifications, Nature 1994 April 28;368(6474):856-9; Lonberg and Huszar, Human antibodies from transgenic mice, Int. Rev. Immunol. 1995;13(l):65-93; Marks et ah, By-passing immunization: building high affinity human antibodies by chain shuffling. Biotechnology (N Y). 1992 July; 10(7):779-83).

Aptamer therapy

Aptamers are macromolecules composed of nucleic acid (e.g., RNA, DNA) that bind tightly to a specific molecular target (e.g., an sPLA2-IIA protein, polypeptide or an epitope thereof). A particular aptamer may be described by a linear nucleotide sequence and is typically about 15-60 nucleotides in length. Without wishing to be bound by any theories, it is contemplated that the chain of nucleotides in an aptamer form intramolecular interactions that fold the molecule into a complex three-dimensional shape, and this three-dimensional shape allows the aptamer to bind tightly to the surface of its target molecule. Given the extraordinary diversity of molecular shapes that exist within the universe of all possible nucleotide sequences, aptamers may be obtained for a wide array of molecular targets, including proteins and small molecules. In addition to high specificity, aptamers have very high affinities for their targets (e.g., affinities in the picomolar to low nanomolar range for proteins). Aptamers are chemically stable and can be boiled or frozen without loss of activity. Because they are synthetic molecules, they are amenable to a variety of modifications, which can optimize their function for particular applications. For example, aptamers can be modified to dramatically reduce their sensitivity to degradation by enzymes in the blood for use in in vivo applications. In addition, aptamers can be modified to alter their biodistribution or plasma residence time.

Selection of aptamers that can bind sPLA2-IIA or a fragment thereof can be achieved through methods known in the art. For example, aptamers can be selected using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method (Tuerk, C, and Gold, L., Science 249:505-510 (1990)). In the SELEX method, a large library of nucleic acid molecules (e.g., 10¹⁵ different molecules) is produced and/or screened with the target molecule (e.g., an sPLA2-IIA protein or an sPLA2-IIA epitope). The target molecule is allowed to incubate with the library of nucleotide sequences for a period of time. Several methods, known in the art, can then be used to physically isolate the aptamer target molecules from the unbound molecules in the mixture, which can be discarded. The aptamers with the highest affinity for the target molecule can then be purified away from the target molecule and amplified enzymatically to produce a new library of molecules that is substantially enriched for aptamers that can bind the target molecule. The enriched library can then be used to initiate a new cycle of selection, partitioning, and amplification. After 5-15 cycles of this iterative selection, partitioning and amplification process, the library is reduced to a small number of aptamers that bind tightly to the target molecule. Individual molecules in the mixture can then be isolated, their nucleotide sequences determined, and their properties with respect to binding affinity and specificity measured and compared. Isolated aptamers can then be further refined to eliminate any nucleotides that do not contribute to target binding and/or aptamer structure, thereby producing aptamers truncated to their core binding domain. S ee Jayasena, S. D. Clin. Chem. 45:1628-1650 (1999) for review of aptamer technology; the entire teachings of which are incorporated herein by reference).

Antisense and Interfering RNA Therapy

Antisense molecules are RNA or single-stranded DNA molecules with nucleotide sequences complementary to a specified mRNA. When a laboratory-prepared antisense molecule is injected into cells containing the normal mRNA transcribed by a gene under study, the antisense molecule can base-pair with the mRNA, preventing translation of the mRNA into protein. The resulting double-stranded RNA or RNA/DNA is digested by enzymes that specifically attach to such molecules. Therefore, a depletion of the mRNA occurs, blocking the translation of the gene product so that antisense molecules find uses in medicine to block the production of deleterious proteins. Methods of producing and utilizing antisense RNA are well known to those of ordinary skill in the art (see, for example, C. Lichtenstein and W. Nellen (Editors), Antisense Technology: A Practical Approach, Oxford University Press (December, 1997); S. Agrawal and S. T. Crooke, Antisense Research and Application (Handbook of Experimental Pharmacology, Volume 131), Springer Verlag (April, 1998); I. Gibson, Antisense and Ribozyme Methodology: Laboratory Companion, Chapman & Hall (June, 1997); J. N. M. MoI and A. R. Van Der Krol, Antisense Nucleic Acids and Proteins, Marcel Dekker; B. Weiss, Antisense Oligonodeoxynucleotides and Antisense RNA Novel Pharmacological and Therapeutic Agents, CRC Press (June, 1997); Stanley et al., Antisense Research and Applications, CRC Press (June, 1993); C. A. Stein and A. M. Krieg, Applied Antisense Oligonucleotide Technology (April, 1998)).

Antisense molecules and ribozymes suitable for inhibiting sPLA2-IIA activity can be designed based on the sequences described above and known in the art. The antisense molecules and ribozymes may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding UGGT. Such DNA sequences maybe incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells, or tissues. RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2'O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept can be extended by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.

RNA interference (RNAi) is a mechanism of post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA), which is distinct from the antisense and ribozyme-based approaches described above. dsRNA molecules are believed to direct sequence-specific degradation of mRNA in cells of various lineages after first undergoing processing by an RNase Ill-like enzyme called DICER (Bernstein et al, Nature 409:363, 2001) into smaller dsRNA molecules comprised of two 21 nt strands, each of which has a 5' phosphate group and a 3' hydroxyl, and includes a 19 nt region precisely complementary with the other strand, so that there is a 19 nt duplex region flanked by 2 nt-3' overhangs. RNAi is thus mediated by short interfering RNAs (siRNA), which typically comprise a double-stranded region approximately 19 nucleotides in length typically with 1-2 nucleotide 3' overhangs on each strand, resulting in a total length typically of between approximately 21 and 23 nucleotides.

It will also be appreciated that siRNAs can have a range of lengths, e.g., the double- stranded portion can range from 15-29 nucleotides. It will also be appreciated that the siRNA can have a blunt end or a 3' overhang at either or both ends. If present, such 3' overhang is often from 1-5 nucleotides in length. siRNA has been shown to downregulate gene expression when transferred into mammalian cells by such methods as transfection, electroporation, or microinjection, or when expressed in cells via any of a variety of plasmid-based approaches. RNA interference using siRNA is reviewed in, e.g., Tuschl, T., Nat. BiotechnoL. 20:446-448, May 2002. See also Yu, J., et al., Proc. Natl. Acad. Sci.. 99(9), 6047-6052 (2002); Sui, G., et al., Proc. Nail. Acad. Sci.. 99(8), 5515-5520 (2002); Paddison, P., et al., Genes and Dev.. 16, 948-958 (2002); Brummelkamp, T. et al., Science, 296, 550-553 (2002); Miyagashi, M. and Taira, K., Nat. Biotech.. 20, 497-500 (2002); Paul, C, et al., Nat. Biotech.. 20, 505-508 (2002). Indeed, in vivo inhibition of specific gene expression by RNAi has been achieved in various organisms including mammals. For example, Song et al., Nature Medicine, 9:347-351 (2003) discloses that intravenous injection of Fas siRNA compounds into laboratory mice with autoimmune hepatitis specifically reduced Fas mRNA levels and expression of Fas protein in mouse liver cells. Several other approaches for delivery of siRNA into animals have also proved to be successful. See e.g., McCaffery et al., Nature, 418:38-39 (2002); Lewis et al., Nature Genetics, 32:107-108 (2002); and Xia et al., Nature Biotech., 20:1006-1010 (2002).

As described in these and other references, the siRNA may consist of two individual nucleic acid strands or of a single strand with a self-complementary region capable of forming a hairpin (stem- loop) structure. A number of variations in structure, length, number of mismatches, size of loop, identity of nucleotides in overhangs, etc., are consistent with effective siRNA-triggered gene silencing. While not wishing to be bound by any theory, it is thought that intracellular processing (e.g., by DICER) of a variety of different precursors results in production of siRNA capable of effectively mediating gene silencing. Generally it is desirable to target exons rather than introns, and it may also be particularly desirable to select sequences complementary to regions within the 3' portion of the target transcript. Generally it is preferred to select sequences that contain approximately equimolar ratio of the different nucleotides and to avoid stretches in which a single residue is repeated multiple times. siRNA may thus comprise RNA molecules typically having a double-stranded region approximately 19 nucleotides in length typically with 1-2 nucleotide 3' overhangs on each strand, resulting in a total length of between approximately 21 and 23 nucleotides. As used herein, siRNA also includes various RNA structures that may be processed in vivo to generate such molecules. Such structures include RNA strands containing two complementary elements that hybridize to one another to form a stem, a loop, and optionally an overhang, preferably a 3' overhang. Typically, the stem is approximately 19 bp long, the loop is about 1-20, preferably about 4-10, and more preferably about 6-8 nucleotides long and/or the overhang is typically about 1-20, and preferably about 2-15 nucleotides long. In certain embodiments of the invention the stem is minimally 19 nucleotides in length and may be up to approximately 29 nucleotides in length. Loops of 4 nucleotides or greater are less likely subject to steric constraints than are shorter loops and therefore may be preferred. The overhang may include a 5 ' phosphate and a 3 ' hydroxyl. The overhang may, but need not, comprise a plurality of U residues, e.g., between 1 and 5 U residues.

The siRNA compounds suitable for the present invention can be designed based on the sPLA2-IIA sequence described above and can be synthesized using conventional RNA synthesis methods. For example, they can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Various applicable methods for RNA synthesis are disclosed in, e.g., Usman et al., J. Am. Chem. Soα, 109:7845- 7854 (1987) and Scaringe et al., Nucleic Acids Res. 18:5433-5441 (1990). Custom siRNA synthesis services are available from commercial vendors such as Ambion (Austin, Tex., USA), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (Rockford, 111., USA), ChemGenes (Ashland, Mass., USA), Proligo (Hamburg, Germany), and Cruachem (Glasgow, UK).

Inventive siRNAs may be comprised entirely of natural RNA nucleotides, or may instead include one or more nucleotide analogs and/or modifications as mentioned above for antisense molecules. The siRNA structure may be stabilized, for example by including nucleotide analogs at one or more free strand ends in order to reduce digestion, e.g., by exonucleases. This may also be accomplished by the inclusion. Alternatively, siRNA molecules may be generated by in vitro transcription of DNA sequences encoding the relevant molecule. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7, T3, or SP6.

The siRNA compounds can also be various modified equivalents of the siRNA structures. As used herein, "modified equivalent" means a modified form of a particular siRNA compound having the same target-specificity (i.e., recognizing the same mRNA molecules that complement the unmodified particular siRNA compound). Thus, a modified equivalent of an unmodified siRNA compound can have modified ribonucleotides, that is, ribonucleotides that contain a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate (or phospodiester linkage). As is known in the art, an "unmodified ribonucleotide" has one of the bases adenine, cytosine, guanine, and uracil joined to the 1 ' carbon of beta-D-ribo-furanose.

Modified siRNA compounds contain modified backbones or non-natural internucleoside linkages, e.g., modified phosphorous-containing backbones and non-phosphorous backbones such as morpholino backbones; siloxane, sulfide, sulfoxide, sulfone, sulfonate, sulfonamide, and sulfamate backbones; formacetyl and thioformacetyl backbones; alkene-containing backbones; methyleneimino and methylenehydrazino backbones; amide backbones, and the like. siRNA may be generated by intracellular transcription of small RNA molecules, which may be followed by intracellular processing events. For example, intracellular transcription is achieved by cloning siRNA templates into RNA polymerase III transcription units, e.g., under control of a U6 or Hl promoter. In one approach, sense and antisense strands are transcribed from individual promoters, which may be on the same construct. The promoters may be in opposite orientation so that they drive transcription from a single template, or they may direct synthesis from different templates. In a second approach siRNAs are expressed as stem-loop structures. The siRNAs of the invention may be introduced into cells by any of a variety of methods. For instance, siRNAs or vectors encoding them can be introduced into cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of aft-recognized techniques for introducing foreign nucleic acid (e.g., DNA or RNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, injection, or electroporation.

Vectors that direct in vivo synthesis of siRNA constitutively or inducibly can be introduced into cell lines, cells, or tissues. In certain preferred embodiments of the invention, inventive vectors are gene therapy vectors (e.g., adenoviral vectors, adeno-associated viral vectors, retroviral or lentiviral vectors, or various nonviral gene therapy vectors) appropriate for the delivery of an siRNA-expressing construct to mammalian cells, most preferably human cells. Thus the present invention includes gene therapy approaches to the treatment of diseases or clinical conditions associated with inflammation in, for example, airway (e.g., airway hyperresponsiveness), digestive, pulmonary or reproductive tract.

The invention includes methods of treating a disease or clinical condition associated with arthritis in, for example, response to infalmmation by administering siRNA compositions comprising siRNA that targets sPLA2-IIA or an sPLA2-IIA receptor. The compositions may be administered parenterally, orally, inhalationally, etc.

Typically, siRNA compositions reduce the level of the target transcript and its encoded protein by at least 2-fold, preferably at least 4-fold, more preferably at least 10-fold or more. The ability of a candidate siRNA to reduce expression of the target transcript and/or its encoded protein may readily be tested using methods well known in the art including, but not limited to, Northern blots, RT-PCR, microarray analysis in the case of the transcript, and various immunological methods such as Western blot, ELISA, immunofluorescence, etc., in the case of the encoded protein. Efficacy may be tested in appropriate animal models or in human subjects. siRNA compounds may be administered to mammals by various methods through different routes. For example, they can be administered by intravenous injection. See Song et al., Nature Medicine, 9:347-351 (2003). They can also be delivered directly to a particular organ or tissue by any suitable localized administration methods. Several other approaches for delivery of siRNA into animals have also proved to be successful. See e.g., McCaffery et al., Nature, 418:38-39 (2002); Lewis et al., Nature Genetics. 32:107-108 (2002); and Xia et al., Nature Biotech., 20:1006-1010 (2002). Alternatively, they may be delivered encapsulated in liposomes, by iontophoresis, or by incorporation into other vehicles such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.

In addition, they may also be delivered by a gene therapy approach, e.g., using a DNA vector from which siRNA compounds in, e.g., small hairpin form (shRNA), can be transcribed directly. Numerous studies have demonstrated that while double-stranded siRNAs are very effective at mediating RNAi, short, single-stranded, hairpin-shaped RNAs can also mediate RNAi, presumably because they fold into intramolecular duplexes that are processed into double-stranded siRNAs by cellular enzymes. Sui et al., Proc Natl Acad Sci USA, 99:5515-5520 (2002); Yu et al., Proc Natl Acad Sci USA, 99:6047-6052 (2002); and Paul et al., Nature Biotech., 20:505-508 (2002)). This discovery has significant and far-reaching implications, since the production of such shRNAs can be readily achieved in vivo by transfecting cells or tissues with DNA vectors bearing short inverted repeats separated by a small number of (e.g., 3 to 9) nucleotides that direct the transcription of such small hairpin RNAs. Additionally, if mechanisms are included to direct the integration of the transcription cassette into the host cell genome, or to ensure the stability of the transcription vector, the RNAi caused by the encoded shRNAs, can be made stable and heritable. Not only have such techniques been used to "knock down" the expression of specific genes in mammalian cells, but they have now been successfully employed to knock down the expression of exogenously expressed transgenes, as well as endogenous genes in the brain and liver of living mice. See generally Hannon, Nature. 418:244- 251 (2002) and Shi, Trends Genet. 19:9-12 (2003); see also Xia et al, Nature Biotech.. 20:1006- 1010 (2002).

Additional siRNA compounds targeted at different sites of the nucleic acids encoding one or more interacting protein members of a protein complex identified in the present invention may also be designed and synthesized according to general guidelines provided herein and generally known to skilled artisans. See e.g., Elbashir, et al. (Nature 411 : 494-498 (2001). For example, guidelines have been compiled into "The siRNA User Guide" which is available at the website of The Rockefeller University, New York, N.Y.

Therapeutic Applications

The invention provides methods of treating various inflammatory and/or autoimmune diseases, disorders and conditions. In some embodiments, methods of the present invention include a step of administering sPLA-V, or variant thereof, to a subject in need thereof. In some embodiments, methods of the present invention include a step of administering sPLA-V, or variant thereof, to a subject in need thereof. In some embodiments, methods of the invention include a step of specifically inhibiting sPLA2-IIA but not sPLA2-V. In some embodiments, methods of the invention include a step of administering specific sPLA2-IIA inhibitors described herein.

For example, the present invention can be used to treat arthritis including, but not limited to, rheumatoid arthritis, spondylitic arthritis, psoriatic arthritis, osteoarthritis. In some embodiments, the present invention can be used to treat inflammatory diseases, disorders or conditions including, but not limited to, rheumatoid arthritis, inflammatory bowel disease, sepsis, septic shock, adult respiratory distress syndrome, pancreatitis, trauma-induced shock, asthma, bronchial asthma, allergic rhinitis, cystic fibrosis, stroke, acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, gout, spondylarthropathris, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, enterapathric spondylitis, Juvenile arthropathy or juvenile ankylosing spondylitis, Reactive arthropathy, infectious or post-infectious arthritis, gonoccocal arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, syphilitic arthritis, Lyme disease, arthritis associated with "vasculitic syndromes", polyarteritis nodosa, hypersensitivity vasculitis, Luegenec's granulomatosis, polymyalgin rheumatica, joint cell arteritis, calcium crystal deposition arthropathris, pseudo gout, non-articular rheumatism, bursitis, tenosynomitis, epicondylitis (tennis elbow), carpal tunnel syndrome, repetitive use injury (typing), miscellaneous forms of arthritis, neuropathic joint disease (charco and joint), hemarthrosis (hemarthrosic), Henoch-Schonlein Purpura, hypertrophic osteoarthropathy, multicentric reticulohistiocytosis, arthritis associated with certain diseases, surcoilosis, hemochromatosis, sickle cell disease and other hemoglobinopathries, hyperlipoproteineimia, hypogammaglobulinemia, hyperparathyroidism, acromegaly, familial Mediterranean fever, Behat's Disease, systemic lupus erythrematosis, and relapsing polychondritis, inflammatory conditions resulting from harmful stimuli, such as pathogens, damaged cells, or irritants, and related diseases or conditions.

In some embodiments, the invention provides methods of treating (e.g., alleviating, ameliorating, relieving, inhibiting, preventing, delaying onset of, reduce severity of, and/or reducing incidence of) one or more symptoms or features of a disease, disorder, and/or condition described herein. Exemplary symptoms or features include, but are not limited to, joint effusion, leukocytic infiltration of synovial fluid, tissue leukocytic infiltration, synovial hyperplasia, synovial pannus, bone erosion, cartilage depletion, pain, swelling, morning stiffness, fatigue, loss of joint range of motion, and combinations thereof.

Typically, sPLA-V or variants thereof, or sPLA2-IIA inhibitors in accordance with the invention are administered at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect. The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). In certain embodiments, the desired dosage may be delivered by a dosing regimen, in which unit doses are administered individually separated by periods of time. sPLA-V or variants thereof, or sPLA2-IIA inhibitors in accordance with the invention may be administered either alone or in combination with one or more other therapeutic agents. By "in combination with," it is not intended to imply that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the invention. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In some embodiments, the invention encompasses the delivery of pharmaceutical compositions in combination with agents that may improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.

It will further be appreciated that therapeutically active agents utilized in combination may be administered together in a single composition or administered separately in different compositions. In general, it is expected that agents utilized in combination with be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (e.g., co-administration of anti-inflammatory agents), or they may achieve different effects (e.g., control of any adverse effects). Anti-inflammatory agents suitable for the present invention include, but are not limited to, NSAID's (Cox-2 selective or non-selective), Methotrexate, Sulfasalazine, Hydroxychloroquine, Leflunomide, Minocycline, Azathioprine, Etanercept, Infliximab, Adalimumab, Rituximab, Abatacept, Anakinra, anti-TNF agents (e.g., pegylated anti-TNF agents), anti-IL-6 receptor antibodies or fragments thereof.

Pharmaceutical Compositions

The present invention further provides pharmaceutical compositions comprising therapeutically active ingredient in accordance with the invention (e.g., sPLA2-V or variants thereof, and/or sPLA2-IIA inhibitors), together with one or more pharmaceutically acceptable excipients. Such pharmaceutical compositions may optionally comprise one or more additional therapeutically-active substances .

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys.

Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.

A pharmaceutical composition in accordance with the invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21^st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.

In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifϊers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.

Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifϊers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents. In certain embodiments for parenteral administration, compositions are mixed with solubilizing agents such an Cremophor^®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S. P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the effect of an active ingredient, it is often desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing compositions with suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and/or fillers or extenders (e.g. starches, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g. glycerol), disintegrating agents (e.g. agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarding agents (e.g. paraffin), absorption accelerators (e.g. quaternary ammonium compounds), wetting agents (e.g. cetyl alcohol and glycerol monostearate), absorbents (e.g. kaolin and bentonite clay), and lubricants (e.g. talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents.

Solid compositions of a similar type may be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

Dosage forms for topical and/or transdermal administration of a composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. Generally, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable excipient and/or any needed preservatives and/or buffers as may be required. Additionally, the present invention contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms may be prepared, for example, by dissolving and/or dispensing the compound in the proper medium. Alternatively or additionally, the rate may be controlled by either providing a rate controlling membrane and/or by dispersing the compound in a polymer matrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Patents 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositions may be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in PCT publication WO 99/34850 and functional equivalents thereof. Jet injection devices which deliver liquid vaccines to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Jet injection devices are described, for example, in U.S. Patents 5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballistic powder/particle delivery devices which use compressed gas to accelerate vaccine in powder form through the outer layers of the skin to the dermis are suitable. Alternatively or additionally, conventional syringes may be used in the classical mantoux method of intradermal administration.

Formulations suitable for topical administration include, but are not limited to, liquid and/or semi liquid preparations such as liniments, lotions, oil in water and/or water in oil emulsions such as creams, ointments and/or pastes, and/or solutions and/or suspensions. Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 nm to about 7 nm or from about 1 nm to about 6 nm. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder and/or using a self propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nm and at least 95% of the particles by number have a diameter less than 7 nm. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nm and at least 90% of the particles by number have a diameter less than 6 nm. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having a boiling point of below 65 ⁰F at atmospheric pressure. Generally the propellant may constitute 50% to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1% to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non- ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).

Pharmaceutical compositions formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations may be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range from about 0.1 nm to about 200 nm.

The formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 μm to 500 μm. Such a formulation is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may, for example, 0.1% to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1/1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid excipient. Such drops may further comprise buffering agents, salts, and/or one or more other of the additional ingredients described herein. Other opthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are contemplated as being within the scope of this invention.

General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21^st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).

Administration sPLA2-V or variants thereof, sPLA2-IIA inhibitors, and/or pharmaceutical compositions thereof may be administered to a subject using any amount and any route of administration effective for treating a disease, disorder, and/or condition (e.g. , a disease, disorder, and/or condition relating to arthritis, autoimmune and/or inflammation). The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular composition, its mode of administration, its mode of activity, and the like. Compositions in accordance with the invention are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

Pharmaceutical compositions in accordance with the present invention may be administered by any route. In some embodiments, pharmaceutical compositions are administered by one or more of a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (e.g. by powders, ointments, creams, gels, lotions, and/or drops), mucosal, nasal, buccal, enteral, vitreal, intratumoral, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; as an oral spray, nasal spray, and/or aerosol, and/or through a portal vein catheter.

In general the most appropriate route of administration will depend upon a variety of factors including the nature of the active ingredient, the condition of the patient (e.g., whether the patient is able to tolerate particular routes of administration), etc. The invention encompasses the delivery of the pharmaceutical compositions by any appropriate route taking into consideration likely advances in the sciences of drug delivery.

Kits

The present invention also provides pharmaceutical packs or kits comprising one or more containers (e.g., vials, ampoules, test tubes, flasks, or bottles) containing one or more ingredients of the inventive pharmaceutical compositions, for example, allowing for the simultaneous or sequential administration of sPLA2-V or variants thereof, and/or sPLA2-IIA inhibitors. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use or sale for human administration. Different ingredients may be supplied in solid (e.g., lyophilized) or liquid form. Each ingredient will generally be suitable as aliquoted in its respective container or provided in a concentrated form. Kits may also include media for the reconstitution of lyophilized ingredients. The individual containers of the kit are preferably maintained in close confinement for commercial sale.

EXAMPLES

Example 1 : Diverse sPLA2 are detected in RA synovial fluids Human synovial fluid analysis

Human synovial fluids from normal and people having RA were obtained. For time- resolved fluorescence immunoassays of sPLA2s, 50 ul of synovial fluid were used for all assays except 5 ul was used for sPLA2-IIA. Assay buffer (50 mM Tris, pH 7.8, 0.9% NaCl, 0.02% Tween-20, 0.05% NaN3, filtered through a 0.45 micron membrane) was added to each well to bring the total volume to 100 ul. Samples were submitted to time -resolved fluorescence immunoassay as described previously ³⁵. For assay callibration, various amounts of each recombinant human sPLA2 (prepared as described, ³⁶ ) were added to assay buffer to generate a standard curve. Blanks were run that contained 100 ul assay buffer alone.

Results

Using recently developed time -resolved immunoassays ³⁵, we quantified the concentration all catalytically active human sPLA2 in these synovial fluid specimens (Figure 1). Interestingly, all sPLA2 iso forms could be detected, although typically in a subgroup of subjects for each isoform. Group HA sPLA2 levels were by far the most highly expressed, with an average expression level of 82 ± 9.7 ng/ml evident in synovial fluid from subjects with RA. Other isoforms with substantial expression included sPLA2-V (11 ± 3.7 ng/ml), -HD (14 ± 9.0 ng/ml) and -XII 31 ± 8.1 ng/ml).

To examine disease-associated differences in sPLA2 isoform expression, we also quantified sPLA2 levels in synovial fluid from healthy volunteers. Here, due to limitations in synovial fluid volumes obtained from healthy individuals, we confined our analyses to sPLA2- IIA and -V isoforms. These isoforms were selected based on their expression levels in RA synovial fluids and on their high degree of homology. While sPLA2-IIA is also detectable in healthy syonvial fluid, the levels are dramatically lower than observed in the RA samples (average 19 ± 3.3 ng/ml). Interestingly, in contrast to the ubiquitous expression of sPLA2-IIA, sPLA2-V was detectable in synovial fluid from only one of the healthy volunteers.

Example 2: sPLA2-IIA contributes to synovial inflammation Mice and Serum transfer protocol and arthritis scoring

We used 6-9 week old male mice for all of our studies. All procedures were approved by the Institutional Animal Care and Use Committee of the Dana-Farber Cancer Institute (Boston, MA). Arthritogenic K/BxN serum was transferred to recipient mice to induce arthritis as described previously³⁷'³⁸. Briefly, serum was administered intraperitoneally on experimental day 0 and 2. Ankle thickness was measured at the malleoli with the ankle in a fully flexed position, using spring-loaded dial calipes (Long Island Indicator Service, NY). The clinical index of arthritis was graded on a scale 0-12 as described previously ³⁷'³⁸. Results concerning mouse arthritis experiments are presented as mean ± SEM. The statistical significance for comparisons between groups was determined using two-way analysis variance, followed by Bonferroni correction using Prism software package 4.00 (GraphPAd Software, San Diego, CA). P values smaller than 0.05 were considered significant.

Results

Having observed expression of numerous sPLA2 isoforms in RA synovial fluid, we proceeded with investigation of sPLA2 isoform contributions to the pathophysiology of inflammatory arthritis. For these in vivo studies, we employed the K/BxN serum transfer model of autoimmune inflammatory arthritis. The progressive distal symmetric erosive polyarthritis observed in K/BxN T-cell receptor (TcR) transgenic mice is generated from an autoreactive transgenic TcR that recognizes a ubiquitous autoantigen, glucose-6-phosphate isomerase (GPI), presented by the major histocompatibility complex (MHC) class II A^g7 molecule ^37~41. These autoreactive T-cells drive high titer arthritogenic autoantibody production; arthritis can be induced in recipient mice by passive transfer of arthritogenic IgG autoantibodies ⁴². Numerous IgG driven effector phase mechanisms that contribute substantially to arthritis pathogenesis have been identified in this model including cellular lineages (neutrophils, mast cells, NK-T cells, others) and soluble mediators (IL- lβ, TNF, complement C5a/C5aR, FcRIII and eicosanoids (LTB4 and PGI2) ⁷'³⁷'^{43 51}. I_n this autoantibody driven model of arthritis, mice lacking sPLA2- IIA display substantial amelioration of clinical signs of arthritis when compared to sPLA2-IIA sufficient WT mice (Figure 2A, B).

Although most closely related by sequence homology, whether human sPLA2-IIA is the ortholog of murine sPLA2-IIA remains speculative. To confirm the pro-inflammatory contribution of sPLA-IIA to synovitis and to further define orthologous activity of the murine and human sPLA2-IIA genes, we assessed K/BxN arthritis severity in mice expressing the human sPLA2-IIA transgene. Because the C57BL/6 mouse strain contains a spontaneous (insertion) mutation in sPLA2-IIA that abrogates expression ⁵², we selected human sPLA2-IIA transgenic mice crossed onto this genetic background. Thus, the only sPLA2-IIA activity in these mice derives from the human transgene. Consistent with a substantial pro-inflammatory contribution from sPLA-IIA to autoimmune arthritis, human group HA sPLA2 transgenic mice display a significantly increased arthritic response to K/BxN serum transfer (Figure 2C).

Histological examination

For histomorphometric analysis, ankle tissues were fixed for 24hours in 4% paraformaldehyde in PBS and decalcified for 72 hours with modified Kristensen's solution. Tissues were then dehydrated , embedded in paraffin, sectioned at 5μm thickness and stained with hematoxylin and eosin. Histological scoring was performed as previously described ⁵³.

Results

Histomorphometric quantification of joint tissues confirms clinical measures of arthritis, with substantial decreases in leukocytic infiltration, synovial pannus, bone erosion and cartilage depletion evident in sPLA2-IIA null mice. Since K/BxN arthritis is reliant on the eicosanoid LTB4 ⁴³, whose synthesis is reliant on PLA2 activity to provide precursor arachidonic acid, and since neutrophil recruitment is a prominent activity of LTB4 in K/BxN arthritis, we note decreased synovial neutrophils in mice lacking sPLA2-IIA. We also see increased inflammatory cell populations, bone erosion and cartilage erosion in human group HA sPLA2 transgenic mice.

Example 3 : Anti-inflammatory activity of sPLA2-V in autoimmune arthritis Mice and Serum transfer protocol and arthritis scoring

We used 6-9 week old male mice for all of our studies. All procedures were approved by the Institutional Animal Care and Use Committee of the Dana-Farber Cancer Institute (Boston, MA). Arthritogenic K/BxN serum was transferred to recipient mice to induce arthritis as described previously³⁷'³⁸. Briefly, serum was administered intraperitoneally on experimental day 0 and 2. Ankle thickness was measured at the malleoli with the ankle in a fully flexed position, using spring-loaded dial calipes (Long Island Indicator Service, NY). The clinical index of arthritis was graded on a scale 0-12 as described previously ³⁷'³⁸. Results concerning mouse arthritis experiments are presented as mean ± SEM. The statistical significance for comparisons between groups was determined using two-way analysis variance, followed by Bonferroni correction using Prism software package 4.00 (GraphPAd Software, San Diego, CA). P values smaller than 0.05 were considered significant.

Results

Having observed a significant, albeit incomplete, amelioration of arthritis in sPLA2-IIA deficient mice, and having demonstrated substantial expression levels of other sPLA2 isoforms in arthritic synovial fluid, we investigated contributions from sPLA2-V and sPLA2-X to inflammatory arthritis. The rationale for focus on these isoforms was substantial structural and substrate homology to sPLA2-IIA in numerous in vitro studies ³⁶ . Examination of sPLA2-X null mice uncovered no demonstrable contribution of this isoform to arthritis severity, even when assessed with varied doses of arthritogenic K/BxN serum (Figure 3 and not shown). In contrast, to our surprise, mice lacking sPL2-V demonstrated substantially more severe autoantibody driven arthritic responses than congenic sPLA2-V sufficient control mice (Figures 3A, 3B).

Histological examination For histomorphometric analysis, ankle tissues were fixed for 24hours in 4% paraformaldehyde in PBS and decalcified for 72 hours with modified Kristensen's solution. Tissues were then dehydrated, embedded in paraffin, sectioned at 5μm thickness and stained with hematoxylin and eosin. Histological scoring was performed as previously described ⁵³.

Results

Histomorphometric analyses confirm clinical measurements with substantially increased leukocytic tissue infiltration, pannus formation and bone and cartilage destruction in sPLA2-V null mice (Figure 3B).

Example 4: Systemic administration of biotherapeutic recombinant sPLA2-V ameliorates arthritis

Recombinant sPLA2

Recombinant mouse and human group V sPLA2 were produced as previously described¹⁸'³⁶.

Results

To confirm the modulating role of sPLA2-V in antibody driven arthritis and to provide in vivo proof of concept for therapeutic use of sPLA2-V in inflammatory arthritis, we produced highly purified functional recombinant sPLA2-V in bacteria and administered parenterally to reconstitute activity in sPLA2-V null mice. Interestingly, sPLA2-V deficient mice treated with recombinant sPLA2-V were substantially protected from K/BxN arthritis (Figure 4 A, B).

Having confirmed the anti-inflammatory activity of sPLA2-V by replacement with biotherapeutic recominbinant protein in sPLA2-V null mice, we hypothesized that endogenous sPLA2-V activity was inadequate to confer protection after administration of high dose K/BxN serum. We thus we further explored the modulating activity of parenterally administered recombinant sPLA2-V in WT mice (sufficient for both sPLA2-IIA and -V). Interestingly, biotherapeutic administration of sPLA2-V resulted in dramatically reduced clinical and histomorphometric indices of arthritis relative to vehicle control in WT mice (Figure 4 C, D). Taken together, these results demonstrate a prominent modulating role for sPLA2-V in inflammatory arthritis. Example 5: sPLA2-V stimulates phagocytic clearance of immune-complexes

Erythrocyte phagocytosis assay

SRBC were opsonized with a subagglutinating concentration of rabbit IgG anti sheep erythrocyte (lOμg/ml). Determination of this concentration was obtained by titrating the IgG to find the maximal dose that does not induce visible agglutination of sRBC after incubation at 37°C. 5μl of SRBC (10% suspension, MP Biomedicals) were washed in Gelatin Veronal Buffer (GVB) by centrifugation at 4000 RPM, for 4 min at 4°C. SRBC were resuspended in ImI of GVB containing lOμg/ml of rabbit IgG anti sRBC and incubated for 20min at 37°C. After washing, sRBC were resuspended in 500μl of 10 mM Hepes-buffered RPMI. Opsonization was verified by immuno-fluorescence staining of IgG-opsonized sRBC (IgG-sRBC) with Cy-3 conjugated donkey anti-rabbit IgG. Alternatively, to generate C3-coated erythrocytes, SRBC were washed as for IgG opsonization, resuspendend in ImI of GVB containing a subagglutinating concentration of rabbit IgM and incubated for 20 min at 37°C. After washing, SRBC were resuspended in 1 ml of GVB containing 10% C5 -depleted human serum (Sigma) and incubated for 20 min at 37°C. During this incubation C3b binds IgM and is then converted to iC3b, the ligand of CR3 ⁵⁵.

Before stimulation macrophages were incubated for 15 min with RPMI containing 150 nM PMA without FBS, to activate CR3 on the surface of the macrophages. The medium was then discarded, and RPMI containing 10% FBS was used as the buffer for stimulation. 50μl of the final suspension (corresponding approximately to 10 sRBC/cell) were added to macrophages in a final volume of 500μl/well. The plate was centrifuged at 500 RPM for 2 min and incubated at 37°C. At the indicated time (5 min to Ih) glass cover-slips were taken off the plate and washed, and non-ingested red cells were lysed with 0.2% NaCl for 7sec followed by addition of 1.6% NaCl to restore physiological concentration (NaCl 0.9%). Cover slips were dried, fixed and stained with Diff-Quick. Phagosomes containing red cells were counted by optical microscopy. The phagocytic index was calculated by dividing the number of phagosomes by the total number of cells in a field, which was multiplied by the percent of phagocytosing cells, as previously described. Results

By what mechanisms does sPLA2-V modulate arthritis? Although numerous plausible sPLA2-V activities could contribute to its impact on inflammatory arthritis, based on recent studies demonstrating reduced zymosan phagocytosis in sPLA2-V deficient macrophages ⁵⁶, we hypothesized that sPLA2-V directed phagocytic uptake of immune complexes by sPLA2-V deficient macrophages could comprise a novel mechanism by which this isoform modulates arthritis activity. We thus monitored immune complex phagocytosis by macrophages isolated from mice sufficient or deficient in sPLA2-V. Indeed, we found sPLA2-V contributes to clearance of immune-complexes by murine macrophages in vitro (Figure 5 A, B). To assess a homologous activity for human sPLA2-V and to extend the relevance of our observations in a murine model to human autoimmune inflammatory arthritis, we exogenously added human sPLA2-V to human macrophages and monitored phagocytosis of human IgG-coated erythrocytes. Based on our murine observations, we expect human sPLA2-V can also trigger human macrophages phagocytosis of immune-complexes.

To demonstrate that sPLA2-V impacts metabolism of immune complexes in vivo, we measured systemic and joint tissue immune complexes as well as C3 fragments on synovial tissue of mice treated with K/BxN serum. Mice, V+ and V-, were injected with K/BxN serum until the inflammation plateau phase was reached (7 days). Cryostat sections from ankle joints were prepared with the tape-capture technique. Accumulated IgG and C3 complement were detected using Texas-red-conjugated anti-mouse IgG (red staining), and FITC-conjugated goat anti-mouse C3 (green staining). Nuclei were counterstained Hoeschst (blue). Fluorescence was detected by fluoresence microscopy. Images were acquired and processed digitally (Photoshop 6.0).

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EQUIVALENTS

The foregoing has been a description of certain non- limiting embodiments of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

In the claims articles such as "a,", "an" and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all, of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. In addition, the invention encompasses compositions made according to any of the methods for preparing compositions disclosed herein.

Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It is also noted that the term "comprising" is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, steps, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, steps, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. Thus for each embodiment of the invention that comprises one or more elements, features, steps, etc., the invention also provides embodiments that consist or consist essentially of those elements, features, steps, etc.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects are excluded are not set forth explicitly herein.

INCORPORATION OF REFERENCES

All publications and patent documents cited in this application are incorporated by reference in their entirety to the same extent as if the contents of each individual publication or patent document were incorporated herein.

Claims

What is claimed is:

1. A method of treating arthritis, the method comprising administering to a subject in need of treatment an effective amount of secretory phospho lipase A2 Group V (sPLA2-V) protein or a variant thereof, such that at least one symptom or feature of the arthritis is reduced in intensity, severity, or frequency, or has delayed onset.

2. The method of claim 1, wherein the arthritis is inflammatory arthritis.

3. The method of claim 1, wherein the arthritis is selected from the group consisting of rheumatoid arthritis, spondylitic arthritis, psoriatic arthritis, juvenile idiopathic arthritis, lupus, reactive arthritis, lyme arthritis, osteoarthritis and combinations thereof.

4. The method of claim 1, wherein the sPLA2-V or the variant thereof comprises an amino acid sequence at least 85% identical to human sPLA2-V.

5. The method of claim 4, wherein the sPLA2-V or the variant thereof comprises human sPLA2- V.

6. The method of claim 4, wherein the variant comprises a tryptophan residue in the sPLA2 interfacial binding site.

7. The method of claim 1, wherein the sPLA2-V or the variant thereof is a fragment of human SPLA2-V.

8. The method of claim 7, wherein the fragment of human sPLA2-V comprises the sPLA2 interfacial binding site.

9. The method of claim 1, wherein the variant of sPLA2-V lacks catalytic activity.

10. The method of claim 9, wherein the variant comprises an amino acid substitution at a position corresponding to position 48 in the human sPLA2-V protein.

11. The method of claim 9, wherein the variant comprises a H48Q substitution.

12. The method of claim 1 , wherein the variant is a mutant sPLA2-IIA protein, wherein the mutant sPLA2-IIA comprises tryptophan substitutions at positions corresponding to positions 3 and/or 31 in the human sPLA2-IIA protein.

13. The method of claim 1, wherein the at least one symptom or feature of the arthritis is selected from the group consisting of joint effusion, leukocytic infiltration of synovial fluid, tissue leukocytic infiltration, synovial hyperplasia, synovial pannus, bone erosion, cartilage depletion, pain, swelling, morning stiffness, fatigue, loss of joint range of motion, and combinations thereof.

14. The method of claim 1, wherein the method further comprises administering an antiinflammatory agent.

15. The method of claim 14, wherein the anti-inflammatory agent is selected from the group consisting ofNSAID's (Cox-2 selective or non-selective), Methotrexate, Sulfasalazine, Hydroxychloroquine, Leflunomide, Minocycline, Azathioprine, Etanercept, Infliximab, Adalimumab, Rituximab, Abatacept, Anakinra

16. The method of claim 14, wherein the anti-inflammatory agent is an anti-TNF agent.

17. The method of claim 14, wherein the anti-inflammatory agent is a pegylated anti-TNF agent.

18. The method of claim 14, wherein the anti-inflammatory agent is an anti-IL-6 receptor antibody or a fragment thereof.

19. The method of claim 1, wherein the method further comprises a step of inhibiting sPLA2- HA.

20. The method of claim 19, wherein the step of inhibiting sPLA2-IIA does not inhibit sPLA2- V.

21. The method of claim 19, wherein the step of inhibiting sPLA2-IIA comprises administering an interfering RNA.

22. The method of claim 20, wherein the interfering RNA is selected from siRNA, shRNA or miRNA.

23. The method of claim 19, wherein the step of inhibiting sPLA2-IIA comprises administering an antibody, or a fragment thereof, that specifically binds sPLA2-IIA.

24. The method of claim 23, wherein the antibody, or a fragment thereof, is selected from the group consisting of intact IgG, F(ab')2, F(ab)2, Fab', Fab, ScFvs, monoclonal antibodies, diabodies, triabodies, tetrabodies, single-domain antibodies and combinations thereof.

25. The method of claim 19, wherein the step of inhibiting sPLA2-IIA comprises administering a small molecule.

26. The method of claim 1, wherein the sPLA2-V is administered parenterally.

27. The method of claim 1, wherein the sPLA2-V is administered intra veneously.

28. The method of claim 1, wherein the effective amount ranges from 0.5 mg/kg to 10 mg/kg.

29. A method of treating inflammatory diseases or conditions, the method comprising administering to a subject in need of treatment an amount of sPLA2-V, or a variant thereof, effective to stimulate phagocytic clearance of immune-complexes.

30. The method of claim 29, wherein the inflammatory diseases or conditions are selected from the group consisting of rheumatoid arthritis, spondylitic arthritis, psoriatic arthritis, osteoarthritis.

31. A method of treating autoimmune diseases or conditions, the method comprising administering to a subject in need of treatment an effective amount of sPLA2-V or a variant thereof, such that at least one symptom or feature of the autoimmune diseases or conditions is reduced in intensity, severity, or frequency, or has delayed onset.

32. The method of claim 31, wherein the autoimmune diseases or conditions are selected from the group consisting of lupus, vasculitis, nephritis.

33. A method of treating arthritis comprising a step of specifically inhibiting sPLA2-IIA but not SPLA2-V.

34. The method of claim 33, wherein the step of specifically inhibiting sPLA2-IIA comprises inhibiting the catalytic activity of sPLA2-IIA.

35. The method of claim 33, wherein the step of specifically inhibiting sPLA2-IIA comprises inhibiting the binding activity of sPLA2-IIA to receptor M-type receptor.

36. The method of any one of claims 33, 34 and 35, wherein the step of inhibiting sPLA2-IIA comprises administering an interfering RNA specific for sPLA2-IIA.

37. The method of claim 36, wherein the interfering RNA is selected from siRNA, shRNA or miRNA.

38. The method of claim 37, wherein the interfering RNA is an siRNA molecule.

39. The method of any one of claims 33, 34 and 35, wherein the step of inhibiting sPLA2-IIA comprises administering an antibody, or a fragment thereof, that specifically binds the sPLA2- IIA protein, but not the sPLA2-V protein.

40. The method of claim 39, wherein the antibody, or a fragment thereof, specifically binds to the catalytic or receptor binding domain of sPLA2-IIA.

41. The method of claim 39, wherein the antibody, or a fragment thereof, is selected from the group consisting of intact IgG, F(ab')2, F(ab)2, Fab', Fab, ScFvs, monoclonal antibodies, diabodies, triabodies, tetrabodies, single-domain antibodies and combinations thereof.

42. The method of claim 41, wherein the antibody is a monoclonal antibody.

43. The method of claim 42, wherein the antibody is a humanized monoclonal antibody.

44. The method of claim 33, wherein the step of inhibiting sPLA2-IIA comprises administering an aptamer that specifically binds the sPLA2-IIA protein, but not the sPLA2-V protein.

45. The method of claim 33, wherein the step of inhibiting sPLA2-IIA comprises administering a small molecule that specifically inhibits sPLA2-IIA activity.

46. The method of claim 45, wherein the small molecule has a structure represented by formula I

wherein

Ri is hydrogen, halogen, Ci-C₆ alkyl, C₃-C₆ cycloalkyl, -0-(Ci-C₆ alkyl), -S-(Ci-C₆ alkyl);

R₂ is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; substituted or unsubstituted, branched or unbranched arylalkyl; substituted or unsubstituted, branched or unbranched heteroarylalkyl; -ORA; -C(=0)RA; -CO₂RA; -CN; -SCN; - SR_A; -SOR_A; -SO₂RA; -NO₂; -N(RA)₂; -NHC(O)RA; -OC(O)RA; -0C(0)0R_A; or -C(RA)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, a Ci-C₆ alkyl moiety, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or hetero arylthio moiety;

Xi is O, S, or NR₃;

R₃ is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; substituted or unsubstituted, branched or unbranched arylalkyl; substituted or unsubstituted, branched or unbranched heteroarylalkyl; C(=0)R_A; or -C(R_A)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, a Ci-C₆ alkyl moiety, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

Each occurrence OfR₄ is independently hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; substituted or unsubstituted, branched or unbranched arylalkyl; substituted or unsubstituted, branched or unbranched heteroarylalkyl; - ORA; -C(=O)R_A; -CO₂RA; -CN; -SCN; -SR_A; -SOR_A; -SO₂R_A; -NO₂; -N(RA)₂; -NHC(O)RA; - OC(O)R_A; -OC(O)OR_A; or -C(R_A)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, a Ci-C₆ alkyl moiety, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety.

47. The method of claim 45, wherein the small molecule has a structure represented by formula II

wherein

R₅ is hydrogen, halogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, -O-(C_rC₆ alkyl), -S-(Ci-C₆ alkyl);

R₆ is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; substituted or unsubstituted, branched or unbranched arylalkyl; substituted or unsubstituted, branched or unbranched heteroarylalkyl; or -C(R_A)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, a Ci-C₆ alkyl moiety, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

X₂ is O, S, Or NR₃;

R₇ is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; substituted or unsubstituted, branched or unbranched arylalkyl; substituted or unsubstituted, branched or unbranched heteroarylalkyl; C(=O)R_A; or -C(R_A)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, a Ci-C₆ alkyl moiety, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; and

Each occurrence of Rg is independently hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; substituted or unsubstituted, branched or unbranched arylalkyl; substituted or unsubstituted, branched or unbranched heteroarylalkyl; - OR_A; -C(=O)R_A; -CO₂R_A; -CN; -SCN; -SR_A; -SOR_A; -SO₂R_A; -NO₂; -N(R_A)₂; -NHC(0)R_A; - OC(O)RA; -OC(O)ORA; or -C(RA)₃; wherein each occurrence of RA is independently a hydrogen, a protecting group, a Ci-C₆ alkyl moiety, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety.

48. The method of claim 45, wherein the small molecule has a structure as shown in Table 5, or a derivative thereof.

49. The method of claim 33, wherein the method further comprises administering an antiinflammatory agent.

50. The method of claim 49, wherein the anti-inflammatory agent is selected from the group consisting ofNSAID's (Cox-2 selective or non-selective), Methotrexate, Sulfasalazine, Hydroxychloroquine, Leflunomide, Minocycline, Azathioprine, Etanercept, Infliximab, Adalimumab, Rituximab, Abatacept, Anakinra.

51. The method of claim 49, wherein the anti-inflammatory agent is an anti-TNF agent.

52. The method of claim 49, wherein the anti-inflammatory agent is a pegylated anti-TNF agent.

53. The method of claim 49, wherein the anti-inflammatory agent is an anti-IL-6 receptor antibody or a fragment thereof.

54. The method of claim 33, wherein the method further comprises administering sPLA2-V or a variant thereof.

55. The method of claim 54, wherein the sPLA2-V or the variant thereof comprises an amino acid sequence at least 85% identical to human sPLA2-V.

56. The method of claim 55, wherein the sPLA2-V or the variant thereof comprises human SPLA2-V.

57. The method of claim 33, wherein the arthritis is inflammatory arthritis.

58. The method of claim 33, wherein the arthritis is selected from the group consisting of rheumatoid arthritis, spondylitic arthritis, psoriatic arthritis, osteoarthritis and combinations thereof.

59. A pharmaceutical composition comprising an effective amount of sPLA2-V, or a variant thereof, wherein the pharmaceutical composition is formulated to treat arthritis, inflammatory diseases or conditions or autoimmune diseases.

60. The pharmaceutical composition of claim 59, wherein the sPLA2-V or the variant thereof comprises an amino acid sequence at least 85% identical to human sPLA2-V.

61. The pharmaceutical composition of claim 59, wherein the sPLA2-V or the variant thereof comprises human sPLA2-V or a fragment thereof.

62. The pharmaceutical composition of claim 59, wherein the pharmaceutical composition further comprises an anti-inflammatory agent.

63. The pharmaceutical composition of claim 62, wherein the anti-inflammatory agent is selected from the group consisting of NSAID's (Cox-2 selective or non-selective), Methotrexate, Sulfasalazine, Hydroxychloroquine, Leflunomide, Minocycline, Azathioprine, Etanercept, Infliximab, Adalimumab, Rituximab, Abatacept, Anakinra.

64. The pharmaceutical composition of claim 62, wherein the anti-inflammatory agent is an anti- TNF agent.

65. The pharmaceutical composition of claim 62, wherein the anti-inflammatory agent is a pegylated anti-TNF agent.

66. The pharmaceutical composition of claim 62, wherein the anti-inflammatory agent is an anti- IL-6 receptor antibody or a fragment thereof.