Pharmaceutical composition comprising mesenchymal cells, human serum albumin and hyaluronic acid, and its use for treating or preventing bone or joint diseases

Abstract

The invention relates to an advanced therapy medicinal product which is intended for use in medicine (especially in orthopaedics, traumatology and sports medicine), in particular for the regeneation of degenerative lesions and cartilage and bone injuries, especially osteoarthritis.

Description

[0001] Pharmaceutical composition of MSC cell suspension and its use for treating or preventing bone or joint diseases

[0002] The invention relates to an advanced therapy medicinal product (ATMP) which is intended for use in medicine (especially in orthopaedics, traumatology and sports medicine), in the treatment or prevention of bone or joint diseases, in particularfor the treatment of degenerative lesions and cartilage and bone injuries, including osteoarthritis (OA).

[0003] In recent years, there has been a growing interest in the use of stem cell-based therapies in regenerative medicine. It is postulated that human mesenchymal stem / stromal cells (MSCs) including adipose tissue-derived MSCs (AT-MSCs) can be used in the regeneration of cartilage and bone defects, including OA.

[0004] Hyaluronic acid (HA) is a substance registered as a medical device, commonly used, among others, in orthopedics to relieve pain in osteoarthritis (i.e. OA), including the knee joint.

[0005] However, the inventors observed that AT-MSCs resuspended in a 0.9% saline solution (NaCI) and then mixed with high molecular weight HA exhibited significantly reduced viability, which may have a significant impact on their pro-regenerative potential. The described practice does not raise any objections of orthopedists, which may result from their ignorance of the impact of HA on the viability of cells, including AT-MSC cells, as well as the widespread use of NaCI in clinical practice.

[0006] The aim of the invention is to provide a medicinal product with a unique composition suitable for the regeneration of degenerative lesions and cartilage and bone injuries, including osteoarthritis, containing hyaluronic acid (HA) and human mesenchymal stem / stromal cells (MSCs), including adipose tissue-derived MSCs (AT-MSCs), with high viability and thus high pro-regenerative potential (named: MesoCellA-Ortho).

[0007] The subject of the invention is a pharmaceutical composition and its medical use, which have been defined in detail in the appended claims.

[0008] Surprisingly, the aforementioned technical problem has been solved by the present invention. The inventors have surprisingly found that the use of a carrier solution which is a NaCI saline solution (preferably at a concentration of 0.9%) supplemented with human serum albumin (HSA) allows to maintain adequate viability of AT-MSC cells after mixing with hyaluronic acid (HA). Thanks to this, the cells maintain adequate viability and, consequently, functionality. The experiments carried out by the inventors showed that a suspension of AT-MSC cells obtained in a saline solution (preferably 0.9% NaCI) containing additional human albumin in a concentration of 5% to 15% maintains a high viability of AT-MSCs after mixing in a ratio of 1:3 with hyaluronic acid (HA) compared to AT-MSCs resuspended in only 0.9% NaCI (without the addition of any proteins). The obtained results regarding the preferred conditions are shown in Figure 1. It is also possible to implement the invention for MSC cells obtained from other tissues, such as: umbilical cord, bone marrow, dental pulp.

[0009] Figure 1 shows the viability of AT-MSCs resuspended in selected carrier solutions after mixing with high molecular weight hyaluronic acid (HA). AT-MSCs were resuspended in one of the analyzed solutions: 0.9% NaCI (control), S5 – 0.9% sodium chloride solution containing 5% human albumin solution, S10 – 0.9% sodium chloride solution containing 10% human albumin solution, S15 – 0.9% sodium chloride solution containing 15% human albumin solution or S10LG – 0.9% sodium chloride solution containing 10% human albumin solution and glucose at a low concentration of 1000 mg / l (LG), and then mixed with HA (1:3). The viability of the AT-MSCs contained in the suspension was assessed immediately after mixing with HA (0 h) and 1 h after mixing with HA, using a method comprising the counting of live cells after staining with trypan blue.

[0010] The product obtained in the exemplary embodiment of the invention consists of 18-22 x 106live human mesenchymal stem / stromal cells derived from adipose tissue (AT-MSCs), which are its active substance. AT-MSCs are isolated from human adipose tissue collected during liposuction from the subcutaneous region of human donors. Then, an isolated so-called stromovascular fraction (SVF) consisting of a heterogeneous cell population, enriched with AT-MSC cells, is preferably cultured for 3 to 5 passages (P3-5) under appropriate conditions. The resulting AT-MSCs are preferably resuspended in 1-2 mL of sterile carrier solution, which consists of: 0.9% NaCI containing 10% human serum albumin (HSA), obtained by mixing 0.9% NaCI solution with 20% HSA solution in a 1:1 ratio. It is also acceptable to resuspend MSC cells in a different solution isotonic to human cells, such as, among others: culture medium, PBS salt solution, Ringer's solution and with a different concentration of albumin or to use other solution containing stabilizing proteins, provided that high viability, stable phenotype, genotype and biological properties of cells characteristic of MSC cells are possible to be maintained. The results obtained in the experimental work that led to the invention unexpectedly prove that HSA increases the viability and functionality of the MSCs comprised in the formulation both before and after its local administration.

[0011] In a preferred embodiment, the manufacturing of the product (suspension of MSCs in a carrier solution) is carried out in accordance with the GMP rules in the manufacturing site - the GMP-certified ATMP Manufacturing Facility, which allows the use of these cells in clinical practice. In an exemplary embodiment, the formulation of the invention is prepared immediately prior to administration to the patient. For this purpose, the MSC cell suspension is mixed with an excipient such as hyaluronic acid (HA). The final formulation containing preferably from 2.0% to 2.5% HSA is thus obtained.

[0012] Detailed description of the invention

[0013] The subject of the present invention is a pharmaceutical composition consisting of a suspension of human mesenchymal stem / stromal cells (MSCs), especially derived from adipose tissue (AT-MSCs) and an excipient. In the method of the invention, the MSC cell suspension is prepared in a solution containing: a solution isotonic to human cells, preferably a saline solution (with 0.9% NaCI) with human albumin (HSA), wherein the excipient is a solution containing hyaluronic acid, wherein the volume ratio of the MSC suspension to the HA solution is preferably from 1:3 to 2:3.

[0014] The MSC cells comprised in the suspension have a viability of at least 80%, express, among others, CD73, CD90 and CD105 markers on their surface; and do not express, among others, CD14, CD19, CD34 and CD45 markers on their surface, have the morphology characteristic of MSCs, are genetically stable, sterile and free from mycoplasmas and bacterial endotoxins. The MSC suspension is preferably obtained in a solution containing: a solution isotonic to human cells, preferably a saline solution (with 0.9% NaCI) and HSA in a concentration of 10% obtained by mixing 0.9% NaCI solution with 20% HSA solution in a 1:1 ratio. It is acceptable to prepare a suspension of MSC cells using a different isotonic solution for human cells such as culture medium, buffered saline solution (PBS), Ringer's solution and with a different concentration of albumin or other solution containing stabilizing proteins, provided that high viability, stable phenotype, genotype and biological properties of cells characteristic of MSC cells are maintained.

[0015] In a preferred embodiment, the dosage unit contains 18-22 x 106live MSC cells in a volume of 1 to 2 mL.

[0016] The excipient is a solution of hyaluronic acid, preferably with a starting concentration of 20 mg / ml.

[0017] In a preferred embodiment, the pharmaceutical composition (when mixed with hyaluronic acid) comprises from 2.0% to 2.5% HSA.

[0018] The pharmaceutical composition of the present application is for use in the treatment or prevention of bone or joint disease, particularly for the regeneration of degenerative lesions and cartilage and bone injuries, including osteoarthritis. Preferably, it is intended for local administration, especially intra-articular administration.

[0019] Example 1. Preparation of AT-MSC cell suspension A schematic diagram of an exemplary embodiment of the method of the invention is shown in Fig. 5, while preferred embodiments of its successive steps are discussed in the example described below.

[0020] Isolation of SVF cells and establishment of a primary culture

[0021] Transfer the collected adipose tissue to centrifuge tubes (e.g. falcon type) and then wait a few minutes until the infiltration fluid separates from the adipose tissue (adipose tissue – upper phase). This step can also be done using a centrifuge. To do this, place the centrifuge tubes containing the tissue in the device and then accelerate the centrifuge to 200xg. After the phases are separated using a serological pipette, remove the infiltration fluid from each falcon tube, then add the saline solution (PBS) with Anti-Anti (containing 2X concentrated solution of antibiotics and an antifungal agent - Antibiotic-Antimycotic: penicillin 200 U / ml, streptomycin 200 μg / ml and amphotericin B 0.5 μg / ml) with a volume equal to the volume of tissue in the falcon tube. Screw the cap on each test tube and then mix by inverting. Wait a few minutes until the phases separate. This step can also be done using a centrifuge (see above). Open the test tubes with the tissue and then, using serological pipettes, remove the saline solution (PBS) with the Anti-Anti (lower phase) in each falcon tube as much as possible, leaving adipose tissue in the falcon tubes. Repeat washing twice. In case the fluid is still strongly colored with erythrocytes, perform additional washes (up to 3 consecutive rwashing cycles). Remove the saline solution (PBS) with the Anti-Anti from the last lipoaspirate wash. To each falcon tube containing adipose tissue, add a volume of collagenase solution using a serological pipette to achieve a final enzyme activity of 0.2 PZ U / ml. Close the test tubes and then incubate in a shaker oven at 200 rpm at 37°C ± 1°C for 35-40 minutes. After incubation, visually assess whether the adipose tissue has been digested. Then spin the test tubes at 370xg, 10 min, at 17 ± 1°C. Using a serological pipette, remove the oily and aqueous phases to leave only the pellet. Perform the operation for each falcon tube. Then add saline solution (PBS) to each falcon tube and pipette the pellets. Prepare a new falcon tube of volume 50 ml. Apply a filter with a pore diameter of 100 pm to the falcon tube, onto which apply a suspension of cells. Wash the tissue fragments remaining on the filter with saline solution (PBS). Fill the collective falcon tube containing the isolated cells with saline solution (PBS) to a volume of 50 ml and spin at a speed of 350xg, 7 min at room temperature.

[0022] After centrifugation, remove the supernatant from the pellet and resuspend the pellet in saline solution (PBS). Then count the cells and determine their viability. Isolated cells should be evenly seeded onto culture flasks in Culture Medium-1 (i.e. aMEM containing 2 lU / mL heparin, 5-10% plate lysate, 1X concentrated Antibiotic-Antimycotic solution). Place the flasks in a culture incubator. Proceed with the culture at 37°C, in an atmosphere of 5% CO2. Control of the primary culture establishment step and the first and subsequent replacement of the culture medium

[0023] Replace the culture medium 24-48 hours after the culture is established. For this purpose, pour out the entire medium. Gently wash the cells with saline solution (PBS). Using a serological pipette, add fresh Culture Medium-1. Conduct microscopic evaluation of cells. Then place the culture flask back in the incubator. Proceed with the culture at 37°C, in an atmosphere of 5% CO2.

[0024] End of primary culture – 1st passage

[0025] Perform the 1st passage 4-5±2 days after the primary culture is established. At this stage the following parameters should be assessed:

[0026] a) symptoms of potential infection in the culture - based on the observation of discoloration and the appearance of turbidity of the culture medium,

[0027] b) percentage of the area occupied by cells (confluence),

[0028] c) cell morphology in the culture.

[0029] After macroscopic and microscopic observation, proceed to the cell passage. Pour out all of the culture medium and wash the cells with saline solution (PBS) using a serological pipette. Pour out or remove the saline solution (PBS) using a serological pipette. Repeat washing twice. Then, using a serological pipette, add the Tryple Select solution to each flasks and distribute it evenly. Incubate culture flasks at 37°C. After approx. 2 minutes, gently hit the side of the flask and assess whether the cells have detached from the substrate. Then add saline solution (PBS). Wash the surface after growing of cells and transfer the entire contents of the culture flask into the centrifuge tube. Spin the cells at 300xg, 5 min, at room temperature. Pour out or remove the supernatant using a serological pipette. Resuspend the pellet in saline solution (PBS). Then count the cells and determine their viability using trypan blue.

[0030] The establishment of secondary culture

[0031] In this step, counted cells should be seeded onto new culture vessels for further culture in Culture Medium-2 (i.e. aMEM comprising 2 lU / mL heparin, 5-10% plate lysate, no antibiotics and no antifungal agent added). Gently rock each bottle to evenly distribute the cell suspension. Proceed with the culture at 37°C, in an atmosphere of 5% CO2.

[0032] Control and replacement of medium in secondary culture

[0033] Macroscopic and microscopic evaluation of AT-MSC cells in the culture should be carried out 24-48 hours after the secondary culture is established. If necessary, replace the medium. Gently pour out or remove all the medium using a serological pipette. Using a serological pipette, add fresh Culture Medium-2. Then place each flask back in the incubator. Proceed with the culture at 37°C, in an atmosphere of 5% CO2 until the required confluence is achieved. When confluence is >70%, cell passage can be performed as described below.

[0034] End of secondary culture and further culture – 2nd and subsequent passages

[0035] Once >70% confluence is reached, perform cell passage. To do this, pour out all the culture medium and wash the cells with saline solution (PBS) using a serological pipette. Repeat washing twice. Then, using a serological pipette, add the Tryple Select solution to each flask and distribute it evenly. After approx. 2 minutes, gently hit the side of the flask and assess whether the cells have detached from the culture surface area. Then add saline solution (PBS) to each culture flask. Wash them and transfer the entire contents of each culture flask into the collective centrifuge tube. Spin the cells at 300xg, 5 min, at room temperature. Pour out or remove the supernatant using a serological pipette. Resuspend the pellet in saline solution (PBS). Then count the cells using Burker’s chamber and determine their viability using trypan blue.

[0036] If a sufficiently high number of cells is obtained (i.e. min. 1.5x106), seed them in a T500 flask (surface area of 500 cm2) in about 100 ml of Culture Medium-2 (per one T500 bottle). Proceed with the culture at 37°C, in an atmosphere of 5% CO2. When the cells reach >70% confluence, passage them again in exactly the same way as described earlier. The culture should be carried out until the required number of cells is reached to allow the release of the final product (preferably culture at 3-5 passage).

[0037] End of AT-MSC cell culture (3-5 passage)

[0038] Proceed with macroscopic and microscopic evaluation of AT-MSC cells in the culture. TFor this purpose, the following should be assessed:

[0039] a) symptoms of potential infection in the culture,

[0040] b) percentage of the area occupied by cells (confluence),

[0041] c) cell morphology in the culture.

[0042] After macroscopic and microscopic evaluation, pour out all the medium from the culture vessels. Perform cell passage. To do this, wash the flask with saline solution (PBS). Pour out the saline solution (PBS). Then, using a serological pipette, add the Tryple Select solution to each flask and distribute it evenly. After approx. 2 minutes, gently hit the side of the flask and assess whether the cells have detached from the culture surface area. After the cells have detached from the culture surface area, pour out the entire contents of the T500 flask into a centrifuge tube. Then wash the flask additionally with saline solution (PBS) and pour it out into the same centrifuge tube. Spin the cells at 300xg, 5 min, at room temperature. Pour out or remove the supernatant using a serological pipette. Resuspend the pellets in saline solution (PBS) and collect them in one collective centrifuge tube. Resuspend the cells in saline solution (PBS) and count them.

[0043] Acceptance criteria for the manufacture of the final product:

[0044] a) cell number:

[0045] no deviations: number of living cells sufficient for product manufacture and quality control tests (according to the protocol of the specific clinical use of the manufactured product)

[0046] ✓ result outside the specification: number of living cells insufficient for product manufacture and quality control tests

[0047] b) cell viability:

[0048] no deviations: ≥ 80%

[0049] result outside the specification: <80% Preparing of MesoCellA-Ortho formulation

[0050] Fill the falcon tube containing AT-MSC cells intended for the final product with saline solution (PBS) to a volume of 50 ml and spin at 300xg, 5 min, at room temperature. After centrifugation, pour out the supernatant and resuspend the pellet containing the desired number of cells, preferably 18-22 x 106live MSC cells, in a volume of 1 to 2 mL of an additive, for example preferably in a solution isotonic to human cells containing 10% human albumin. Collect a specific volume of cell suspension (containing the appropriate number of live AT-MSC cells) and transfer to the container closures system for packing of the product. Close the container closures system. Immediately before administration, mix the suspension of MSC cells with the hyaluronic acid solution. The prepared composition should be administered in a syringe with a needle, preferably 0 1.2.

[0051] At the appropriate steps of culturing of AT-MSCs and preparing of the product, take samples for quality control tests.

[0052] As a result an AT-MSC suspension having the characteristics listed in Table 1 is obtained. Table 1. Specification of MSC cell suspension

[0053] Test Limits or range Method Interpretation Parameters tested before the last passage (before cell collection)

[0054] Typical for MSCs

[0055] (elongated, spindle- Microscopic

[0056] Cell morphology Correct

[0057] shaped, fibroblast-like evaluation

[0058] cells)

[0059] Mycoplasma spp.* Negative (≥40CT) Ph. Eur. 2.6.7 Correct

[0060] LAL

[0061] Endotoxins ≤ 1.0 IU / mL Correct

[0062] (Ph. Eur. 2.6.14)

[0063] Parameters tested after the last passage (immediately before the formulation of the final product)

[0064] 18-22x106viable Light microscopy

[0065] Cell count Correct

[0066] cells (Ph. Eur. 2.7.29)

[0067] Light microscopy

[0068] Cell viability ≥80% Correct

[0069] (Ph. Eur. 2.7.29)

[0070] Identity / CD73, CD90 ≥ 90% Flow cytometry

[0071] Correct Phenotype** CD105 ≥ 80% (Ph. Eur. 2.7.24)

[0072]

[0073] CD14, CD19, CD34,

[0074] CD45 ≤5%

[0075] Parameters tested in the final product (in the final cell suspension)

[0076] Direct inoculation

[0077] Sterility * Sterile (Ph. Eur. 2.6.1 or Correct

[0078] 2.6.27)

[0079]

[0080] * Due to the short shelf life of the product, the prepared formulation is released before the quality control tests are completed.

[0081] ** Antigen expression is calculated with reference to isotype controls.

[0082] Example 2. Pharmaceutical composition.

[0083] The suspension of AT-MSC cells obtained according to Example 1 in a carrier solution containing NaCI (preferably at a baseline concentration of 0.9%) and human serum albumin (HSA) (preferably at a final concentration of 5-15%, in particular 10%) is mixed with a solution of an excipient, such as high molecular weight hyaluronic acid (HA), to obtain the final formulation. It is preferred to use a 20 mg / ml HA solution (HA concentration before mixing with the cell suspension).

[0084] The cell suspension is mixed with the HA solution in a volume ratio of 1:3 to 2:3.

[0085] Preferably, the finally obtained pharmaceutical composition contains from 1% to 3% HSA, particularly preferably from 2.0% to 2.5% HSA.

[0086] Preferably, the volume of the cell suspension for preparing a single dose of the formulation for injection is in the range of 1-2 mL. This volume ensures proper mixing of the cell solution with the excipient (HA). At the same time, it is possible to maintain a high viability of AT-MSCs, which affects their functionality. The proposed dose volume is also a convenient volume for administration to the articular space (to the best medical knowledge of the inventors).

[0087] In an exemplary embodiment, the fixed therapeutic dose is 18-22 x 106AT-MSCs / 1 vial. At the same time, cell viability is at least 80%, wherein AT-MSCs identity is confirmed (presence of CD73, CD90, CD105 markers on the cell surface; simultaneous absence of CD14, CD19, CD34, CD45 antigens on the cell surface), and they have morphology characteristic of MSC cells. The sterility of the final product, which is also free of mycoplasmas and bacterial endotoxins, is also preserved.

[0088] In an exemplary embodiment, by using a carrier solution for AT-MSCs consisting of: 0.9% NaCI saline solution containing an additional 10% human serum albumin (HSA) obtained by mixing 0.9% NaCI with 20% HSA solution in a 1:1 volume ratio, a high viability of AT-MSCs was ensured, both before and after mixing with the excipient (HA). Figure 2 shows the viability of AT-MSC cells resuspended in solutions obtained using two types of HSA. The viability of AT-MSCs resuspended in 0.9% NaCI containing 10% human albumin after mixing with high molecular weight hyaluronic acid is shown; in a ratio of 1:3, directly (0 h), and 1 and 2 h after obtaining the formulation. The human albumin solution used to prepare the cell solution was manufactured by Grifols (HSA Grifols) or CSL Behring (HSA Behring). The viability was assessed by microscopic evaluation after staining the cells with trypan blue.

[0089] Example 3. Biological properties of the pharmaceutical composition.

[0090] Administration of the medicinal product described in Example 2 allows for effective regeneration of cartilage and bone defects and injuries arising during the course of osteoarthritis.

[0091] During preclinical studies conducted in a large model of mechanical injury of cartilage and bone in vivo (in pigs), animals treated with the pharmaceutical composition of the invention were observed to have twice the incidence of hyaline cartilage formation compared to animals treated with a carrier solution mixed with HA (without active substance, i.e., without AT-MSC cells) or animals not treated with any medicinal product. The pro-regenerative effect of AT-MSCs on cartilage tissue was experimentally confirmed, promoting not only the healing process, but also the formation of hyaline cartilage representing a normal type of cartilage on the surface of the articular bone.

[0092] Safety and pro-regenerative potential of AT-MSCs in a large preclinical model of mechanical injury of cartilage and bone in vivo (in pigs)

[0093] Safety and pro-regenerative potential of AT-MSCs was assessed by intra-articular administration of the developed medicinal product (of the present invention) in a large preclinical model of mechanical injury of cartilage and bone in vivo (in pigs). The animals were subjected to a surgical procedure, which resulted in a mechanical injury of cartilage and bone in the femur, in the knee joint, after which formulations marked as: " HA" - carrier solution (i.e.

[0094] 0.9% NaCI solution containing 10% HSA solution), then mixed with high molecular weight HA (without active substance, i.e. without AT-MSC cells), " AT-MSCs" - composition according to the invention (AT-MSC cells suspended in a carrier solution and then mixed with high molecular weight HA) prepared according to the description of the invention, Control (self-healing) -animals left to heal spontaneously.

[0095] Figure 3 shows the results of the assessment of the pro-regenerative potential of AT-MSC cells in a large preclinical model of cartilage and bone injury in vivo (in pigs). Figure 3 A. Comparison of 3D reconstruction of the cartilage and bone block taken from the femur by computed microtomography for selected animals. 3D visualization of the cartilage and bone structure (cross-section) made for the following groups of animals, which were administered with formulations marked as: " HA" - carrier solution (i.e. 0.9% NaCI solution containing 10% HSA solution), then mixed with high molecular weight HA (without active substance, i.e. without AT-MSC cells), " AT-MSCs" - composition according to the invention (AT-MSC cells resuspended in a carrier solution and then mixed with high molecular weight HA) prepared according to the description of the invention, Control (self-healing) - animals left to heal spontaneously. Figure 3 B. Quantitative assessment of damaged joint regeneration measured by cartilage defect volume [mm3], bone defect volume [mm3], and cartilage thickness over bone defect compared to cartilage thickness over healthy bone [mm].

[0096] Figure 4 shows the results of the assessment of the safety and pro-regenerative potential of AT-MSCs in a preclinical model of mechanical injury of cartilage and bone in vivo (in pigs). Groups of animals, which were administered with formulations marked as: " HA" - carrier solution (i.e. 0.9% NaCI solution containing 10% HSA solution), then mixed with high molecular weight HA (without active substance, i.e. without AT-MSC cells), " AT-MSCs" - composition according to the invention (AT-MSC cells resuspended in a carrier solution and then mixed with high molecular weight HA) prepared according to the description of the invention, Control (self-healing) - animals left to heal spontaneously. Figure 4 A. Histological staining of cartilage and bone sections taken from the femoral healing area. Figure 4 B. The type of newly formed tissue in the femoral healing area assessed by histological staining method.

[0097] After a 6-month follow up period, fully healed tissue defects (Figure 3A-B, 4A) filled with newly formed hyaline cartilage were observed twice as often in animals treated with formulations containing AT-MSC cells (compared to the control group), as shown by histological and microtomographic studies (Figure 3-4). Importantly, no unfilled cartilage and bone defects were observed in all animals treated with products containing AT-MSC cells (Figure 3B), confirming the pro-regenerative potential of the composition of the present invention. Interestingly, control animals administered with excipient only (carrier solution mixed with high molecular weight HA) or control animals showed limited healing of cartilage and bone defects, which were mostly filled with fibrous cartilage, fatty tissue, or unfilled (Figure 4B). Thus, data obtained from a study in pigs indicate a pro-regenerative effect of AT-MSC cells on cartilage tissue, promoting not only the healing process, but also the formation of hyaline cartilage representing a normal type of cartilage tissue on the surface of the articular bone.

[0098] In addition to the pro-regenerative capacity of AT-MSC cells (in the form of the investigational product containing 18-22x106AT-MSC cells resuspended in excipients and mixed with high molecular weight HA) with respect to the control product (excipients mixed with high molecular weight HA), the safety of these products was assessed by macroscopic and microscopic observations. In 7 of 7 animals treated with intra-articular knee administration of formulations containg AT-MSC cells, no tumors or other potential side effects associated with the administered products were observed, confirming the safety profile of products containg AT-MSC cells. A summary of the safety profile of the product containing AT-MSC cells as an active substance, subsequently administered in HA, is shown in Table 2.

[0099] Table 2. Safety assessment of the product containing AT-MSC cells as an active substance (i.e. a cell -based medicinal product) in a preclinical model of cartilage and bone injury in a large animal model in vivo (in pigs).

[0100] Macro- and microscopic observations

[0101] Other potential side effects Animal Administered Presence of neoplasms related to the administered number product [0-1] product

[0102] [0-1]

[0103] micro-CT 0 micro-CT 0 1 AT-MSCs

[0104] histology 0 histology 0 micro-CT 0 micro -CT 0 2 AT-MSCs

[0105] histology 0 histology 0 micro-CT 0 micro-CT 0 3 AT-MSCs

[0106] histology 0 histology 0 micro-CT 0 micro-CT 0 4 AT-MSCs

[0107] histology 0 histology 0 micro-CT 0 micro-CT 0 5 AT-MSCs

[0108] histology 0 histology 0 micro-CT 0 micro-CT 0 6 AT-MSCs

[0109] histology 0 histology 0 micro-CT 0 micro-CT 0 7 AT-MSCs

[0110] histology 0 histology 0

[0111] micro-CT - micro-CT - 1 HA

[0112] histology 0 histology 0 micro-CT 0 micro-CT 0 2 HA

[0113] histology 0 histology 0 micro-CT 0 micro-CT 0 3 HA

[0114] histology 0 histology 0 micro-CT 0 micro-CT 0 4 HA

[0115] histology 0 histology 0

[0116]

[0117] micro-CT 0 micro-CT 0 5 HA

[0118] histology 0 histology 0 micro-CT 0 micro-CT 0 6 HA

[0119] histology 0 histology 0

[0120]

[0121] Legend (markings of groups of animals according to the administered formulation): " HA" -carrier solution (i.e. 0.9% NaCI solution containing 10% HSA solution), then mixed with high molecular weight HA (without active substance, i.e. without AT-MSC cells), " AT-MSCs" -composition according to the invention (AT-MSC cells resuspended in a carrier solution and then mixed with high molecular weight HA) prepared according to the description of the invention; 0 - no neoplastic changes / other potential side effects; 1 - presence of neoplastic changes / other potential side effects; micro-CT - microtomography. Example 4. Clinical study.

[0122] A pharmaceutical composition of the MSC cell suspension according to the invention (hereinafter also referred to as MesoCellA-Ortho) was used in a phase I / II clinical study entitled "Randomized, double-blind, controlled phase I / II study to evaluate the safety and efficacy of the advanced therapy medicinal product MesoCellA-Ortho in patients with osteoarthritis after intra-articular knee administration" (BioMiStem-CT; NCT05081921), in order to assess the safety and efficacy of the developed medicinal product.

[0123] The clinical trial included patients aged 40-70 years with confirmed osteoarthritis of the 2nd / 3rd degree on the Kellgren-Lawrence scale, whose pain intensity was not lower than 3 (on a 10-point NRS numerical scale). In addition, these patients had no contraindications to liposuction and the use of biological therapy, stem cells, platelet-rich plasma and autologous whole blood. Two products were used in this study:

[0124] - MesoCellA-Ortho - composition according to the invention (AT-MSC cells resuspended in a carrier solution and then mixed with high molecular weight hyaluronic acid (HA)) prepared according to the description of the invention;

[0125] - Control product - carrier solution (i.e. 0.9% NaCI solution containing 10% HSA solution), then mixed with high molecular weight HA (without active substance, i.e. without AT-MSC cells); marked as " HA" in Figures 7-11 and ‘‘Hyaluronic acid therapy” in Tables 3, 4 and 6

[0126] The conducted clinical study was randomized and double-blind. A total of 192 patients received a therapy based on MesoCellA-Ortho product and a control product (hyaluronic acid), while 191 patients completed the clinical study. MesoCellA-Ortho was received by 97 patients, while the control product was received by 95 patients.

[0127] The clinical study design is shown in Figure 6.

[0128] The primary endpoints of this study in terms of determining the safety profile of the medicinal product included the measurement of the number and frequency of adverse events, including serious adverse events throughout the duration of the study.

[0129] Particular attention was paid to the occurrence of, among others, swelling of the knee joint, allergic reactions.

[0130] All adverse events were reported regardless of their association with the administration of MesoCellA-Ortho medicinal product / control product.

[0131] Table 3 shows the results of the evaluation of knee joint swelling summarized during the patient's site visits (Visits 1-12). No statistically significant differences between patient groups were observed. The percentage of patients who did not experience knee joint swelling (marked as “none”) at Visit 6 was comparable between the groups: 91.75% in the MesoCellA-Ortho group versus 89.47% in the hyaluronic acid group. By contrast, at Visit 12, the percentage of patients who did not experience knee joint swelling was 91.67% in the MesoCellA-Ortho group versus 80.00% in the hyaluronic acid group, suggesting a safety profile for MesoCellA-Ortho therapy.

[0132] Table 3. Presence of knee joint swelling at subsequent patient visits to the study site. Visit 6 -during the visit, patients were administered with MesoCellA-Ortho formulation or a control formulation - hyaluronic acid. All collected data were analyzed. N=95-97 patients / group, p-statistical significance.

[0133] MesoCellA-Ortho Hyaluronic

[0134] Total

[0135] Parameter Level therapy acid;|||| iiiii |f number

[0136] therapy iiiii Knee joint

[0137] swelling n 97 95 192 CA 1.02 2 0.3083 (Visit 1)

[0138] 83 (85.57%) 73 156

[0139] None

[0140] (76.84%) (81.25%)

[0141] 11 (11.34%) 21 32

[0142] Mild

[0143] (22.11%) (16.67%)

[0144] Moderate 3 (3.09%) 1 (1.05%) 4 (2.08%)

[0145] Knee joint

[0146] swelling n 97 95 192 CA 0.43 2 0.6706 (Visit 6)

[0147] 89 (91.75%) 85 174

[0148] None

[0149] (89.47%) (90.63%)

[0150] Mild 6 (6.19%) 8 (8.42%) 14 (7.29%)

[0151] Moderate 2 (2.06%) 2 (2.11%) 4 (2.08%)

[0152] Knee joint

[0153] swelling n 96 95 191 CA 0.83 3 0.4093 (Visit 11)

[0154] 84 (87.50%) 79 163

[0155] None

[0156] (83.16%) (85.34%)

[0157] 10 (10.42%) 14 24

[0158] Mild

[0159] (14.74%) (12.57%)

[0160] Moderate 2 (2.08%) 1 (1.05%) 3 (1.57%)

[0161] Severe 0 (0.00%) 1 (1.05%) 1 (0.52%)

[0162] Knee joint n 96 95 191 CA 1.31 3 0.1889

[0163]

[0164] swelling MesoCellA-Ortho Hyaluronic

[0165] Total

[0166] Parameter Level therapy acid iiiii number

[0167] therapy ■ BBiii II (Visit 12)

[0168] 88 (91.67%) 76 164

[0169] None

[0170] (80.00%) (85.86%)

[0171] 5 (5.21%) 18 23

[0172] Mild

[0173] (18.95%) (12.04%)

[0174] Moderate 2 (2.08%) 1 (1.05%) 3 (1.57%)

[0175] Severe 1 (1.04%) 0 (0.00%) 1 (0.52%)

[0176]

[0177] There was 1 case of allergic reaction in the group of patients who received MesoCellA-Ortho medicinal product. There was also 1 case of allergic reaction in the group of patients who received control product. No significant differences were observed in the incidence of allergic reactions between the MesoCellA-Ortho therapy receiving group and the hyaluronic acid therapy receiving group.

[0178] Table 4 shows the results of the presence of allergic reactions in both patient groups.

[0179] Table 4. Presence of allergic reactions after administration of MesoCellA-Ortho and control formulation - hyaluronic acid. Yes - the occurrence of an allergic reaction. No - the absence of an allergic reaction. All collected data were analyzed. N=95-97 patients / group, p- statistical significance.

[0180] Hyaluronic

[0181] MesoCellA-Ortho Total

[0182] Parameter Level acid iBiiii therapy number OB l therapy Bliii i Allergic

[0183] n 97 95 192 F - - 1.0000 reaction

[0184] yes 1 (1.03%) 1 (1.05%) 2 (1.04%)

[0185] 94 190

[0186] no 96 (98.97%)

[0187] (98.95%) (98.96%)

[0188] Number of

[0189] allergic n 97 95 192 F - - 1.0000 reactions

[0190] 94 190

[0191] 0 96 (98.97%)

[0192] (98.95%) (98.96%)

[0193] 1 1 (1.03%) 1 (1.05%) 2 (1.04%)

[0194]

[0195] Further, all adverse events that occurred in the group of patients treated with MesoCellA-Ortho product, the occurrence of which may be associated with the administration of this product, are presented.

[0196] Table 5 presents information on all adverse events that occurred in the group of patients treated with MesoCellA-Ortho product, the occurrence of which may be associated with the administered medicinal product. Knee joint pain and swelling, along with reduced range of motion (ROM) in the knee joint, occurred only in one patient who already had knee joint swelling before the administration of the product, but met all clinical study inclusion criteria. After this patient underwent an appropriate medical procedure, these symptoms were resolved.

[0197] Table 5. Adverse events that occurred in the group of patients treated with MesoCellA-Ortho product, the occurrence of which may be associated with the administration of the investigational medicinal product. For the analysis, data from 96 patients who received MesoCellA-Ortho product, were used.

[0198] Adverse events in the group of patients treated with MesoCellA-Ortho

[0199] Causality criterion: possible association with the investigational product

[0200] Adverse event Number / Frequency Severity criterion Intensity criterion Knee joint pain 1* / 96 (1.04%) Serious Moderate Knee joint swelling 1* / 96 (1.04%) Serious Moderate Decrease of ROM 1* / 96 (1.04%) Serious Moderate

[0201] (*) Patient with BMI>30, before product administration - knee joint swelling, increased Joint fluid volume; patient met all study inclusion criteria

[0202]

[0203] In the BioMiStem-CT clinical trial, no other serious adverse events were reported that could be a consequence of the administration of MesoCellA-Ortho product.

[0204] In conclusion, the obtained results presented in Tables 3-5 indicate a high safety profile of the MesoCellA-Ortho medicinal product based on the pharmaceutical composition comprised by the present application.

[0205] The primary endpoints of the BioMiStem-CT clinical trial in terms of determining the effectiveness profile of the medicinal product included the measurement of the positive effect of MesoCellA-Ortho therapy in knee joint osteoarthritis in the subjective assessment of the patient's function and pain. For this purpose, the following scales were used: i) KOOS (Knee Injury and Osteoarthritis Outcome Score) scale - subjective assessment of knee joint function, and ii) SF (The Short Form (36)) scale - subjective assessment of quality of life.

[0206] The results obtained using the KOOS scale indicate that patients treated with MesoCellA-Ortho experience a significant reduction in knee pain in relation to patients who were treated with hyaluronic acid (HA) (Figure 7). The reduction of pain sensation in these patients is accompanied by a significant improvement in their activity of daily living (Figure 8).

[0207] These results confirm the data obtained using the SF-36 questionnaire. Patients treated with MesoCellA-Ortho product experience a significant reduction in the level of perceived knee joint pain as well as a significant improvement in physical fitness in relation to patients who received a hyaluronic acid formulation (Figures 9 and 10). Importantly, the overall parameter - the physical dimension of the quality of life is significantly greater in the group of patients treated with MesoCellA-Ortho medicinal product (Figure 11), which indicates that the administration of MesoCellA-Ortho significantly increases the physical dimension of the quality of life of patients suffering from osteoarthritis.

[0208] The results of the conducted clinical tests are summarized in the following figures.

[0209] Figure 7 shows an assessment of the magnitude of pain experienced by patients who received MesoCellA-Ortho formulation and a control formulation (hyaluronic acid, HA) using the KOOS scale at individual visits to the study site. N=95-96 patients / group. (*) p<0.05.

[0210] Figure 8 shows an assessment of ability to perform daily activities by patients who received MesoCellA-Ortho formulation and a control formulation (hyaluronic acid, HA) using the KOOS scale at individual visits to the study site. N=95-96 patients / group. (*) p<0.05.

[0211] Figure 9 shows an assessment of the magnitude of pain experienced by patients who received MesoCellA-Ortho formulation and a control formulation (hyaluronic acid, HA) using the SF-36 questionnaire at Visits 1 and 12 to the study site. N=95-96 patients / group. (*) p<0.05.

[0212] Figure 10 shows an assessment of physical functioning ability by patients who received MesoCellA-Ortho formulation and a control formulation (hyaluronic acid, HA) using the SF-36 questionnaire at Visits 1 and 12 to the study site. N=95-96 patients / group. (*) p<0.05 Figure 11 shows an assessment of physical dimension of the quality of life based on responses from patients who received MesoCellA-Ortho formulation and a control formulation (hyaluronic acid, HA) using the SF-36 questionnaire at Visits 1 and 12 to the study site. N=95-96 patients / group. (*) p<0.05

[0213] Reduction of pain and improvement in the ability of daily physical functioning was particularly felt by obese patients (BMI>30) who received MesoCellA-Ortho medicinal product (Table 6). Table 6. Efficacy study of MesoCellA-Ortho medicinal product using the SF-36 questionnaire in a subgroup of obese patients (BMI > 30) not suffering from type 2 diabetes mellitus. The table presents only the values of parameters significantly differentiating the two groups of patients.

[0214] Parameter MesoCellA-Ortho Hyaluronic acid Statistical therapy therapy significance level

[0215] SF-36 questionnaire: Physical 21.15 (±23.88) 10.26 (±21.85) p=0.0464 Functioning (PF) (calculated as

[0216] value difference: Visit 12- Visit 1)

[0217] SF-36 questionnaire: Bodily Pain 27.85 (±23.31) 9.21 (±19.57) p=0.0003

[0218] (BP) (calculated as value

[0219] difference: Visit 12- Visit 1)

[0220] SF-36 questionnaire: Bodily pain 11.23 (±9.40) 3.71 (±7.89) p=0.0003

[0221] - standard based assessment

[0222] (BP_NBS) (calculated as value

[0223] difference: Visit 12- Visit 1)

[0224] SF-36 questionnaire: The 7.84 (±8.83) 2.87 (±6.65) p=0.0064 physical dimension of the quality

[0225] of life (PCS) (calculated as value

[0226] difference: Visit 12- Visit 1)

[0227] SF-36 questionnaire: Physical 71.03 (±23.40) 60.39 (±16.76) p=0.0238 Functioning (PF) (analyzed at

[0228] Visit 12 between groups)

[0229] SF-36 questionnaire: Bodily pain 68.87 (±24.04) 49.33 (±19.17) p=0.0003

[0230] (BP) (analyzed at Visit 12

[0231] between groups)

[0232] SF-36 questionnaire: Physical 46.45 (±8.96) 42.38 (±6.41) p=0.0247 Functioning - standard based

[0233] assessment (PF_NBS)

[0234] (analyzed at Visit 12 between

[0235] groups)

[0236] SF-36 questionnaire: Bodily pain 49.45 (±9.69) 41.57 (±7.73) p=0.0003

[0237] - standard based assessment

[0238] (BP_NBS) (analyzed at Visit 12

[0239] between groups)

[0240] SF-36 questionnaire: The 46.87 (±8.75) 42.12 (±6.47) p=0.0081 physical dimension of the quality

[0241] of life (PCS) (analyzed at Visit 12

[0242]

[0243] between groups)

[0244] In conclusion, the administration of MesoCellA-Ortho formulation results in a statistically significant reduction of knee joint pain and an improvement in the ability of daily functioning, thus increasing the physical dimension of the quality of life in patients, including obese patients. The secondary endpoints of the BioMiStem-CT clinical trial in terms of determining the effectiveness profile of the treatment included the use of, among others, magnetic resonance imaging (MRI) for the qualitative and quantitative assessment of articular cartilage and subchondral bone. MRI analysis of the knee joints showed slightly lower T2 relaxation time values in the MesoCellA-Ortho group compared to the hyaluronic acid group, which may indicate that the progression of osteoarthritis has stopped and the recovery process has begun. The results of the analyses indicate a reduction in the T2 relaxation time, especially within the medial tibia, in the group of patients who were treated with MesoCellA-Ortho formulation. Reducing the T2 relaxation time may reflect the initiation of the regeneration process of the damaged articular cartilage. The study identified so-called Responders, i.e. patients who received MesoCellA-Ortho medicinal product and in whom a beneficial effect of the administered dproduct on the integrity of articular cartilage was found. In the next phase III clinical study, it is planned to include patients whose clinical characteristics are the same as those of the Responder patients.

[0245] Figure 12 summarizes the results of the T2 relaxation time analysis based on the MRI. For each group of patients, for each anatomical area shown in the graph, the average ROI value was calculated and presented as a bar on the graph. The value for each ROI area included in the analysis has been presented as a separate point. Results are presented as % change at Visit 12 compared to values at Visit 3 (**) p<0.01.

[0246] In conclusion, the presented results indicate a high safety profile of the developed MesoCellA-Ortho medicinal product, as well as its significant impact on the reduction of knee joint pain and the improvement of the ability of daily functioning. In addition, the use of MesoCellA-Ortho formulation affects the integrity of articular cartilage, especially in the group of patients referred to as Responders.

Claims

Claims1. A pharmaceutical composition consisting of a suspension of human mesenchymal stem / stromal cells (MSCs), especially derived from adipose tissue (AT-MSCs) and an excipient, wherein the MSC suspension is prepared in a carrier solution (HA) comprising: a solution isotonic to human cells, preferably a saline solution (with 0.9% NaCI) with human albumin (HSA), wherein the excipient is a solution comprising hyaluronic acid, wherein the volume ratio of the MSC suspension to the HA solution is from 1:1 to 1:10, preferably from 1:3 to 2:3.

2. The pharmaceutical composition of claim 1, characterised in that the MSC cells comprised in the suspension: have a viability of at least 80%, express, among others, CD73, CD90 and CD105 markers on cell surface; and do not express, among others, CD14, CD19, CD34 and CD45 markers on cell surface, have the morphology characteristic of MSCs, are sterile and free from mycoplasmas and bacterial endotoxins.

3. The pharmaceutical composition of claim 1, characterised in that the MSC suspension is preferably obtained in a solution comprising: a solution isotonic to human cells, preferably a saline solution (with 0.9% NaCI) and HSA in a concentration of from 1 % to 20%, preferably in a concentration of 10% HSA obtained by mixing 0.9% NaCI solution with 20% HSA solution in a 1:1 ratio.

4. The pharmaceutical composition of claim 1, characterized in that the excipient is a hyaluronic acid solution at a concentration of 2-20 mg / mL, preferably 12-15 mg / mL.

5. The pharmaceutical composition of claim 1, characterised in that it comprises from 1 % to 10% HSA, preferably from 2.0% to 2.5% HSA.

6. The pharmaceutical composition of claims 1-5 for use in the treatment or prevention of bone or joint disease, particularly for the regeneration of degenerative lesions and cartilage and bone injuries, preferably osteoarthritis.

7. The pharmaceutical composition for use according to claim 6, characterized in that it is intended for local administration, preferably intra-articular administration.

8. The pharmaceutical composition for use according to claim 6, characterized in that the dosage unit comprises 2-50 x 106live MSC cells, preferably 18-22 x 106live MSC cells.

9. The pharmaceutical composition for use according to claim 6, characterized in that the volume of the dosage unit (MSC cell suspension) is from 0.5 ml to 4 ml, preferably from 1 to 2 ml.

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