WO2010111240A2 - Methods for treating inflammatory arthritis - Google Patents

Abstract

Provided herein are, inter alia, methods of treating subjects suffering from inflammatory arthritis and identifying candidate compounds for treating inflammatory arthritis.

Description

METHODS FOR TREATING INFLAMMATORY ARTHRITIS

CLAIM OF PRIORITY

This application claims the benefit of U.S. Provisional Patent Application Serial No. 61/162,514, filed on March 23, 2009, and U.S. Provisional Patent Application Serial No. 61/299,681, filed on January 29, 2010. The entire contents of these prior applications are hereby incorporated by reference.

TECHNICAL FIELD

This invention relates to methods for treating inflammatory arthritis and methods for identifying compounds for treating inflammatory arthritis.

BACKGROUND Arthritis comprises more than 100 different rheumatic diseases and conditions that affect the joints, the tissues which surround the joint and other connective tissue. It is one of the most common causes of disability in the United States. Typically, arthritis is characterized by pain and stiffness in and around one or more joints. Some types of arthritis are inflammatory disorders. For example, rheumatoid arthritis (RA) is characterized by the inflammation of the synovial membrane of multiple joints.

Although the definitive causes are unknown, RA is believed to be the result of a faulty immune response.

SUMMARY

The present invention is based, at least in part, on the discovery that platelets play an important role in the pathophysiology of inflammatory arthritis by producing proinflammatory microparticle, and that the collagen receptor glycoprotein VI (GPVI) is a key mediator of platelet microparticle production. Accordingly, the present invention provides, inter alia, novel methods for treating inflammatory arthritis in a subject and methods for identifying candidate compounds for treating inflammatory arthritis in a subject.

In one aspect, provided herein are uses of a glycoprotein VI (GPVI) antagonist for the treatment of inflammatory arthritis in a subject. The GPVI antagonists can include antagonists known in the art (e.g., the GPVI antibodies) and antagonists identified by the screening methods described herein. Inflammatory arthritis can include rheumatoid arthritis, psoriatic arthritis, IBD-associated inflammatory arthritis, spondylitis, gouty arthritis, pseudogout (also called CPPD arthritis), juvenile idiopathic arthritis (JRA), Sjogren's arthritis, Lupus arthritis (also called Jaccoud's arthritis), virally induced arthritis, and Lyme arthritis.

In another aspect, the present invention includes uses of a glycoprotein VI (GPVI) antagonist for the preparation of a medicament for the treatment of inflammatory arthritis in a subject. In yet another aspect, described herein are methods for treating inflammatory arthritis in a subject, the method comprising: (a) identifying a subject suffering from inflammatory arthritis; and (b) administering to the subject a therapeutically effective amount of a GPVI antagonist.

The GPVI antagonist useful for the uses and methods provided herein include antibodies that bind specifically to a GPVI polypeptide. GPVI antagonists also include antagonists that, e.g., inhibit binding between a GPVI polypeptide and a GPVI agonist, inhibit GPVI signaling, inhibit expression of a GPVI polypeptide, inhibit expression of a procoagulant surface on platelets or platelet microparticles. The GPVI agonist can inhibit binding between GPVI and collagen, laminin, collagen-related peptide (CRP), and/or convulxin.

In another aspect, the present invention provides methods of identifying a candidate compound for treating inflammatory arthritis in a subject, the method comprising: (a) providing a sample comprising a GPVI polypeptide; (b) contacting the sample with a GPVI agonist and a test compound; and (c) detecting binding between the GPVI agonist and the GPVI polypeptide; wherein a decrease in the binding in the presence of the test compound as compared to a control indicates that the test compound is a candidate compound for treating inflammatory arthritis.

Cell or cell-free samples can be used in the screening methods provided herein. The sample can further comprise a Fc receptor γ-chain (FcR γ-chain). A test compound that decreases binding between GPVI and the ligand can be further tested for its ability to decreases GP VI -induced production of platelet microparticles. In one aspect, described herein are methods of identifying a candidate compound for treating inflammatory arthritis in a subject, the method comprising: (a) providing a platelet; (b) contacting the platelet with a GPVI agonist and a test compound; and (c) detecting production of platelet microparticles; wherein a decrease in the level of microparticles produced in the presence of the test compound as compared to a control indicates that the test compound is a candidate compound for treating inflammatory arthritis.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

Figures 1(A)-(C) are exemplary human glycoprotein VI (GPVI) amino acid and nucleic acid sequences. (A) An exemplary human GPVI amino acid sequence

(Accession No. NP 057447.4; SEQ ID NO:1). (B) The various functional domains within SEQ ID NO:1 are shown. (C) An exemplary human GPVI nucleic acid sequence (Accession No. NM 016363.4; SEQ ID NO:8).

Figures 2(A)-(E) are scatter and dot plots showing that platelet microparticles are abundant in inflammatory SF. Cells in freshly isolated RA SF were stained with lineage markers: CD 15 (neutrophils), CD3 (T cells), CD 14 (monocytes and macrophages) and CD41 and CD42b (platelets) or the appropriate isotype controls and analyzed by flow cytometry. (A) Forward by side-scatter profiles of events in RA SF. Populations identified by further gating and lineage marker staining are labeled. (B) Representative histogram of CD41+ (black fill) platelet MPs resident in RA SF. Events were gated based on the forward scatter parameters indicated in (A). (Grey fill=isotype control). Data are representative of profiles from eight RA patients. (C) Flow cytometric quantification of CD41+ platelet MPs (<lμm as determined by size calibration beads) in RA and OA SF after removal of leukocytes by centrifugation (n= 20 donors per group). (D) Flow cytometric quantification of platelet (CD41+) MPs in SF from juvenile idiopathic arthritis (JIA, n=6), psoriatic arthritis (PA, n=19) and gout (n=14). (E) Flow cytometric quantification of MPs in RA SF derived from indicated cell types (n=20 donors). Figures 3(A)-(F) are graphs showing data demonstrating that platelets are involved in arthritis development. (A) Arthritis severity after K/BxN serum transfer in mice administered a platelet-depleting antibody (triangles) or isotype control (squares). n=10 mice/group. Data are mean ± SEM pooled from two independent experiments. P < 0.001. Arrows = parenteral administration of platelet-depleting antibody; arrowheads = K/BxN serum administration. (B) Histomorphometric quantification of arthritis severity in ankle joints of platelet-depleted and control mice at experiment termination. n=10 mice/group. *P<0.01. (C-F) Arthritis severity was measured after administration of K/BxN serum in mice (C) deficient in thromboxane synthase (Tbxasl-/-), treated daily with (D) the thromboxane A2 (TP) antagonist SQ 29,548 or (E) ADP:P2Y12 inhibitor clopidogrel or in mice deficient in (F) GPIb (Gplba -/-). Data are mean ± SEM, n=10 mice/group. C, D, F; P = NS, E; PO.001. Figures 4 (A)-(F) are scatter plots, bar and line graphs and a reproduction of a scanning electron micrograph showing that platelets form MPs and participate in arthritis pathophysiology via stimulation of the collagen receptor GPVI. (A) Representative flow cytometry forward and side scatter plots of CD41+ mouse platelets incubated in presence or absence of FLS. (B) Examination of candidate stimuli of murine platelet MP formation upon co-culture with FLS. Mouse platelets incubated in presence of cyclooxygenase inhibitor salicylic acid (ASA), isolated from mice treated with ADP:P2Y12 inhibitor clopidogrel, or from the indicated gene targeted mice were co-incubated with mouse FLS. MP formation was quantified by flow cytometry. Data are mean ± SEM, n=3 independent experiments in duplicate. (C) Scanning electron micrograph of human platelets exhibiting MP budding when incubated in the presence of FLS. Arrows indicate the edge of the fibroblast-like synoviocyte. Upper and lower panels are 9800 and 69,270 X magnifications respectively. (D) Human platelets form MPs when incubated with FLS and when exposed to a GPVI specific peptide ligand (CRP) but not in the presence of a related control peptide (GPP). (E) Arthritis severity after K/BxN serum transfer was quantified in GPVI null (Gp6 -/-) (triangles) or wild-type control (squares) mice. Data are mean ± SEM pooled from three independent experiments. n=25 mice/group. P < 0.001. (F) Histomorphometric quantification of arthritis severity in ankle tissues from GPVI-null (Gp6 -/-) and WT mice at experimental termination. Data are mean ± SEM. n=25 mice/group. *P= 0.019, **P< 0.05, ***P<0.01.

Figures 5(A)-(G) are line and bar graphs representing data showing that MPs activate FLS in an IL-I dependent manner. (A) MPs generated by collagen stimulation of human platelets were co-incubated with human FLS and cytokine release was quantified by Proteome Profilertm. Data are representative of 3 independent experiments. (B) MPs isolated from RA SF were co-incubated with FLS and supernatants were assayed for IL-8 release by ELISA. (C) Mouse platelet MPs generated by collagen stimulation of platelets from indicated genotypes were co-incubated with mouse FLS and supernatants were assayed for KC release by ELISA. (D) Mouse MPs generated by collagen stimulation of WT platelets were co-incubated with IL-IRl null (Illrl -/-) FLS and supernatants were assayed for KC release by ELISA. Recombinant TNF (10 ng/ml) was added to FLS to induce KC release as a positive control. (E) Mouse platelet MPs were co-incubated with FLS in the presence of IL-I neutralizing antibodies and supernatants were assayed for KC release by ELISA. (F) Potency of human MP stimulation of FLS. FLS were exposed to graded concentrations of IL-I β, TNF or platelet MPs and IL-8 release was quantified in culture supernatants by ELISA. (G) Human platelet MPs were co-incubated with FLS in the presence of IL-I neutralizing antibodies and supernatants were assayed for IL-8 release by ELISA. Data for B to G are mean ± SEM of 3 independent experiments.

Figures 6(A)-(E) are scatter plots, graphs and immunostains showing that platelet microparticles, and not platelets, are detected in RA SF. (A) RA SF cells were stained with the platelet marker anti-CD41, the RBC marker antiglycophorin A or appropriate isotype controls and analyzed cytofluorometrically. n=S RA patients. (B) Cells in freshly isolated RA SF were stained with lineage markers: CD 15 (neutrophils), CD3 (T- lymphocytes), CD 14 (monocytes and macrophages) and CD41 and CD42b (platelets) or the appropriate isotype controls and analyzed by flow cytometry. Shown is a flow cytometry forward and side scatter plot of RA SF to which freshly isolated human platelets were added (left panel). For comparison, the non-'spiked' sample is provided in the right panel. Please note: the right panel is identical to data presented in Figure 2A. (C) CD41+ MP from RA SF, unstimulated platelets and platelet microparticles generated by stimulating platelets with collagen (as labeled) were stained for CD42b (black fill) or the appropriate isotype controls (grey fill) and examined cytofluorometrically. (D) Cytospin preparations of RA SF leukocytes either alone (left) or spiked with intact platelets from peripheral blood (right) were labeled with nuclear dye Draq5 (blue) and stained with the platelet marker anti-CD41 (red). Arrows on the left identify punctate CD41+ microparticles of <1 μm diameter found associated with leukocytes whereas arrows on the right identify intact platelets (2-4μm diameter) spiked into the cell population, one of which is outlined with a dotted line. Results are representative of 4 independent experiments. (E) RA and OA SF were stained with the platelet marker CD41 and the leukocyte marker CD45 or appropriate isotype controls and examined for co-expression of CD45 and CD41.

RA and 5 OA donors.

Figure 7 is a set of bar graphs showing cytokines and chemokines expression by MPs and MP-stimulated FLS. Human platelets were stimulated with collagen in vitro to generate MPs. FLS and MPs were cultured in DMEM for 5 hours at 37°C either alone or in combination. Supernatants were analyzed for presence of indicated cytokines and chemokines in the Proteome Profϊlertm. Results are mean values of pooled data from 3 independent experiments performed in duplicate.

Figure 8 is a bar graph showing RA SF MPs stimulate IL-6 release by FLS. MP (200 000 MP/ μl) isolated from RA SF were added to FLS for 5h and supernatants were assayed for IL-6 release using a sandwich ELISA. Data are mean ± SEM of 3 independent experiments using MP from 3 different donors.

Figure 9 is a dot graph showing that IL- lα is detected on the surface of platelet MPs from RA SF. CD41+ MPs in SF from RA patients (n=5) were analyzed by flow cytometry for the presence of surface bound IL- lα or IL - lβ. Figure 10 is a bar graph showing data demonstrating reduced platelet MP stimulation by FLS deficient in collagen type IV. FLS isolated firm mice deficient in collagen type I (α2 chain), collagen type IV (α5 chain) and their appropriate congenic WT controls were cultured to confluence in 6-well plates and co-incubated with WT platelets. MP generation was monitored by flow cytofluorometry as described under methods. Data are mean ± SEM of 3 independent experiments.

DETAILED DESCRIPTION

Platelets are highly abundant hematopoietic cells, outnumbering leukocytes in the peripheral circulation by almost two orders of magnitude (Gartner and Strum, Edition: 5, illustrated by Lippincott Williams & Wilkins, (2006)). The role of platelets in hemostasis and wound repair after vascular injury is well known (George, Lancet 355:1531 (2000)). Their role in inflammation has been studied in atherosclerosis, a chronic inflammatory disease of the blood vessels in which platelets release a broad range of inflammatory mediators that support endothelial cell activation, leukocyte adhesion and transmigration, monocyte maturation, and elaboration of cytokines and reactive oxygen species (for review, see Davi and Patrono, N EnglJ Med 357:2482 (2007). Data described herein demonstrate that platelets play an important role in the pathophysiology of inflammatory arthritis via proinflammatory microparticle production. Further, data described herein reveal that the collagen receptor glycoprotein VI (GPVI) is a key mediator of platelet microparticle production. Accordingly, the present invention provides, inter alia, novel methods for treating inflammatory arthritis in a subject and methods for identifying candidate compounds for treating inflammatory arthritis in a subject.

I. Inflammatory Arthritis

As used herein, the term "inflammatory arthritis" or "inflammatory joint disorder" refers to one of a number of disorders that involves inflammation in one or more joints in the patient. Examples of inflammatory arthritis include, but are not limited to rheumatoid arthritis, psoriatic arthritis, IBD-associated inflammatory arthritis, spondylitis, gouty arthritis, pseudogout (also called CPPD arthritis), juvenile idiopathic arthritis (JRA), Sjogren's arthritis, Lupus arthritis (also called Jaccoud's arthritis), virally induced arthritis, and Lyme arthritis. A diagnosis of inflammatory arthritis can be made, e.g., by a board certified Rheumatologist, based on knowledge in the art, for example, by applying ACR (American College of Rheumatology) clinical classification criteria.

II. Glycoprotein VI Glycoprotein VI (GPVI) is a 37-kD immunoglobulin superfamily member expressed exclusively by megakaryocytes and platelets. It is the major receptor responsible for platelet activation by collagen. GPVI includes two extracellular immunoglobulin (Ig)-like domains. These Ig-like domains are connected by a glycosylated stem of approximately 60 amino acids to a transmembrane domain, which is associated with a disulfide-linked Fc receptor (FcR)γ-chain homodimer. Glycosylation accounts for the apparent mass on electrophoresis of ~60kDa. The cytoplasmic domain of human GPVI includes binding sites for various signaling molecules. Upon binding of a ligand to GPVI, each (FcR)γ-chain is phosphorylated on two conserved tyrosines in the immunoreceptor, tyrosine -based activation motif (ITAM), leading to binding and activation of the tyrosine kinase Syk, and initiation of downstream signaling events. For a review of GPVI structure and signaling, see, e.g., Smethurst et al., Blood, 103(3); 903- 911 (2004); and Watson et al., Journal of Thrombosis and Haemostasis, 3: 1752-1762 (2005).

GPVI has been shown to recognize glycine-proline-hydroxyproline (GPO) repeat motifs in the triple helical structure of collagen (Morton et al Biochem J 306:337-334 (1995); Kehrel et al., Blood, 91 :491-499 (1998)). For example, a synthetic triple-helical collagen-related peptide (CRP), containing 10 GPO repeats, is a potent and specific GPVI agonist. A crystal structure of the collagen-binding domain of human GPVI (e.g., residues 21-203) has been determined (Horii et al., Blood, 108(3): 936-942 (2006)). In addition to collagen and CRP, GPVI ligands or agonists include laminin (Inoue et al Blood 107:1405-12 (2006)) and convulxin (Polgar et al, J Biol Chem, 272:13576-83 (1997)).

GPVI polypeptides or biologically active fragments thereof, and nucleic acids encoding full-length GPVI polypeptides or biologically active fragments thereof are useful for the methods described herein (e.g., treatment and screening methods). GPVI polypeptides and nucleic acids encoding them are readily obtained by one of ordinary skill in the art without undue experimentation. For example, SEQ ID NO:1 is an exemplary amino acid sequence of a full-length human GPVI polypeptide (Accession No. NP 057447.4; shown in Figure IA). Full-length GPVI nucleic acids include human GPVI nucleic acid sequence, such as SEQ ID NO: 2 (Accession No. NM 016363.4; shown in Figure IB). A biologically active or functional fragment of GPVI can be a fragment that includes, for example, the extracellular collagen-binding domain of GPVI. There are two common alleles of GPVI: the most abundant form A, having 0.85 frequency, and the second most abundant form, B, having 0.13 frequency (Joutsi- Korhonen et al, Blood, 101 :4372-4379 (2003)). The latter form is reported to have preserved collagen-binding activity, but lower intrinsic signalling activity through its impaired ability to bind src-family kinases in its cytoplasmic domain (Trifϊro et al.Blood, 114:1893-99 (2009).

The terms "protein" and "polypeptide" both refer to any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Thus, the terms "GPVI protein" and "GPVI polypeptide" include full- length naturally occurring isolated proteins, as well as recombinantly or synthetically produced polypeptides that correspond to the full-length naturally occurring proteins, or to a fragment of the full-length naturally occurring or synthetic polypeptide.

Fragments of a protein can be produced by any of a variety of methods known to those skilled in the art, e.g., recombinantly, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid that encodes the polypeptide. Expression of such mutagenized DNA can produce polypeptide fragments. Digestion with "end-nibbling" endonucleases can thus generate DNAs that encode an array of fragments. DNAs that encode fragments of a protein can also be generated, e.g., by random shearing, restriction digestion, chemical synthesis of oligonucleotides, amplification of DNA using the polymerase chain reaction, or a combination of the above-discussed methods. Fragments can also be chemically synthesized using techniques known in the art, e.g., conventional Merrifield solid phase FMOC or t-Boc chemistry. For example, peptides of the present invention can be arbitrarily divided into fragments of desired length with no overlap of the fragments, or divided into overlapping fragments of a desired length.

GPVI polypeptides useful in the methods and compositions described herein include, but are not limited to, recombinant polypeptides and natural polypeptides. Also included are nucleic acid sequences that encode forms of GPVI polypeptides in which naturally occurring amino acid sequences are altered or deleted. Certain nucleic acids of the present invention may encode polypeptides that are soluble under normal physiological conditions.

In some embodiments, a GPVI polypeptide useful in the present methods (e.g., in screening methods) is at least about 90%, 95%, 99%, or 100% identical to an amino acid sequence described herein (e.g., to a human sequence). In some embodiments, a nucleic acid encoding a GPVI useful in the present methods (e.g., in screening methods) is at least about 90%, 95%, 99%, or 100% identical to a nucleic acid sequence described herein (e.g., to a human sequence). The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In the present methods, the percent identity between two amino acid sequences can determined using the Needleman and Wunsch (J. MoI. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available on the world wide web at gcg.com), using the default parameters, e.g., a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

Also within the invention are nucleic acids encoding fusion proteins, and the fusion proteins themselves, in which a GPVI polypeptide is fused to an unrelated polypeptide, also referred to herein as a "heterologous polypeptide" or a "non-GPVI polypeptide" (e.g., a marker polypeptide or a fusion partner) to create a fusion protein. For example, the polypeptide can be fused to a hexa-histidine tag or a FLAG tag to facilitate purification of bacterially expressed polypeptides or to a hemagglutinin tag or a FLAG tag to facilitate purification of polypeptides expressed in eukaryotic cells. The invention also includes, for example, isolated fusion polypeptides (and the nucleic acids that encode these polypeptides) that include a first portion and a second portion, where the first portion includes, e.g., a GPVI polypeptide, and the second portion includes an immunoglobulin constant (Fc) region or a detectable marker (e.g., β-galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol acetyltransferase, beta- glucuronidase, exo-glucanase, and/or glucoamylase).

A nucleic acid encoding a mammalian, e.g., human, GPVI amino acid sequence can be amplified from human cDNA by conventional PCR techniques, using primers upstream and downstream of the coding sequence. GPVI polypeptides or fragments thereof can be produced and isolated using methods described herein or known in the art. The GPVI nucleic acids described herein include both RNA and DNA, including genomic DNA and synthetic (e.g., chemically synthesized) DNA. Nucleic acids can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be a sense strand or an antisense strand. Nucleic acids can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases. The term "isolated nucleic acid" means a DNA or RNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated GPVI nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequences that are immediately contiguous to the GPVI nucleic acid coding sequence. The term includes, for example, recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

The term "purified nucleic acid" or "purified polypeptide" refers to a nucleic acid or polypeptide that is substantially free of cellular or viral material with which it is naturally associated, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an isolated nucleic acid fragment is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state.

The compositions and methods described herein can also include the use of genetic constructs (e.g., vectors and plasmids) that include a GPVI nucleic acid described herein, operably linked to a transcription and/or translation sequence to enable expression, e.g., expression vectors. A selected nucleic acid, e.g., a DNA molecule encoding a GPVI polypeptide, is operably linked to another nucleic acid molecule, e.g., a promoter, when it is positioned either adjacent to the other molecule or in the same or other location such that the other molecule can direct transcription and/or translation of the selected nucleic acid. These genetic constructs are useful for, e.g., the therapeutic and treatment methods described herein or testing the activity of a GPVI polypeptide.

Various engineered cells, e.g., transformed host cells, which contain a GPVI nucleic acid described herein, can be useful for the methods (e.g., the screening methods) described herein. A transformed cell is a cell into which (or into an ancestor of which) has been introduced, by means of standard techniques, a nucleic acid encoding a GPVI polypeptide. Both prokaryotic and eukaryotic cells are included, e.g., mammalian cells (e.g., CHO cells), fungi (such as yeast), and bacteria (such as Escherichia coif), and the like.

III. Methods for Treating Inflammatory Arthritis

Data described herein demonstrate that activation of platelets via GPVI, resulting in platelet microparticle generation, plays an important role in the pathogenesis of inflammatory arthritis. Accordingly, provided herein are novel methods for treating inflammatory arthritis in a subject by administering a GPVI inhibitor or antagonist to the subject.

The term "patient" or "subject" is used throughout the specification to describe an animal, human or non-human, rodent or non-rodent, to whom treatment or diagnosis according to the methods of the present invention is provided. Veterinary and non- veterinary applications are contemplated. The term includes, but is not limited to, birds, reptiles, amphibians, and mammals, e.g., humans, other primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep and goats. Typical patients or subjects include humans, farm animals, and domestic pets such as cats and dogs.

The term "treat(ment)" is used herein to describe delaying the onset of, inhibiting, or alleviating the detrimental effects of a condition, e.g., inflammatory arthritis. The terms "effective amount" and "effective to treat," as used herein, refer to an amount or a concentration of a compound utilized for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome. Effective amounts of compound for use in the present treatment methods include, for example, amounts that, e.g., block binding between GPVI and a GPVI ligand or agonist, inhibit production of platelet mircoparticles, inhibit GPVI signaling, decrease joint inflammation, or generally improve the clinical condition, symptoms, quality of life, or prognosis of a patient diagnosed with inflammatory arthritis.

GPVI Antagonists

The methods described herein include the use of GPVI antagonists. GPVI antagonists can include agents or compounds (e.g., an antibody, a small molecular compound, and a peptide) that block a function of GPVI, for example, GPVFs ability to bind to one of its natural or synthetic ligands, to mediate downstream signaling events, to induce platelet aggregation, to express a procoagulant surface, and to mediate platelet microparticle production. GPVI antagonists useful in the methods described herein include direct GPVI antagonists that act directly on GPVI (e.g., an antibody that binds specifically to GPVI and a small molecular compound that targets GPVI directly) and indirect GPVI antagonist that act indirectly on GPVI (e.g., an agent that targets a signaling molecule downstream or upstream of GPVI). GPVI antagonists that target GPVI directly, for example, those that target the extracellular portion of GPVI, are particularly useful in the treatment methods described herein, as these antagonists are less likely to have undesirable side effects. This consideration reflects the restriction of GPVI to the platelet lineage. A number of GPVI antagonists are known in the art. For example, antibodies that bind specifically to and antagonize GPVI have been generated (Walker et al, Platelets 20(4):268-76 (2009); O'Connor et al, J Biol Chem 281 :33505 (2006); Smethurst et al, Blood 103(3):903 (2004); O'Connor et al., J Thromb Haemost 4:869 (2005); WO2003054020; WO2008049928; WO02080968; CA2567394; JP2007295926; U.S. Pat. Appl. Pub. Nos. US20070207155, US2007025992, US2008050380, and US2007207155; and U.S. Patents 7,645,592, 7,611,707, 7,597,888, 7,291,714, 7,101,549, 6,998,469, 6,989,144, 6,383,779, and 6,245,527).

In addition, there are numerous methods useful for identifying, designing, and assaying candidate GPVI antagonists (e.g., the methods described herein). For example, antibodies that bind specifically to GPVI can be generated by methods known in the art (e.g., the methods described herein). Those of ordinary skill in the art can also design screens for identifying compounds or agents that block the binding between GPVI and its ligand, e.g., collagen.

GPVI Antibodies As discussed above, antibodies that specifically bind to and antagonize GPVI can be used in the treatment methods provided herein. An antibody "specifically binds" to a particular antigen, e.g., a GPVI polypeptide, when it binds to that antigen, and binds to a significantly lesser extent (e.g., with significantly lower affinity or not at all) to other molecules in a sample, e.g., a biological sample that includes a GPVI polypeptide. The antibodies described herein include monoclonal antibodies, polyclonal antibodies, humanized or chimeric antibodies, monospecific antibodies, single chain antibodies, Fab fragments, F(ab')2 fragments, and molecules produced using a Fab expression library. As used herein, the term "antibody" refers to a protein comprising at least one, e.g., two, heavy (H) chain variable regions (abbreviated herein as VH), and at least one, e.g., two, light (L) chain variable regions (abbreviated herein as VL). The VH and VL regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" (FR). The extent of the framework region and CDR's has been precisely defined (see, Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia et al., J. MoI. Biol. 196:901-917 (1987)). Each VH and VL is composed of three CDR's and four FRs, arranged from amino- terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.

An anti-GPVI antibody can further include a heavy and light chain constant region, to thereby form a heavy and light immunoglobulin chain, respectively. The antibody can be a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are interconnected by, e.g., disulfide bonds. The heavy chain constant region is comprised of three domains, CHl, CH2, and CH3. The light chain constant region is comprised of one domain, CL. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system. A "GPVI binding fragment" of an antibody refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to a GPVI polypeptide or a portion thereof. Examples of GPVI polypeptide binding fragments of an anti-GPVI antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341 :544-546 (1989)), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., Science 242:423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such single chain antibodies are also encompassed within the term "GPVI binding fragment" of an antibody. These antibody fragments can be obtained using conventional techniques known to those with skill in the art.

To produce antibodies, GPVI polypeptides (or antigenic fragments (e.g., fragments of GPVI that appear likely to be antigenic by criteria such as high frequency of charged residues) or analogs of such polypeptides), e.g., those produced by standard recombinant or peptide synthetic techniques (see, e.g., Ausubel et al., supra), can be used. In general, the polypeptides can be coupled to a carrier protein, such as KLH, as described in Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N. Y.) at Unit 2.10, mixed with an adjuvant, and injected into a host mammal. A "carrier" is a substance that confers stability on, and/or aids or enhances the transport or immunogenicity of, an associated molecule. For example, nucleic acids encoding GPVI or fragments thereof can be generated using standard techniques of PCR, and can be cloned into a pGEX expression vector (Ausubel et al., supra). Fusion proteins can be expressed in E. coli and purified using a glutathione agarose affinity matrix as described in Ausubel, et al., supra.

Typically, to produce antibodies, various host animals are injected with GPVI polypeptides. Examples of suitable host animals include rabbits, mice, guinea pigs, rats, and fowl. Various adjuvants can be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete adjuvant), adjuvant mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such procedures result in the production of polyclonal antibodies, i.e., heterogeneous populations of antibody molecules derived from the sera of the immunized animals. Antibodies can be purified from blood obtained from the host animal, for example, by affinity chromatography methods in which the GPVI polypeptide antigen is immobilized on a resin.

Monoclonal antibodies (mAbs), which are homogeneous populations of antibodies to a particular antigen, can be prepared using GPVI polypeptides and standard hybridoma technology (see, e.g., Kohler et al., Nature, 256:495 (1975); Kohler et al., Eur. J. Immunol, 6:511 (1976); Kohler et al., Eur. J. Immunol., 6:292 (1976); Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY (1981); Ausubel et al., supra).

Typically, monoclonal antibodies are produced using any technique that provides for the production of antibody molecules by continuous cell lines in culture, such as those described in Kohler et al., Nature, 256:495 (1975), and U.S. Patent No. 4,376,110; the human B-cell hybridoma technique (Kosbor et al., Immunology Today, 4:72 (1983); Cole et al., Proc. Natl. Acad. Sci. USA, 80:2026, (1983)); and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, (1983)). Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. The hybridomas producing the mAbs of this invention can be cultivated in vitro or in vivo.

Once produced, polyclonal or monoclonal antibodies can be tested for recognition, e.g., specific recognition, of GPVI in an immunoassay, such as a Western blot or immunoprecipitation analysis using standard techniques, e.g., as described in Ausubel et al., supra. Antibodies that specifically bind to a GPVI polypeptide, or conservative variants thereof, are useful in the invention. For example, such antibodies can be used in an immunoassay to detect a GPVI polypeptide in a sample, e.g., plasma or serum.

Alternatively or in addition, an antibody can be produced recombinantly, e.g., produced by phage display or by combinatorial methods as described in, e.g., Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. Bio/Technology 9:1370-1372 (1991); Hay et al., Hum Antibod Hybridomas 3:81-85 (1992); Huse et al., Science 246:1275-1281 (1989); Griffths et al., EMBO J 12:725-734 (1993); Hawkins et al., J MoI Biol 226:889-896 (1992); Clackson et al., Nature 352:624- 628 (1991); Gram et al., PNAS 89:3576-3580 (1992); Garrad et al., Bio/Technology 9:1373-1377 (1991); Hoogenboom et al, Nuc Acid Res 19:4133-4137 (1991); and Barbas et al., PNAS 88:7978-7982 (1991).

Anti-GPVI antibodies can be fully human antibodies (e.g., an antibody made in a mouse which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or non-human antibodies, e.g., rodent (mouse or rat), goat, primate (e.g., monkey), camel, donkey, porcine, or fowl antibodies.

An anti-GPVI antibody can be one in which the variable region, or a portion thereof, e.g., the CDRs, are generated in a non-human organism, e.g., a rat or mouse. The anti-GPVI polypeptide antibody can also be, for example, chimeric, CDR-grafted, or humanized antibodies. The anti-GPVI polypeptide antibody can also be generated in a non-human organism, e.g., a rat or mouse, and then modified, e.g., in the variable framework or constant region, to decrease antigenicity in a human.

Techniques developed for the production of "chimeric antibodies" (Morrison et al., Proc. Natl. Acad. ScL, 81 :6851 (1984); Neuberger et al., Nature, 312:604 (1984); Takeda et al., Nature, 314:452 (1984)) can be used to splice the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.

Alternatively, techniques described for the production of single chain antibodies (see, e.g., U.S. Patents 4,946,778; 4,946,778; and 4,704,692) can be adapted to produce single chain antibodies against a GPVI polypeptide. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognize and bind to specific epitopes can be generated by known techniques. For example, such fragments can include but are not limited to F(ab')2 fragments, which can be produced by pepsin digestion of the antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of F(ab')2 fragments. Alternatively, Fab expression libraries can be constructed (Huse et al., Science, 246:1275 (1989)) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

Pharmaceutical Compositions and Methods of Administration Included herein are pharmaceutical compositions (e.g., comprising an antibody) for the treatment of inflammatory arthritis. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including epidermal and transdermal, and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, or intranasal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion. In addition, the pharmaceutical compositions of the present invention may be administered to one or more joints of a subject by intraarticular and/or periarticular injection.

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifϊers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes, which can be prepared according to methods known in the art. The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifϊers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like. Dosing is generally dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from one to several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual composition, and can generally be estimated based on EC50S found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 μg to 100 g per kg of body weight, e.g., 1-10 mg per kg and 50 mg per kg, and may be given once or more daily, weekly, monthly or yearly. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the composition is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, e.g., once or more daily or yearly.

In one embodiment, the present invention contemplates delivering a pharmaceutical composition within a medium comprising a microparticle. Preferably, microparticles comprise liposomes, nanoparticles, microspheres, nanospheres, microcapsules, and nanocapsules. Preferably, some microparticles contemplated by the present invention comprise poly(lactide-co-glycolide), aliphatic polyesters including, but not limited to, poly-glycolic acid and poly-lactic acid, hyaluronic acid, modified polysacchrides, chitosan, cellulose, dextran, polyurethanes, polyacrylic acids, psuedo- poly(amino acids), polyhydroxybutrate -related copolymers, polyanhydrides, polymethylmethacrylate, poly(ethylene oxide), lecithin and phospholipids.

In one embodiment, the present invention contemplates liposomes capable of attaching and releasing the compositions described herein. Liposomes are microscopic spherical lipid bilayers surrounding an aqueous core that are made from amphiphilic molecules such as phospholipids. Water soluble compounds can be entrapped in the core and lipid-soluble compounds can be dissolved in the shell-like bilayer. Liposomes have a special characteristic in that they enable water soluble and water insoluble chemicals to be used together in a medium without the use of surfactants or other emulsifϊers. For example, liposomes form spontaneously by forcefully mixing phosopholipids in aqueous media. Water soluble compounds are dissolved in an aqueous solution capable of hydrating phospholipids. Upon formation of the liposomes, therefore, these compounds are trapped within the aqueous liposomal center. The liposome wall, being a phospholipid membrane, holds fat soluble materials such as oils. Liposomes provide controlled release of incorporated compounds. In addition, liposomes can be coated with water soluble polymers, such as polyethylene glycol to increase the pharmacokinetic half- life. Ultra high-shear technology can be used to refine liposome production, resulting in stable, unilamellar (single layer) liposomes having specifically designed structural characteristics. These unique properties of liposomes, allow the simultaneous storage of normally immiscible compounds and the capability of their controlled release. Cationic and anionic liposomes, as well as liposomes having neutral lipids, can be used for comprising the compositions described herein. Preferably, cationic liposomes comprise negatively-charged materials by mixing the materials and fatty acid liposomal components and allowing them to charge-associate. The choice of a cationic or anionic liposome depends upon the desired pH of the final liposome mixture. Examples of cationic liposomes include LIPOFECTIN® and LIPOFECT AMINE.

A medium comprising liposomes that provide controlled release of compounds can be used in the methods described herein. Preferably, liposomes that are capable of controlled release: i) are biodegradable and non-toxic; ii) carry both water and oil soluble compounds; iii) solubilize recalcitrant compounds; iv) prevent compound oxidation; v) promote protein stabilization; vi) control hydration; vii) control compound release by variations in bilayer composition such as, but not limited to, fatty acid chain length, fatty acid lipid composition, relative amounts of saturated and unsaturated fatty acids, and physical configuration; viii) have solvent dependency; iv) have pH-dependency and v) have temperature dependency. The compositions of liposomes may be broadly categorized into two classifications. Conventional liposomes are generally mixtures of stabilized natural lecithin (PC) that may comprise synthetic identical-chain phospholipids that may or may not contain glycolipids.

Special liposomes may comprise: i) bipolar fatty acids; ii) the ability to attach antibodies for tissue-targeted therapies; iii) coated with materials such as, but not limited to lipoprotein and carbohydrate; iv) multiple encapsulation and v) emulsion compatibility.

Liposomes may be easily made in the laboratory by methods such as, but not limited to, sonication and vibration. Alternatively, compound-delivery liposomes are commercially available. For example, Collaborative Laboratories, Inc. East Setauket, NY) is known to manufacture custom designed liposomes for specific delivery requirements.

Microspheres and microcapsules are useful due to their ability to maintain a generally uniform distribution, provide stable controlled compound release and are economical to produce and dispense. Preferably, an associated delivery gel or the compound-impregnated gel is clear or, alternatively, said gel is colored for easy visualization by medical personnel. It is intended that the terms "microspheres, microcapsules and microparticles" (i.e., measured in terms of micrometers) are synonymous with their respective counterparts "nanospheres," "nanocapsules" and

"nanoparticles" (i.e., measured in terms of nanometers). Further, the terms "micro/nanosphere," "micro/nanocapsule" and "micro/nanoparticle" are used interchangeably, as discussed herein.

Microspheres may be obtainable commercially (e.g., PROLEASE^®, Alkermes

(Cambridge, MA)). For example, a freeze dried GPVI antagonist is homogenized in a suitable solvent and sprayed to manufacture microspheres in the range of 20 to 90 μm. Techniques are then followed that maintain sustained release integrity during phases of purification, encapsulation and storage. See Scott et al., Nature Biotechnology, Volume

16:153-157 (1998).

Modification of the microsphere composition by the use of biodegradable polymers can provide an ability to control the rate of release of, e.g., GPVI antagonists. Miller et al., J. Biomed. Mater. Res., 11:711-719 (1977). Alternatively, a sustained or controlled release microsphere preparation is prepared using an in- water drying method, where an organic solvent solution of a biodegradable polymer metal salt is first prepared. Subsequently, a dissolved or dispersed medium of, e.g., a GPVI antagonist, is added to the biodegradable polymer metal salt solution. The weight ratio of, e.g., a GPVI antagonist, to the biodegradable polymer metal salt may for example be about 1 : 100000 to about 1 :1, preferably about 1 :20000 to about 1 :500 and more preferably about 1 : 10000 to about 1 :500. Next, the organic solvent solution containing the biodegradable polymer metal salt and, e.g., a GPVI antagonist, is poured into an aqueous phase to prepare an oil/water emulsion. The solvent in the oil phase is then evaporated off to provide microspheres. Finally, these microspheres are then recovered, washed and lyophilized. Thereafter, the microspheres may be heated under reduced pressure to remove the residual water and organic solvent.

Other methods useful in producing microspheres that are compatible with a biodegradable polymer and, e.g., a GPVI antagonist, mixture are: i) phase separation during a gradual addition of a coacervating agent; ii) an in- water drying method or phase separation method, where an antiflocculant is added to prevent particle agglomeration and iii) by a spray-drying method.

The methods described herein can include the use of a medium comprising a microsphere or microcapsule capable of delivering a controlled release of a compound for a duration of approximately, e.g., between 1 day and 6 months. Controlled release microcapsules may be produced by using known encapsulation techniques such as centrifugal extrusion, pan coating and air suspension. Many techniques to produce such microspheres/microcapsules can be engineered to achieve particular release rates. For example, OLIOSPHERE^® (Macromed) is a controlled release microsphere system. These particular microsphere's are available in uniform sizes ranging between 5 - 500 μm and composed of biocompatible and biodegradable polymers. Specific polymer compositions of a microsphere control the drug release rate such that custom-designed microspheres are possible, including effective management of the burst effect. PROMAXX^® (Epic Therapeutics, Inc.) is a protein-matrix drug delivery system. The system is aqueous in nature and is adaptable to standard pharmaceutical drug delivery models. In particular, PROMAXX^® is a bioerodible protein microsphere that can deliver both small and macromolecular compounds, and may be customized regarding both microsphere size and desired release characteristics.

A microsphere or microparticle can comprise a pH sensitive encapsulation material that is stable at a pH less than the pH of the internal mesentery. The typical range in the internal mesentery is pH 7.6 to pH 7.2. Consequently, the microcapsules should be maintained at a pH of less than 7. However, if pH variability is expected, the pH sensitive material can be selected based on the different pH criteria needed for the dissolution of the microcapsules. The encapsulated compound, therefore, will be selected for the pH environment in which dissolution is desired and stored in a pH preselected to maintain stability. Examples of pH sensitive material useful as encapsulants are

EUDRAGIT^® L-IOO or S-IOO (Rohm GMBH), hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate phthalate, and cellulose acetate trimellitate. In one embodiment, lipids comprise the inner coating of the microcapsules. In these compositions, these lipids may be, but are not limited to, partial esters of fatty acids and hexitiol anhydrides, and edible fats such as triglycerides. See, e.g., Patent No. 5,364,634 (herein incorporated by reference).

A microparticle can comprise a gelatin, or other polymeric cation having a similar charge density to gelatin (i.e., poly-L-lysine) and is used as a complex to form a primary microparticle. A primary microparticle is produced as a mixture of the following composition: i) Gelatin (60 bloom, type A from porcine skin), ii) chondroitin 4-sulfate (0.005% - 0.1%), iii) glutaraldehyde (25%, grade 1), and iv) l-ethyl-3-(3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC hydrochloride), and ultra-pure sucrose (Sigma Chemical Co., St. Louis, Mo.). The source of gelatin is not thought to be critical; it can be from bovine, porcine, human, or other animal source. Typically, the polymeric cation is between 19,000-30,000 daltons. Chondroitin sulfate is then added to the complex with sodium sulfate, or ethanol as a coacervation agent.

Following the formation of a microparticle, a GPVI antagonist can be directly bound to the surface of the microparticle or is indirectly attached using a bridge or spacer. The amino groups of the gelatin lysine residues are easily derivatizable to provide sites for direct coupling of a compound. Alternatively, spacers (i.e., linking molecules and dramatizing moieties on targeting ligands) such as avidin-biotin are also useful to indirectly couple targeting ligands to the microparticles. Stability of the microparticle can be controlled by the amount of glutaraldehyde-spacer crosslinking induced by the EDC hydrochloride. A controlled release medium can also empirically determined by the final density of glutaraldehyde-spacer crosslinks.

IV. Screening Methods

Also provided herein are novel methods for identifying candidate compounds for treating inflammatory arthritis is a subject by screening for GPVI antagonists.

Screens

Screens for compounds that antagonize GPVI can be performed by identifying from a group of test compounds those that interfere with a function of GPVI, e.g., those that block GPVI ligand binding, inhibit platelet microparticle production, inhibit expression of a procoagulant surface on platelet or platelet microparticle, or inhibit GPVI signaling. Such compounds are candidate compounds for treating inflammatory arthritis. Assays disclosed herein can be carried out in whole cell preparations and/or in ex vivo cell-free systems. For example, cultured cells can be engineered to express full-length GPVI or a fragment of GPVI that retains ligand-binding activity, e.g., the extracellular domain of GPVI (see, e.g., Smethurst et al, Blood 103:903-11 (2004)). The cells can be contacted with a GPVI ligand or agonist (e.g., the synthetic ligand CRP) and a test compound. Methods known in the art (such as the methods described below) can then be used to detect binding between GPVI and the ligand. Test compounds that block binding between GPVI and the ligand are candidate compounds for treating inflammatory arthritis. A method useful for high throughput screening of compounds capable of modulating protein-protein interactions is described in Lepourcelet et al., Cancer Cell, 5: 91-102 (2004), which is incorporated herein by reference in its entirety. Typically, a first protein is provided, e.g., a GPVI polypeptide or a fragment thereof (for example, an extracellular fragment). A second protein is provided that is different from the first protein and which is optionally labeled. The second protein can be, e.g., a GPVI - specific ligand or agonist, e.g., collagen-related peptide (CRP). A test compound is provided. The first protein, second protein, and test compound are contacted with each other. The amount of the second protein bound to the first protein is then determined, e.g., by detecting the label bound to the second protein. A change in protein-protein interaction (e.g., binding) between the first protein and the second protein, e.g., as assessed by the amount of label bound, is indicative of the usefulness of the test compound in inhibiting protein-protein interactions between the first and second proteins. In some embodiments, the change is assessed relative to the same reaction without addition of the test compound. Those of ordinary skill in the art can readily design screens for assaying protein-protein binding in vitro and in vivo. In certain embodiments, the first or second protein is attached to a solid support.

Solid supports include, e.g., resins such as agarose, beads, and multiwell plates. In certain embodiments, the method includes a washing step after the contacting step, so as to separate bound and unbound label.

The solid support to which the first or second protein is attached can be, e.g., SEPHAROSE™ beads, scintillation proximity assay (SPA) beads (microspheres that incorporate a scintillant) or a multiwell plate. SPA beads can be used when the assay is performed without a washing step, e.g., in a scintillation proximity assay. SEPHAROSE™ beads can be used when the assay is performed with a washing step. The second protein can be labeled with any label that will allow its detection, e.g., a radiolabel, a fluorescent agent, biotin, a peptide tag, or an enzyme fragment. The second protein can also be radiolabeled, e.g., with ¹²⁵I or ³H.

In certain embodiments, the enzymatic activity of an enzyme chemically conjugated to, or expressed as a fusion protein with, the first or second protein, can be used to detect bound protein. A binding assay in which a known immunological method is used to detect bound protein is also included.

In certain other embodiments, the interaction of a first protein and a second protein is detected by fluorescence resonance energy transfer (FRET) between a donor fluorophore covalently linked to a first protein (e.g., a fluorescent group chemically conjugated to a peptide disclosed herein, or a variant of green fluorescent protein (GFP) expressed as a GFP chimeric protein linked to a peptide disclosed herein) and an acceptor fluorophore covalently linked to a second protein, where there is suitable overlap of the donor emission spectrum and the acceptor excitation spectrum to give efficient nonradiative energy transfer when the fluorophores are brought into close proximity through the protein-protein interaction of the first and second protein. Alternatively, both the donor and acceptor fluorophore can be conjugated at each end of the same peptide. The free peptide has high FRET efficiency due to intramolecular FRET between donor and acceptor sites causing quenching of fluorescence intensity. Upon binding, the intramolecular FRET of the peptide-dye conjugate decreases, and the donor signal increases. In another embodiment, fluorescence polarization (FP) is used to monitor the interaction between two proteins. For example, a fluorescently labeled peptide will rotate at a fast rate and exhibit low fluorescence polarization. When bound to a protein, the complex rotates more slowly, and fluorescence polarization increases.

In other embodiments, the protein-protein interaction is detected by reconstituting domains of an enzyme, e.g., beta-galactosidase (see Rossi et al., Proc. Natl. Acad. Sci. USA, 94:8405-8410 (1997)). In still other embodiments, the protein-protein interaction is assessed by fluorescence ratio imaging (Bacskai et al., Science, 260:222-226 (1993)) of suitable chimeric constructs of a first and second protein, or by variants of the two-hybrid assay (Fearon et al, Proc. Nat'l. Acad. Sci USA, 89:7958-7962 (1992); Takacs et al, Proc. Natl. Acad. Sci. USA, 90:10375-10379 (1993); Vidal et al, Proc. Nat'l. Acad. Sci. USA, 93: 10315-10320 (1996); Vidal et al, Proc. Nat'l Acad. Sci USA, 93: 10321-10326 (1996)) employing suitable constructs of first and second protein tailored for a high throughput assay to detect compounds that inhibit the first protein/second protein interaction. These embodiments have the advantage that the cell permeability of compounds that act as modulators in the assay is assured. Label-free methods available in the art can also be used to detect protein-protein binding, for example, SRU BIND^® technology (SRU Biosystems, Woburn, MA) and surface plasma resonance.

Candidate compounds for treating inflammatory arthritis can also be identified by screening for test compounds that decrease or inhibit expression of GPVI. Levels of GPVI protein or mRNA in cells can be determined by conventional methods, e.g., immunoblotting, RT-PCR, or northern blotting. Test compounds that decrease platelet microparticle production can also be candidate compounds for treating inflammatory arthritis. Platelets can be isolated from blood samples, e.g., from human, using methods known in the art, e.g., the methods described in the example. Isolated platelets can be contacted with test compounds and microparticle production can be determined by the methods described herein or known in the art.

Screening methods that detect test compounds that inhibit the expression of a procoagulant surface on platelets or platelet microparticles can also be used to identify candidate compounds for treating inflammatory arthritis. A procoagulant surface can be assayed by methods known in the art, e.g., using flow cytometry to measure the binding of fluorescent Annexin V or of fluorescent anti-phosphatidylserine antibodies to the procoagulant surface of platelets or platelet microparticles (Dachary-Prigent, J et al., Blood, 81 :2554-65 (1993)). Alternatively, the expression of a procoagulant surface on an adherent activated platelet or platelet fragment can be detected using fluorescent Annexin V or fluorescent anti-phosphatidylserine antibodies using a suitable microscope (Siljander, PR-M et al, Blood, 103: 1333-41 (2004)).

The screening methods provided herein can be performed with platelets or cultured cells engineered to express GPVI, e.g., mammalian cells such as HEK293, COS or CHO cells. Candidate compounds can be retested, e.g., in vitro, or tested on animals, e.g., animals that are models for inflammatory arthritis. Candidate compounds that are positive in a retest can be considered "lead" compounds to be further optimized and derivatized, or may be useful therapeutic or diagnostic compounds themselves.

Libraries of Test Compounds In certain embodiments, screens disclosed herein utilize libraries of test compounds. As used herein, a "test compound" can be any chemical compound, for example, a macromolecule (e.g., a polypeptide, a protein complex, a glycoprotein, a polysaccharide, an antibody, or a nucleic acid) or a small molecule (e.g., an amino acid, a nucleotide, or an organic or inorganic compound). A test compound can have a formula weight of less than about 10,000 grams per mole, less than 5,000 grams per mole, less than 1,000 grams per mole, or less than about 500 grams per mole. The test compound can be naturally occurring (e.g., from an herb or other natural product), synthetic, or can include both natural and synthetic components. Examples of test compounds include peptides, peptidomimetics (e.g., peptoids, retro-peptides, inverso peptides, and retro- inverso peptides), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, and organic or inorganic compounds, e.g., heteroorganic or organometallic compounds.

Test compounds can be screened individually or in parallel. An example of parallel screening is a high throughput drug screen of large libraries of chemicals. Such libraries of candidate compounds can be generated or purchased, e.g., from Chembridge Corp., San Diego, CA. Libraries can be designed to cover a diverse range of compounds. For example, a library can include 500, 1000, 10,000, 50,000, or 100,000 or more unique compounds. Alternatively, prior experimentation and anecdotal evidence can suggest a class or category of compounds of enhanced potential. A library can be designed and synthesized to cover such a class of chemicals. The synthesis of combinatorial libraries is well known in the art and has been reviewed (see, e.g., Gordon et al, J. Med. Chem., 37:1385-1401 (1994); Hobbes et al, Ace. Chem. Res., 29:114 (1996); Armstrong, et al., Ace. Chem. Res., (1996) 29:123; Ellman, Ace. Chem. Res., (1996) 29:132; Gordon et al., Ace. Chem. Res., 29:144 (1996); Lowe, Chem. Soc. Rev., 309 (1995); Blondelle et al., Trends Anal. Chem., 14:83 (1995); Chen et al., J. Am. Chem. Soc, 116:2661 (1994); U.S. Patents No: 5,359,115, 5,362,899, and 5,288,514; PCT Publication Nos. WO92/10092, WO93/09668, WO91/07087, WO93/20242, and WO94/08051).

Libraries of compounds can be prepared according to a variety of methods, some of which are known in the art. For example, a "split-pool" strategy can be implemented in the following way: beads of a functionalized polymeric support are placed in a plurality of reaction vessels; a variety of polymeric supports suitable for solid-phase peptide synthesis are known, and some are commercially available (for examples, see, e.g., Bodansky, "Principles of Peptide Synthesis," 2nd edition, Springer- Verlag, Berlin (1993)). To each aliquot of beads is added a solution of a different activated amino acid, and the reactions are allowed to proceed to yield a plurality of immobilized amino acids, one in each reaction vessel. The aliquots of derivatized beads are then washed, "pooled" (i.e., recombined), and the pool of beads is again divided, with each aliquot being placed in a separate reaction vessel. Another activated amino acid is then added to each aliquot of beads. The cycle of synthesis is repeated until a desired peptide length is obtained. The amino acid residues added at each synthesis cycle can be randomly selected; alternatively, amino acids can be selected to provide a "biased" library, e.g., a library in which certain portions of the inhibitor are selected non-randomly, e.g., to provide an inhibitor having known structural similarity or homology to a known peptide capable of interacting with an antibody, e.g., the an anti-idiotypic antibody antigen binding site. It will be appreciated that a wide variety of peptidic, peptidomimetic, or non-peptidic compounds can be readily generated in this way.

The "split-pool" strategy can result in a library of peptides, e.g., modulators, which can be used to prepare a library of test compounds of the invention. In another illustrative synthesis, a "diversomer library" is created by the method of DeWitt et al. (Proc. Natl. Acad. Sci. U.S.A., 90:6909 (1993)). Other synthesis methods, including the "tea-bag" technique of Houghten (see, e.g., Houghten et al., Nature, 354:84-86 (1991)) can also be used to synthesize libraries of compounds according to the subject invention.

Libraries of compounds can be screened to determine whether any members of the library can inhibit or antagonize GPVI, and, if so, to identify the inhibitory compound. Methods of screening combinatorial libraries have been described (see, e.g., Gordon et al., J Med. Chem, supra). Exemplary assays useful for screening libraries of test compounds are described above.

Test compounds can also include antibodies, e.g. antibodies that bind to GPVI or a GPVI ligand or agonist. Antibodies suitable for screening in the methods disclosed herein include known antibodies as well as new antibodies (e.g., as discussed herein) that selectively bind to, for example, GPVI polypeptides or fragments thereof.

Medicinal Chemistry

Once a compound (or agent) of interest has been identified, standard principles of medicinal chemistry can be used to produce derivatives of the compound. Derivatives can be screened for improved pharmacological properties, for example, efficacy, pharmaco-kinetics, stability, solubility, and clearance. The moieties responsible for a compound's activity in the assays described above can be delineated by examination of structure-activity relationships (SAR) as is commonly practiced in the art. A person of ordinary skill in pharmaceutical chemistry could modify moieties on a candidate compound or agent and measure the effects of the modification on the efficacy of the compound or agent to thereby produce derivatives with increased potency. For an example, see Nagarajan et al., J. Antibiot. 41 :1430-8 (1988). Furthermore, if the biochemical target of the compound (or agent) is known or determined, the structure of the target and the compound can inform the design and optimization of derivatives. Molecular modeling software is commercially available (e.g., Molecular Simulations, Inc.) for this purpose.

EXAMPLE

Data described in this example demonstrate that the collagen receptor glycoprotein VI (GPVI) is a key trigger for platelet microparticle generation in arthritis pathophysiology.

Materials and Methods

Human synovial fluid analysis. Human knee synovial fluids were obtained as discarded material from patients with various arthritides undergoing diagnostic or therapeutic arthrocentesis. Arthritis diagnosis was ascertained by an American Board of Internal Medicine certified Rheumatologist and/or by review of laboratory, radiologic and clinic notes and by applying ACR classification criteria (Arnett et al., Arthritis Rheum, 31 :315 (1988)). All studies received Institutional Review Board approval.

Cytofluorometric analysis of platelet-leukocyte interaction in freshly obtained SF was assessed as described previously with slight modifications (Joseph et al., Br J Haematol, 115:451 (2001); Brown et al., Am J Respir Cell MoI Biol, 18:100 (1998)). PE-conjugated anti-CD41 (clone M148, Abeam), FITC-conjugated anti-CD15 (clone VIMC6), CD3 (clone S4.1), CD14 (clone TuK4), glycophorin A (clone CLB-ery-1) (Caltag Laboratories), PerCP-conjugated anti-CD45 (clone 2DlBeckton Dickinson) and the appropriate IgG isotype controls in 20μl HBSS were added to 75μl SF and incubated for 10 minutes at room temperature (RT). Blood contamination was excluded via red blood cell marker glycophorin A. Cells were next incubated in 84μl fixative (500μl 10% paraformaldehyde+ 600μl HBSS 1OX + 900μl H₂O) for 10 min at RT. Cells were finally diluted with lOOOμl HBSS and analyzed cytofluorometrically (BD FACSCanto).

Microscopic analysis of platelet-leukocyte interaction was performed as follows; lOμl fresh SF diluted in lOOμl HBSS was subjected to cytospin preparation of leukocytes onto a glass slide. Cells were rinsed with PBS and fixed in 4% paraformaldehyde in PBS. Cells were next washed in PBS and slides incubated in 5% horse and bovine serum containing 0.02% sodium azide and 10 μM Draq5™ for 45 minutes at RT. PE- conjugated anti-CD41, and FITC-conjugated anti-CD 15 (2 μg/slide) in blocking buffer (5% horse and 5% bovine serum and 0.02% sodium azide) were added for 1 hour at RT and then thoroughly washed with PBS. Coverslips were mounted in polyvinyl-based media (Vinol) and analyzed by fluorescence microscopy.

MP quantification in SF. Human platelet MP were detected using CD41 as a marker (clone M 148) (Pastakia et al, Clin MoI Pathol, 49:M17 (1996); Trappenburg et al., Haematologica, (2009). For these experiments, choice of an appropriate marker is important. Platelet MP partial loss of CD61 (GPIIIa) expression has been previously described (Kim et al., Blood Coagul Fibrinolysis, 13:393 (2002)). Indeed, in this study, lower amounts of MP (~58 000 MP/ μl RA SF) was found when probing MP using an anti-CD61 (clone PM6/13). Furthermore, CD61+ MP was not detected when using another anti-CD61 monoclonal antibody (Y2/51) (AbD Serotec), in congruence with a previous report that described an absence of CD61 MP in RA SF using this antibody (Berckmans et al., Arthritis Rheum, 46:2857 (2002)). Concentrations of MP present in SF were determined using SF cleared of leukocytes by centrifugation at 600 x G for 30 minutes performed twice. SF (30μl) was next incubated in presence of antibodies for 20 minutes and diluted to 800μl with PBS. To quantify SF MP concentration, known amounts of 15 μm polystyrene microsphere Polybead ^®, (Polysciences Inc) were added to FACS tubes just prior analysis. Size of MP was determined using lμm beads (Fluka, Sigma). FACS analysis was performed using settings where the threshold was lowered to 200 and FSC and SSC gates were drawn to include events smaller than lμm (Kim et al., Blood Coagul Fibrinolysis, 13:393 (2002)). Phosphatidylserine exposure was monitored using annexin- V-FITC labeling according to the instructions from the manufacturer (BD Pharmingen) and the platelet markers CD42b (clone AK2, Abeam) and CD62P (clone AK-4, BD Pharmingen) were identified by cytofluorometry. Membrane-associated IL lα and β on SF MP was detected using FITC-conjugated anti-ILlα (clone 3405, R&D Systems) and IL lβ (ASlO, Abeam). Mice. 6-9 week old mice were used for all of the studies. All procedures were approved by the Institutional Animal Care and Use Committee of the Dana-Farber Cancer Institute (Boston, MA). Mice were housed in the specific pathogen free animal facility of the Dana-Farber Cancer Institute. C57BL/6J, thromboxane A synthase 1 null (Yu et al, Blood, 104:135 (2004)), GPIIIa null (Hodivala-Dilke et al, J Clin Invest, 103:229 (1999)), ILlRl null (Labow et al., J Immunol, 159:2452 (1997)), collagen type Iα2 null (Chipman et al., Proc Natl Acad Sci, USA 90:1701 (1993)) and collagen type IVα5 null mice (Fox et al., Cell, 129:179 (2007)) were obtained from Jackson Laboratory (Bar Harbor, ME). FcRγ null (Takai et al., Cell, 76:519 (1994)) and cox-1 (Langenbach et al., Cell, 83:483 (1995)) null mice were obtained from Taconic (Hudson, NY). GPVI null, GPIb and ILla/b null mice were generated and maintained as described (Kato et al., Blood, 102:1701 (2003); Horai et al., J Exp Med, 187:1463 (1998); Bergmeier et al., Proc Natl Acad Sci, USA, 103:16900 (2006); Kato et al., Blood, 104:2339 (2004)). When drug inhibitors were used in vivo, the following regimen were followed: Clopidogrel (20mg/kg) diluted in 1% methyl-cellulose was administrated by gavage daily and the thromboxane A2 antagonist SQ 29,548 (Cayman) (10mg/kg) was injected daily via tail vein. Inhibitors were injected from day 0 beginning 2 hours prior K/BxN serum injection. Platelet depletion was achieved by injecting 4μg/g of depleting antibody (mixture of rat monoclonal antibodies against CD42b) or isotype control per mouse as described by the manufacturer (Emfret Analytics). These antibodies were administered 30 minutes prior to K/BxN serum injection and at day 4.

Serum transfer protocol and arthritis scoring. Arthritogenic K/BxN serum was transferred to recipient mice via intraperitoneal injection (75 μl K/BxN serum) on experimental day 0 and 2 (at day 0 only in platelet depletion experiments in order to avoid internal bleeding) to induce arthritis as described (Chen et al., Arthritis Rheum, 58: 1354 (2008); Korganow et al., Immunity, 10:451 (1999)). Ankle thickness was measured at the malleoli with the ankle in a fully flexed position, using a spring-loaded dial caliper (Long Island Indicator Service, NY). The clinical index of arthritis was graded on a scale 0-12 as described previously. (Chen et al; Korganow et al.)

Histological examination. For histomorphometric analysis, ankle tissues were fixed for 24 hours in 4% paraformaldehyde in PBS and decalcified for 72 hours with modified Kristensen's solution. Tissues were then dehydrated, embedded in paraffin, sectioned at 5 μm thickness and stained with hematoxylin and eosin. Histological scoring was performed in a blinded manner as previously described (Chen et al., J Exp Med, 203:837 (2006); Pettit et al., Am J Pathol, 159:1689 (2001))

Platelet isolation. Mouse blood was drawn by cardiac puncture using ACD anticoagulant (acid citrate dextrose: 0.085M sodium citrate, 0.0702M citric acid, 0.11 IM dextrose, pH 4.5). Blood was diluted by addition of 400μl Tyrode's buffer pH 6.5 (134mM NaCl, 2.9mM KCl, 0.34mM Na₂HPO₄, 12mM NaHCO₃, 2OmM HEPES, ImM MgCl₂, 5mM glucose, 0.5mg/ml BSA) and centrifuged at 600 x G for 3 minutes. Platelet-rich plasma (PRP) was further centrifuged for 2 minutes at 400 x G to pellet contaminating RBC. This was thereafter centrifuged for 5 minutes at 1300 x G to pellet platelets. The role of P2Y12 receptors in microparticle release was investigated ex vivo by isolating platelets from mice treated by gavage with clopidogrel (30mg/kg) daily for 3 consecutive days and 2 hours before blood drawing. Platelets were obtained from healthy human volunteers using ACD as coagulant and isolated as described as above. Platelets were resuspended in Tyrode's buffer at pH 7.4 and quantified cytofluorometrically using anti-CD41 staining (human CD41 : clone M148 and mouse CD41 : MWReg30 (BD Pharmingen)) and known amounts of 15 micron polystyrene microsphere Polybeads ^®.

FLS co-culture with platelets. Primary human FLS were obtained by collagenase digest of synovial tissues obtained at time of joint surgery of RA patients. These cells were passaged in basal medium between 4-8 times for these experiments (Lee et al., Science, 315:1006 (2007)). Mouse primary FLS were obtained by collagenase digest of isolated ankle synovial tissues and passaged 4-8 times in basal medium (Lee et al.). Platelets in DMEM (30xl06/ml) were added to washed FLS in 6-well plates at 37⁰C for 2h. MP release was quantified cytofluorometrically using anti-CD41 or corresponding isotype antibody control and known amounts of 15 micron polystyrene microsphere Polybeads ®. Scanning electron microscopy. Human primary RA FLS (Lee et al.) were cultured to near confluence on 10mm glass coverslips in 12-well plates prior to the removal of all media and gentle rinsing with fresh media to remove non-adherent cells. Human platelets in DMEM (30xl06/ml) were then added to the culture and incubated for 3 hours at 37⁰C. The cells/platelets were fixed in 2.5% glutaraldehyde (Electron

Microscopy Science, Fort Washington, PA) buffered in 0.1M sodium cacodylate buffer (pH 7.4) for 2 hours at RT followed by rinsing in the same buffer (3 times for 15 min each). Postfixation was carried out in 1% osmium tetroxide buffered in 0. IM sodium cacodylate (pH 7.4) for 1 hour at RT. The samples were rinsed again in 0.1 M cacodylate buffer (4 times for 15 min each) and dehydrated through a graded series of ethanol solutions (50, 70, 80, 90, 95, 100%). The samples were then critical-point dried (Tousimis Auto Samdri 815 Series A), mounted on stubs, sputter-coated with Platinum- Palladium (Cressington 208 HR) and finally analyzed using a Zeiss EVO 50 SEM operated at an accelerating voltage of 3OkV. Preparation of MPs and detection of ILl. Mouse and human platelets in

Tyrode's buffer pH 7.4 containing ImM CaCl₂ were stimulated with 5μg/ml collagen (Horm, Nycomed Arzenmittel, Munich, Germany) for 5 hours at RT and centrifuged 1300 x G for 5 minutes. The residual supernatant, which contained the MPs, was further centrifuged at 1300 x G for 5 minutes to remove residual intact platelets. This MP containing supernatant was subsequently centrifuged at 21, 000 x G for 90 minutes at 18⁰C in conical ultra-clear centrifuge tubes (Beckman) using a sw55Ti rotor in a Beckman centrifuge. Pelleted MP were subsequently resuspended in Hepes-Tyrode's buffer (137mM NaCL, 2.8mM KCl, ImM MgCl₂, 12mM NaHCO₃, 0.4mM Na²HPO₄, 0.35% BSA, 1OmM HEPES, 5.5 mM glucose, pH 7.4). MP concentration was monitored by FACS using anti-CD41 staining and known amounts of 15 micron polystyrene microsphere Polybead ^®. To monitor IL-I content, MPs were resuspended in PBS containing 0.1% NP-40 and 0.1% Triton XlOO, sonicated, and lysates monitored for IL- lα and IL- lβ by ELISA (R&D Systems).

FLS stimulation by MPs. Human and mouse primary FLS were grown to semiconfluence in 6-well plates over at least 5 days. Prior to addition of MPs, FLS were washed with DMEM and then incubated in the presence of indicated amounts of MPs in DMEM for 5 hours at 37°C. As controls, FLS were also stimulated with recombinant IL- lβ or TNF (R&D Systems). Supernatants were next assayed for presence of released cytokine using the Proteome ProfϊlerTM (R&D Systems). Specific MP -mediated release of cytokines by FLS was calculated based on cytokines released from FLS and MP individually and in combination. For quantification, images were captured using a

Multilmagc Light Cabinet (Alpha Innotech, San Lcandro,CA) and spot densitometry was performed using Cherai Imager 4400 software (Alpha Innotech). Results were confirmed using and IL-6, IL-8 or KC ELISA kits (R&D Systems). To block IL-I activity using anti-ILl neutralizing antibodies, MPs were diluted to indicated concentrations in DMEM and incubated with indicated concentrations of either anti-mouse or anti-human IL- lα

(AF-400-NA or clone 4414 respectively, R&D Systems) or IL-I β (clone 1400.24.17 from Endogen or 8516 from R&D Systems, respectively).

Statistical analysis. Results of mouse arthritis experiments are presented as mean ±SEM. The statistical significance for comparisons between groups was determined using two-way ANOVA, 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 Whether platelets could participate in inflammatory arthritis was investigated. Of the inflammatory arthritides, rheumatoid arthritis (RA) is the most common (Helmick et al., Arthritis Rheum, 58: 15 (2008)). RA manifests as chronic inflammation of the synovial lining of the joint, resulting in pain, swelling and ultimately destruction of cartilage and bone (Lee and Weinblatt, Lancet, 358:903 (2001)). Although evidence has implicated lymphocytes, innate immune cells such as neutrophils and mast cells, and synovial tissue cells in the evolution of RA, to date platelets have no known functional role in RA.

Whether platelets are present in RA synovial fluids (SF) was determined by use of the platelet-specific marker CD41 (GPIIb/α2b from the platelet-specific integrin GPIIbIIIa/α2bβ3) (Pastakia et al., Clin MoI Pathol, 49:M17 (1996); Valant et al., Br J Haematol, 100:24 (1998). Flow cytometric analyses detected a substantial number of CD41 -positive events in RA SF (Fig. 2, A and B). Unexpectedly, the majority of CD41- positive events were smaller in size than intact leukocytes or platelets (Fig. 6), suggesting that these particles were platelet microparticles (MP). Platelet MPs are intact vesicles (0.2- lμm in diameter) that form by budding from the membranes of activated platelets (Perez-Pujol et al, Cytometry, A71 :38 (2007); Thiagarajan and Tait, J Biol Chem,

266:24302 (1991); Wolf, Br J Haematol, 13:269 (1967)). On average, just under 2X10⁵ CD41⁺ MPs per microliter of SF were found in samples from patients with RA (Fig. 2C). In contrast to RA SF, where MPs were present in all samples, CD41⁺ MPs were undetectable in 19 out of 20 osteoarthritis (OA) SF samples (Fig. 2C). Platelet MPs in other inflammatory arthritides were also detected (Fig. 2D).

The presence of MPs originating from other hematopoeitically-derived cell types was further investigated. Consistent with previous observations (Berckmans et al., Arthritis Rheum, 46:2857 (2002), MPs expressing neutrophil, T cell or macrophage markers were present in RA SF, albeit in substantially smaller amounts than observed for platelet MPs (Fig. 2E). Recognizing that platelets can adhere to migrating leukocytes (Levene and Rabellino, Blood, 67:207 (1986); Joseph et al., Br J Haematol, 115:451 (2001)), the presence of platelets associated with SF leukocytes was also analyzed. A substantial number of SF leukocytes co-stained with CD41 and the leukocyte marker CD45 (Figs. 6D and 6E). Microscopic examination revealed that CD41 staining in these cells was restricted to discrete particles of MP size adherent to the leukocyte surface; intact platelets associated with SF leukocytes could not be detected (Fig. 6D).

The K/BxN serum transfer model of inflammatory arthritis was employed to explore the pathophysiologic importance of platelets and platelet MPs in inflammatory arthritis in vivo. The progressive distal symmetric erosive polyarthritis observed in K/BxN T-cell receptor transgenic mice results from T cell recognition of a ubiquitous autoantigen, glucose-6-phosphate isomerase (GPI), presented by major histocompatability complex (MHC) class II I-A^g7, driving high-titer arthritogenic autoantibody production (reviewed in Kyburz and Corr, Springer Semin Immunopathol, 25:79 (2003)). Arthritis can also be induced by passive transfer of immunoglobulin (Ig)G autoantibodies from K/BxN mice into wild type mice. Numerous effector mechanisms have been implicated in the pathogenesis of the IgG-driven effector phase of K/BxN serum-transfer arthritis, including neutrophils, mast cells, Fc gamma receptors, and soluble mediators (e.g., interleukin (IL)-I, tumor necrosis factor (TNF), complement C5a/C5a receptor, eicosanoids and the mast cell protease tryptase) (Kyburz and Corr; Chen et al, Arthritis Rheum, 58:1354 (2008); Shin et al, J Immunol, 182:647 (2009)). To study the role of platelets in this system, K/BxN serum transfer arthritis was initiated in animals treated with a platelet-depleting antibody regimen that rapidly (within 60 minutes) reduces platelets number by >95% for at least six days (Iannacone et al., Nat Med, 11 : 1167 (2005)). Platelet-depleted mice exhibited a striking reduction in arthritis as assessed by clinical scoring and by histological analysis (Figs. 3A and 3B). These findings demonstrate platelets are required for inflammatory arthritis development in vivo.

To gain further insight into the link between platelets and joint inflammation, the mechanisms by which platelets are activated to release MP in the context of arthritis were investigated. Platelets can be triggered via several pathways, many of which have already been targeted for the prevention of thrombosis. The pathways studied in this example were thromboxane A2 stimulation of its receptor (TP) on platelets (blocked by TP antagonist SQ 29548), ligation of the P2Y12 receptor by ADP (inhibited by clopidogrel) and GPIb-IX, a platelet membrane glycoprotein complex that binds to von Willebrand factor. Data showed that interference with these pathways (by using genetically deficient mice and pharmacologic blockade) did not impede development of joint inflammation, suggesting that these pathways do not regulate platelet MP generation in inflammatory arthritis (Figs. 3C-3G).

As the concentration of platelet-derived MPs within RA SF (2X10⁵/μl) greatly exceeds that in RA peripheral blood (600/μl) (Knijff-Dutmer et al., Arthritis Rheum, 46: 1498 (2002)), it was hypothesized that platelet activation occurs locally. The vasculature of the joint is in intimate contact with fibroblast- like synoviocytes (FLS) and the extracellular matrix (ECM) elaborated by these cells (Lee et al., Textbook of Rheumatology. St. Louis: W. B. Saunders. 7th edition, 175 (2004)). The interaction of platelets with synoviocytes and their ECM was modeled in vitro. Mouse platelets co- incubated with primary mouse FLS promptly released MPs (Fig. 4A). To determine the trigger for MP release, MP formation was assessed after treatment with specific pharmacologic inhibitors or using platelets isolated from mice deficient in candidate genes. Consistent with the in vivo results described above, cyclooxygenase (Ptgsl^'1'), thromboxane, GPIIbIIIa

and ADP-P2Y12 pathways were found to be dispensable (Fig. 4B). Whether the ECM generated by primary cultured FLS could be the relevant stimulus, particularly collagen, was investigated. Previous studies have demonstrated that collagen can activate platelets to form MPs (Perez-Pujol et al., Cytometry, A71 :38 (2007) and that glycoprotein VI (GPVI) is the predominant collagen receptor on platelets (Nieswandt et al., J Biol Chem, 275:23998 (2000). Using platelets lacking either FcR-γ- chain {Fcerlg^1') or GPVI {Gpό^'1'), it found that generation of MPs by primary FLS was mediated predominantly via this pathway (Fig. 4B). After confirming that human platelets also released MPs upon coincubation with primary human FLS (Figs. 4, C and D), the relevance of GPVI in MP release in man was validated using the GPVI specific agonist collagen-related peptide (CRP) (Smethurst et al., J Biol Chem, 282:1296 (2007) (Fig. 4D). Further characterization of collagen-stimulated platelet MPs showed that their phenotype is congruent with that of platelet MPs from SF and distinct from intact platelets (Table 1). Whether GPVI is relevant for platelet activation in vivo was determined by administering K/BxN serum to Gp6^~'^~ and control mice and assessing the development of synovitis. Both clinical and histomorphometric assessment confirmed that arthritis in Gp6^{~ '} mice was significantly reduced (Figs. 4, E and F). These results confirm that activation of platelets via the collagen receptor GPVI - a pathway resulting in MP generation - plays an important role in the pathogenesis of arthritis.

Having demonstrated platelet participation in inflammatory arthritis in vivo, experiments were carried out to identify platelet MP effector activities that contribute to joint inflammation. The most abundant cell in the pathologic rheumatoid pannus tissue is the FLS (Lee et al., Textbook of Rheumatology. St. Louis: W. B. Saunders. 7th edition, 175 (2004)). This lineage plays a substantial role in the perpetuation of joint inflammation and in the destruction of cartilage (Lee et al., Textbook of Rheumatology. St. Louis: W. B. Saunders. 7th edition, 175 (2004); Lee et al., Science, 315:1006 (2007)). The capacity of collagen-stimulated human platelet MPs to elicit a range of cytokines and chemokines from FLS was surveyed. Prominent production of the broadly inflammatory cytokine IL-6 and the neutrophil chemoattractant IL-8 was observed (Fig. 5A and Fig. 7). Consistent with this observation, incubation of MPs isolated from RA SF induced FLS to release substantial quantities of both cytokines (Fig. 5B and Fig. 8). MP stimulation of IL-8 by FLS was studied further, since SF from the inflammatory arthritides is rich in neutrophils. To elucidate a mechanism by which platelet MPs stimulate FLS, a genetic approach using MPs generated from mice deficient in specific candidate genes was employed. Platelet MPs generated from mice lacking both IL- lα and IL- lβ (Illa/b^'^ were incapable of stimulating murine FLS to produce the murine IL-8 ortholog KC (Fig. 5C). Furthermore, FLS generated from mice deficient in the IL-I receptor {Illrl^'1') were unresponsive to platelet MPs, though release of KC remained intact after TNF stimulation (Fig. 5D). By contrast, MPs from mouse platelets deficient in prostaglandin synthesis capacity (PtgsT ^") retained their ability to stimulate KC production from FLS (Fig. 5C).

TABLE 1. Markers expressed on platelets and platelet microparticles

Marker RA SF MP Collagen- Ionophore- Platelets induced MP induced MP

CD41 + + + +

Annexin V + + + -

CD42b - - - (low) +

CD62P - - - +*

* CD62P is found on surface of platelets after stimulation with cross-linked collagen- related peptide (0.5μg/ml) or ionophore (lμM) in Tyrode's buffer containing 5 μM CaCl₂ for 60 min at room temperature.

Platelets exhibit membrane-associated IL-I activity (Lindemann et al., J Cell Biol, 154:485 (2001). This study confirmed that both forms of this cytokine were present in wild-type murine MPs, although IL- lα was predominant (IL- lα, 87 ± 7 pg/mg protein; IL- lβ, 2 ± 0.2 pg/mg protein). Blocking both forms of IL-I using neutralizing antibodies was necessary to fully blunt FLS activation by MPs (Fig. 5E). Similar results were obtained in the human system. Platelet MPs from RA SF expressed surface IL- lα, which, as in murine MPs, predominated over IL- lβ (Fig. 9). Similarly, human platelet

MPs elicited by in vitro collagen stimulation expressed both IL- lα and IL- lβ (19.1 vs 0.1 pg/mg protein) and triggered RA FLS to release IL-8 in a dose-dependent manner, indeed more robustly than either IL-I β or TNF (Fig. 5F). Data suggest that both forms of IL-I participate in human FLS stimulation because neutralization of platelet MP IL-I activity required blocking antibodies against both IL- lα and IL- lβ (Fig. 5G). Together, these results suggest that platelet MPs likely contribute to joint inflammation via mechanisms including highly potent IL-I -mediated activation of resident synoviocytes.

These surprising results provide an explanation for several disparate previous observations. Radiolabeled platelets localize to inflamed joints (Farr et al., Ann Rheum Dis 42:545 (1983)) and RA SF displays appreciable levels of soluble platelet proteins (Ginsberg et al., Arthritis Rheum 21 :994 (1978)), yet intact platelets are rare in arthritic SF. Whereas SF MP levels exceed those in the circulation of RA patients by several orders of magnitude (Knijff-Dutmer et al., Arthritis Rheum 46:1498 (2002)), platelet activation appears to be primarily an articular process, wherein MPs disseminate platelet- derived cytokines into the arthritic joint. Subsynovial capillaries exhibit fenestrations and are prone to enhanced permeability after stimulation (Binstadt et al., Nat Immunol 7:284 (2006); Schumacher, Ann Clin Lab Sci 5:489 (1975)). Although applicants do not wish to be bound by theory, it is hypothesized that circulating platelets contact ECM via these fenestrations when local conditions favor permeability activating GPVI. Alternatively, platelet "sampling" of ECM via fenestrations may be routine, activating platelets as ECM constituents are modified. Thus, synovium is enriched for collagen type IV (Poduval et al., Arthritis Rheum 56:3959 (2007)), and FLS deficient in collagen type IV demonstrated reduced capacity to stimulate platelet MP release (Fig. 10).

The results described herein provide compelling evidence that platelets, via GPVI, play an amplifying role in the pathophysiology of inflammatory arthritis, liberating pro- inflammatory MPs that represent the most abundant cellular element in SF. The observation that membrane-associated MP IL-I is unusually difficult to antagonize (Fig. 5) may help to explain the limited effect of IL-I blockade in RA (Mertens and Singh, Cochrane Database Syst Rev, CD005121 (2009)), and the prominence of IL-I α within MP poses potential constraints on the efficacy of IL-lβ-specific agents. The relevance of the results described herein for human disease is substantial. Since mice or humans lacking GPVI remain healthy (Nieswandt and Watson, Blood 102:449 (2003)), antagonism of this receptor represents a novel therapeutic approach for inflammatory arthritis.

Claims

WHAT IS CLAIMED IS:

1. Use of a glycoprotein VI (GPVI) antagonist for the treatment of inflammatory arthritis in a subject.

2. Use of a glycoprotein VI (GPVI) antagonist for the preparation of a medicament for the treatment of inflammatory arthritis in a subject.

3. A method for treating inflammatory arthritis in a subject, the method comprising:

(a) identifying a subject suffering from inflammatory arthritis; and

(b) administering to the subject a therapeutically effective amount of a GPVI antagonist.

4. The use of claim 1 or claim 2, or the method of claim 3, wherein the GPVI antagonist is an antibody that binds specifically to a GPVI polypeptide.

5. The use of claim 1 or claim 2, or the method of claim 3, wherein the GPVI antagonist inhibits binding between a GPVI polypeptide and a GPVI agonist.

6. The use of claim 1 or claim 2, or the method of claim 3, wherein the GPVI antagonist inhibits GPVI signaling.

7. The use of claim 1 or claim 2, or the method of claim 3, wherein the GPVI antagonist inhibits expression of a GPVI polypeptide.

8. The use of claim 4 or the method of claim 4, wherein the antibody binds specifically to the extracellular domain of GPVI.

9. The use of claim 5 or the method of claim 5, wherein the GPVI agonist is selected from the group consisting of collagen, laminin, collagen-related peptide (CRP), and convulxin.

10. A method of identifying a candidate compound for treating inflammatory arthritis in a subject, the method comprising:

(a) providing a sample comprising a GPVI polypeptide;

(b) contacting the sample with a GPVI agonist and a test compound; and

(c) detecting binding between the GPVI agonist and the GPVI polypeptide; wherein a decrease in the binding in the presence of the test compound as compared to a control indicates that the test compound is a candidate compound for treating inflammatory arthritis.

11. A method of identifying a candidate compound for treating inflammatory arthritis in a subject, the method comprising:

(a) providing a platelet;

(b) contacting the platelet with a GPVI agonist and a test compound; and

(c) detecting production of platelet microparticles; wherein a decrease in the level of microparticles produced in the presence of the test compound as compared to a control indicates that the test compound is a candidate compound for treating inflammatory arthritis.

12. The method of claim 10, wherein the sample further comprises a Fc receptor γ-chain (FcR γ-chain).

13. The method of claim 10, wherein the sample is a cell.

14. The method of claim 10, the method further comprising determining whether the test compound decreases GPVI-induced production of platelet microparticles.

15. The method of claim 10, wherein the GPVI agonist is selected from the group consisting of collagen, CRP, laminin, and convulxin.