PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING OSTEOARTHRITIS

Abstract

The present invention provides a pharmaceutical composition for prevention or treatment of osteoarthritis including cells or cell groups having a specific size or less, a kit for preparing a cell therapeutic agent, and a method for manufacturing a therapeutic agent for osteoarthritis. Mixed cells, which are selected to a specific size or less as an active ingredient of the present invention, of transformed mammalian cells with TGF- and untransformed mammalian cells minimize aggregation between chondrocytes and have beneficial therapeutic effects. In addition, when administering the selected mixed cells having the specific size or less to a patient, patient compliance may be improved, and it is easy to manage quality in therapeutic agent manufacturing facilities or hospitals.

Claims

1. A pharmaceutical composition for prevention or treatment of osteoarthritis, comprising a transformed mammalian cell population, wherein cells or cell groups included in the population have a particle diameter D.sub.90 of less than 200 m.

2. The composition according to claim 1, wherein the transformed mammalian cell population is further treated by irradiation.

3. The composition according to claim 1, further comprising an untransformed mammalian cell population in addition to the untransformed mammalian cell population.

4. The composition according to claim 3, wherein the cells or cell groups included in the untransformed mammalian cell population have a particle diameter D.sub.90 of less than 300 m.

5. The composition according to claim 3, wherein the untransformed mammalian cell population and the transformed mammalian cell population are mixed in a mixing ratio of 1 to 10:1 based on the number of cells.

6. The composition according to claim 1, wherein the mammalian cell is a chondrocyte or a chondroprogenitor cell.

7. The composition according to claim 1, wherein the mammalian cell is transformed with a transforming growth factor beta (TGF-).

8. A kit for preparation of a cell therapeutic agent, comprising a mammalian cell population transformed with a transforming growth factor beta (TGF-), wherein cells or cell groups included in the population have a particle diameter D.sub.90 of less than 200 m.

9. The kit according to claim 8, further comprising an untransformed mammalian cell population in addition to the untransformed mammalian cell population.

10. The kit according to claim 9, wherein the cells or cell groups included in the untransformed mammalian cell population have a particle diameter D.sub.90 of less than 300 m.

11. The composition according to claim 9, wherein the untransformed mammalian cell population and the transformed mammalian cell population are mixed in a mixing ratio of 1 to 10:1 based on the number of cells.

12. A method for manufacturing an osteoarthritis therapeutic agent, comprising the steps of: (1) preparing a transformed mammalian cell population with TGF- and an untransformed mammalian cell population, respectively; (2) selecting cells or cell groups having a particle diameter D.sub.90 of less than 200 m included in the transformed mammalian cell population with TGF- in the above step (1); and cells or cell groups having a particle diameter D.sub.90 of less than 300 m included in the untransformed mammalian cell population, respectively; (3) filling a separate vial with both of the selected cell populations in step (2), respectively; and (4) mixing the selected cell populations in the vial in step (3).

13. The method according to claim 12, wherein the mixing in step (4) is to mix the transformed mammalian cell population and the untransformed mammalian cell population in step (2) in a mixing ratio or 1 to 10:1 based on the number of cells.

14. The method according to claim 12, wherein the transformed mammalian cell population with TGF- in step (1) is further irradiated.

Description

DESCRIPTION OF DRAWINGS

[0064] FIG. 1 is views illustrating vials before and after chondrocytes passed through a cell strainer.

[0065] FIG. 2A is photographs illustrating microscopic observation results before and after untransformed cells passed through the cell strainer; and FIG. 2B is photographs illustrating microscopic observation results before and after transformed cells passed through the cell strainer.

[0066] FIG. 3A is photographs illustrating microscopic observation results before and after untransformed cells passed through the cell strainer over time, respectively; and FIG. 3B is photographs illustrating microscopic observation results before and after transformed cells passed through the cell strainer over time, respectively.

[0067] FIG. 4A is a graph illustrating results of von Frey filament tests when an MIA-induced osteoarthritis animal model was treated with the selected mixed cells; and FIG. 4B a graph illustrating results of von Frey filament tests in an area under the curve (AUC) when treating the MIA-induced osteoarthritis animal model with the selected mixed cells.

[0068] FIG. 5 is photographs illustrating results of H & E staining analysis of cartilage tissues when treating the MIA-induced osteoarthritis animal model with the selected mixed cells.

BEST MODE

[0069] Hereinafter, the following embodiments are provided to more concretely describe the present invention. It will be apparent to those skilled in the art that the scope of the present invention in regard to the objects of invention is not limited by these embodiments.

Example 1. Preparation of Cell Therapeutic Agent

[0070] The cell therapeutic agent used in this example of the present invention is a transformed cell population so as to express TGF-1 (NCBI Reference Sequence: NM_000660.6) (first population; hereinafter referred to as TC) and a normal cell population without transformation using the above gene (second population; hereinafter referred to as HC).

[0071] The TC could be prepared by injecting cDNA of TGF-1 into cells according to a known method. For instance, the cDNA of TGF-1 is inserted into a known vector having a resistant gene such as ampicillin or neomycin (for example, pCI (containing ampicillin resistant gene) from Promega Co.) to construct a vector containing cDNA of TGF-1, followed by injecting the same into chondrocytes according to a known method such as a calcium phosphate method or a lipofectin method, thus to prepare TC. Otherwise, the TC may be prepared using a gene delivery vehicle such as retroviral vectors, lentiviral vectors, and the like.

[0072] The HC and TC are human-derived chondrocytes, wherein HC is a normal chondrocyte while TC is a transformed chondrocyte to secrete TGF-1. A method for construction of HC and TC has been disclosed in known documents [Cytotherapy, 2012 February; 14 (2): 247-256) and U.S. Pat. Nos. 7,005,127 and 7,282,200.

[0073] A mixing ratio of HC and TC was 3:1 based on the number of cells and was applied to the following examples.

[0074] The prepared TC and HC were filled in a vial, respectively, then frozen and prepared/stored for use as a mixed cell-based therapeutic agent. At this time, the TC was inactivated by irradiation before or after freezing.

Example 2. Selection of Cell Types Having Efficiency

[0075] The present inventors have intended to identify effectiveness of the mixed cell, which is the cell therapeutic agent prepared in Example 1, according to the cell aggregates type and size.

[0076] For this purpose, a cell strainer was prepared for each pore size, and then subjected to a series of quality control (QC) for determining whether HC cells and TC cells were aggregated.

[0077] 2-1. Identification of Cell Aggregation in HC Cells Depending Upon the Pore Size of Cell Strainer

[0078] The present inventors have cultured HC cells and collect a cell suspension. Some of the cells were not filtered using a cell strainer, and the cells were filled in a vial and inspected for foreign substance. The remaining cell suspension was sieved with cell strainers having pore sizes of 200 and 300 m, respectively, followed by inspection of foreign substances after cell filtration using the cell strainers.

[0079] The results are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Non-use of cell HC cell strainer 200 m 300 m Cell aggregation after Yes No Yes using a cell strainer

[0080] That is, in order to determine whether or not the cell aggregation phenomenon of HC cells depending upon the pore size of the cell strainer, after passing the cell suspension through the cell strainer to completely remove cell aggregates, whether or not the cell aggregate was reduced has been checked. For the HC cells, the cells passing through 200 m cell strainer were all filtered out of the cell aggregate as shown in FIG. 1, and therefore, were passed for foreign substance inspection after filling.

[0081] Further, as shown in FIG. 2A, it was observed under a microscope that the cell aggregate disappeared after application of 200 m cell strainer.

[0082] On the other hand, after application of 300 m cell strainer, it was visibly confirmed that irregular and large aggregate as shown in FIG. 1 were not removed but remained.

[0083] Accordingly, in a case of HC cell, it could be seen that cells having a particle diameter D.sub.90 of less than 300 m, which can pass through the 300 m cell strainer, in particular, cells having a particle diameter D.sub.90 of less than 200 m are preferable.

[0084] 2-2. Identification of Cell Aggregation in TC Cells Depending Upon the Pore Size of Cell Strainer

[0085] The present inventors have cultured TC cells and collect a cell suspension. Some of the cells were not filtered using a cell strainer, and the cells were charged in a vial and inspected for foreign substance. The remaining cell suspension was sieved with cell strainers having pore sizes of 70, 100 and 200 m, respectively, followed by inspection of foreign substance after cell filtration using the cell strainers.

[0086] The results are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Non-use of cell TC cell strainer 70 m 100 m 200 m Cell aggregation Yes No No Yes after using a cell strainer

[0087] That is, in order to determine whether or not the cell aggregation phenomenon of TC cells depending upon the pore size of the cell strainer, after passing the cell suspension through the cell strainer to completely remove cell aggregation, whether or not the cell aggregation was reduced has been checked. For TC cells, the cells passing through 70 m and 200 m cell strainers were all filtered out of the cell aggregate as shown in FIG. 1, and therefore, were passed for foreign substance inspection after filling.

[0088] Further, as shown in FIG. 2B, it was observed under a microscope that the cell aggregate disappeared after application of 100 m cell strainer.

[0089] On the other hand, after application of 200 m cell strainer, it was visibly confirmed that irregular and large aggregate shown in FIG. 1 were not removed but remained.

[0090] Accordingly, in a case of TC cell, it could be seen that cells having a particle diameter D.sub.90 of less than 200 m, which can pass through the 200 m cell strainer, in particular, cells having a particle diameter D.sub.90 of less than 100 m are preferable.

Example 3. Identification of Further Aggregation Prevention after Application of Cell Strainer

[0091] The present inventors have determined whether or not the cells before and after passing through the cell strainer prepared in Example 2 have a change in cell aggregation type and size over time, and whether or not the application of the strainer has been helpful to prevent further aggregation.

[0092] For this purpose, a cell strainer was prepared for each pore size and used for filtration. Then, the vial was left at room temperature to determine whether or not the aggregation of HC and TC cells was changed over time.

[0093] 3-1. Identification of Cell Aggregation in HC Cells Over Time after Application of Cell Strainer

[0094] The present inventors have cultured HC cells and collect a cell suspension. Some cells were filled in a vial without application of a cell strainer and observed cell aggregation over time. The remaining cell suspension was sieved with a cell strainer having a pore size of 200 m and the cells were filled in the vial, followed by determining cell aggregation phenomenon over time.

[0095] In order to determine whether or not the cell aggregation phenomenon of HC cells depending upon the pore size of the cell strainer, after passing the cell suspension through the cell strainer to completely remove cell aggregates, whether or not the cell aggregate was reduced has been checked.

[0096] According to the results, as shown in FIG. 3A, it was observed under a microscope that, after application of the 200 m cell strainer at 0 hour, the cell aggregate was removed. Further, it was also observed under a microscope that the cell aggregates was no longer generated even after 3 hours has elapsed.

[0097] On the other hand, when the cell strainer was not applied, it was observed under a microscope that irregular and large aggregates remained without being removed.

[0098] Furthermore, the agglomerates still remained even after the time has elapsed.

[0099] 3-2. Identification of Cell Aggregation in TC Cells Over Time after Application of Cell Strainer

[0100] The present inventors have cultured TC cells and collect a cell suspension. Some cells were filled in a vial without application of a cell strainer and observated cell flocculation over time. The remaining cell suspension was sieved with a cell strainer having a pore size of 100 m, and the cells were filled in the vial, followed by determining cell aggregation phenomenon over time.

[0101] In order to determine whether or not the cell aggregation phenomenon of TC cells depending upon the pore size of the cell strainer, after passing the cell suspension through the cell strainer to completely remove cell aggregate, whether or not the cell aggregate was reduced has been checked.

[0102] According to the results, as shown in FIG. 3B, it was observed under a microscope that, after application of the 100 m cell strainer at 0 hour, the cell aggregate was removed. Further, it was also observed under a microscope that the cell aggregate was no longer generated even after 3 hours has elapsed.

[0103] On the other hand, as shown in FIG. 3B, when the cell strainer was not applied, it was observed under a microscope that irregular and large aggregates remained without being removed. Furthermore, the aggregates still remained even after the time has elapsed.

Example 4. Identification of Osteoarthritis Therapeutic Effects by Treatment Using Selected Mixed Cells in MIA Osteoarthritis Model

[0104] 4-1. Confirmation of the Effect of Pain Relief

[0105] The present inventors have intended to determine effectiveness of the mixed cell therapeutic agent according to the present invention by: administering the selected mixed cells having a specific size or less defined in Example 2, that is, the mixed cells, which include HC cells having a particle diameter D.sub.90 of less than 300 m and TC cells having a particle diameter D.sub.90 of less than 200 m in a ratio of 3:1, to a rat animal model with osteoarthritis induced by MIA administration; and comparing analgesic effects therein.

[0106] In other words, in order to examine therapeutic effects of the selected mixed cells, the present inventors have prepared an MIA-induced osteoarthritis animal model, and then treated the model with the selected mixed cells, in order to observe a change in pain.

[0107] First, 6-week-old male rats (SPARGUE-DAWLEY, 200 to 225 g, Nara Biotech, Korea) were used for animal modeling. Animal experiments were conducted under approval by Institutional Animal Care and Use Committee in Kolon Life Science (IACUC No. KLS IACUC 2013-04) and under the supervision of a veterinary surgeon.

[0108] In order to induce arthritis, 50 l of MIA (monosodium iodoacetate, Sigma, USA) solution with a concentration of 60 mg/mL was administered into the joint cavity in the left knee of a rat using a 31 G syringe.

[0109] 2 weeks after the MIA administration, objects with developed osteoarthritis were subjected to administration of a vehicle (CS-10) and the mixed cells, respectively, into the joint cavity in the left knees.

TABLE-US-00003 TABLE 3 Dosage of Number MIA Administered of Animal administration substance Group Gender animals code (mg/ knee) (50 L/knee) G1 M 6 1481-1486 3 Vehicle (CS- 10) G2 M 6 1505-1510 3 2.8 10.sup.5 cells

[0110] Thereafter, von Frey filament test was performed. This test was conducted using 50% up & down threshold method which was established in 1980 by Dixon (Chaplan S R et al., Quantitative assessment of tactile allodynia in the rat paw, Journal of Neuroscience Methods, 1994, 53: 55-63; and Dixon W. J., Efficient analysis of experimental observations, Annual Reviews Pharmacology Toxicology, 1980, 20: 441-62). Using a total of nine (9) von Frey filaments with N values of 0.4, 0.6, 1, 2, 4, 6, 8, and 15 grams (g), respectively, pain response was examined and a threshold value was calculated according to predetermined patterns.

[0111] A certain period of time (0, 7, 14, 21, 28, 35, 42, 49 and 56 days) passing after the administration of the mixed cells and the control substance, von Frey-filament test values were measured.

[0112] According to the results, as shown in FIGS. 4A and 4B, the von Frey filament measurement values at the certain period of time (0, 7, 14, 21, 28, 35, 42, 49 and 56 days) after the administration of the mixed cells have demonstrated that the mixed cell administration group exhibited statistically significant analgesic effects, compared to the control group, that is, the CS-10 administration group (p<0.05). When these von Frey filament results were expressed as AUC value (area under the curve), which is used as a performance evaluation index, the mixed cell administration group showed statistically significant results, as compared to the control group, that is, the CS-10 administration group (p<0.05).

[0113] 4-2. Identification of Cartilage Structural Improvement Effects

[0114] The present inventors have implemented comparison of cartilage structural improvement between a control group (vehicle) and the selected mixed cells having a specific size or less as prepared in Example 2, which were administered to a rat model with osteoarthritis induced by MIA administration, respectively, thereby determining effectiveness of the mixed cell therapeutic agent.

[0115] For this purpose, the animal model prepared as in Example 4-1 was sacrificed 56 days after the experiment, and then H & E staining analysis was implemented.

[0116] According to the result, as shown in FIG. 5, no cartilage structural improvement was observed in the control group (vehicle), that is, the CS-10 administration group, whereas cartilage structural improvement was observed in the mixed cell administration group.

[0117] Consequently, the mixed cells selected according to the present invention, wherein HC cells having a particle diameter D.sub.90 of less than 300 m and TC cells having a particle diameter D.sub.90 of less than 200 m are mixed in a ratio of 3:1, may exhibit excellent pain alleviation and cartilage formation effects even in the absence of aggregated cells. Therefore, the mixed cells may be used as an excellent therapeutic agent for osteoarthritis.