Use of LINK-TSG6 for the treatment of osteoarthritis pain

Abstract

The present invention relates to a LINK_TSG6 polypeptide for use in the treatment of osteoarthritis pain or pain associated with osteoarthritis, in particular in the treatment of osteoarthritis pain. Osteoarthritis is caused by the deterioration of cartilage in the joints and is characterized by pain and loss of mobility in the affected joints. Osteoarthritis pain results from multiple factors including bone marrow lesions, wear of the bone below the joint, synovitis, effusions, and innervation of normally insensitive tissues. LINK_TSG6 is a short recombinant peptide containing the LINK module of human TSG-6.

Description

[Technical field]

[0001] This application claims priority to UK Patent Application No. 2100761.2, filed on 20 January 2021, the contents and elements of which are incorporated herein by reference for all purposes.

[0002] The present invention relates to the treatment of osteoarthritis pain, particularly but not exclusively to the treatment of osteoarthritis pain using LINK_TSG6 polypeptides. [Background technology]

[0003] Osteoarthritis (OA) is the most common form of arthritis worldwide. It is a long-term chronic disease caused by the deterioration of cartilage in the joints and is characterized by pain and loss of mobility in the affected joints (e.g., knees, hips, and hands). The World Health Organization (WHO) estimates that 9.6% of men and 18% of women over 60 years of age suffer from symptomatic OA, of which 80% have limited movement and 25% are unable to perform major activities of daily living. Multiple factors such as age, sex, obesity, physical inactivity, genetic factors, bone density, previous joint injury or trauma, or joint diseases such as gout or rheumatoid arthritis may contribute to the development of OA. The prevalence of OA is also increasing due to an aging population and an increase in associated factors such as obesity.

[0004] There are no disease-modifying therapies for OA, and current treatments are limited to pain reduction and surgical intervention, primarily joint replacement. Patients with OA are undergoing joint replacement at an increasingly younger age, requiring multiple revision surgeries, usually with poor outcomes, and requiring pain management.

[0005] Existing pain treatments for OA include acetaminophen (paracetamol), opiate analgesics (painkillers) such as codeine, and nonsteroidal anti-inflammatory drugs (NSAIDs). All of these have limitations or side effects. Acetaminophen can cause liver damage and is not suitable for those with existing liver symptoms. Narcotic painkillers are effective in treating moderate to severe OA pain, but can cause side effects such as dizziness and nausea and can be habit forming. In addition to reducing pain, NSAIDs can reduce swelling and inflammation caused by OA, but taking NSAIDs increases the risk of gastrointestinal disorders, heart attack, stroke, and heart failure, and therefore oral NSAIDs are often recommended to be prescribed for the shortest time and at the lowest effective dose, as outlined by Altman and Barthel (Drugs. 2011;71(10):1259-1279).

[0006] Topical treatments for OA pain include capsaicin cream, which treats pain by inducing reversible nerve degeneration. However, capsaicin can cause significant pain early in treatment, which can lead to discontinuation of treatment; one review found that among 1,556 subjects, 54% of patients treated with topical capsaicin experienced adverse reactions (compared to 15% of patients treated with placebo), and 13% of patients treated with capsaicin discontinued treatment due to these effects (Altman and Barthel, Drugs. 2011;71(10):1259-1279). In addition, there is a risk of side effects due to nerve damage, especially for diabetic patients.

[0007] Steroid injections can be used to treat OA pain by reducing swelling and inflammation. These may be administered intra-articularly (into a joint), but there is some emerging evidence that intra-articular steroid injections can cause side effects such as accelerated OA progression and bone loss (Kompel et al., Radiology. 2019;293(3):656-663).

[0008] Therefore, new treatments are needed to relieve osteoarthritis pain. Yang et al., 2020 (J Neuroinflammation 17(1):154) described the use of intrathecally injected bone marrow stem cells (BMSCs) in rats that secrete TSG-6 and reduce neuropathic pain caused by chronic constriction injury. This study also showed that intrathecal injection of exogenous TSG-6 attenuated CCI-induced neuropathic pain.

[0009] The present invention has been devised in light of the above considerations. Summary of the Invention [Means for solving the problem]

[0010] The present disclosure relates to LINK-TSG6 polypeptides for use in the treatment of osteoarthritis pain. As used herein, the abbreviation OA means osteoarthritis, and OA pain means osteoarthritis pain, or pain associated with osteoarthritis.

[0011] As shown in the examples, LINK_TSG6 can reduce osteoarthritis pain in a dose-dependent manner. LINK_TSG6 was effective in reducing osteoarthritis pain when administered subcutaneously and intra-articularly at both high and low doses, and in both monthly and weekly dosing schedules.

[0012] Administration of LINK_TSG6 reduced OA pain in a partial medial meniscectomy model of OA and OA pain, as demonstrated by reduction in "direct pain" caused by flexion of the injured joint, improved thermal and mechanical sensitivity, and improved weight bearing.

[0013] The analgesic effect of LINK_TSG6 was observed in the ipsilateral limb (i.e., the side where surgery was performed), but also in the contralateral limb (i.e., the side of the body opposite the limb where surgery was performed). This indicates that LINK_TSG6 has analgesic properties and may be useful for both direct administration to the affected osteoarthritic joint and administration to more distant sites. Furthermore, LINK_TSG6 may be suitable not only for direct pain resulting from osteoarthritis of the joint, but also for the treatment of referred pain, such as pain at a site distant from the osteoarthritic joint.

[0014] In a first aspect, the present disclosure provides LINK_TSG6 for use in medicine, particularly for use in the treatment of osteoarthritis pain.Also described is a method for treating OA pain, comprising administering LINK_TSG6, and the use of LINK_TSG6 in the manufacture of a medicament for the treatment of osteoarthritis pain.The treatment may be for a subject in need of treatment.

[0015] In preferred aspects, LINK_TSG6 is administered subcutaneously or intra-articularly. Treatment may be less frequent, such as once a week or once a month. In some aspects, treatment involves subcutaneous administration of 10 mg to 150 mg per month, 20 mg to 140 mg per month, 30 mg to 130 mg per month, 40 mg to 120 mg per month, or 50 mg to 110 mg per month. In some aspects, treatment involves subcutaneous administration of about 50 mg per month, about 60 mg per month, about 70 mg per month, about 80 mg per month, 90 mg per month, or about 100 mg per month of LINK_TSG6. In other aspects, treatment involves intra-articular administration of between 2 mg to 30 mg, 3 mg to 28 mg, 4 mg to 25 mg, 5 mg to 23 mg, 6 mg to 20 mg, or 10 mg to 20 mg of LINK_TSG6 per month. In some aspects described herein, the treatment can include intra-articular administration of at least 3, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, or at least 180 mg of LINK_TSG6.

[0016] In some aspects, LINK_TSG6 is administered to an osteoarthritis joint. In some cases, it is administered distal to an osteoarthritis joint. In some cases, a patient or subject may have multiple osteoarthritis joints, and LINK_TSG6 is administered to one of the osteoarthritis joints. In some cases, LINK_TSG6 is administered to some of the osteoarthritis joints.

[0017] In some cases, LINK_TSG6 is used to treat direct pain, i.e., LINK_TSG6 is used to treat joint pain resulting from osteoarthritis of the joint. LINK_TSG6 may alternatively or additionally be used to treat referred pain, which is pain felt at a location other than the site of osteoarthritis.

[0018] In some aspects, LINK_TSG6 is administered once every 7 days, once every 10 days, once every 14 days, once every 21 days, once every 30 days, once every 45 days, once every 60 days, once every 90 days, or less than once every 90 days. The analgesic (pain reducing) effect can last for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 days, or more.

[0019] In some aspects disclosed herein, the LINK_TSG6 polypeptide comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9. In some aspects, the LINK_TSG6 polypeptide comprises, consists of, or consists essentially of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9.

[0020] In a further aspect, the present disclosure provides a composition comprising LINK_TSG6 formulated for subcutaneous administration. The composition can comprise at least 50 mg, at least 60 mg, at least 70 mg, at least 80 mg, at least 90 mg, or at least 100 mg of LINK_TSG6 per dose.

[0021] In a further aspect, the present disclosure provides a composition comprising LINK_TSG6 formulated for intra-articular administration, the composition can comprise at least 1 mg / ml, at least 2 mg / ml, at least 3 mg / ml, at least 4 mg / ml, at least 5 mg / ml, at least 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml, about 20 mg / ml, or greater than about 20 mg / ml of LINK_TSG6.

[0022] The present invention includes combinations of the described aspects and preferred features unless such combinations are clearly impermissible or explicitly avoided. BRIEF DESCRIPTION OF THE DRAWINGS Embodiments and experiments illustrating the principles of the present invention are described with reference to the accompanying drawings. [Brief description of the drawings]

[0023] [Figure 1-1] FIG. 1 shows sequences relevant to the present disclosure. [Figure 1-2] FIG. 1 shows sequences relevant to the present disclosure. [Figure 1-3] FIG. 1 shows sequences relevant to the present disclosure. [Figure 1-4] FIG. 1 shows sequences relevant to the present disclosure. [Figure 1-5] FIG. 1 shows sequences relevant to the present disclosure. [Figure 1-6] FIG. 1 shows sequences relevant to the present disclosure. [Figure 1-7] FIG. 1 shows sequences relevant to the present disclosure. [Diagram 2] Figure 1. Link_TSG6 reduces direct OA joint pain. Subcutaneous (sc) Link_TSG6 treatment, A. weekly (top panel; high 55 μg, low 13.75 μg per dose) or B. monthly (bottom panel; high 220 μg, low 55 μg per dose), dose-dependently reduces pain associated with knee flexion in a murine partial medial meniscectomy (pMx) model of OA as assessed by vocalization. Significance was determined using one-way ANOVA with Tukey's post-hoc test: *p<0.05, **p<0.01, ***p<0.001 compared to corresponding vehicle controls. [Figure 3A]Link_TSG6 reduces heat sensitivity. A. Weekly subcutaneous (sc) Link_TSG6 treatment reduces referential heat sensitivity (assessed by paw withdrawal latency to a cold (10°C) stimulus) in the ipsilateral (operated) paw (front) using a murine partial meniscectomy (pMx) model of OA. A significant analgesic effect is also evident in the contralateral paw (rear). B. Monthly sc Link_TSG6 treatment reduces referential heat sensitivity in the ipsilateral (front) paw in a murine pMx model of OA. A significant analgesic effect is also evident in the contralateral paw (rear). Significance was determined using one-way ANOVA with Tukey's post-hoc test: *p<0.05, **p<0.01, ***p<0.001 compared to corresponding vehicle controls. [Figure 3B] Link_TSG6 reduces heat sensitivity. A. Weekly subcutaneous (sc) Link_TSG6 treatment reduces referential heat sensitivity (assessed by paw withdrawal latency to a cold (10°C) stimulus) in the ipsilateral (operated) paw (front) using a murine partial meniscectomy (pMx) model of OA. A significant analgesic effect is also evident in the contralateral paw (rear). B. Monthly sc Link_TSG6 treatment reduces referential heat sensitivity in the ipsilateral (front) paw in a murine pMx model of OA. A significant analgesic effect is also evident in the contralateral paw (rear). Significance was determined using one-way ANOVA with Tukey's post-hoc test: *p<0.05, **p<0.01, ***p<0.001 compared to corresponding vehicle controls. [Figure 4A]Link_TSG6 reduces mechanical sensitivity. A. Weekly subcutaneous (sc) Link_TSG6 treatment reduces the referential mechanical sensitivity (measured by paw withdrawal threshold) in the ipsilateral (surgical) paw (front) using a murine partial meniscectomy (pMx) model of OA. A significant analgesic effect is also evident in the contralateral paw (rear). B. Monthly Link_TSG6 treatment reduces the referential mechanical sensitivity in the ipsilateral (front) paw using a murine pMx model of OA. A significant analgesic effect is also evident in the contralateral paw (rear). Significance was determined using one-way ANOVA with Tukey's post-hoc test: *p<0.05, **p<0.01, ***p<0.001 compared to corresponding vehicle control. [Figure 4B] Link_TSG6 reduces mechanical sensitivity. A. Weekly subcutaneous (sc) Link_TSG6 treatment reduces the referential mechanical sensitivity (measured by paw withdrawal threshold) in the ipsilateral (surgical) paw (front) using a murine partial meniscectomy (pMx) model of OA. A significant analgesic effect is also evident in the contralateral paw (rear). B. Monthly Link_TSG6 treatment reduces the referential mechanical sensitivity in the ipsilateral (front) paw using a murine pMx model of OA. A significant analgesic effect is also evident in the contralateral paw (rear). Significance was determined using one-way ANOVA with Tukey's post-hoc test: *p<0.05, **p<0.01, ***p<0.001 compared to corresponding vehicle control. [Figure 5A]Link_TSG6 improves weight bearing. A. Link_TSG6 treatment (once a week) dose-dependently improves weight bearing (measured by dual channel weight average) in a murine pMx model of OA when using either subcutaneous (sc) (pre) or intra-articular (ia) (post) administration. B. Link_TSG6 treatment (once a month) dose-dependently improves weight bearing in a murine pMx model of OA when using either subcutaneous (sc) (pre) or intra-articular (ia) (post) administration. Significance was determined using one-way ANOVA with Tukey's post-hoc test: *p<0.05, **p<0.01, ***p<0.001 compared to corresponding vehicle control. [Figure 5B] Link_TSG6 improves weight bearing. A. Link_TSG6 treatment (once a week) dose-dependently improves weight bearing (measured by dual channel weight average) in a murine pMx model of OA when using either subcutaneous (sc) (pre) or intra-articular (ia) (post) administration. B. Link_TSG6 treatment (once a month) dose-dependently improves weight bearing in a murine pMx model of OA when using either subcutaneous (sc) (pre) or intra-articular (ia) (post) administration. Significance was determined using one-way ANOVA with Tukey's post-hoc test: *p<0.05, **p<0.01, ***p<0.001 compared to corresponding vehicle control. [Figure 6A]Figure 1. Link_TSG6 reduces tactile allodynia in ACLTpMMx rats. OA was induced in the right knee of male Lewis rats by ACLT and pMMx (30%). Link_TSG6 treatment (40 g) or vehicle was administered ia on days 7, 14, and 21 after surgery. After 28 days, tactile allodynia was assessed by determining the paw withdrawal threshold to a von Frey hair applied to the hind paw of (A) the unoperated control leg and (B) the operated leg. Mean values ​​± SD are indicated by horizontal lines (n=15). Data were analyzed by one-way ANOVA with Tukey's post-hoc test; *p<0.05, ***p<0.001, ****p<0.0001 vs vehicle control. [Figure 6B] Figure 1. Link_TSG6 reduces tactile allodynia in ACLTpMMx rats. OA was induced in the right knee of male Lewis rats by ACLT and pMMx (30%). Link_TSG6 treatment (40 g) or vehicle was administered ia on days 7, 14, and 21 after surgery. After 28 days, tactile allodynia was assessed by determining the paw withdrawal threshold to a von Frey hair applied to the hind paw of (A) the unoperated control leg and (B) the operated leg. Mean values ​​± SD are indicated by horizontal lines (n=15). Data were analyzed by one-way ANOVA with Tukey's post-hoc test; *p<0.05, ***p<0.001, ****p<0.0001 vs vehicle control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Aspects and embodiments of the present invention will now be described with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0025] TSG-6 (tumor necrosis factor stimulated gene-6) TSG-6 is a secreted protein composed of two modular domains. TSG-6 is not normally constitutively expressed in adult tissues, but rather is induced in response to inflammatory mediators. During inflammation, TSG-6 is an endogenous protector of tissues. Many of the immunomodulatory and tissue protective effects of mesenchymal stem / stromal cells (MSCs) are mediated by the secretion of TSG-6.

[0026] Recombinant full-length TSG-6 protein has been shown to have anti-inflammatory and tissue-protective effects in a wide range of disease models, including atherosclerosis, myocardial infarction, hypertrophic scars, colitis, autoimmune diabetes, rheumatoid arthritis, traumatic brain injury, or acute lung injury.

[0027] Full-length TSG-6 is difficult to produce, insoluble and prone to aggregation. As disclosed herein, these drawbacks are not present in a short recombinant peptide, LINK_TSG6, containing the LINK module of human TSG-6. This short polypeptide is easier to produce, more soluble and more stable in solution than full-length TSG-6.

[0028] TSG-6 is constitutively expressed in a few tissues, but generally, it is upregulated wherever there is inflammation. In most cases, TSG-6 displays anti-inflammatory and tissue protective properties, but it has been suggested to play an occasional role in disease pathology, for example, in the lung. Although TSG-6 is formed by a wide range of cell types, it has been found to be produced by mesenchymal stem / stromal cells (MSCs) in response to inflammatory signals and mediates many of their immunomodulatory and repair activities, leading to a large body of publications on the therapeutic effects of this intriguing molecule across a wide range of disease models.

[0029] TSG-6 is a relatively small protein, with a molecular weight of only about 35–38 kDa, and composed mainly of two modular domains (Link and CUB_C). Given its size, TSG-6 has a surprising number of activities, including regulation of immune and stromal cell functions, as well as contribution to the formation, organization, and remodeling of the extracellular matrix. Possibly underlying its diverse range of functions is the ability of TSG-6 to regulate matrix organization and control the binding of matrix molecules to cell surface receptors and extracellular signaling factors (e.g., chemokines). In this regard, TSG-6 interacts with numerous ligands, such as glycosaminoglycans (GAGs), proteoglycan (PG) core proteins, and other matrix components, and directly binds to multiple chemokines and bone morphogenetic proteins (BMPs). One particularly unusual function of TSG-6 is its role as an enzyme that catalyzes the covalent modification of the nonsulfated GAG hyaluronan (HA) by the so-called heavy chains (HCs) of the inter-alpha inhibitor (lαi) family of proteoglycans. This process, mediated by the full-length TSG-6 protein and not by the LINK_TSG6 polypeptide, which contains only a fragment of TSG-6, leads to the formation of HC·HA complexes that are essential for mammalian ovulation and fertilization, as well as in many other situations (e.g., inflammation) where HC·HA provides tissue protection or contributes to pathological processes.

[0030] The sites and contexts of TSG-6 expression, its structure and ligand-binding properties, and how these together are responsible for its diverse biology and therapeutic potential at the molecular level are reviewed in Day & Milner (Matrix Biology (2019) 78–79, 60–83).

[0031] The LINK_TSG6 polypeptide disclosed herein comprises the Link module of human or mammalian TSG-6. In some embodiments, the TSG-6 polypeptide comprises or consists essentially of an amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:5. The Link module corresponds to residues 37-128 of SEQ ID NOs:2 and 5 and is set forth in SEQ ID NO:7.

[0032] The Link module has hyaluronic acid (HA)-binding activity, chondroitin sulfate-binding activity, aggrecan-binding activity, inter-alpha inhibitor (Iαi)-binding activity, heavy chain (HC) 1-binding activity, HC2-binding activity, HC3-binding activity, bikunin-binding activity, versican-binding activity, dermatan sulfate-binding activity, pentraxin-3-binding activity, thrombospondin-1-binding activity, thrombospondin-2-binding activity, fibronectin-binding activity, heparin / heparan sulfate-binding activity, and TSG -6 is involved in the RANKL-binding activity, bone morphogenetic protein (BMP)-2-binding activity, BMP-4-binding activity, BMP-5-binding activity, BMP-6-binding activity, BMP-7-binding activity, BMP-13-binding activity, BMP-14-binding activity, CXCL4-binding activity, CXCL6-binding activity, CXCL8-binding activity, CXCL11-binding activity, CXCL12-binding activity, CCL2-binding activity, CCL5-binding activity, CCL7-binding activity, CCL19-binding activity, CCL21-binding activity, or CCL27-binding activity.

[0033] LINK_TSG6 has hyaluronic acid (HA)-binding activity, chondroitin sulfate-binding activity, aggrecan-binding activity, inter-alpha inhibitor (Iαi)-binding activity, heavy chain (HC) 1-binding activity, HC2-binding activity, HC3-binding activity, bikunin-binding activity, versican-binding activity, dermatan sulfate-binding activity, pentraxin-3-binding activity, thrombospondin-1-binding activity, thrombospondin-2-binding activity, fibronectin-binding activity, heparin / heparan sulfate-binding activity, RANKL-binding activity, and bone marrow-binding activity. It may be a fragment of TSG-6 that exhibits one or more of morphogenetic protein (BMP)-2 binding activity, BMP-4 binding activity, BMP-5 binding activity, BMP-6 binding activity, BMP-7 binding activity, BMP-13 binding activity, BMP-14 binding activity, CXCL4 binding activity, CXCL6 binding activity, CXCL8 binding activity, CXCL11 binding activity, CXCL12 binding activity, CCL2 binding activity, CCL5 binding activity, CCL7 binding activity, CCL19 binding activity, CCL21 binding activity, or CCL27 binding activity.

[0034] US Patent Application Publication No. 2015 / 0057229 describes the use of LINK_TSG6 in inhibiting cartilage degradation. The LINK domain of TSG-6 (LINK_TSG6) may be the region from the N-terminus of full-length TSG-6 to the CUB_C domain. Thus, the LINK_TSG6 protein may lack all or part of the CUB_C domain. In a preferred aspect, the LINK_TSG6 polypeptide may include the N-terminal region of the CUB_C domain.

[0035] The LINK domain may contain the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9. The LINK_TSG6 polypeptide comprises, consists of, or consists essentially of (i) the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9, or (ii) an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 7 or 9.

[0036] LINK_TSG6 is preferably a polypeptide comprising, consisting of, or consisting of (i) the amino acid sequence of SEQ ID NO: 7 or 9, or (ii) an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 7 or 9.

[0037] In some cases, LINK_TSG6 consists of the amino acid sequence of SEQ ID NO:9 and optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 additional amino acids at the C-terminus, N-terminus, or C-terminus and N-terminus, respectively. In some cases, LINK_TSG6 consists of the amino acid sequence of SEQ ID NO:7 and optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 additional amino acids at the C-terminus, N-terminus, or C-terminus and N-terminus, respectively.

[0038] Thus, the LINK_TSG6 polypeptide is (a) the amino acid sequence of SEQ ID NO:7; (b) a variant thereof having at least 50% identity to the amino acid sequence of SEQ ID NO: 7 and having RANKL binding activity; or (c) It may comprise a fragment of either (a) or (b) having CXCL4-binding activity, CXCL6-binding activity, CXCL8-binding activity, CXCL11-binding activity, CXCL12-binding activity, CCL2-binding activity, CCL5-binding activity, CCL7-binding activity, CCL19-binding activity, CCL21-binding activity, or CCL27-binding activity.

[0039] The LINK_TSG6 polypeptide may consist or consist essentially of the sequence shown in SEQ ID NO:7. SEQ ID NO: 9 shows a recombinant polypeptide comprising the Link module of TSG-6 (LINK_TSG6). Thus, the TSG-6 polypeptide used in the present invention is preferably (a) the amino acid sequence of SEQ ID NO:9; (b) a variant thereof having at least 50% identity to the amino acid sequence of SEQ ID NO: 9 and having RANKL binding activity; or (c) It may comprise a fragment of either (a) or (b) having CXCL4-binding activity, CXCL6-binding activity, CXCL8-binding activity, CXCL11-binding activity, CXCL12-binding activity, CCL2-binding activity, CCL5-binding activity, CCL7-binding activity, CCL19-binding activity, CCL21-binding activity, or CCL27-binding activity.

[0040] The LINK_TSG6 polypeptide preferably consists or consists essentially of the sequence shown in SEQ ID NO:9. Amino acid identity can be calculated using a suitable algorithm. For example, the UWGCG package provides the BESTFIT program, which can be used to calculate homology (e.g., used with default settings) (Devereux et al. (1984) Nucleic Acids Research 12, 387-395). The PILEUP and BLAST algorithms can be used to calculate homology and align sequences (such as to identify equivalent or corresponding sequences (usually with default settings)), as described, for example, in Altschul (1993) J. Mol. Evol. 36, 290-300; Altschul et al. (1990) J. Mol. Biol. 215, 403-10.

[0041] Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in the database sequences. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find HSPs that contain them. The word hits are extended in both directions along each sequence for as long as the cumulative alignment score can be increased. Extension of the word hits in each direction is stopped when the cumulative alignment score falls below 0 due to the accumulation of one or more negative scoring residue alignments by an amount X from the maximum achieved value, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as default a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89, 10915-10919), alignment (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0042] The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90, 5873-5787. One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which defines a measure of the probability that a match between two polynucleotide or amino acid sequences would occur by chance. For example, a sequence is considered to be similar to another sequence if the minimum sum probability in the comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0043] The variant sequence typically differs by at least 1, 2, 5, 10, 20, 30, 50 or more mutations, which may be amino acid substitutions, deletions or insertions. For example, there may be 1-50, 2-30, 3-20, or 5-10 amino acid substitutions, deletions or insertions. The modified polypeptides generally may retain CXCL4 binding activity, CXCL6 binding activity, CXCL8 binding activity, CXCL11 binding activity, CXCL12 binding activity, CCL2 binding activity, CCL5 binding activity, CCL7 binding activity, CCL19 binding activity, CCL21 binding activity or CCL27 binding activity, preferably in a dose-dependent manner. The substitutions are preferably conservative substitutions, for example according to the following table. Amino acids in the same block in the second column, and preferably in the same row in the third column, may be substituted for each other.

[0044] [Table 1]

[0045] The LINK_TSG6 polypeptide used in the present invention is typically at least 10, such as at least 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or more amino acids in length, up to 100, 150, 200 or 250 amino acids in length, so long as it retains the CXCL4-, CXCL6-, CXCL8-, CXCL11-, CXCL12-, CCL2-, CCL5-, CCL7-, CCL19-, CCL21- or CCL27-binding activity of TSG-6. Preferably, the polypeptide comprises the sequence shown in SEQ ID NO:7. Fragments of the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:9 preferably contain residues shown to be essential for hyaluronic acid binding in Mahoney et al. (2001) J. Biol. Chem. 276, 22764-22771 and Blundell et al. (2003) J. Biol. Chem. 278, 49261-49270. Fragments of the amino acid sequences of SEQ ID NO:2 or 5 preferably contain residues Lys-46 and / or Tyr-47 and / or Tyr-94 and / or Phe-105 and / or Tyr-113 of SEQ ID NO:2 or 5. Most preferably, fragments of SEQ ID NO:2 or 5 contain residues Lys-46, Tyr-47, Tyr-94, Phe-105, and Tyr-113, respectively, of SEQ ID NO:2 or 5.

[0046] Fragments of the amino acid sequence of SEQ ID NO: 7 may be used in the present invention. Such fragments preferably contain residues Lys-10 and / or Tyr-11 and / or Tyr-58 and / or Phe-69 and / or Tyr-77 of SEQ ID NO: 7. Most preferably, the fragment of SEQ ID NO: 7 contains each of residues Lys-10, Tyr-11, Tyr-58, Phe-69, and Tyr-77 of SEQ ID NO: 7.

[0047] The fragment of the amino acid sequence of SEQ ID NO:9 preferably contains residues Lys-11 and / or Tyr-12 and / or Tyr-59 and / or Phe-70 and / or Tyr-78 of SEQ ID NO:9. Most preferably, the fragment of SEQ ID NO:9 contains residues Lys-11, Tyr-12, Tyr-59, Phe-70, and Tyr-78 of SEQ ID NO:9. The TSG-6 polypeptides used in the present invention may be chemically modified, for example post-translationally modified. For example, they may be glycosylated, phosphorylated, or contain modified amino acid residues. They may be modified by adding histidine residues to aid purification, or by adding transmembrane sequences to facilitate insertion into the cell membrane. Such modified polypeptides are included within the scope of the term "polypeptide" as used herein.

[0048] TSG-6 polypeptides are involved in the regulation of HA, chondroitin sulfate, aggrecan, inter-alpha inhibitor (Iαi), heavy chain (HC) 1, HC2, HC3, bikunin, versican, dermatan sulfate, pentraxin-3, thrombospondin-1, thrombospondin-2, heparin / heparan sulfate, fibronectin, RANK, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-13, BMP-14, CXCL4, C Suitable assays for determining the ability to bind to XCL6, CXCL8, CXCL11, CXCL12, CCL2, CCL5, CCL7, CCL19, CCL21, and CCL27 are well known in the art (Getting et al. (2002) J. Biol. Chem. 277, 51068-51076; Mahoney et al. (2005) J. Biol. Chem. 280, 27044-27055; Salustri et al. (2004) Development 131, 1577~1586;Parkar et al. (1997) FEBS Lett.410, 413~417;Parkar et al. (1998) FEBS Lett.428, 171~176;Mahoney et al. (2001) J.Biol.Chem.276, 22764~22771;Nentwich et al. (2002) J.Biol Chem.211, 15354~15362;Kuznetsova et al. (2005) J.Biol.Chem.280, 30899~30908;Dyer et al. (2014) J.Immunol 192, 2177~2185;Dyer et al. (2016) J.Biol.Chem.291, 12627~12640;Mahoney et al. (2008) J.Biol.Chem 283, 25952~25962;Marson et al. (2009) Glycobiology 19, 1537~1546; Kuznetsova et al. (2006) J.Cell Sci.119, 4499~4509; Kuznetsova et al. (2008) Matrix Biology 27, 201~210), and Day & Milner. (2019) Matrix Biology.78~79, 60~83. )

[0049] The TSG-6 polypeptide for use in the present invention may be in substantially isolated form.It will be understood that the polypeptide may be mixed with a carrier or diluent that does not interfere with the intended purpose of the polypeptide and still be considered to be substantially isolated.The polypeptide for use in the present invention may also be in substantially purified form, which generally comprises the polypeptide in a preparation, and more than 50% by weight of the polypeptide in the preparation, for example 80%, 90%, 95% or more than 99% by weight, is the polypeptide of the present invention.

[0050] The LINK_TSG6 polypeptide for use in the present invention may be a naturally occurring or non-naturally occurring polypeptide. The polypeptide may be isolated from any suitable organism that expresses the TSG-6 polypeptide. The TSG-6 polypeptide may be isolated from a human or another suitable mammal, such as a primate, rat or mouse. Alternatively, the TSG-6 polypeptide may be isolated from a fish or amphibian. The polypeptide for use in the present invention may also be prepared as a fragment of such an isolated polypeptide.

[0051] In addition, the LINK_TSG6 polypeptide can also be produced by synthetic or recombinant means. For example, recombinant LINK_TSG6 polypeptide can be produced by transfecting an expression vector comprising a nucleotide sequence encoding the polypeptide operably linked to an appropriate control sequence into cells in culture, culturing the cells, and extracting and purifying the LINK_TSG6 polypeptide produced by the cells. Methods for recombinant production of polypeptides are well known in the art (e.g., Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rd ed., Cold Harbour Laboratory Press). Preferably, the LINK_TSG6 polypeptide is produced in bacteria such as E. coli.

[0052] The amino acid sequence of the LINK_TSG6 polypeptide for use in the present invention may be modified to include non-naturally occurring amino acids or to increase the stability of the compound. If the polypeptide is produced by synthetic means, such amino acids may be introduced during production. The polypeptide may also be modified after synthesis or recombinant production.

[0053] In some aspects, the LINK_TSG6 polypeptide used herein is not contained within a fusion protein. That is, LINK_TSG6 is not fused to another peptide or polypeptide. In some cases, LINK_TSG6 is not a fusion protein with a growth factor such as IGF-1.

[0054] The LINK_TSG6 polypeptides for use in the present invention may also be produced using D-amino acids. In such cases, the amino acids are linked in reverse sequence from C to N. This is the convention in the art for producing such polypeptides.

[0055] A number of side chain modifications are known in the art and can be made to the side chains of the LINK_TSG6 polypeptide as long as the polypeptide retains the ability to reduce osteoarthritis pain.

[0056] Osteoarthritis The present disclosure relates to the treatment of osteoarthritis pain. Osteoarthritis pain is pain associated with osteoarthritis. Osteoarthritis pain is caused by osteoarthritis. In other words, osteoarthritis can cause pain and stiffness in a patient's joints.

[0057] Osteoarthritis (OA) is the most common form of arthritis worldwide. It is a long-term chronic disease caused by the deterioration of articular cartilage and is characterized by pain and loss of mobility in the affected joints. Although almost any joint can be affected by osteoarthritis, the disease most often causes problems in the knees, hips, and small joints of the hands and wrists. For example, the joints of the hands and wrists may be the joints of the fingers and thumbs (interphalangeal joints), the joints at the base of the fingers or thumbs (metacarpophalangeal joints), the base of the hand (carpometacarpal joints), or the wrist (scaphotrapezoid, intercarpal, and midcarpal / radiocarpal joints). Other joints that may be affected include those of the spine, shoulder, elbow, ankle (including the talocrural and subtalar, and tibiofibular joints), and small joints of the feet (e.g., metatarsophalangeal, proximal interphalangeal, and distal interphalangeal joints).

[0058] OA may be diagnosed based on a description of symptoms, including the location and pattern of any pain experienced. This may include a history of joint pain that worsens with movement or time of day. Symptom descriptions may include experiencing stiffness, especially after waking in the morning, and limited joint movement. Diagnosis may also take into account a family history of OA.

[0059] OA is usually diagnosed through a physical exam to check for symptoms such as tender joints, joint stiffness and limited joint movement, changes in gait, clicking or crepitation when moving a joint, swelling in bones or deformed joints such as Heberden's or Bouchard's nodes, joint instability, excess fluid, or weakness of the muscles supporting the joints.

[0060] In some cases, the diagnosis of OA may involve the use of x-rays of the affected joint to identify symptoms such as narrowing of the joint space between adjacent bones, calcium deposits within the joint, and osteophyte formation (growths that occur where bones meet within the joint). In some cases, magnetic resonance imaging (MRI) may also be used to diagnose OA. MRI can image soft tissues, including cartilage and bone marrow, and can show bone marrow lesions and cartilage destruction.

[0061] OA severity is often characterized by "stages." The four stages of OA progression are 1-mild, 2-mild, 3-moderate, or 4-severe. Stage 0 represents "normal" knee health (no signs of joint damage or known functional impairment). OA does not develop at a uniform rate. OA may worsen over weeks, months, years, or decades. OA progression depends on many factors, including age, genetics, overall health and activity level, weight / BMI, and how much the affected joint is used. In many cases, the level of pain is related to structural changes in the joint. However, some patients experience chronic pain even without substantial structural changes such as cartilage loss.

[0062] Patients with stage 1 (mild) OA develop minor structural changes and usually experience little or no pain. In stage 1, there may be minor growth of bone spurs in the affected joints.

[0063] Patients with stage 2 (mild) OA may experience stiffness, discomfort, and in some cases, pain in the affected joints. Stiffness and discomfort may be worse during prolonged sitting, in the morning, or after training. Prominent bone spurs may be detected by x-rays, and cartilage destruction may begin. Stage 2 OA may require treatment to reduce pain or other treatments such as exercise therapy, weight loss, or wearing joint supports or braces.

[0064] Stage 3 (moderate) OA occurs when there is obvious cartilage erosion in the affected joint. The spaces between the bones in the joint narrow, and bone spurs may develop or continue to develop between the bones in the joint. The shape of the joint may appear misshapen or curved. The joint may be inflamed, and patients with stage 3 OA may experience frequent pain and discomfort during daily activities such as walking, running, kneeling, or extending the affected joint. Stiffness and discomfort may increase after prolonged sitting or in the morning. Patients are more likely to seek pain reduction.

[0065] Patients with stage 4 (severe) OA experience the most advanced form of OA. Stage 4 may be characterized by little or no cartilage and a significant reduction in the joint space between the bones. Synovial fluid within the joint may decrease, increasing friction, pain, and discomfort when moving the affected joint. In some cases, however, synovial fluid within the joint increases with edema. The growth of bone spurs is likely to be multiplied. The shape of the joint may appear distorted or crooked. Typically, the pain of stage 4 OA is greater and is described as chronic pain, i.e., severe and excruciating pain, and the pain of stage 4 OA may also make daily activities such as walking and using stairs difficult.

[0066] Osteoarthritis pain OA pain is pain associated with OA. OA pain can be viewed in a biopsychological framework, where biological, psychological, and social factors all contribute. Chemical mediators are released into the inflamed joint and sensitize primary afferents, so that normally innocuous movements (such as increased physical activity, high-heeled shoes, or weather changes) evoke painful responses. This is the neurophysiological basis of allodynia (pain in response to normally non-painful stimuli). Over time, increased neuronal activity can trigger plastic changes in the central nervous system by a process known as "wind-up," during which the firing rate of second-order neurons in the spinal cord increases, thus enhancing the transmission of pain information to the somatosensory cortex.

[0067] This phenomenon of central sensitization intensifies the sensation of pain and may even result in pain responses from areas of the body distant from the inflamed joint (ie, referred pain). Structural determinants of OA pain likely involve multiple pathways. Because articular cartilage is aneural (has no nerve tissue) and avascular (lacks blood vessels), OA pain is unlikely to arise directly from damaged cartilage. Instead, other sources of symptom generation within the joint include the subchondral bone, periosteum, periarticular ligaments, periarticular musculoskeletal spasms, synovium, and joint capsule, all of which are richly supplied with nerves and contribute to nociception in OA. OA pain may be associated with structural causes such as bone marrow lesions, wear of the subarticular bone, synovitis (inflammation of the synovial membrane), and effusions (excessive accumulation of synovial fluid in or around the knee joint). Such structural causes may be detected using MRI. A previous study by Lo et al. revealed a strong association of bone marrow lesions and effusions with pain in osteoarthritis (Lo et al., Arthritis & Rheumatism. 2008;56(9):S790. Abstract).

[0068] In some cases, OA joint pain may be due to neural plasticity: for example, normally insensitive tissue such as cartilage may become innervated in OA patients, meaning that normally insensitive cartilage can become a source of pain.

[0069] Constitutional factors that predispose to symptoms include self-efficacy, pain catastrophizing, and the social context of arthritis (e.g., social support, pain communication), all of which are important considerations in understanding the pain experience.

[0070] OA pain may be described as "tingling" and may be worse after moving the affected joint. In some cases, OA pain is worse at the end of the day. OA pain may increase or decrease after eating certain foods. OA pain is not always proportional to the stage of OA, and in some cases, patients may experience severe pain with a small amount of joint damage. Pain from moderate and severe OA can seriously affect quality of life. OA pain can cause sleep problems, leading to fatigue and depression. In some cases, OA pain that varies from day to day may cause more sleep problems because it is difficult to predict or adapt to the pain. Lack of sleep may increase sensitivity to OA pain. In some cases, OA patients may find it beneficial to join support groups for people with chronic pain.

[0071] Osteoarthritis pain usually worsens over time, but in some cases it may wax and wane, not necessarily in a linear progression. Periods of moderate to severe pain may be followed by periods of improvement. OA pain may sometimes worsen at different times of the year or may be sensitive to weather. This may be due to the influence of air pressure and ambient temperature (McAlindon et al., 2007. Am J Med. 120(5):429-434). In some cases, osteoarthritis pain may be worse in the fall or winter, or periods of high air pressure or low ambient temperature may be accompanied by intense pain. In some cases, applying hot or cold compresses to the affected area may temporarily relieve OA pain.

[0072] Reducing OA pain may also be important for surgical outcomes after joint replacement. For example, knee arthroplasty (knee replacement) pain outcomes may be influenced by preoperative pain perception. In some cases, preoperative pain catastrophizing (the belief that the pain will get worse and that there is nothing that can be done about it) is a predictor of poor postoperative pain outcomes (Riddle et al., 2010, Clin Orthop Relat Res. 468(3):798-806).

[0073] Osteoarthritis pain is different from neuropathic pain. Neuropathic pain is pain caused by injury or disease affecting the somatosensory nervous system and may involve abnormal sensations called dysesthesias, or pain from normally painless stimuli (allodynia). It may include continuous and / or episodic (paroxysmal) components. In contrast, osteoarthritis pain is nociceptive pain. Nociceptive pain is pain that is transmitted by the nervous system to the brain, rather than arising in the nervous system itself.

[0074] In certain aspects disclosed herein, osteoarthritis pain is not neuropathic pain, nor osteoarthritis pain with neuropathic components.In certain aspects disclosed herein, the patient does not have neuropathic pain.In some cases, OA pain is or includes referred pain.Referred pain may be pain in a body part that is distant from the osteoarthritis joint.

[0075] OA pain can be measured using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and the Medical Outcomes Study Short-Form 36 (SF-36) scales.

[0076] The WOMAC is a questionnaire that breaks down OA pain into five dimensions: while walking, using stairs, in bed, sitting or lying down, or standing upright. In addition to pain, the WOMAC also includes questions about two dimensions of stiffness after first getting up and later in the day, as well as assessment of 17 aspects of "physical function" when completing activities such as going down stairs, going up stairs, rising from a sitting position, standing, bending, walking, getting in and out of a car, shopping, putting on socks, taking off socks, getting out of bed, lying in bed, getting in and out of the bath, sitting, sitting and standing on the toilet, heavy and light housework. The questions in the test are scored on a scale of 0 to 4, corresponding to none (0), mild (1), moderate (2), severe (3), and extreme (4). The scores for each subscale are summed, which can range from 0 to 20 for pain, 0 to 8 for stiffness, and 0 to 68 for physical function. Typically, the scores for all three subscales are summed to a total WOMAC score out of 96, with higher scores indicating more pain, stiffness, and limited physical function. A reduction in the WOMAC pain score, or a reduction in the total WOMAC score, can suggest successful treatment of OA pain.

[0077] The SF-36 includes questions about how much physical pain the patient has had in the past 4 weeks (on a scale of 1-6, where 1-none, 2-very mild, 3-mild, 4-moderate, 5-severe, 6-very severe) and how much pain has interfered with normal work (including both outside work and housework), with 1-not at all, 2-a little, 3-moderate, 4-a lot, and 5-extremely. Questions may also include whether the patient's health condition limits activities such as running, lifting, participating in sports, pushing a vacuum cleaner, climbing stairs, bending, kneeling, or stooping, walking various distances, or bathing or dressing (for each activity, 1-limits a lot, 2-limits a little, or 3-does not limit at all).

[0078] Qualitative aspects of OA pain can be assessed using scales such as the Short-Form McGill Pain Questionnaire (MPQ-SF). The MPQ-SF includes a "sensory subscale" and an "affective subscale." Sensory subscale terms may include, for example, throbbing, shooting, stabbing, sharp, tingling, gnawing, hot, burning, tingling, oppressive, painful, and cracking. Emotional subscale terms may include, for example, "exhausting-debilitating," "feeling sick," "frightening," and "punishing-cruel." Each term or aspect of pain is scored using a scale of 0-none, 1-mild, 2-moderate, or 3-severe.

[0079] In some cases, OA pain can be measured using questionnaires such as the Measure of Intermittent and Constant Osteoarthritis Pain (ICOAP) (https: / / www.oarsi.org / research / outcome-measures). The ICOAP measures constant OA pain separately from "pain that comes and goes". Constant OA pain can be measured by questions including how intense the pain is, how much it has affected sleep, how much it has affected overall quality of life, and how much the pain has caused frustration or annoyance, or upset or worry. Each dimension is scored as 0-not at all, 1-mild, 2-moderate, 3-severe, or 4-extreme. Infrequent OA pain or "comes and goes" OA pain can also be measured by intensity, frequency (0-never, 1-rarely, 2-occasionally, 3-frequently, 4-very frequently), impact on sleep, impact on quality of life, and how much the OA pain has caused frustration or annoyance, or upset or worry. Each dimension except frequency is scored as 0-never, 1-mild, 2-moderate, 3-severe, or 4-extreme. These variables can be measured, for example, over the past few days or over the past week. In some cases, they can be measured over a period longer than one week.

[0080] patient The method disclosed herein may include a step of evaluating a subject or patient for evidence of OA and osteoarthritis pain. The patient may have osteoarthritis. In other words, the patient may have been previously diagnosed with osteoarthritis. The patient may have previously been treated for osteoarthritis pain, for example, the patient may have previously been administered acetaminophen (paracetamol), NSAIDs, or opiates / opioid painkillers to treat osteoarthritis pain. In some cases, the patient does not suffer from other types of pain. For example, the patient does not have or suffer from neuropathic pain.

[0081] The patient may be male or female. The patient may be of any age. For example, the patient may be at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more years old.

[0082] The subject to be treated may be any animal or human. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, such as a dog, cat, horse, or cow. The subject may be male or female. In some cases, osteoarthritis pain in non-human mammals may be characterized by behavioral changes such as shuffling, biting or snapping, whining when touched, or reduced play. The subject may be a patient. The therapeutic use may be human or animal (veterinary use). The subject or patient may be a subject or patient in need of treatment for OA pain. The subject or patient has or is suspected of having osteoarthritis. The subject or patient has pain that is or is suspected of being osteoarthritis pain. In other words, the pain experienced by the patient or subject is caused or suspected of being caused by the osteoarthritis of the subject / patient. The subject / patient may have one or more osteoarthritic joints (i.e., a relationship formed between two or more bones that have one or more symptoms of osteoarthritis). The subject or patient may have a clinical diagnosis of osteoarthritis. The subject / patient may have stage I, stage II, stage III, or stage IV osteoarthritis. The subject / patient most likely has stage II, stage III, or stage IV osteoarthritis. The subject or patient may be self-diagnosed as having osteoarthritis.

[0083] treatment Disclosed herein is a method for treating or preventing osteoarthritis pain. The treatment of osteoarthritis pain may result or be aimed at reducing or eliminating OA pain. The method may include reducing one or more of the following: pain severity, pain frequency, OA pain's impact on sleep, pain's impact on quality of life, the extent to which pain causes frustration or irritation, anxiety or upset, and reducing pain when walking, standing, sitting, using stairs, doing housework, dressing, or bathing. The treatment may cause complete or partial resolution of OA pain. The treatment may cause partial or complete relief of pain symptoms, for example, at least 1 day after administration, at least 2 days after administration, at least 3 days after administration, at least 4 days after administration, at least 5 days after administration, at least 6 days after administration, at least 7 days after administration, at least 1 week after administration, at least 10 days after administration, at least 2 weeks after administration, at least 3 weeks after administration, or at least 1 month after administration. Partial or complete alleviation of symptoms may occur at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days after administration. Preferably, partial or complete alleviation of symptoms occurs at least one week after administration.

[0084] After treatment, the patient may have a reduction in the WOMAC pain score in one or more of the five aspects, or a reduction in the WOMAC pain score in two or more, three or more, four or more, or all five aspects of the WOMAC pain score. In some cases, the reduction in the score in one or more aspects may be more than one point, or more than two, three, or four points. For example, treatment may cause a reduction in at least one aspect of the WOMAC score by at least one point, or a reduction in each aspect of at least two aspects of the WOMAC score by at least one point, or a reduction in each aspect of at least three aspects of the WOMAC score by at least one point, or a reduction in each aspect of at least four aspects of the WOMAC score by at least one point, or a reduction in each aspect of all five aspects of the WOMAC score by at least one point. Treatment may cause a reduction of at least 2 points in at least one aspect of the WOMAC score, or a reduction of at least 2 points in one aspect of at least two aspects of the WOMAC score, or a reduction of at least 2 points in one aspect of at least three aspects of the WOMAC score, or a reduction of at least 2 points in one aspect of at least four aspects of the WOMAC score, or a reduction of at least 2 points in one aspect of all five aspects of the WOMAC score.

[0085] Treatment may result in a reduction in the WOMAC pain score to 0. A patient may have an overall WOMAC score of 96, for example, a decrease of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, or a complete decrease in the WOMAC total score to 0.

[0086] In some cases, the patient may experience a one or more point decrease in SF-36 pain score after treatment, for example, a decrease in the score for how much pain the patient has experienced in the past 4 weeks or since treatment. This may be a decrease of 1, 2, 3, 4, or 5 points. The patient may also experience a one or more point decrease on the SF-36 scale of how much pain has interfered with normal work. In some cases, there may be a two, three, or four point decrease. A one or more point decrease may be recorded for bilateral pain (i.e., how much pain the patient is experiencing and how much this pain has interfered with normal work).

[0087] After treatment, patients may experience improvement in the qualitative aspects of OA pain, as assessed using the MPQ-SF. This improvement may be quantified as a reduction in the score of one or more of the eleven aspects of the "sensation subscale". The improvement may be in one, two, three, four, five, six, seven, eight, nine, ten, or all eleven aspects of the sensory subscale. In some cases, the reduction in the score of one or more aspects may be one or more points, two or more points, or three points. In some cases, the improvement may be a reduction in the score of one or more of the four aspects of the "emotional subscale". The improvement may be in one, two, three, or all four aspects of the sensory subscale. There may be an improvement in one or more aspects of both the sensory and emotional subscales.

[0088] After treatment, the patient may experience an improvement in intermittent and / or steady pain as measured using ICOAP. This improvement may be quantified as a reduction in the score of one or more aspects of steady pain. In some cases, the improvement may be a reduction in two or more, three or more, four or more aspects of steady pain, or a reduction in the score across all aspects of steady pain. The reduction in score may be more than one point, such as 2, 3, or 4 points. It may be one or more aspects, or the sum of the steady pain score may be reduced to 0. The patient may also experience an improvement in intermittent pain, such as a reduction in frequency or intensity score. In some cases, the improvement may be a reduction in two or more, three or more, four or more, five or more aspects of intermittent pain, or a reduction in the score across all aspects of intermittent pain. The reduction in score may be more than one point, such as 2, 3, or 4 points. It may be one or more aspects, or the sum of the intermittent pain score may be reduced to 0.

[0089] After treatment, patients may have reduced pain scores using another clinically appropriate measure of pain intensity, frequency, or impact on daily activities and behavior, or mental health.

[0090] Route of administration The methods described herein can include administration of LINK_TSG6 by any route, such as intra-articular, local, parenteral, systemic, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, subcutaneous, oral, or transdermal routes of administration, including injection.

[0091] In a preferred aspect, treatment involves subcutaneous or intra-articular administration. In some cases, the treatment includes administration of LINK_TSG6 to a site distant from the site of osteoarthritis pain. The distal site may be any suitable site distant from the site of osteoarthritis pain. The distal site is typically a site suitable for subcutaneous administration. In some cases, LINK_TSG6 may be administered subcutaneously to the abdomen, arm, thigh, buttocks, or lower back. For example, the upper outer region of the arm, the front and outer region of the thigh, the upper outer region of the buttocks or lower back. The distal site may be a site within the same limb as the site of osteoarthritis pain, such as near the opposite end of the bone of the osteoarthritis joint. For example, if the site of osteoarthritis pain is an osteoarthritis knee joint, LINK_TSG6 may be administered near the top of the femur, thigh, or lower back of the same leg as the osteoarthritis knee. In some cases, the distal site is a contralateral site (i.e., a site on the body opposite to the site of osteoarthritis pain), such as the opposite leg to the leg containing the osteoarthritis knee. In some cases, the distal site is a different limb than the site of the osteoarthritis pain, such as the arm, or the abdomen.

[0092] In some cases, the treatment includes intra-articular (ia) administration of LINK_TSG6. The ia administration may include administration to an osteoarthritic joint. The ia administration may include administration to multiple osteoarthritic joints. For example, ia administration to multiple different types of joints, such as either or both of the knee or hip, or one or more knee joints and one or more hip joints. Administration to both knee joints can be performed at substantially the same time, such as during the same appointment with a physician. Administration may include injection under ultrasound or fluoroscopy. In some cases, intra-articular administration does not include administration of a fusion protein comprising LINK_TSG6 or administration of a LINK_TSG6 nucleic acid for expression in the joint.

[0093] In some aspects described herein, administration is not intrathecal. Administration may be once a week (one time). Preferably, the week is a calendar week (i.e., one administration every 7 days on average). Administration may be once a month (once a calendar month, or once every 30 days on average). In some preferred cases, administration is monthly (once every 30 days on average). In some cases, treatment includes administering LINK_TSG6 less frequently than once a week, such as once every 2 weeks, once every 3 weeks, or once a month. As used herein, the term "average" refers to the mathematical average.

[0094] In some cases, the administration is on average once every 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. Preferably, the administration is on average once every 7, 14, 21, or 30 days. In some cases, the administration is on average every 30 days.

[0095] The subject can be evaluated for osteoarthritis pain at any time before, during, or after administration of a therapeutically effective amount of LINK_TSG6. Dosage Administration is preferably in a "therapeutically effective amount", which is sufficient to show benefit to an individual. The actual amount administered, the rate of administration, and the duration of administration will depend on the nature and severity of the disease being treated. The prescription of treatment, e.g., the determination of dosage, etc., is within the responsibility of general practitioners and other physicians, and typically takes into account the disorder being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to the specialist. Examples of the techniques and procedures mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, published by Lippincott, Williams & Wilkins.

[0096] As disclosed herein, LINK_TSG6 was effective in reducing osteoarthritis pain when administered subcutaneously or intra-articularly at both high and low doses. Several methods for determining human equivalent doses (HEDs) based on mouse dosages are known in the art and can be used to determine appropriate dosages for use in the methods, treatments, and compositions described herein.

[0097] In certain aspects, dosages may be adjusted for administration to humans based on comparisons of mouse and human body surface areas, particularly for subcutaneous administration. Alternatively, dosages may be adjusted based on weight comparisons and drug concentrations, or based on "virtual space" considerations, particularly for intra-articular administration. See, in particular, Nair and Jacob (Journal of basic and clinical pharmacy. 2016;7(2), 27-31), WO 2015085706, and Emami et al., 2018, the contents of which are incorporated herein in their entirety.

[0098] Thus, in some aspects, the treatment involves administration of 3 mg to 180 mg per month, 10 mg to 150 mg per month, 20 mg to 140 mg per month, 30 mg to 130 mg per month, 40 mg to 120 mg per month, or 50 mg to 110 mg per month. In some aspects, the treatment involves subcutaneous administration of about 50 mg per month, about 60 mg per month, about 70 mg per month, about 80 mg per month, or 90 mg per month, or about 100 mg per month of LINK_TSG6.

[0099] In some aspects described herein, the treatment comprises subcutaneous administration of 3 mg to 180 mg per month, 10 mg to 150 mg per month, 20 mg to 140 mg per month, 30 mg to 130 mg per month, 40 mg to 120 mg per month, or 50 mg to 110 mg per month. In some aspects, the treatment comprises subcutaneous administration of about 2 mg per month, about 3 mg per month, about 4 mg per month, about 5 mg per month, about 10 mg per month, about 20 mg per month, about 30 mg per month, about 40 mg per month, about 50 mg per month, about 60 mg per month, about 70 mg per month, about 80 mg per month, or about 90 mg per month, or about 100 mg per month of LINK_TSG6.

[0100] Given that many OA patients are overweight or obese, higher doses are likely to be required, and higher doses may be required if the treatment is administered less frequently than monthly (e.g., once every 8 weeks).

[0101] For intra-articular administration, the dosage can be adjusted based on the weight comparison between mice and humans, based on the methods described in WO2015085706. In some aspects, treatment involves administration of 0.5 mg to 15 mg, 1 mg to 14 mg, 2 mg to 13 mg, 3 mg to 12 mg, 4 mg to 11 mg, 5 mg to 10 mg, 6 mg to 9 mg, or about 8 mg of LINK_TSG6 per week.

[0102] In some aspects, the treatment involves administration of 0.5 mg to 50 mg, 0.5 mg to 45 mg, 1 mg to 40 mg, 2 mg to 35 mg, 5 mg to 30 mg, 10 mg to 20 mg of LINK_TSG6, e.g., at least 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, or 45 mg of LINK_TSG6 per week.

[0103] formulation The medicaments and pharmaceutical compositions according to aspects of the present invention may be formulated for administration by many routes, including, but not limited to, subcutaneous, intraarticular, parenteral, intravenous, intraarterial, intramuscular, oral, and nasal. The medicaments and compositions may be formulated in fluid or solid form. Fluid formulations may be formulated for administration by injection into selected areas of the human or animal body. For example, fluid formulations may be formulated for administration by subcutaneous injection into the abdomen.

[0104] Preferably, the medicaments and pharmaceutical compositions according to aspects of the present invention are formulated for subcutaneous or intra-articular administration. Optionally, LINK_TSG6 is formulated with one or more other agents known to be useful in the treatment of OA. For example, an intra-articular formulation of LINK_TSG6 may further include one or more of corticosteroids, hyaluronic acid, or platelet-rich plasma (see Ayhan E. et al., (2014) World J Orthop. 5(3):351-361. doi:10.5312 / wjo.v5.i3.351).

[0105] Pharmaceutical compositions can be prepared using pharma- ceutically acceptable "carriers" that are composed of materials that are considered safe and effective. "Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe," e.g., physiologically acceptable when administered to humans, and do not usually cause allergic or similar undesirable reactions, such as stomach upset, loss or alteration of taste (ageusia). In some aspects, the term refers to molecular entities and compositions that have been approved by U.S. federal or state government regulatory authorities as GRAS listings under Sections 204 and 409 of the Federal Food, Drug, and Cosmetic Act, subject to premarket review and approval by the FDA, or similar listings in the United States Pharmacopeia, or another generally accepted pharmacopoeia, for use in animals, particularly humans.

[0106] The term "carrier" refers to diluents, binders, lubricants, and disintegrants. Those skilled in the art are familiar with such pharmaceutical carriers and how to formulate pharmaceutical compositions using such carriers.

[0107] The pharmaceutical compositions provided herein may include one or more excipients, such as solvents, dissolution enhancers, suspending agents, buffers, tonicity agents, antioxidants, or antimicrobial preservatives. When used, the excipients of the composition do not adversely affect the stability, bioavailability, safety, and / or efficacy of the active ingredient, i.e., LINK_TSG6, used in the composition. Thus, a person skilled in the art will understand that a composition is provided in which there is no incompatibility between any of the components of the dosage form. The excipients may be selected from the group consisting of buffers, solubilizers, tonicity agents, chelating agents, antioxidants, antimicrobial agents, and preservatives.

[0108] The features disclosed in the foregoing description, the following claims, or the accompanying drawings and, where appropriate, expressed in a specific form thereof, or in a means for performing a disclosed function, or in a method or step for obtaining a disclosed result, may be utilized separately or in any combination of such features to realize the invention in various of its forms.

[0109] While the present invention has been described in conjunction with the exemplary embodiments set forth above, many equivalent modifications and variations will be apparent to those skilled in the art given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes may be made to the described embodiments without departing from the spirit and scope of the invention.

[0110] For the avoidance of doubt, any theoretical explanations provided herein are provided for the purpose of enhancing the understanding of the reader, and the inventors do not wish to be bound by any of these theoretical explanations.

[0111] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Throughout this specification, including the following claims, unless the context requires otherwise, the words "comprisie" and "include", and variations such as "comprises", "icomprising" and "including", will be understood to mean the inclusion of a specified integer, or step, or group of integers or steps, but not to the exclusion of any other integer, or step, or group of integers or steps.

[0112] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, ranges may be expressed as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, using the antecedent "about," it will be understood that the particular value forms another embodiment. The term "about" with respect to numerical values ​​is optional and means, for example, + / - 10%. EXAMPLES

[0113] Example 1 Mouse pMx model To test whether Link_TSG6 reduces direct pain caused directly by OA, partial medial meniscectomy (pMx) (Knights, Gentry & Bevan, 2012 -292 Pain 153, 281) was performed in one of the stifle joints of 10-week-old C57BL / 6 male mice, producing progressive degenerative joint disease and concomitant evoked pain behavior. Under general anesthesia, the leg was held in an extended position and a medial parapatellar skin incision (~3 mm) and arthrotomy were performed using a scalpel blade. The collateral ligaments were left intact and the patella was not displaced. Using a light microscope and an angled 23-gauge needle, the attachment of the medial meniscus to the edge of the tibial plateau was removed and approximately half of the medial meniscus (~1 mm of tissue) was removed. The skin incision was closed with wound closure clips. Sham-operated animals underwent the same skin incision and arthrotomy, but the meniscus was left intact. Link_TSG6 (dissolved in PBS; "vehicle") was administered by subcutaneous (sc; in the nape of the neck) or intra-articular (ia) injection at high (220 μg sc / month; 40 μg ia / month) or low (55 μg sc / month; 10 μg ia / month) doses, once per week or once per month, from 1 week to 12 weeks after surgery. Vehicle was administered to the control group using the same route and frequency. All groups contained 10 mice.

[0114] Pain responses (relative mechanical / thermal sensitivity in the foot to knee flexion and weight bearing on the operated leg) were assessed weekly over the 12-week treatment period. Data were analyzed using one-way ANOVA with Tukey's post-hoc test ( * p<0.05, ** p<0.01, *** p<0.0001).

[0115] Example 2 Link_TSG6 directly reduces OA joint pain. Knee flexion was used to assess direct pain in arthritic or sham-operated knees. Mice were gently restrained with one hand. The thigh was placed in a fixed position and the researcher's thumb and index finger were used to passively flex and extend the knee through its full range of motion 10 times. The number of audible calls for each limb was recorded.

[0116] As shown in Figure 2, subcutaneous administration of Link_TSG6 (high dose once a month) reduces direct OA joint pain (assessed by pain response to flexion of the arthritic knee) to sham levels in the mouse pMx model. Low and high doses administered once a week also have significant therapeutic effects.

[0117] Example 3 Link TSG6 reduces heat sensitivity. Cold sensitivity was used to assess referred pain to the paw by methods described in (Gentry, Stoakley et al. (2010) Mol Pain 6:4) using a commercially available cold plate (Ugo Basile; Milan, Italy). The cold plate was set according to previously determined calibration curve data using a surface temperature probe to correlate the set temperature to the actual surface temperature. The cold plate was allowed to stabilize at the set temperature for 5 minutes prior to testing. Paw withdrawal latency was determined with the cold plate set at 10°C. Animals were lightly restrained and each hind paw was placed in turn on the surface of the cold plate. The endpoint was paw withdrawal, recorded in seconds as the latency to withdraw the ipsilateral (operated leg) or contralateral (non-operated leg) paw. A maximum cutoff of 30 seconds was used for each paw.

[0118] Link_TSG6 subcutaneous treatment dose-dependently reduces thermal sensitivity in the murine pMx model (assessed by paw withdrawal threshold to cold (10 °C) stimulation), with weekly (Figure 3A) and monthly (Figure 3B) treatments producing similar effects in the ipsilateral (surgically operated) paw. Here, thermal sensitivity is reduced below sham levels, indicating that Link_TSG6 has analgesic effects, acting at least in part via the CNS. Consistent with this, significant analgesic effects are also evident in the contralateral paw in both weekly (Figure 3A) and monthly (Figure 3B) treatments.

[0119] Example 4 Link_TSG6 reduces mechanical sensitivity. Mechanical hyperalgesia, as an index of referred pain to the paw, was assessed by measuring the paw withdrawal threshold to increasing pressure stimuli applied to the dorsal surface of the hindpaw using an analgesia measuring apparatus (model 7200, Ugo Basile; Milan, Italy) with a dome-shaped probe and a cut-off of 150 grams (Randall and Selitto (1957) Arch. Int. Pharmacodyn. CXI(4):409-419). Paw withdrawal thresholds (in grams) were measured in the ipsilateral and contralateral paws.

[0120] Link_TSG6 subcutaneous treatment dose-dependently reduces mechanical sensitivity (assessed by paw withdrawal threshold to mechanical pressure), with weekly (Figure 4A) and monthly (Figure 4B) treatments producing similar effects in the ipsilateral (surgically operated) paw. Here, mechanical sensitivity is reduced below sham levels, indicating that Link_TSG6 has an analgesic effect, acting at least in part via the CNS. Consistent with this, significant analgesic effects are also evident in the contralateral paw in both weekly (Figure 3A) and monthly (Figure 3B) treatments.

[0121] Example 5 Link TSG6 improves weight bearing in a dose-dependent manner. The difference in weight borne by the ipsilateral compared to the contralateral hind paw was measured using a dual channel weight averager (Linton Instrumentation; Diss, Norfolk, UK). Mice were individually placed into Perspex chambers designed for hind paw rearing, with each paw placed on a separate transducer pad. The transducer pads record the animal's weight distribution onto each paw for 3 seconds. Animals were acclimated to the apparatus at least twice before measurements were taken. Results are expressed as weight bearing difference (contralateral minus ipsilateral) in grams.

[0122] Link_TSG6 (once per week) dose-dependently improves weight bearing (combined direct and referred pain effects) on the "arthritic" (operated) leg (Figure 5A). This effect was observed using subcutaneous (top) and intra-articular delivery (bottom). A dose-dependent effect of Link_TSG6 on weight bearing was also seen with monthly dosing (Figure 5B), with the higher dose (sc) maintaining weight bearing differences close to sham levels throughout the 12-week study period, and higher intra-articular administration also produced significant effects reaching sham levels at later time points.

[0123] Example 6 Link_TSG6 suppresses tactile allodynia in the ACLTpMMx model. We investigated whether the reduction in joint damage in Link_TSG6-treated animals was associated with an effect on OA symptoms. ACLTpMMx rats (12-week-old male Lewis rats with OA induced by anterior cruciate ligament transection with partial (30%) medial meniscectomy) were treated with Link_TSG6 (40 μg / week, ia) and at 4 weeks, touch-evoked pain (tactile allodynia) was assessed in the paws of the surgically destabilized leg (right) and the non-operated control leg (left) as an index of secondary hyperalgesia. Paw withdrawal responses in the non-operated leg (with and without Link_TSG6 treatment) were essentially identical to those of sham-operated control animals (Figure 6A). However, sensitivity to punctate mechanical stimuli was significantly increased in the ACLTpMMx-operated leg (treated with vehicle). This response was significantly attenuated by intra-articular injection of Link_TSG6 (Figure 6B). There was no correlation between joint lesion area and the degree of tactile allodynia (data not shown), which is consistent with human OA, where a significant proportion of individuals with extensive radiation damage do not experience pain.

[0124] Example 7 A method for extrapolating intra-articular administration from mice to humans Several methods have been proposed in the art for extrapolating intra-articular doses in mice to humans. In particular, WO2015085706 describes a method based on comparison of body weight and drug concentration, and Emami et al. (2018, Int J Toxicol. 37(1):4-27) describe a method based on virtual space considerations.

[0125] 7.1 Dosage based on weight comparison Equivalent human doses were calculated using a method based on weight comparisons, as described in WO2015085706. Human dosing can be extrapolated from animal doses based on established relationships between metabolism, body size, and body weight in various species. Thus, the following formula can be used to scale up doses from one species to another using the ratio of 3 / 4 power of body weight: Dose(human) = Dose(mouse) x [Human weight] 0.75 [Mouse weight] 0.75 As described in WO2015085706, a typical human is believed to weigh 70 kg. The mice used in the study are believed to weigh approximately 20 g each.

[0126] Using the formula above and based on these body weights, an intra-articular monthly low dose of 10 μg / month would correspond to approximately 4.56 mg per human per month: 4,555μg(4.56mg)=10μg×

[70] 0.75 [0.02] 0.75 Based on these body weights, an intra-articular monthly high dose of 40 μg / month would correspond to approximately 18.2 mg per human per month: 18,202μg(18.20mg)=40μg×

[70] 0.75 [0.02] 0.75 Based on these calculations, a low dose may be 1.14 mg per person per week, or a high dose may be 4.55 mg per person per week. Higher doses may be required if the dosing frequency is less frequent, such as once every 8 weeks.

[0127] A typical injection volume for use in humans is believed to be 2-10 ml (see WO2015085706). Therefore, a typical drug concentration that would be effective in human subjects was calculated as follows: For 2ml injections: Low dose (monthly) = 2.28mg / ml. High dose (monthly) = 9.11mg / ml. For a 10ml injection: Low dose (monthly) = 0.46mg / ml. High dose (monthly) = 1.82mg / ml. For 2ml injection: Low dose (weekly) = 0.57mg / ml. High dose (weekly) = 2.28mg / ml. For a 10ml injection: Low dose (weekly) = 0.114mg / ml. High dose (weekly) = 0.46mg / ml.

[0128] Calculations were performed for 70 kg, but considering that many OA patients are overweight or obese, higher doses are likely to be required, therefore, adjusted doses were calculated based on other weights: 60 kg, 80 kg, 90 kg, 100 kg, 110 kg, and 120 kg, creating a range of values.

[0129] Examples of monthly dosages are calculated for human weights: 60 kg (low dose 4.05 mg, high dose 16.21 mg); 80 kg (low 5.03 mg, high 20.12 mg); 90 kg (low 5.50 mg, high 21.98 mg); 100 kg (low 5.95 mg, high 23.78 mg); 110 kg (low 6.39 mg, high 25.55 mg), and 120 kg (low 6.82 mg, high 27.27 mg).

[0130] Thus, the monthly dose range based on low and high dose data in mice and extrapolation to human body weight may be 4.05 mg to 27.27 mg. The weekly dose range based on low and high dose data in mice and extrapolation to human body weight may be 1.01 mg to 13.64 mg. Higher doses can be extrapolated if administered less frequently than once a month.

[0131] 7.2 Dosage based on "virtual space" considerations The human equivalent dose for intra-articular administration was calculated using a virtual space method (based on Emami et al., Int J Toxicol. 2018;37(1):4-27. doi:10.1177 / 1091581817741840). This virtual space method takes into account the difference in the size of the intra-articular space between mice and humans and adjusts the dose so that the drug concentration in each joint cavity is equivalent. A hypothetical example illustrating this dose adjustment method is shown in Emami et al., 2018, Figure 7.

[0132] The calculation of the equivalent dose based on virtual space considerations is based on the assumption that the administration site (e.g., the intra-articular space of a joint) is a virtual fluid-filled container into which the formulation is introduced and diffuses throughout, reaching a steady-state concentration where the drug is present for some time, before finally being removed from the area. Therefore, the size of the virtual space in the mouse OA model was estimated and then adjusted to the virtual space where the drug is administered, i.e., the virtual space within the joint of an OA patient. As the steady-state concentration of Link_TSG6 in synovial fluid is unknown (neither in mice nor in humans), we used the concentration at the time of injection to adjust from mouse to human doses, and assumed that the clearance kinetics and steady-state values ​​are the same in these two species.

[0133] To estimate the virtual space of the mouse model, mouse synovial fluid volume was considered to be 3-5 μl (based on Xu et al. (2017) Bone Res. 5:17012). Human synovial fluid volume in OA patients was considered to be between 0.56 and 71.71 ml (median 3.05 ml) (cited from Kraus et al. (2007) Osteoarthritis & Cartilage. 15;1217-1220). Therefore, in the exemplary calculation, it was assumed that the mouse SF volume was 4 μl and the human SF volume in OA patients was 3 ml. In the pMx study, 5 μl was injected into the mouse knee joint per injection. Equivalent doses were calculated based on injection volumes of 2-10 ml considered to be typically used in humans. (See patent text of WO2015085706).

[0134] 7.2.1 Monthly dose Assuming a synovial fluid volume (SF) in mice of 4 μl SF, then injecting 5 μl, the total volume is 9 μl. The intra-articular low dose was 10 μg / month, administered in a total volume of 9 μl, or 1.11 mg / ml. The intra-articular high dose was 40 μg / month, administered in a total volume of 9 μl, or 4.44 mg / ml.

[0135] As mentioned above, SF volumes are known to vary between 0.56 and 71.71 ml (median 3.05 ml) in OA patients. Considering a median SF of approximately 3 ml, an injection of 2 ml would result in a total volume of 5 ml, meaning that a dose of 1.11 mg / ml would require a 2 ml injection of 2.78 mg / ml (5.56 mg) or an equivalent lower dose in humans would require a 10 ml injection of 1.44 mg / ml (14.4 mg).

[0136] A higher dose equivalent to 4.44mg / ml would require a 2ml injection of 11.1mg / ml or a 10ml injection of 5.77mg / ml (i.e. 22.2mg in 2ml or 57.7mg in 10ml).

[0137] If the amount of human SF is different, it can be adjusted accordingly. For different amounts of human SF, the dose ranges of 2 ml and 10 ml injection volumes were also calculated.

[0138] [Table 2]

[0139] 7.2.2 Weekly dose Assuming a synovial fluid volume (SF) in mice of 4 μl SF, if 5 μl is injected, then the total volume is 9 μl. The intra-articular low dose was administered at 2.5 μg / week in a total volume of 9 μl, or 0.28 mg / ml. The intra-articular high dose was administered at 10 μg / month in a total volume of 9 μl, or 1.11 mg / ml.

[0140] Using the method outlined in 6.2.1 above, the human equivalent weekly dose is calculated as follows:

[0141] [Table 3]

[0142] 7.2.3 Dose range Therefore, the equivalent dose range for 1-30 ml SF (and 2 or 10 ml injections) based on low and high dose data in mice could be 3.34 mg to 177.6 mg per month. Therefore, the weekly dose range for 1-30 ml SF (and 2 or 10 ml injections) based on low and high dose data in mice could be 0.84 mg to 44.4 mg. Higher doses can be extrapolated for less frequent than monthly dosing. References Several publications have been cited above in order to more fully describe and disclose the present invention and the state of the art to which it pertains. These references are provided in their entirety below. Each of these references is incorporated herein in its entirety.

[0143] [Table 4]

Claims

1. A pharmaceutical composition comprising LINK_TSG6 for use in treating osteoarthritis pain in a patient in need thereof.

2. The pharmaceutical composition of claim 1, wherein the LINK_TSG6 is administered subcutaneously or intraarticularly.

3. 3. The pharmaceutical composition of claim 1 or claim 2, wherein the LINK_TSG6 is administered once a week or less frequently.

4. 3. The pharmaceutical composition of claim 1 or claim 2, wherein the osteoarthritis pain is referred pain.

5. 4. The pharmaceutical composition of claim 3, wherein the pharmaceutical composition is administered once every 7 days, once every 10 days, once every 14 days, once every 21 days, once every 30 days, once every 45 days, once every 60 days, once every 90 days, or less than once every 90 days.

6. 3. The pharmaceutical composition of claim 1 or claim 2, wherein the LINK_TSG6 polypeptide comprises, consists of, or consists essentially of (i) the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9, or (ii) an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:

9.

7. A pharmaceutical composition comprising LINK_TSG6, the pharmaceutical composition being formulated for subcutaneous administration.

8. 8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition comprises 10 mg to 150 mg of LINK_TSG6 per dose.

9. A pharmaceutical composition comprising LINK_TSG6, the pharmaceutical composition being formulated for intra-articular administration.

10. 10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition comprises 1 mg to 180 mg of LINK_TSG6 per dose.

11. Use of LINK-TSG6 in the manufacture of a medicament for the treatment of osteoarthritis pain.

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