Combination therapy comprising a composition of mesenchymal stromal cells and hyaluronic acid for use in the treatment of osteoarthritis

Abstract

The present disclosure relates to the intra-articular administration of a composition comprising pooled mesenchymal stromal cells and a combination of medium molecular weight hyaluronic acid under ultrasound guidance for the treatment of osteoarthritis.The present disclosure also provides a method for increasing the level of anti-inflammatory markers and reducing the level of disease progression markers in human subjects suffering from osteoarthritis by using the combination.Therefore, the present invention also relates to a kit comprising the composition and hyaluronic acid itself for the above use and treatment.

Description

[Technical Field]

[0001] The present disclosure relates to the field of combination therapy for the treatment of osteoarthritis.More specifically, the present disclosure relates to the ultrasound-guided intra-articular administration of a composition comprising pooled mesenchymal stromal cells in combination with medium-molecular-weight hyaluronic acid for the treatment of osteoarthritis.The present disclosure also provides a method for increasing the level of anti-inflammatory markers and reducing the level of disease progression markers in human subjects suffering from osteoarthritis by using the combination.Therefore, the present invention also relates to a kit comprising the composition and hyaluronic acid itself for the above use and treatment.

[0002] Background of the disclosure Osteoarthritis (OA) is a degenerative joint disease that can affect many tissues in the joint. Historically, osteoarthritis was generally known as a condition of "wear and tear" associated with aging. However, it is now understood to be a disease of the entire joint, including bone, cartilage, ligaments, fat, and the tissue lining the joint (synovium).

[0003] Symptoms of OA often progress slowly and worsen over time and include pain, stiffness, tenderness, loss of flexibility, a grating sensation, bone spurs and swelling.

[0004] There is currently no cure for OA, but medications, assistive devices, and other non-drug therapies can help relieve pain. As a last resort, damaged joints may be surgically stabilized or replaced with ones made from a combination of metal, plastic, and / or ceramic.

[0005] Therefore, current OA treatments focus on symptom management. The most effective type of treatment varies greatly depending on the severity of the symptoms and their location. Mild cases of OA can be treated with a combination of non-pharmacological (e.g., physical therapy) and pharmacological therapies to reduce pain and inflammation. However, as the disease progresses, more aggressive treatments become necessary. The most common medications used to treat OA include analgesics; nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, naproxen, and celecoxib; anti-irritants such as products containing capsaicin, menthol, and lidocaine; corticosteroids; and platelet-rich plasma (PRP) therapy.

[0006] Although some patients experience temporary relief, the effectiveness of these interventions varies and is highly controversial. In more advanced or severe cases of OA, surgical replacement is the only viable treatment option. Therefore, current OA treatments aim only to reduce pain, maintain mobility, and minimize disability. However, none of these approaches actually cause significant regeneration of damaged cartilage or provide long-term relief without the need for regular therapeutic intervention. The present disclosure provides compositions and methods aimed at solving this unmet need. Summary of the Invention

[0007] The present disclosure relates to a method of treating osteoarthritis in a human subject, the method comprising administering to the subject a combination of: a composition comprising a protein-free, serum-free, animal component-free formulation of approximately 20 to 30 million mesenchymal stromal cells, a plurality of electrolyte solutions, and dimethyl sulfoxide (DMSO); and Medium molecular weight hyaluronic acid The method includes administering

[0008] In some embodiments of the present disclosure, the composition does not contain human serum albumin (HSA); and the mesenchymal stromal cells are bone marrow-derived pooled mesenchymal stromal cells obtained by pooling mesenchymal stromal cells from at least three donors.

[0009] In some embodiments of the present disclosure, the composition comprises a protein-free, serum-free, animal component-free formulation of about 20-30 million mesenchymal stromal cells, about 1 ml of a plurality of electrolyte solutions, and about 1 ml of about 2.5%-5% dimethyl sulfoxide (DMSO).

[0010] In some embodiments of the present disclosure, administration of the composition or hyaluronic acid or both is ultrasound-guided via an intra-articular route.

[0011] In some embodiments of the present disclosure, the combination provides the subject with an improvement in an osteoarthritis symptom selected from the group consisting of pain and stiffness; and improves or maintains each cartilage quality parameter in the subject compared to the subject before administration of the combination.

[0012] The present invention also relates to a kit comprising a combination product or combination as described above.

[0013] In some embodiments of the present invention, the combination product or kit comprises a composition as described above together with about 2 ml of hyaluronic acid.

[0014] In some embodiments of the present disclosure, such combinations are employed for use in methods of treating osteoarthritis in human subjects.

[0015] In some embodiments of the present disclosure, such combinations are used for use in a method of increasing levels of interleukin-10 (IL-10), an anti-inflammatory marker, and / or decreasing levels of cross-linked C-telopeptide of type II collagen (CTX-II), a disease progression marker, in a human subject suffering from osteoarthritis.

[0016] Accordingly, the present disclosure also relates to a method for increasing the level of interleukin-10 (IL-10), an anti-inflammatory marker, and / or decreasing the level of cross-linked C-telopeptide of type II collagen (CTX-II), a disease progression marker, in a human subject suffering from osteoarthritis, comprising administering to the subject a combination as defined above.

[0017] In some embodiments of the present disclosure, the level of the marker is increased and / or decreased compared to the subject prior to administration of the combination.

[0018] In some embodiments of the present disclosure, administration of the composition or hyaluronic acid or both is ultrasound-guided via an intra-articular route.

[0019] So that the present disclosure may be readily understood and put into practice, reference will now be made to exemplary embodiments, which are illustrated in conjunction with the accompanying drawings, and which, together with the following detailed description, are incorporated in and constitute a part of this specification and serve to further illustrate the embodiments and explain various principles and advantages. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 shows TSP-2 secretion levels for unprimed Stempeucel® (in the absence of IFN-γ and TNF-α) and primed Stempeucel® (in the presence of IFN-γ and TNF-α). [Figure 2]Figure 2 shows the secretion levels of TSP-2 and PGE-2 in the primed state (in the presence of IFN-γ and TNF-α); the secretion levels of TSP-2 and PGE-2 in the unprimed state; and the TNF-α levels in the primed samples. [Figure 3] FIG. 3 shows the relevance of TSP-2 as a potential indicator for determining the efficacy of stempeucel® for the indication of OA. DETAILED DESCRIPTION OF THE INVENTION

[0021] Unless otherwise defined, all terms used in this disclosure, including technical and scientific terms, have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. For further guidance, definitions of several terms are included to facilitate a better understanding of this disclosure.

[0022] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0023] As used herein, the terms "comprising," "comprises," or "comprised of" are synonymous with "including," "includes," "containing," or "contains," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0024] The recitation of numerical ranges by endpoints includes not only the recited endpoints but also all numbers and fractions subsumed within each range.

[0025] As used herein, the term "about" when referring to a measurable value such as a parameter, amount, duration, etc., is meant to encompass a variation of no more than ±10%, preferably no more than ±5%, more preferably no more than ±1%, and even more preferably no more than ±0.1% from the specified value, insofar as such variation is appropriate for the practice of the present disclosure. It is to be understood that the value referred to by the modifier "about" is itself specifically and preferably disclosed.

[0026] As used herein, terms such as "treat" or "treatment" when used in reference to the disease osteoarthritis in this disclosure are intended to refer to the ordinary meaning of the term as known to those skilled in the art. This term is used broadly in this disclosure to refer to the management of osteoarthritis after a diagnosis to provide partial or complete relief to patients suffering from one or more symptoms commonly associated with osteoarthritis. This term is also used to refer to a medical regimen that can cure, eliminate, or reduce the level of osteoarthritis or its associated clinical / biological markers, or slow the progression of such a disease. Those skilled in the art will understand the manner and mechanisms for determining whether a regimen has led to successful treatment; an example of such a mechanism is comparing the rate of change in symptoms from baseline before and after administration of a treatment regimen.

[0027] To meet the need in the art for patients suffering from osteoarthritis (OA) and to fill a gap that exists in the current course of treatment for such patients, the present disclosure provides a combination therapy for treating osteoarthritis in a human subject, the method comprising administering to the subject a combination of: a composition comprising a protein-free, serum-free, animal component-free formulation of approximately 20 to 30 million mesenchymal stromal cells, a plurality of electrolyte solutions, and dimethyl sulfoxide (DMSO); and Medium molecular weight hyaluronic acid The method includes administering

[0028] In some embodiments, the compositions used as part of the combination therapy of the present disclosure do not include human serum albumin (HSA).

[0029] In some embodiments, the mesenchymal stromal cells that form the core component of the compositions of the present disclosure are bone marrow-derived pooled mesenchymal cells obtained by pooling mesenchymal stromal cells from at least three donors.

[0030] In some embodiments, administration of the composition or hyaluronic acid or both as part of the combination therapy of the present disclosure is ultrasound-guided via an intra-articular route.

[0031] Thus, more specifically, the present disclosure provides a method of treating osteoarthritis in a human subject, comprising administering to the subject a combination of: a composition comprising a protein-free, serum-free, animal component-free formulation of approximately 20 to 30 million mesenchymal stromal cells, a plurality of electrolyte solutions, and dimethyl sulfoxide (DMSO); and Medium molecular weight hyaluronic acid administering The method includes administering the composition, wherein the composition does not contain human serum albumin (HSA); the mesenchymal stromal cells are bone marrow-derived pooled mesenchymal cells obtained by pooling mesenchymal stromal cells from at least three donors; and administering the composition, hyaluronic acid, or both, via an intra-articular route under ultrasound guidance.

[0032] In some embodiments, the osteoarthritis treated by the combination therapy of the present disclosure is grade II or grade III osteoarthritis.

[0033] In some embodiments, the composition comprises about 25 million pooled mesenchymal stromal cells.

[0034] In some embodiments, the multiple electrolyte solution present in the combination therapy composition of the present disclosure is Plasmalyte A.

[0035] In some embodiments, the multiple electrolyte solutions are present in the combination therapy compositions of the present disclosure in an amount of about 1 ml.

[0036] In some embodiments, DMSO is present in the disclosed combination therapy composition in an amount of about 1 ml.

[0037] In some embodiments, DMSO is present in the disclosed combination therapy composition as a solution having a concentration of about 2.5%.

[0038] In some embodiments, DMSO is present in the disclosed combination therapy compositions as a solution having a concentration of about 5%.

[0039] Thus, in some embodiments, the combination therapy of the present disclosure comprises administering a composition comprising about 20-30 million mesenchymal stromal cells, about 1 ml of a plurality of electrolyte solutions, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 2.5%-5% dimethyl sulfoxide (DMSO); and about 2 ml of hyaluronic acid, wherein the composition does not contain human serum albumin (HSA).

[0040] In some embodiments, the combination therapy of the present disclosure comprises administering a composition consisting of about 20-30 million mesenchymal stromal cells, about 1 ml of a plurality of electrolyte solutions, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 2.5%-5% dimethyl sulfoxide (DMSO); and about 2 ml of hyaluronic acid, wherein the composition does not contain human serum albumin (HSA).

[0041] Thus, in some embodiments, the combination therapy of the present disclosure comprises administering a composition comprising about 25 million mesenchymal stromal cells, about 1 ml of a plurality of electrolyte solutions, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 2.5% to 5% dimethyl sulfoxide (DMSO); and about 2 ml of hyaluronic acid, wherein the composition does not contain human serum albumin (HSA).

[0042] In some embodiments, the combination therapy of the present disclosure comprises administering a composition consisting of about 25 million mesenchymal stromal cells, about 1 ml of a plurality of electrolyte solutions, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 2.5% to 5% dimethyl sulfoxide (DMSO); and about 2 ml of hyaluronic acid, wherein the composition does not contain human serum albumin (HSA).

[0043] In some embodiments, the combination therapy of the present disclosure comprises administering a composition consisting of about 25 million mesenchymal stromal cells, about 1 ml of a multiple electrolyte solution, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 2.5% dimethyl sulfoxide (DMSO); and about 2 ml of hyaluronic acid, wherein the composition does not contain human serum albumin (HSA).

[0044] In some embodiments, the combination therapy of the present disclosure comprises administering a composition consisting of about 25 million mesenchymal stromal cells, about 1 ml of a multiple electrolyte solution, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 5% dimethyl sulfoxide (DMSO); and about 2 ml of hyaluronic acid, wherein the composition does not contain human serum albumin (HSA).

[0045] In some embodiments, it is the composition that is administered via a single intra-articular administration. Alternatively, in some embodiments, it is hyaluronic acid that is administered via a single intra-articular administration.

[0046] Thus, if only one of the composition or hyaluronic acid is administered via ultrasound-guided intra-articular administration, the other component is administered via conventional single-dose intra-articular administration.

[0047] In some embodiments, both the composition and the hyaluronic acid are administered via ultrasound-guided intra-articular administration.

[0048] In some embodiments, both the composition and hyaluronic acid are administered via ultrasound-guided intra-articular injection, injected one after the other through the same needle.

[0049] Ultrasound-guided administration is a non-invasive, portable, radiation-free administration method that provides dynamic assessment. Some of its advantages are: Real-time visualization, - Helps in effective pre-treatment planning; Helps reduce complications; and · Less time spent administering.

[0050] In some embodiments, ultrasound-guided administration was more accurate than anatomical guidance (95.8% vs. 77.8%, P<0.001), with an odds ratio of 6.4 (95% confidence interval 2.9-14). Thus, ultrasound guidance significantly improves injection accuracy in the targeted intra-articular joint space of large joints, including the knee.

[0051] Nevertheless, regardless of how the composition and hyaluronic acid are administered, the administration remains a single administration for both the composition and hyaluronic acid, and no repeated administration is required for a period of about 6 months from the first administration.Therefore, a single administration of the composition and hyaluronic acid remains sufficient for treating osteoarthritis according to the present disclosure for up to about 6 months.

[0052] In some embodiments, repeated administration is not required for up to about 12 months from the initial administration. Thus, a single administration of the composition and hyaluronic acid remains sufficient for treating osteoarthritis according to the present disclosure for up to about 12 months. In some embodiments, the composition and hyaluronic acid are administered simultaneously as part of the combination therapy of the present disclosure.

[0053] In some embodiments, the composition and hyaluronic acid are administered sequentially as part of a combination therapy of the present disclosure. In such cases, administration of the hyaluronic acid can follow or precede administration of the composition.

[0054] In some embodiments, when the composition and hyaluronic acid are administered sequentially as part of a combination therapy of the present disclosure, the hyaluronic acid is administered immediately after administration of the composition.

[0055] In some embodiments, when the composition and hyaluronic acid are administered sequentially as part of a combination therapy of the present disclosure, the hyaluronic acid is administered between about 1 second and about 30 minutes after administration of the composition, or within 1800 seconds of administration of the composition.

[0056] In some embodiments, when the composition and hyaluronic acid are administered sequentially as part of a combination therapy of the present disclosure, the hyaluronic acid is administered within 60 seconds of administration of the composition.

[0057] In some embodiments, when the composition and hyaluronic acid are administered sequentially as part of a combination therapy of the present disclosure, the hyaluronic acid is administered within 30 seconds of administration of the composition.

[0058] In some embodiments, when the composition and hyaluronic acid are administered sequentially as part of a combination therapy of the present disclosure, the hyaluronic acid is administered within 10 seconds of administration of the composition.

[0059] In some embodiments, the amount of hyaluronic acid administered as part of the combination therapy of the present disclosure is about 2 ml.

[0060] In some embodiments, the hyaluronic acid administered comprises 20 mg of hyaluronic acid administered in 2 ml of buffered saline having a pH of 6.8 to 7.5.

[0061] The combination therapy of the present disclosure provides a subject with an improvement in a symptom of osteoarthritis selected from the group consisting of pain and stiffness; and improves or maintains each cartilage quality parameter in the subject compared to the subject prior to administration of the combination.

[0062] In some embodiments, the cartilage quality parameter is selected from the group comprising articular cartilage quality such as areas of altered composition and ultrastructure, cartilage thickness, cartilage morphology, and cartilage volume.

[0063] In some embodiments, improvement in one or more osteoarthritis symptoms is measured by comparing the change in a subject's baseline values ​​before and after treatment with a combination therapy of the present disclosure.

[0064] In some embodiments, improvement in one or more osteoarthritis symptoms is measured by comparing the change in baseline values ​​of a subject treated with a combination therapy of the present disclosure to another subject with the same or similar level of disease but not treated with a combination therapy of the present disclosure.

[0065] In some embodiments, the osteoarthritis symptoms improved by the combination therapy of the present disclosure are measured by a score selected from the group including the WOMAC composite index score, the WOMAC pain index score, the WOMAC stiffness index score, the WOMAC physical function index score, and the VAS score, or any combination thereof. As is well understood in the osteoarthritis field, the WOMAC or The Western Ontario and McMaster Universities Osteoarthritis Index score is a widely used, proprietary, standardized set of questionnaires used by medical professionals to assess the status of patients with osteoarthritis of the knee and hip, including joint pain, stiffness, and physical function.

[0066] Thus, in some embodiments, the osteoarthritis symptoms improved by the combination therapy of the present disclosure are measured by the WOMAC Composite Index score.

[0067] In some embodiments, the combination therapy of the present disclosure improves the mean percent change from baseline in WOMAC composite index score by about 26% over a span of about 6 months.

[0068] In some embodiments, the combination therapies of the present disclosure demonstrate a mean difference in percent change from baseline in WOMAC composite index score of at least about 20% at 6 months.

[0069] In some embodiments, the combination therapy of the present disclosure demonstrates a mean difference in percent change from baseline in WOMAC composite index score of about 23% at 6 months.

[0070] In some embodiments, the combination therapy of the present disclosure demonstrates a mean difference in percent change from baseline in WOMAC composite index score of at least about 40% at 12 months.

[0071] In some embodiments, the combination therapy of the present disclosure demonstrates a mean difference in percent change from baseline in WOMAC composite index score of about 45% at 12 months.

[0072] Similarly, in some embodiments, the osteoarthritis symptoms improved by the combination therapy of the present disclosure are measured by the WOMAC Pain Index score.

[0073] In some embodiments, the combination therapy of the present disclosure improves the mean percent change from baseline in WOMAC Pain Index scores by about 27% over a span of about 6 months.

[0074] In some embodiments, the combination therapies of the present disclosure demonstrate a mean difference in percent change from baseline in WOMAC Pain Index scores of at least about 20% at 6 months.

[0075] In some embodiments, the combination therapy of the present disclosure demonstrates a mean difference in percent change from baseline in WOMAC Pain Index scores of about 26% at 6 months.

[0076] In some embodiments, the combination therapies of the present disclosure demonstrate a mean difference in percent change from baseline in WOMAC Pain Index scores of at least about 40% at 12 months.

[0077] In some embodiments, the combination therapy of the present disclosure demonstrates a mean difference in percent change from baseline in WOMAC Pain Index scores of about 46% at 12 months.

[0078] Furthermore, in some embodiments, the osteoarthritis symptoms improved by the combination therapy of the present disclosure are measured by the WOMAC Stiffness Index score.

[0079] In some embodiments, the combination therapy of the present disclosure improves the mean percent change from baseline in WOMAC Stiffness Index score by about 11% over a span of about 6 months.

[0080] In some embodiments, the combination therapies of the present disclosure demonstrate a mean difference in percent change from baseline in WOMAC Stiffness Index scores of at least about 25% at 6 months.

[0081] In some embodiments, the combination therapy of the present disclosure demonstrates a mean difference in percent change from baseline in WOMAC Stiffness Index scores of about 31% at 6 months.

[0082] In some embodiments, the combination therapies of the present disclosure demonstrate a mean difference in percent change from baseline in WOMAC Stiffness Index scores of at least about 50% at 12 months.

[0083] In some embodiments, the combination therapy of the present disclosure demonstrates a mean difference in percent change from baseline in WOMAC Stiffness Index scores of about 55% at 12 months.

[0084] Furthermore, in some embodiments, the osteoarthritis symptoms improved by the combination therapy of the present disclosure are measured by the WOMAC Physical Function Index score.

[0085] In some embodiments, the combination therapy of the present disclosure improves the mean percent change from baseline in WOMAC Physical Function Index score by about 27% over a span of about 6 months.

[0086] In some embodiments, the combination therapies of the present disclosure demonstrate a mean difference in percent change from baseline in WOMAC Physical Function Index scores of at least about 15% at 6 months.

[0087] In some embodiments, the combination therapy of the present disclosure demonstrates a mean difference in percent change from baseline in WOMAC Physical Function Index scores of about 22% at 6 months.

[0088] In some embodiments, the combination therapies of the present disclosure demonstrate a mean difference in percent change from baseline in WOMAC Physical Function Index scores of at least about 40% at 12 months.

[0089] In some embodiments, the combination therapy of the present disclosure demonstrates a mean difference in percent change from baseline in WOMAC Physical Function Index scores of about 45% at 12 months.

[0090] Furthermore, in some embodiments, the osteoarthritis symptoms improved by the combination therapy of the present disclosure are measured by VAS score.

[0091] In some embodiments, the combination therapy of the present disclosure improves the mean percent change from baseline in VAS score by about 26% over a span of about 6 months.

[0092] In some embodiments, the combination therapies of the present disclosure demonstrate a mean difference in percent change from baseline in VAS scores of at least about 10% at 6 months.

[0093] In some embodiments, the combination therapy of the present disclosure demonstrates a mean difference in percent change from baseline in VAS scores of about 17% at 6 months.

[0094] In some embodiments, the combination therapies of the present disclosure demonstrate a mean difference in percent change from baseline in VAS scores of at least about 35% at 12 months.

[0095] In some embodiments, the combination therapy of the present disclosure demonstrates a mean difference in percent change from baseline in VAS scores of about 41% at 12 months.

[0096] Thus, in some embodiments, the combination therapy of the present disclosure improves the mean percent change from baseline in the WOMAC composite index score and / or the WOMAC pain index score and / or the WOMAC stiffness index score and / or the WOMAC physical function index score and / or the VAS score by at least about 10% over a span of about 6 months.

[0097] Similarly, in some embodiments, the combination therapies of the present disclosure demonstrate a mean difference in percent change from baseline in WOMAC composite index score and / or WOMAC pain index score and / or WOMAC stiffness index score and / or WOMAC physical function index score and / or VAS score of at least about 15% at 6 months.

[0098] Similarly, in some embodiments, the combination therapies of the present disclosure demonstrate a mean difference in percent change from baseline in WOMAC composite index score and / or WOMAC pain index score and / or WOMAC stiffness index score and / or WOMAC physical function index score and / or VAS score of at least about 40% at 12 months.

[0099] Thus, in some embodiments, the combination therapy of the present disclosure demonstrates a mean difference in percent change from baseline (where the p-value for the mean difference is 0.001 or less) of (any one or combination of the following): · At least 20.0% at 6 months on the WOMAC composite index, WOMAC pain index, WOMAC stiffness index, and / or WOMAC physical function index; · At least 40.0% at 12 months on the WOMAC composite index, WOMAC pain index, WOMAC stiffness index, and / or WOMAC physical function index; · at least 35.0% at 12 months on a VAS measure and at least 10.0% at 6 months on a VAS measure, if selected; · At least 25.0% at 6 months on the WOMAC pain index and / or WOMAC stiffness index; · WOMAC stiffness index of at least 30.0% at 6 months; · At least 45.0% at 12 months on the WOMAC composite index, WOMAC pain index, WOMAC stiffness index, and / or WOMAC physical function index; · At least 50.0% at 12 months on the WOMAC stiffness index; · At least 55.0% at 12 months on the WOMAC stiffness index; · At least 40.0% at 12 months on a VAS measure and at least 15.0% at 6 months on a VAS measure, if selected; · 20.0% to 35.0% at 6 months for the WOMAC composite index, WOMAC pain index, WOMAC stiffness index, and / or WOMAC physical function index; · 20.0% to 30.0% at 6 months for the WOMAC composite index, WOMAC pain index, and / or WOMAC physical function index; · 40.0% to 60.0% at 12 months for the WOMAC composite index, WOMAC pain index, WOMAC stiffness index, and / or WOMAC physical function index; 40.0% to 50.0% at 12 months on the WOMAC composite index, WOMAC pain index, and / or WOMAC physical function index; and - 35.0% to 45.0% at 12 months for VAS index, 10.0% to 20.0% at 6 months for optional VAS index.

[0100] In some embodiments, the combination therapy of the present disclosure improves a parameter of cartilage quality selected from the group including articular cartilage quality such as areas of altered composition and ultrastructure, cartilage thickness, cartilage morphology, and cartilage volume.

[0101] In some embodiments, the combination therapy of the present disclosure increases the level of the anti-inflammatory marker interleukin-10 (IL-10) in a subject compared to the subject prior to administration of the combination.

[0102] In some embodiments, the combination therapy of the present disclosure reduces the level of cross-linked C-telopeptide of type II collagen (CTX-II), a marker of disease progression, in a subject compared to the subject before administration of the combination.

[0103] Thus, in some embodiments, the combination therapy of the present disclosure increases the level of interleukin-10 (IL-10), an anti-inflammatory marker, and decreases the level of cross-linked C-telopeptide of type II collagen (CTX-II), a disease progression marker, in a subject compared to the subject prior to administration of the combination.

[0104] In some embodiments, a combination therapy of the present disclosure increases the level of the anti-inflammatory marker interleukin-10 (IL-10) in a subject by about 0.051 pg / mol over a 365-day period when compared to the marker level in the subject before administration of the combination, whereas the level of IL-10 in the absence of the combination therapy decreased by about 0.228 pg / mol with disease progression in the subject.

[0105] Thus, in some embodiments, if, for comparison, the starting level of IL-10 is considered to be 0 on day 0, the combination therapy of the present disclosure improved IL-10 levels by about 5% over a 365 day period, compared to a reduction in IL-10 levels of about 22% in subjects who were not administered the combination therapy of the present disclosure.

[0106] In some embodiments, the combination therapy of the present disclosure reduces the level of cross-linked C-telopeptide of type II collagen (CTX-II), a marker of disease progression, in a subject by about 44.784 ng / mmol over a 90-day period when compared to the marker level in the subject before administration of the combination, whereas the level of CTX-II in the absence of the combination therapy increased by about 83.612 ng / mmol with disease progression in the subject.

[0107] Thus, in some embodiments, for comparison, if the starting level of CTX-II is considered to be 0 on day 0, the combination therapy of the present disclosure reduced CTX-II levels by about 44% over a 90-day period, compared to an increase in CTX-II levels of about 83% in subjects who were not administered the combination therapy of the present disclosure.

[0108] Without wishing to be bound by any particular theory, the anticipated mechanism of action of the current combination therapy for OA is as follows: The disclosed combination has the potential to secrete various paracrine factors, including PGE-2 and TSP-2, which play key roles as anti-inflammatory and pro-chondrogenic signals, respectively. When the combination was administered in an in vitro inflammatory environment in the presence of IFN-γ and TNF-α (mimicking the pro-inflammatory environment in the knee joints of OA patients), high levels of PGE-2 and low levels of TSP-2 were secreted. The PGE-2 secreted from this combination inhibits further synthesis of pro-inflammatory cytokines and modifies the inflammatory environment. As the level of pro-inflammatory cytokines decreases, the secretion of TSP-2 gradually increases. TSP-2 is known to stimulate chondrogenic progenitor cells and promote their differentiation into mature chondrocytes. At the same time, the primed MSCs as part of this combination still retain the potential to differentiate into chondrocytes. Furthermore, hyaluronic acid promotes MSC migration and adhesion. Hyaluronic acid used in conjunction with MSC compositions acts as a scaffold and aids in homing of cells to the defect site, and therefore the combination of both paracrine and chondrocyte differentiation methods can be used as a treatment option for OA.

[0109] Thus, as will be apparent to one of skill in the art from reading this disclosure, the significance of this disclosure lies in the specificity or selection of features that result in the combination therapy of the present disclosure. Accordingly, the positive effects and improvements seen in human subjects suffering from osteoarthritis after treatment with the combination therapy of the present disclosure are a direct result of the amalgamation of features including: a specific number of pooled mesenchymal stromal cells present in the composition; The number of donors from which the pooled mesenchymal stromal cells are derived; The availability of protein-free, serum-free, and animal component-free formulations of several electrolyte solutions and dimethyl sulfoxide (DMSO); Absence of human serum albumin (HSA) medium molecular weight hyaluronic acid, and A single administration of the composition or hyaluronic acid, or both, via an ultrasound-guided intra-articular route.

[0110] Here, to facilitate the administration of the above-mentioned combination therapy, the present disclosure accordingly also provides a combination product or kit comprising the two components used in the combination therapy.

[0111] Accordingly, the present disclosure also provides a combination product or kit for treating osteoarthritis in a human subject, comprising the following combination: a composition comprising about 20 to 30 million mesenchymal stromal cells, about 1 ml of a plurality of electrolyte solutions, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 2.5% to 5% dimethyl sulfoxide (DMSO); and Approximately 2ml of hyaluronic acid Including, The composition relates to a combination product or kit, wherein the composition does not contain human serum albumin (HSA); and the mesenchymal stromal cells are pooled mesenchymal cells obtained by pooling mesenchymal stromal cells from at least three donors.

[0112] In some embodiments, the amount of hyaluronic acid administered as part of the combination therapy of the present disclosure is about 2 ml.

[0113] In some embodiments, the hyaluronic acid administered comprises 20 mg of hyaluronic acid administered in 2 ml of buffered saline having a pH of 6.8 to 7.5.

[0114] In some embodiments, the combination product or kit further comprises instructions for administering the composition and hyaluronic acid during the course of a combination therapy of the present disclosure.

[0115] In some embodiments, a composition of the combination therapy, product, or kit of the disclosure comprises about 25 million pooled mesenchymal stromal cells.

[0116] Thus, in some embodiments, the composition of a combination therapy, product, or kit of the disclosure comprises about 20 million, 21 million, 22 million, 23 million, 24 million, 25 million, 26 million, 27 million, 28 million, 29 million, or 30 million pooled mesenchymal stromal cells (including any number of cells therebetween).

[0117] Thus, the composition of the disclosed combination therapy, product, or kit includes about 25 million mesenchymal stromal cells, about 1 ml of a plurality of electrolyte solutions, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 2.5% to 5% dimethyl sulfoxide (DMSO).

[0118] Additionally, the composition of the disclosed combination therapy, product, or kit includes a protein-free, serum-free, animal component-free formulation of about 25 million mesenchymal stromal cells, about 1 ml of a plurality of electrolyte solutions, and about 1 ml of about 2.5% dimethyl sulfoxide (DMSO).

[0119] Additionally, the composition of the disclosed combination therapy, product, or kit includes a protein-free, serum-free, animal component-free formulation of about 25 million mesenchymal stromal cells, about 1 ml of a plurality of electrolyte solutions, and about 1 ml of about 5% dimethyl sulfoxide (DMSO).

[0120] As will be appreciated by those skilled in the art, the terms mesenchymal stromal cells or mesenchymal stem cells are used interchangeably and are generally abbreviated as MSCs within the scope of this disclosure.

[0121] In some embodiments, the MSCs present in the composition of the combination therapy, product, or kit of the disclosure are bone marrow-derived MSCs (BM-MSCs).

[0122] Alternatively, the MSCs present in the compositions of the combination therapies, products, or kits of the present disclosure are derived from a source selected from the group including, but not limited to, adipose tissue, Wharton's jelly, and dental pulp, or any combination thereof.

[0123] Thus, the source of the MSCs is not a limitation on their presence in the compositions of the combination therapies, products, or kits of the present disclosure, and one of skill in the art can use MSCs from any source known to one of skill in the art.

[0124] In some embodiments, the MSCs present in the compositions of the combination therapies, products, or kits of the present disclosure are allogeneic. Allogeneic MSCs refer to MSCs that are derived from an individual / donor other than the recipient, but that belong to the same species.

[0125] In some embodiments, the MSCs used as part of the compositions herein are derived from the bone marrow of multiple healthy donors in accordance with statutory requirements with appropriate informed consent and authorization. Alternatively, the MSCs are bone marrow-derived MSCs obtained by ex vivo culture or cell banking.

[0126] In some embodiments, MSCs used as part of the compositions herein are individually isolated from the bone marrow of at least three donors and subjected to various processes to preserve or cryopreserve the cells. The MSCs used in the compositions herein are pooled from these individual MSCs. Prior to pooling, each individual donor's MSCs are subcultured to obtain a master cell bank composition (MCB). The MCB, containing the cultured MSCs isolated from each donor, is cryopreserved in a reagent such as fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO). In some embodiments, the MCB contains individual donor MSCs cryopreserved in approximately 90% FBS and approximately 10% DMSO. The MSCs are then pooled to prepare the cell compositions used in the combination therapy, product, or kit of the present disclosure. Pooling may be defined as combining / mixing MSCs from at least three donors from the MCB during the manufacturing process. The pooled MSCs are then passaged to arrive at a working cell bank composition (WCB). The WCB therefore contains pooled MSCs, which are further subjected to passaging / culture and washing processes to remove any xenogeneic or other impurities present, resulting in a final composition or investigational medicinal product (IMP) comprising pooled allogeneic MSCs for use as part of a combination therapy, product or kit in the present disclosure.

[0127] Thus, the MSCs thus obtained are mixed with about 1 ml of a multiple electrolyte solution, which is Plasmalyte A, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 2.5% to 5% dimethyl sulfoxide (DMSO) to obtain the composition used in the present disclosure.

[0128] In some embodiments, the 5% dimethyl sulfoxide (DMSO) used as part of the compositions herein is commercially available as CryoStor5 (CS5).

[0129] In some embodiments, those skilled in the art will understand that DMSO is commonly used to cryopreserve mesenchymal stromal cell preparations or compositions such as those used in the present disclosure. Therefore, if a mesenchymal stromal cell preparation is freshly prepared for administration, DMSO need not necessarily be included in the composition of the present disclosure. Thus, if administration of the combination or composition of the present disclosure is performed using freshly prepared mesenchymal stromal cells and the mesenchymal stromal cells do not need to be cryopreserved, the combination or composition need not contain DMSO. All such freshly prepared formulations of mesenchymal stromal cells containing multiple electrolyte solutions are also contemplated within the scope of the combinations, compositions, and kits of the present disclosure.

[0130] While the foregoing process represents one method by which the pooled MSC composition of the present disclosure can be obtained, those skilled in the art will appreciate that there are several other alternative methods by which a similar composition comprising pooled MSCs, about 1 ml of a multiple electrolyte solution, such as Plasmalyte A, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 2.5%-5% dimethyl sulfoxide (DMSO) can be prepared. All such compositions and respective methods for producing such compositions are within the scope of the present disclosure, so long as the final composition contains a total of about 20-30 million mesenchymal stromal cells pooled in equal proportions from at least three donors, about 1 ml of a multiple electrolyte solution, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 2.5%-5% dimethyl sulfoxide (DMSO).

[0131] In some embodiments, the pooling of MSCs to arrive at the compositions of the present disclosure is similar to or identical to the method described in PCT / IB2010 / 055424, which is incorporated by reference in its entirety, however, as noted above, the method described therein is only one way in which the compositions of the present disclosure may be prepared.

[0132] In contrast, the hyaluronic acid present as part of the combination therapy, product or kit of the present disclosure is a medium molecular weight hyaluronic acid.

[0133] In some embodiments, the molecular weight of the hyaluronic acid present as part of a combination therapy, product, or kit of the present disclosure ranges from about 500,000 daltons to about 730,000 daltons.

[0134] Thus, in some embodiments, the molecular weight of the hyaluronic acid present as part of a combination therapy, product, or kit of the present disclosure is about 500,000 daltons, 550,000 daltons, 600,000 daltons, 650,000 daltons, 700,000 daltons, or 730,000 daltons.

[0135] In some embodiments, the amount of hyaluronic acid administered as part of the combination therapy of the present disclosure is about 2 ml.

[0136] In some embodiments, the hyaluronic acid administered comprises 20 mg of hyaluronic acid administered in 2 ml of buffered saline having a pH of 6.8 to 7.5.

[0137] Thus, as will be apparent to one of skill in the art from reading this disclosure, the significance of this disclosure lies in the specificity or selection of features that result in the combination product or kit of the present disclosure. Accordingly, the positive effects and improvements seen in human subjects suffering from osteoarthritis after treatment with the combination therapy of the present disclosure are a direct result of the use of a combination product or kit that incorporates features including: a specific number of pooled mesenchymal stromal cells present in the composition; The number of donors from which the pooled mesenchymal stromal cells are derived; The availability of protein-free, serum-free, and animal component-free formulations of several electrolyte solutions and dimethyl sulfoxide (DMSO); Absence of human serum albumin (HSA) medium molecular weight hyaluronic acid, and The amount of electrolyte solutions, dimethyl sulfoxide (DMSO), and hyaluronic acid present in the combination product or kit.

[0138] Thus, in some embodiments, a combination product or kit of the present disclosure comprises a composition comprising about 20-30 million mesenchymal stromal cells, about 1 ml of a plurality of electrolyte solutions, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 2.5%-5% dimethyl sulfoxide (DMSO); and about 2 ml of hyaluronic acid, wherein the composition does not contain human serum albumin (HSA).

[0139] In some embodiments, a combination product or kit of the present disclosure comprises a composition consisting of about 20-30 million mesenchymal stromal cells, about 1 ml of a plurality of electrolyte solutions, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 2.5%-5% dimethyl sulfoxide (DMSO), and about 2 ml of hyaluronic acid, wherein the composition does not contain human serum albumin (HSA).

[0140] In some embodiments, the combination product or kit of the present disclosure comprises a composition consisting of about 20-30 million mesenchymal stromal cells, about 1 ml of a plurality of electrolyte solutions, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 2.5% dimethyl sulfoxide (DMSO), and about 2 ml of hyaluronic acid, wherein the composition does not contain human serum albumin (HSA).

[0141] In some embodiments, a combination product or kit of the present disclosure comprises a composition consisting of about 20-30 million mesenchymal stromal cells, about 1 ml of a plurality of electrolyte solutions, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 5% dimethyl sulfoxide (DMSO), and about 2 ml of hyaluronic acid, wherein the composition does not contain human serum albumin (HSA).

[0142] Thus, in some embodiments, a combination product or kit of the present disclosure comprises a composition comprising a protein-free, serum-free, animal component-free formulation of about 25 million mesenchymal stromal cells, about 1 ml of a plurality of electrolyte solutions, and about 1 ml of about 2.5% to 5% dimethyl sulfoxide (DMSO), and about 2 ml of hyaluronic acid, wherein the composition does not include human serum albumin (HSA).

[0143] In some embodiments, the combination product or kit of the present disclosure comprises a composition consisting of about 25 million mesenchymal stromal cells, about 1 ml of a plurality of electrolyte solutions, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 2.5% to 5% dimethyl sulfoxide (DMSO), and about 2 ml of hyaluronic acid, wherein the composition does not contain human serum albumin (HSA).

[0144] In some embodiments, a combination product or kit of the present disclosure comprises a composition consisting of about 25 million mesenchymal stromal cells, about 1 ml of a plurality of electrolyte solutions, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 2.5% dimethyl sulfoxide (DMSO), and about 2 ml of hyaluronic acid, wherein the composition does not contain human serum albumin (HSA).

[0145] In some embodiments, a combination product or kit of the present disclosure comprises a composition consisting of about 25 million mesenchymal stromal cells, about 1 ml of a plurality of electrolyte solutions, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 5% dimethyl sulfoxide (DMSO), and about 2 ml of hyaluronic acid, wherein the composition does not contain human serum albumin (HSA).

[0146] Particular features of the combination therapies, products and kits of the present disclosure provide several advantages summarized below: Cell viability is maintained despite the presence of multiple electrolyte solutions and low volumes of protein-, serum-, and animal component-free DMSO formulations. The overall volume of the composition and hyaluronic acid is reduced, thereby reducing the total volume of intra-articular injection and making it more convenient to administer to the patient; Additionally, the presence of multiple electrolyte solutions and low amounts of protein-, serum-, and animal component-free DMSO formulations ensures a reduced number of serious adverse events and toxicity to cells; Product stability is not compromised as the product is stable for up to 18 months. Reduced cryopreservation-induced cellular stress and damage; and The absence of HSA makes the composition serum-free.

[0147] As mentioned above, the combination product or kit of the present disclosure is for use in the treatment of osteoarthritis. Accordingly, the present disclosure relates to such uses of the combination therapy of the present disclosure.

[0148] Accordingly, the present disclosure also provides a combination of: a composition comprising a protein-free, serum-free, animal component-free formulation of approximately 20 to 30 million mesenchymal stromal cells, a plurality of electrolyte solutions, and dimethyl sulfoxide (DMSO); and Medium molecular weight hyaluronic acid, Regarding The composition does not contain human serum albumin (HSA); the mesenchymal stromal cells are bone marrow-derived pooled mesenchymal cells obtained by pooling mesenchymal stromal cells from at least three donors; and the composition or hyaluronic acid or a combination thereof is administered via an intra-articular route by ultrasound-guided administration.

[0149] In some embodiments of the use of the combination, the osteoarthritis is grade II or grade III osteoarthritis.

[0150] In some embodiments relating to the use of a combination, the electrolyte solution is Plasmalyte A.

[0151] In some embodiments relating to the use of a combination, each of the composition and hyaluronic acid is administered via a single intra-articular injection.

[0152] In some embodiments relating to the use of the combination, at least one or both of the composition and hyaluronic acid are administered via ultrasound-guided intra-articular injection.

[0153] In some embodiments relating to combined use, the composition and hyaluronic acid are administered simultaneously or sequentially.

[0154] In some embodiments relating to the use of a combination, the hyaluronic acid is administered between 30 seconds and 30 minutes after administration of the composition, or within 1800 seconds of administration of the composition.

[0155] In some embodiments relating to the use of the combination, the combination provides the subject with an improvement in a symptom of osteoarthritis selected from the group consisting of pain and stiffness; and improves or maintains each cartilage quality parameter in the subject compared to the subject before administration of the combination.

[0156] In some embodiments relating to the use of the combination, the combination improves the mean percent change from baseline in at least one of the WOMAC composite index score, or the WOMAC pain index score, or the WOMAC stiffness index score, or the WOMAC physical function index score, or the VAS score by at least about 10% over a span of about 6 months.

[0157] In some embodiments relating to the use of the combination, the combination demonstrates a mean difference in percent change from baseline of at least one of the WOMAC composite index score, or the WOMAC pain index score, or the WOMAC stiffness index score, or the WOMAC physical function index score, or the VAS score of at least about 15% at 6 months.

[0158] In some embodiments relating to the use of the combination, the combination demonstrates a mean difference in percent change from baseline of at least one of the WOMAC composite index score, or the WOMAC pain index score, or the WOMAC stiffness index score, or the WOMAC physical function index score, or the VAS score of at least about 40% at 12 months.

[0159] In some embodiments of combined use, the composition comprises 25 million mesenchymal stromal cells.

[0160] In some embodiments of the use of the combination, the combination increases the level of interleukin-10 (IL-10), an anti-inflammatory marker, and decreases the level of cross-linked C-telopeptide of type II collagen (CTX-II), a disease progression marker, in the subject compared to the subject before administration of the combination.

[0161] Accordingly, the present disclosure also provides the following combination for use in a method of increasing the level of interleukin-10 (IL-10), an anti-inflammatory marker, in a human subject suffering from osteoarthritis: a composition comprising a protein-free, serum-free, animal component-free formulation of approximately 20 to 30 million mesenchymal stromal cells, a plurality of electrolyte solutions, and dimethyl sulfoxide (DMSO); and Medium molecular weight hyaluronic acid When the combination is administered to a subject, the level is elevated compared to the subject before administration of the combination.

[0162] In some embodiments relating to the use of the combination, administration of the composition or hyaluronic acid or both is a single ultrasound-guided administration via an intra-articular route.

[0163] The present disclosure also provides a combination of: a composition comprising a protein-free, serum-free, animal component-free formulation of approximately 20 to 30 million mesenchymal stromal cells, a plurality of electrolyte solutions, and dimethyl sulfoxide (DMSO); and Medium molecular weight hyaluronic acid When the combination is administered to a subject, the level is reduced compared to the subject before administration of the combination.

[0164] In some embodiments relating to the use of the combination, administration of the composition or hyaluronic acid or both is a single ultrasound-guided administration via an intra-articular route.

[0165] Accordingly, the present disclosure also provides a method for increasing the level of interleukin-10 (IL-10), an anti-inflammatory marker, and decreasing the level of cross-linked C-telopeptide of type II collagen (CTX-II), a disease progression marker, in a human subject suffering from osteoarthritis, comprising the following combination: a composition comprising a protein-free, serum-free, animal component-free formulation of approximately 20 to 30 million mesenchymal stromal cells, a plurality of electrolyte solutions, and dimethyl sulfoxide (DMSO); and Medium molecular weight hyaluronic acid When the combination is administered to a subject, the levels increase and decrease, respectively, compared to the subject before administration of the combination.

[0166] In some embodiments relating to the use of the combination, administration of the composition or hyaluronic acid or both is a single ultrasound-guided administration via an intra-articular route.

[0167] Accordingly, the present disclosure also relates to a method for increasing the level of interleukin-10 (IL-10), an anti-inflammatory marker, in a human subject suffering from osteoarthritis, said method comprising administering to the subject the above combination; wherein the level is increased compared to the subject before administration of the above combination.

[0168] The present disclosure also relates to a method for reducing the level of cross-linked C-telopeptide of type II collagen (CTX-II), a marker of disease progression, in a human subject suffering from osteoarthritis, the method comprising administering the above combination to the subject; the level is reduced compared to the subject before administration of the combination.

[0169] Accordingly, the present disclosure also relates to a method for increasing the level of interleukin-10 (IL-10), an anti-inflammatory marker, and decreasing the level of cross-linked C-telopeptide of type II collagen (CTX-II), a disease progression marker, in a human subject suffering from osteoarthritis, the method comprising administering the above combination to the subject; the levels are increased and decreased, respectively, compared to the subject before administration of the combination.

[0170] In some embodiments of the methods of modulating (increasing and / or decreasing) the levels of the described biomarkers, administration of the composition or hyaluronic acid or both is a single ultrasound-guided administration via an intra-articular route.

[0171] Although only a few of the embodiments have been specifically described in the context of combination uses or methods of modulating the levels of the described biomarkers, all other aforementioned embodiments described within this disclosure in the context of describing methods of treating osteoarthritis, combination therapies, combination products, or kits are likewise applicable to the combination uses contemplated herein, and it is for the sake of brevity that not all such embodiments will be described again here.

[0172] It should be understood that the foregoing description is illustrative and not limiting. While considerable emphasis has been placed herein on particular features of the present disclosure, it will be understood that various modifications may be made, and that many changes may be made in the preferred embodiments without departing from the principles of the present disclosure. Those skilled in the art will recognize that the embodiments herein may be practiced with modification within the spirit and scope of the embodiments described herein. Similarly, additional embodiments and features of the present disclosure will be apparent to those skilled in the art based on the description provided herein.

[0173] Descriptions of well-known / conventional methods / steps and techniques are omitted so as not to unnecessarily obscure the embodiments herein. Furthermore, the disclosure herein provides examples illustrating the above-described embodiments, and specific aspects are employed to illustrate the embodiments of the present disclosure. The examples used herein for such illustration are intended merely to facilitate understanding of how the embodiments may be implemented and to further enable those skilled in the art to implement the embodiments. Therefore, the following examples should not be construed as limiting the scope of the embodiments herein.

[0174] Example Example 1 - Efficacy and Safety of a Single Intra-Articular Administration of the Combination Therapy of the Present Disclosure

[0175] the purpose Main Objective To evaluate the efficacy of a single intra-articular injection of a composition of the present disclosure in patients with knee osteoarthritis, the composition comprising 25 million mesenchymal stromal cells, approximately 1 ml of a multiple electrolyte solution, and approximately 1 ml of 5% dimethyl sulfoxide (DMSO), the composition being free of HSA. Secondary Objectives To evaluate the safety and tolerability of the above composition after a single intra-articular injection in patients with knee osteoarthritis.

[0176] Patient Eligibility Patients who met the following criteria were eligible for enrollment in this study: 1. Male or female patients (both included) aged 40-65 years. 2. Patients with a history of primary osteoarthritis of the knee characterized by pain requiring the use of analgesics. 3. Patients self-reported difficulty with at least one of the following activities due to knee pain: lifting and carrying groceries, walking 400 meters, getting in and out of a chair, rising from a squatting or cross-legged position, or going up and down stairs. 4. Patients who had been receiving analgesics for OA for 6 weeks based on investigator feedback. 5. Patients with radiologically qualified x-ray knee joints showing radiological evidence of grade 2-3 osteoarthritis according to the radiological criteria of Kellgren and Lawrence (based on the central radiologist's report). 6. Patients willing to refrain from other stromal cell therapies for the two-year study period. 7. Female patients of childbearing age who are willing to use common contraceptive methods during the study period. 8. Patients willing to provide written informed consent including audio-video consent.

[0177] Protocol One hundred forty-six patients were randomized 1:1 to receive either the stromal cell (composition) or placebo group: 73 patients received a single dose of 25 million pooled MSCs in 1 ml CryoStor CS5 + 1 ml PlasmaLyte A followed by 2 ml hyaluronic acid (Stempeucel® group), and 73 patients received a single intra-articular injection of 2 ml placebo (1 ml CryoStor CS5 + 1 ml PlasmaLyte A) followed by 2 ml hyaluronic acid (placebo group).

[0178] The study consisted of nine visits: Visit 1 - Patient Screening and Clinical Evaluation (Days -21 to 0) Visit 2 - Randomization, baseline activity and IMP administration (Day 0) Visit 3-7 days (1 week) ± 3 days Telephone follow-up on days 15±3 Visit 4-30 days (1 month) ±7 days Telephone follow-up on days 60±7 Visit 5 - 90 days (3 months) ± 7 days Visit 6 - 180 days (6 months) ± 7 days Telephone follow-up at 270 days (9 months) ± 7 days Visit 7 - 365 days (12 months) ± 14 days Visit 8 - 540 days (18 months) ± 30 days Visit 9 - 730 days (24 months) ± 30 days

[0179] Evaluation criteria Primary endpoint To evaluate the change from baseline to one year in the Western Ontario and McMaster Universities Osteoarthritis (WOMAC) osteoarthritis composite index score compared to the placebo group.

[0180] Secondary endpoints: To assess the change from baseline to two years in the Western Ontario and McMaster Universities Osteoarthritis (WOMAC) osteoarthritis composite index score compared to placebo. To evaluate the change from baseline to 1-year and 2-year follow-up compared to the placebo group: WOMAC OA pain index score WOMAC OA stiffness index score WOMAC OA physical function index score Patient-assessed osteoarthritis pain using the VAS score [osteoarthritis-pain visual analog scale (VAS)] Magnetic resonance imaging (T2 mapping) to assess the quality of articular cartilage (showing areas of compositional and ultrastructural changes) Magnetic resonance imaging to measure cartilage thickness and cartilage morphology at the midpoint of 20 defined subregions across two compartments of the knee (medial and lateral femoro-tibial joints). Magnetic resonance imaging measurements to assess cartilage volume.

[0181] Exploratory endpoints : Biomarker assessment: (IL-10 (serum) and CTX-II (urine))

[0182] Administration The compositions were administered intra-articularly under ultrasound guidance using a blinded 5 ml syringe under all sterile precautions and a screenshot of the needle position in the intra-articular space of the knee joint was recorded.

[0183] Intra-articular injections were primarily performed as medial or lateral parapatellar injections (injections into the patellofemoral joint) with a 2.0-inch (5.1 cm) 20-gauge needle by PI / Co-PI, although the optimal joint location and needle insertion site relative to the affected knee may vary according to the anatomic and pathological conditions. Patients were hospitalized for 24 hours between procedures to monitor for acute local or systemic (systolic) side effects or toxicity, if any.

[0184] After the IMP injection, the syringe was removed from the needle (but not removed from the injection site), and medium molecular weight hyaluronic acid was injected from the same needle under ultrasound guidance, resulting in only one needle insertion into the joint.

[0185] result Of the total 146 patients, 65 patients in the stempeucel® group and 68 patients in the placebo group completed the 12-month follow-up period. A total of 13 patients withdrew during the 1-year follow-up period due to death (1 patient in the stempeucel® group), loss to follow-up (3 patients in the stempeucel® group and 1 patient in the placebo group), or voluntary withdrawal (4 patients each in the stempeucel® group and placebo group). Both groups were comparable in terms of baseline parameters and demographic details. No major protocol deviations were observed during the study. The primary analysis cohort was the mITT cohort for efficacy endpoints and the safety cohort for safety endpoints.

[0186] Efficacy Results The primary efficacy outcome was calculated in the mITT and PP cohorts. The WOMAC index is used to assess subjects with hip or knee OA using 24 parameters.

[0187] For comparison, mean differences and p-values ​​were used.

[0188] mean difference : The mean difference measures the absolute difference between the means of two different groups. It shows how much difference there is between the means of the experimental group (Stempeucel®) and the control group (placebo).

[0189] p-value : The p-value is a statistical measure that indicates whether an effect is statistically significant. By convention, if the p-value is less than 0.05 (i.e., there is less than a 5% chance that the result occurred by chance), then there is probably a real difference between the treatments. If the p-value is 0.001 or less (there is less than a 0.1% chance that the result occurred by chance), then the result is considered highly significant. If the p-value indicates that there is a high probability that there is a difference between the treatments, then the confidence interval indicates how large the difference in effect is. Therefore: · The p-value is a statistical measurement used to test a hypothesis against observed data. · The p-value measures the probability of obtaining the observed results given that the null hypothesis is true. · The smaller the p-value, the greater the statistical significance of the observed difference. Generally, a p-value of 0.05 or less is considered statistically significant. P-values ​​can be used as an alternative to, or in addition to, a preselected confidence level for a hypothesis test.

[0190] WOMAC Composite Index Score: WOMAC composite index scores between treatment groups were comparable at baseline. WOMAC composite index scores decreased significantly over the course of the study in the stempeucel® group.

[0191] The mean WOMAC composite index score showed statistically significant improvements (p-values ​​<0.0001) with stempeucel® compared to placebo at days 180 and 365. Furthermore, in the stempeucel® group, mean WOMAC composite index scores showed statistically significant improvements within treatment groups (p-values ​​<0.0001) as early as day 30, with symptomatic improvements consistent through day 365. In contrast, the placebo group showed a worsening of mean WOMAC composite index scores after day 180. The mean percent change from baseline in WOMAC composite index scores showed statistically significant improvements (p-values ​​<0.0001) with stempeucel® compared to placebo at days 180 and 365. [Table 1]

[0192] WOMAC Pain Index Scores: WOMAC Pain Index Scores between treatment groups were comparable at baseline. WOMAC Pain Index Scores decreased significantly over the course of the study in the stempeucel® group.

[0193] Mean WOMAC pain index scores showed statistically significant improvements (p-values ​​<0.0001) with stempeucel® compared to placebo at days 180 and 365. Furthermore, within the treatment group, mean WOMAC pain index scores showed statistical significance of <0.0001 as early as day 30 in the stempeucel® group, with symptomatic improvement remaining consistent through day 365. In contrast, the placebo group showed a worsening of mean WOMAC pain index scores after day 180. Mean percent change from baseline in WOMAC pain index scores showed statistically significant improvements (p-values ​​<0.0001) with stempeucel® compared to placebo at days 180 and 365. [Table 2]

[0194] WOMAC Stiffness Index Scores: WOMAC Stiffness Index scores between treatment groups were comparable at baseline. WOMAC Stiffness Index scores decreased significantly over the course of the study in the stempeucel® group.

[0195] Mean WOMAC Stiffness Index scores showed statistically significant improvements (p-values ​​<0.0001) with stempeucel® compared to placebo at days 180 and 365. Furthermore, in the stempeucel® group, mean WOMAC Stiffness Index scores achieved statistical significance within treatment groups at <0.0001 as early as day 30, demonstrating consistent symptom improvement through day 365. In contrast, the placebo group experienced a worsening of mean WOMAC Stiffness Index scores after day 180. Mean percent change from baseline in WOMAC Stiffness Index scores showed statistically significant improvements (p-values ​​<0.0001) with stempeucel® compared to placebo at days 180 and 365. [Table 3]

[0196] WOMAC Physical Function Index Scores: WOMAC Physical Function Index scores between treatment groups were comparable at baseline. WOMAC Physical Function Index scores decreased significantly over the course of the study in the stempeucel® group.

[0197] The mean WOMAC Physical Function Index score showed statistically significant improvements (p-values ​​<0.0001) with stempeucel® compared to placebo at days 180 and 365. Furthermore, in the stempeucel® group, mean WOMAC Physical Function Index scores showed statistical significance within treatment groups at <0.0001 as early as day 30, with symptomatic improvements consistent through day 365. In contrast, the placebo group showed a worsening of mean WOMAC Physical Function Index scores after day 180. The mean percent change from baseline in WOMAC Physical Function Index scores showed statistically significant improvements (p-values ​​<0.0001) with stempeucel® compared to placebo at days 180 and 365. [Table 4]

[0198] Pain assessment by VAS score: VAS scores between treatment groups were comparable at baseline. VAS scores decreased significantly over the study period in the stempeucel® group.

[0199] Mean VAS scores showed statistically significant improvements (p-values ​​<0.0001) with stempeucel® compared to placebo at days 180 and 365. Furthermore, in the stempeucel® group, mean VAS scores achieved statistical significance within treatment groups at <0.0001 as early as day 30, with improvements consistent through day 365. In contrast, the placebo group experienced a worsening of mean VAS scores after day 180. Mean percent change from baseline in VAS scores showed statistically significant improvements (p-values ​​<0.0001) with stempeucel® compared to placebo at days 180 and 365. [Table 5]

[0200] Magnetic resonance imaging measurements (T2 mapping) to assess articular cartilage quality (showing areas of altered composition and ultrastructure): Medial T2 mapping assesses the state of the cartilage matrix and identifies biochemical changes associated with early stages of OA. The T2 relaxation time of articular cartilage is a function of both the water content and collagen ultrastructure of the tissue. Measurement of the spatial distribution of T2 relaxation times reveals areas of increased or decreased water content, which correlates with cartilage damage. Increased water content and damage to the collagen network lead to elevated T2 values.

[0201] To understand the quality of the articular cartilage, changes from baseline in magnetic resonance imaging measurements (T2 mapping) were assessed (showing areas of altered composition and ultrastructure). During the study period, superficial, intermediate, and deep cartilage scores were measured for both the medial and lateral femoral-tibial compartments, as well as the mean cartilage score.

[0202] Medial FT compartment (femoral condyle) area Table 6 below shows the results of the analysis of T2 mapping of the medial FT compartment (femoral condyle) in the mITT cohort.

[0203] For deep cartilage, the mean (SD) scores at baseline were 35.7 (7.68) ms in the stempeucel® group and 39.2 (8.04) ms in the placebo group. At day 180, the mean (SD) scores were 37.8 (8.72) ms in the stempeucel® group and 39.2 (8.07) ms in the placebo group. The mean (SD) change from baseline in deep cartilage score at day 180 was an increase of 1.6 (6.77) ms in the stempeucel® group and a decrease of 0.4 (7.94) ms in the placebo group; the between-group comparison was found to be statistically non-significant with a p-value of 0.4436. At day 365, the mean (SD) scores were 36.1 (7.79) ms in the stempeucel® group and 47.0 (75.12) ms in the placebo group. The mean (SD) change from baseline in deep cartilage score at Day 365 was a decrease of 0.6 (7.29) ms in the stempeucel® group and an increase of 10.8 (79.53) ms in the placebo group; a between-group comparison was found to be statistically non-significant with a p-value of 0.7064. Furthermore, the mean deep cartilage score at Day 180 was statistically significant at <0.0001 in the stempeucel® group and statistically non-significant at 0.7643 in the placebo group; and at Day 365 was statistically non-significant at 0.6188 in the stempeucel® group and statistically significant at <0.0001 in the placebo group. Therefore, it can be concluded that cartilage deterioration was observed in the placebo group at the deep cartilage level, with the mean score increasing from 39.2 ms at baseline to 47.0 ms at Day 365, but the difference between groups was not significant. [Table 6]

[0204] Magnetic resonance imaging measurements to measure cartilage thickness and cartilage morphology at the midpoint of a given subregion spanning two compartments of the knee (medial femoro-tibial joint and lateral femoro-tibial joint): Lateral femoro-tibial compartment: The Stempeucel® group had a significant mean increase of 1.762 (p=0.0003) at Day 365, and the placebo group also had a significant mean increase of 1.973 (p=0.0008) at Day 365. Mean values ​​were not significant between groups at Day 365.

[0205] Medial femoro-tibial compartment - The stempeucel® group had a significant increase in mean value of 1.292 (p-value=0.0289) at Day 365, and the placebo group also had a significant increase in mean value of 1.867 (p-value=0.0113) at Day 365. Mean values ​​were not significant between groups at Day 365.

[0206] Total score - The stempeucel® group had a significant mean increase of 3.054 (p-value=0.0015) at day 365, and the placebo group also had a significant mean increase of 3.840 (p-value=0.0010) at day 365. The mean values ​​were not significant between groups at day 365. Therefore, there was no change in cartilage thickness at day 365 in either group (Table 7 below). [Table 7]

[0207] Cartilage volume: Lateral femoro-tibial compartment: The Stempeucel® group had a significant mean increase of 107.2 (p<0.0001) at Day 365, and the placebo group had a significant mean increase of 143.7 (p<0.0001) at Day 365. Mean values ​​were not significant between groups at Day 365.

[0208] Medial femoro-tibial compartment - The stempeucel® group had a significant mean increase of 60.0 at Day 365 (p=0.0195) and the placebo group had a significant mean increase of 102.8 at Day 365 (p=0.0007). Mean values ​​were not significant between groups at Day 365.

[0209] Total Score - The stempeucel® group had a significant mean increase of 167.3 at Day 365 (p<0.0001), and the placebo group also had a significant mean increase of 246.5 at Day 365 (p<0.0001). Mean differences were not significant between groups at Day 365. At Day 180, the stempeucel® group had a significant mean decrease of 53.9 (p<0.0001), and the placebo group had a significant mean decrease of 271.6 (p<0.0001). The difference between groups approached significance at Day 180 (p=0.0555) (Table 8).

[0210] Additional analysis of the total cartilage volume score using generalized estimating equations (GEE) revealed differences in total cartilage volume values ​​between the two groups at one year. The stempeucel® group had a mean total cartilage volume increase of 34.07 compared to the placebo group, regardless of time, which was not statistically significant (Table 8A below, respectively).

[0211] GEE analysis demonstrated differences in cartilage volume in the lateral femoral-tibial compartment between the two groups at 1 year. The stempeucel® group experienced a 25.97 increase in cartilage volume over time compared to the placebo group, which was not statistically significant. The stempeucel® group experienced a 0.26 increase in cartilage volume over time compared to placebo, which was not statistically significant.

[0212] GEE analysis revealed differences in total cartilage volume between the two groups over one year. The stempeucel® group had a mean increase of 34.07 in total cartilage volume over time compared to the placebo group, which was not statistically significant. The stempeucel® group had a decrease of 1.61 in total cartilage volume over time compared to the placebo group, which was not statistically significant. [Table 8]

[0213] Biomarkers: Interleukin 10 (IL-10) In the stempeucel® group, there was a significant mean increase of 0.376 pg / mol at day 30 (p=0.0313), a significant increase of 0.323 pg / mol at day 90 (p=0.0625), and a significant increase of 0.051 pg / mol at day 365 (p=0.0625). In the placebo group, there was a significant increase of 0.267 pg / mol at day 30 (p=0.0313), a significant decrease of 0.342 pg / mol at day 90 (p=0.0625), and a significant decrease of 0.228 pg / mol at day 365 (p=0.0156). However, the differences between groups were not significant at any time point. Thus, IL-10 levels increased by 0.051 pg / mol in the stempeucel® group and decreased by 0.228 pg / mol in the placebo group at day 365. The mean difference between groups was 0.28 pg / mol, which was not significant (p=0.4546) (Table 9).

[0214] Details are as follows: The mean (SD) serum levels of IL-10 at baseline were 0.083 (0.44) pg / ml in the stempeucel® group and 0.558 (4.60) pg / ml in the placebo group. The mean (SD) serum levels of IL-10 at day 7 were 0.185 (0.91) pg / ml in the stempeucel® group and 0.578 (2.32) pg / ml in the placebo group. The change from baseline in mean (SD) serum levels of IL-10 at day 7 was an increase of 0.101 (1.03) pg / ml in the stempeucel® group and 0.012 (5.24) pg / ml in the placebo group. When comparing between groups, the mean difference (95% CI) for stempeucel® compared to placebo was 0.09 (-1.16, 1.34), which was not statistically significant (p=0.2405).

[0215] The mean (SD) serum levels of IL-10 at day 30 were 0.462 (2.00) pg / ml in the stempeucel® group and 0.842 (5.01) pg / ml in the placebo group. The change from baseline in mean (SD) serum levels of IL-10 at day 30 was an increase of 0.376 (2.07) pg / ml in the stempeucel® group and 0.267 (6.94) pg / ml in the placebo group. When comparisons were made between groups, the mean difference (95% CI) for stempeucel® compared with placebo was 0.11 (-1.61, 1.82), which was not statistically significant (p=0.9800). The mean (SD) serum levels of IL-10 at day 90 were 0.415 (1.59) pg / ml in the stempeucel® group and 0.305 (1.18) pg / ml in the placebo group. Furthermore, the change from baseline in mean (SD) serum levels of IL-10 at day 90 was an increase of 0.323 (1.62) pg / ml in the stempeucel® group and a decrease of 0.342 (4.26) pg / ml in the placebo group. Comparisons between groups showed a mean difference (95% CI) of 0.66 (-0.45, -1.78) in favor of stempeucel® compared with placebo, which was not statistically significant (p=0.6137). The mean (SD) serum levels of IL-10 at day 180 were 0.029 (0.22) pg / ml in the stempeucel® group and 0.114 (0.87) pg / ml in the placebo group. The change from baseline in mean (SD) serum levels of IL-10 at day 180 was a decrease of 0.079 (0.56) pg / ml in the stempeucel® group and a decrease of 0.562 (5.21) pg / ml in the placebo group. When compared between groups, the mean difference (95% CI) for stempeucel® compared with placebo was 0.48 (-0.91, 1.87), which was not statistically significant (p=0.4519).

[0216] The mean (SD) serum levels of IL-10 at Day 365 were 0.143 (0.48) pg / ml in the stempeucel® group and 0.586 (2.74) pg / ml in the placebo group. Similarly, the change from baseline in mean (SD) serum levels of IL-10 at Day 365 was an increase of 0.051 (0.71) pg / ml in the stempeucel® group and a decrease of 0.228 (6.33) pg / ml in the placebo group. Comparisons between groups revealed a mean difference (95% CI) of 0.28 (-1.48, 2.03) for stempeucel® compared with placebo, which was not statistically significant (p=0.4546). Overall, no statistical significance of <0.0001 was observed between treatment groups in mean serum levels of IL-10.

[0217] Furthermore, no statistically significant difference of <0.0001 was observed within the treatment groups for mean serum levels of IL-10 (except day 30 for both groups [p-value = 0.0313] and day 365 for the placebo group [p-value = 0.00156]).

[0218] Therefore, it can be concluded that the serum IL-10 level in the placebo group decreased by 0.228 pg / ml at 365 days of follow-up, which was statistically significant (p=0.0156). In the stempeucel group, the serum IL-10 level increased by 0.051 pg / ml at 365 days, which was not significant (p=0.0625). However, the difference between the groups was not significant at 365 days.

[0219] C-telopeptide of type II collagen (CTX-II) (urine)The mean value decreased by 44.784 ng / mmol at day 90 in the stempeucel® group, which was significant (p<0.0001), whereas the placebo group increased by 83.612 ng / mmol at day 90 (p<0.0001). The mean value increased by 32.568 ng / mmol at day 365 in the stempeucel® group and 49.354 ng / mmol in the placebo group. The difference between the groups was not significant at any time point. Additional ad hoc analysis using GEE methods revealed differences in CTX II values ​​between the two groups at 1 year. The stempeucel® group showed a 7.79 ng / mmol decrease in CTX II levels compared with the placebo group, regardless of time, but this was not statistically significant (Table 9). [Table 9]

[0220] Additional analyses performed on the CTX II biomarker: GEE analysis revealed a difference in CTX-II levels between the two groups over one year. The results are as follows: [Table 10]

[0221] In the stempeucel® group, CTX-II levels decreased by 7.79 ng / mmol compared to the placebo group regardless of time, but this was not statistically significant (Tables 10 and 10A).

[0222] Safety Results: A total of 82 adverse events (AEs) were reported in 44 patients. 49 AEs were observed in 24 patients in the stempeucel® group and 33 AEs were observed in 20 patients in the placebo group. All AEs observed in both treatment groups were treatment-emergent, except for three AEs in the placebo group (pyrexia, vomiting, and anemia) that occurred before administration of study drug. Most AEs were mild and unrelated to study drug. No patients in either treatment group withdrew from the study due to AEs.

[0223] conclusion In summary, efficacy data demonstrated significant improvements in patient-reported outcomes, including WOMAC and VAS scores, in the stempeucel® group. The study's primary endpoint, the WOMAC composite index score, remained significant in the stempeucel® group compared to the placebo group from the 6-month follow-up through to the 12-month follow-up (p<0.0001). Similar results were observed for WOMAC subscores (pain, stiffness, and physical function) and VAS scores, which remained significant in the stempeucel® group compared to the placebo group from the 6-month follow-up through to the 12-month follow-up (p<0.0001).

[0224] T2 mapping, performed on MRI to look at cartilage quality (hydration and collagen matrix), showed no deterioration of deep cartilage in the medial compartment of the knee in the stemeucel® group compared to the placebo group (which showed a significant gradual deterioration of cartilage within the group). There was a 34.07 mcg increase in mean total cartilage volume in the stemeucel® group compared to the placebo group, regardless of time, but this was not statistically significant.

[0225] Biomarker analysis showed that IL-10 levels (an anti-inflammatory marker) increased by 0.051 pg / mol in the stempeucel® group and decreased by 0.228 pg / mol in the placebo group (mean difference 0.28 pg / mol), which was not significant, at day 365. Additionally, CTX II levels (a measure of urinary C-terminal cross-linked telopeptide type II collagen, which indicates disease progression) decreased by 7.79 ng / mmol in the stempeucel® group compared with the placebo group, regardless of time, but this was not statistically significant.

[0226] Based on the above findings, it can be concluded that stempeucel® is safe and effective in the treatment of KL grade II and grade III knee osteoarthritis.

[0227] Example 2 -TSP-2 secretion in the presence of inflammatory cytokines (IFN-γ and TNF-α) the purpose To evaluate the potential for TSP-2 secretion by stempeucel® under inflammatory conditions.

[0228] Priming of MSCs with inflammatory cytokines is performed to mimic the clinical condition of OA, and stempeucel® is administered intra-articularly into knee joints with high levels of inflammation.

[0229] Eight batches of Stempeucel® (CS5 at 25 million cells / ml) were seeded in duplicate into T75 flasks at a cell concentration of 1 million cells in 10 ml of complete medium. One set of cultures was primed with IFN-γ (10 ng / ml) and TNF-α (15 ng / ml) 24 hours after seeding and further incubated for up to 96 hours after seeding.

[0230] The other set was cultured for 96 h without priming with IFN-γ and TNF-α and is hereafter referred to as “unprimed.”

[0231] Conditioned medium samples were collected from both primed and unprimed cultures at 48, 72, and 96 hours after seeding and analyzed for TSP-2 by ELISA.

[0232] result TSP-2 concentrations in unprimed cells varied between 4 and 16 ng / ml, 12 and 30 ng / ml, and 17 and 29 ng / ml after 48, 72, and 96 hours, respectively (Figure 1, Table 11). Priming resulted in decreased TSP-2 concentrations (ranging from 2 to 7 ng / ml) in all batches at all time points. There was an average 5-fold difference in TSP-2 secretion between primed and unprimed (Stempeucel®) cells at these time points. [Table 11]

[0233] conclusion Stempeucel® primed with inflammatory cytokines (IFN-γ and TNF-α) reduced TSP-2 secretion levels compared to unprimed Stempeucel®.

[0234] Example 3 - Analysis of the ability of stempeucel® to secrete TSP-2 and PGE-2 in the presence and absence of inflammatory cytokines the purpose To evaluate the ability of stempeucel® to secrete PGE-2 (an anti-inflammatory molecule) in an inflammatory environment.

[0235] Protocol Stempeucel® (25 million cells / ml CS5) from five batches were seeded in duplicate at a cell concentration of 1 million cells per T75 flask in 10 ml of complete medium.

[0236] One set of cultures was primed with IFN-γ (10 ng / ml) and TNF-α (15 ng / ml) 24 h after seeding and further incubated for 96 h after seeding, while the other set was cultured for 96 h after seeding without the aforementioned priming and was designated "unprimed."

[0237] Conditioned medium samples were collected from both primed and unprimed cultures at 48, 72, and 96 hours after seeding and analyzed by ELISA for TSP-2, PGE2, and TNF-α.

[0238] result The levels of PGE2, TSP-2, and TNF-α were analyzed in five batches of Stempeucel® under inflammatory conditions and compared with the unprimed condition. The amount of PGE2 secreted from the unprimed samples was relatively lower than that of the primed samples (Table 12). The primed samples secreted as much as 24 ng / ml, compared with 0.3 ng / ml in the unprimed condition (Figure 2). An inverse correlation was observed between PGE2 and TSP-2 secretion, with TSP-2 secretion being higher in the unprimed condition than in the primed condition. In the unprimed condition, MSCs secreted no or only trace amounts of TNF-α. When priming was performed with the addition of IFN-γ and TNF-α to the culture medium, a gradual decrease in TNF-α levels was observed over time. [Table 12]

[0239] conclusion Priming stempeucel® with proinflammatory cytokines, such as IFN-γ and TNF-α, increased the secretion of PGE2 (an anti-inflammatory molecule) and simultaneously decreased the secretion level of TSP-2. In contrast, unprimed stempeucel® decreased PGE2 levels and increased TSP-2 levels. These data demonstrate that when stempeucel® is administered to the knee joints of OA patients, the injected stempeucel® induces the production of anti-inflammatory molecules, such as PGE2, in the surrounding inflammatory environment, thereby reducing the inflammatory environment and further increasing the secretion of TSP-2.

[0240] Furthermore, the chondrogenic differentiation potential and sGAG secretion of primed stempeucel® were also analyzed. The results showed that chondrocytes differentiated from stempeucel® primed with inflammatory cytokines (IFN-γ and TNF-α) were able to induce higher expression of proteoglycan, namely sGAG (one of the important components of cartilage, accounting for approximately 3-6% of the total cartilage matrix).

[0241] An overall schematic of the relevance of TSP-2 as a potential indicator for determining the efficacy of stempeucel® for OA indications is further shown in FIG.

[0242] Example 4 – Effect of hyaluronic acid on the performance of MSCs the purpose To evaluate the effect of hyaluronic acid on the performance of MSCs to treat OA.

[0243] Protocol A comparison was made between existing literature studies using mesenchymal stromal cells alone for osteoarthritis and the disclosed combination having a composition including a protein-free, serum-free, animal component-free formulation of about 20-30 million mesenchymal stromal cells, about 1 ml of a multiple electrolyte solution, and about 1 ml of about 2.5%-5% dimethyl sulfoxide (DMSO), along with about 2 ml of medium molecular weight hyaluronic acid.

[0244] All comparisons revealed that the disclosed combination worked much better for treating OA, and showed significant improvements in WOMAC composite index, stiffness, pain levels, and WOMAC total score when compared to 2 ml of mesenchymal stromal cells only formulation without hyaluronic acid.

[0245] The foregoing description of specific embodiments reveals the general nature of the embodiments herein, and by applying current knowledge, others may readily modify and / or adapt such specific embodiments for various uses without departing from the general concept; therefore, such adaptations and modifications should, and are intended to, be understood within the meaning and range of equivalents of the disclosed embodiments. It should be understood that the phraseology or terminology used herein is for purposes of description and not limitation. Thus, while the embodiments of the present disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that modifications can be made to the embodiments herein within the spirit and scope of the embodiments described herein.

[0246] Throughout this specification, the terms "combination thereof" or "any combination thereof" are used interchangeably and are intended to have the same meaning as commonly known in the art of patent disclosure.

[0247] With respect to the embodiments characterized herein, each embodiment is intended to be read independently as well as in combination with other embodiments. For example, for embodiment 1 listing three alternatives A, B, and C, embodiment 2 listing three alternatives D, E, and F, and embodiment 3 listing three alternatives G, H, and I, unless otherwise specified, the specification will be understood to expressly disclose embodiments corresponding to the combinations A,D,G; A,D,H; A,D,I; A,E,G; A,E,H; A,E,I; A,F,G; A,F,H; A,F,I; B,D,G; B,D,H; B,D,I; B,E,G; B,E,H; B,E,I; B,F,G; B,F,H; B,F,I; C,D,G; C,D,H; C,D,I; C,E,G; C,E,H; C,E,I; C,F,G; C,F,H; C,F,I.

[0248] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present disclosure and is not to be construed as an admission that any or all of such matters form part of the prior art or were common general knowledge in the art relevant to the present disclosure as existing at any time before the priority date of this application.

Claims

1. For use in a method of treating osteoarthritis in a human subject, a composition comprising about 20 to 30 million mesenchymal stromal cells, about 1 ml of a plurality of electrolyte solutions, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 2.5% to 5% dimethyl sulfoxide (DMSO); and Approximately 2 ml of medium molecular weight hyaluronic acid A combination of The composition does not contain human serum albumin (HSA); the mesenchymal stromal cells are bone marrow-derived pooled mesenchymal stromal cells obtained by pooling mesenchymal stromal cells from at least three donors; and the composition or the hyaluronic acid or the combination is administered via an intra-articular route by ultrasound-guided administration.

2. 2. The combination for use according to claim 1, wherein the osteoarthritis is grade II or grade III osteoarthritis.

3. 2. The combination for use according to claim 1, wherein said plurality of electrolyte solutions is Plasmalyte A.

4. The combination for use according to claim 1, wherein each of the composition and hyaluronic acid is administered via a single intra-articular injection.

5. 2. The combination for use according to claim 1, wherein at least one or both of the composition and hyaluronic acid are administered via ultrasound-guided intra-articular injection.

6. The combination for use according to claim 1, wherein the composition and hyaluronic acid are administered simultaneously or sequentially.

7. The combination for use according to claim 6, wherein the hyaluronic acid is administered from about 1 second to about 30 minutes after administration of the composition.

8. 8. The combination for use according to any one of claims 1 to 7, wherein the combination provides the subject with an improvement in symptoms of osteoarthritis selected from the group consisting of pain and stiffness; and improves or maintains cartilage quality in the subject compared to the subject before administration of the combination.

9. 9. The combination for use according to claim 8, wherein the combination improves the mean percent change from baseline or placebo in at least one of the WOMAC composite index score, or the WOMAC pain index score, or the WOMAC stiffness index score, or the WOMAC physical function index score, or the VAS score by at least about 10% over a span of about 6 months.

10. 9. The combination for use of claim 8, wherein the combination demonstrates a mean difference in percent change from baseline or placebo of at least about 15% at 6 months for at least one of the WOMAC composite index score, or the WOMAC pain index score, or the WOMAC stiffness index score, or the WOMAC physical function index score, or the VAS score.

11. 9. The combination for use according to claim 8, wherein the combination demonstrates a mean difference in percent change from baseline or placebo of at least about 20% at 12 months for at least one of the WOMAC composite index score, or the WOMAC pain index score, or the WOMAC stiffness index score, or the WOMAC physical function index score, or the VAS score.

12. 8. The combination for use according to any one of claims 1 to 7, wherein the combination increases the level of interleukin-10 (IL-10), an anti-inflammatory marker, in the plasma of the subject, and decreases the level of cross-linked C-telopeptide of type II collagen (CTX-II), a disease progression marker, in the subject, compared to the subject before administration of the combination.

13. 2. The combination for use according to claim 1, wherein the composition comprises 25 million mesenchymal stromal cells.

14. A combination of: a composition comprising about 20 to 30 million mesenchymal stromal cells, about 1 ml of a plurality of electrolyte solutions, and about 1 ml of a protein-free, serum-free, animal component-free formulation of about 2.5% to 5% dimethyl sulfoxide (DMSO); and Approximately 2 ml of medium molecular weight hyaluronic acid A kit comprising: The composition does not contain human serum albumin (HSA); and the mesenchymal stromal cells are bone marrow-derived pooled mesenchymal stromal cells obtained by pooling mesenchymal stromal cells from at least three donors.

15. For use in a method of increasing the level of interleukin-10 (IL-10), an anti-inflammatory marker, and / or decreasing the level of cross-linked C-telopeptide of type II collagen (CTX-II), a disease progression marker, in a human subject suffering from osteoarthritis. a composition comprising a protein-free, serum-free, animal component-free formulation of approximately 20-30 million mesenchymal stromal cells, a plurality of electrolyte solutions, and dimethyl sulfoxide (DMSO); and ・Medium molecular weight hyaluronic acid wherein the composition does not contain human serum albumin (HSA); the mesenchymal stromal cells are pooled mesenchymal stromal cells derived from bone marrow obtained by pooling mesenchymal stromal cells from at least three donors; and when the combination is administered to a subject, the level is increased and / or decreased compared to the subject before administration of the combination.

16. 16. The combination for use according to claim 15, wherein the administration of the composition or the hyaluronic acid or both is by ultrasound-guided administration via an intra-articular route.

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