Mesenchymal stem cell exosome and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to application of a mesenchymal stem cell exosome in preparation of a medicine for treating premature ovarian failure. The exosome provided by the invention can effectively repair the damaged degree of the ovary and improve the ovarian function.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of mesenchymal stem cell exosomes in the preparation of premature ovarian failure, especially chemotherapy-induced premature ovarian failure. Background Technology

[0002] Chemotherapy drugs are an important treatment for cancer, but they can also significantly impact ovarian function, leading to premature ovarian failure (POF). POF refers to the decline in ovarian function in women before the age of 40, manifested as decreased hormone levels and impaired fertility. Chemotherapy drugs exert toxicity on the ovaries through multiple mechanisms, affecting egg reserve and function. First, chemotherapy drugs can directly damage follicular cells in the ovary. Many chemotherapy drugs, such as cyclophosphamide, fluorouracil, and paclitaxel, can affect cell division and proliferation, leading to a decrease in the number and quality of follicles. This damage may be temporary, but in some cases, ovarian dysfunction may be irreversible. Second, chemotherapy can trigger ovarian ischemia and inflammation, further exacerbating the decline in ovarian function. After chemotherapy, some patients may experience menopausal symptoms such as irregular menstruation and hot flashes, indicating impaired ovarian function. Furthermore, different types of chemotherapy regimens have varying degrees of impact on the ovaries. High-dose and long-term chemotherapy generally causes more significant ovarian damage than short-term and low-dose chemotherapy. In addition, the patient's age, ovarian reserve, and the specific combination of chemotherapy drugs all affect the recovery of ovarian function. Some common treatment options include hormone replacement therapy (HRT), oocyte cryopreservation, and ovarian preservation surgery. HRT can alleviate symptoms caused by declining ovarian function by supplementing estrogen and progesterone, but it may increase the risk of certain cancers (such as breast cancer), may not be suitable for some patients, especially those with a history of blood clots or cardiovascular disease, and does not restore fertility. Oocyte cryopreservation involves freezing eggs or embryos before chemotherapy to provide options for future fertility; however, the freezing process involves hormone stimulation, which may lead to further ovarian damage in some cases. Ovarian preservation surgery can reduce the direct damage of chemotherapy to the ovaries, but the surgery itself carries risks such as infection and bleeding, and it cannot completely eliminate the systemic effects of chemotherapy drugs.

[0003] Mesenchymal stem cells (MSCs) have attracted widespread attention in regenerative medicine and the treatment of various diseases in recent years, including their potential role in premature ovarian failure (POF). They may promote the repair and regeneration of ovarian cells by secreting growth factors and cytokines. These cells can protect and repair damaged ovarian tissue, potentially helping to restore normal ovarian function. On the other hand, MSCs may help improve hormone levels and alleviate menopausal symptoms caused by POF (such as hot flashes and irregular menstruation). MSCs also possess immunomodulatory properties, which can reduce the inflammatory response that may be involved in POF and protect ovarian tissue. Nevertheless, although MSCs have relatively good immunomodulatory properties, they may still trigger immune responses in some cases, leading to adverse reactions. Sufficient data on the long-term safety and efficacy of stem cell therapy are still lacking, and unknown long-term risks may exist.

[0004] Mesenchymal stem cells are rich in a variety of bioactive molecules, and exosomes are small vesicles secreted by them, which have functions such as intercellular communication, promoting tissue repair and regulating immunity. Summary of the Invention

[0005] Purpose of the invention

[0006] In view of the problems or needs existing in the prior art, the purpose of this invention is to provide an exosome of mesenchymal stem cells and to develop the application of this exosome in the preparation of a drug for treating premature ovarian failure.

[0007] Solution

[0008] To achieve the above objectives, the present invention has obtained mesenchymal stem cell exosomes that highly express certain marker miRNAs or proteins through extensive screening.

[0009] Specifically, the present invention provides the following technical solution:

[0010] In a first aspect of the invention, a mesenchymal stem cell exosome is provided, which highly expresses the following microRNAs: hsa-miR-21-5p, hsa-miR-199a-3p, hsa-miR-221-3p, hsa-miR-146a-5p, hsa-miR-100-5p, hsa-miR-320a, hsa-miR-92a-3p, hsa-miR-423-5p, hsa-miR-199a-5p, hsa-miR-29a-3p, said microRNAs having the sequences shown in SEQ ID No. 1 to 10; or proteins: A2 macroglobulin (A2M), complement C3 (C3), immunoglobulin δ-1 heavy chain (Immunoglobulin δ-1 heavy chain). The protein contains the following sequences: immunoglobulin λ-1 light chain, immunoglobulin heavy constant μ (IGHM), immunoglobulin heavy constant alpha 1 (IGHA1), immunoglobulin λ constant 2 (IGLC2), complement factor H (CFH), haptoglobin (HP), and complement C4-A (C4-A), wherein the protein has the sequences shown in SEQ ID Nos. 11 to 20. In a preferred embodiment, the microRNA has the sequences shown in SEQ ID Nos. 1 to 10, and the protein has the sequences shown in SEQ ID Nos. 11 to 20.

[0011] In a preferred embodiment, the microRNA is expressed at a level of not less than 50% in the total exosome RNA, more preferably not less than 54%.

[0012] In another preferred embodiment, the protein is expressed at a level of not less than 60% of the total exosome protein, more preferably not less than 65%, and even more preferably not less than 67%.

[0013] In a second aspect of the invention, the use of the aforementioned mesenchymal stem cell exosomes in the preparation of a medicament for treating premature ovarian failure is provided. Preferably, the premature ovarian failure is chemotherapy-induced premature ovarian failure.

[0014] In a third aspect of the invention, a composition is provided comprising mesenchymal stem cell exosomes as described in any of the preceding claims. In this composition, the number of active exosomes is not less than 1.5 × 10⁻⁶. 6 The composition is preferably in injectable form and is administered intravenously.

[0015] Beneficial effects

[0016] The mesenchymal stem cell exosomes provided by this invention can effectively repair ovarian damage and improve ovarian function, with significant improvements in multiple biochemical and hormonal indicators. Attached Figure Description

[0017] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0018] Figure 1 The morphological observation and biomarker analysis of exosomes prepared in Example 1 of this invention are as follows: (A): The extracted exosomes contain universal biomarker proteins: HSC70, TSG101 and CD9; (B): Representative results of transmission electron microscopy of exosomes; (C): Images of exosomes CD-61 (green) and CD-63 (red) under a super-resolution confocal microscope: exosomes numbered 1, 3, 5, 7, 9, and 10 are in red and green; exosome number 4 is red; exosomes numbered 2, 6, and 8 are green.

[0019] Figure 2 To investigate the effects of exosome treatment on serum (A)E2, (B)FSH, (C)LH, and (D)AMH levels in mice in Example 2 of this invention, a one-way ANOVA was performed with n=8. Control group: control group; Model group: POF model group; High: high-dose group; Medium: medium-dose group; Low: low-dose group. Compared with the model group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0020] Figure 3 Example 2 of this invention illustrates the effect of exosome treatment on the expression level of Ki67 in mouse ovarian tissue. (A): Control group; (B): Model group; (C): High-dose group; (D): Medium-dose group; (E): Low-dose group.

[0021] Figure 4 Example 2 of this invention illustrates the effect of exosome treatment on MVH expression levels in mouse ovarian tissue. (A): Control group; (B): Model group; (C): High-dose group; (D): Medium-dose group; (E): Low-dose group.

[0022] Figure 5 This is a bar chart showing the biochemical indicators of mouse blood samples in Example 3 of the present invention, analyzed by one-way ANOVA, n = 8-10. Control: control group; Exosomes: exosome group; Model: POF model group; High: high-dose group; Medium: medium-dose group; Low: low-dose group. Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Detailed Implementation

[0023] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0024] Unless otherwise stated, the present invention will be implemented using conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, all of which are within the scope of the art.

[0025] To facilitate a better understanding of this invention, certain technical terms are specifically defined as follows. Unless otherwise expressly defined elsewhere in this document, the technical terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] The term “about” when used in conjunction with a numeric value means to encompass a range of numeric values ​​having a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as these variations are suitable for carrying out the disclosed methods.

[0027] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0028] As used herein, the term “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” should be interpreted inclusively, that is, including at least one in the quantity or element list, but also including more than one, and optionally, additional unlisted items. Only when explicitly indicated by the opposite terms, such as “only one” or “exactly one” or when “consisting of” is used in the claims, will it refer to only one number or one element of the list.

[0029] The term "percentage (%) amino acid sequence identity," or simply "identity," is defined as the percentage of identical amino acid residues in a candidate amino acid sequence to a reference amino acid sequence after aligning the amino acid sequences (and, where necessary, introducing gaps) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine percentage amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0030] Example 1: Preparation of exosomes

[0031] Human umbilical cord mesenchymal stem cells (cultured and provided by Shenzhen Xinyilun Biotechnology Co., Ltd.) were cultured in α-MEM medium containing 10% fetal bovine serum until approximately 50% confluence was achieved. Cells at approximately 50% confluence were then replaced with phenol red-free DMEM medium (containing 10% fetal bovine serum) and cultured for 48 hours at 37°C and 5% CO2. Cell culture medium was collected every 12 hours for a total of four times to facilitate large-scale exosome production. The collected medium was first centrifuged at 1300 rpm for 10 minutes at 4°C, then filtered through a 0.22 μm vacuum filter / storage bottle system to remove large non-exosome particles, including cells, cell debris, microvesicles, and apoptotic bodies. Finally, exosomes were isolated using ultracentrifugation (10000g, 4°C for 30 minutes; the supernatant was then centrifuged again at 100000g, 4°C for 90 minutes; the supernatant was discarded; the remaining precipitate was resuspended in PBS and centrifuged again at 100000g, 4°C for 90 minutes). Preliminary quality control was conducted through morphological observation and detection of marker proteins and CD markers (such as CD63 and CD61) (see [link to relevant documentation]). Figure 1 All exosomes should exhibit a microvesicle morphology, possess the universal marker proteins HSC70, TSG101, and CD9, and be positive for CD63 and CD61, indicating that the extracted and isolated exosomes are exosomes. Proteomics and miRNA analysis were then performed using LC-MS / MS and small RNA sequencing.

[0032] The expression of exosomal marker microRNAs was quantitatively analyzed by small RNA sequencing (conducted by Hong Kong Chengqi Medical Biotechnology Co., Ltd.): hsa-miR-21-5p, hsa-miR-199a-3p, hsa-miR-221-3p, hsa-miR-146a-5p, hsa-miR-100-5p, hsa-miR-320a, hsa-miR-92a-3p, hsa-miR-423-5p, hsa-miR-199a-5p, hsa-miR-29a-3p. The expression level of the above microRNAs accounted for no less than 50% of the total RNA expression in exosomes, preferably no less than 54.14%.

[0033] hsa-miR-21-5p sequence (SEQ ID No. 1): 5'-UAGCUUAUCAGACUGAUGUUGA-3'

[0034] hsa-miR-199a-3p sequence (SEQ ID No.2): 5'-ACAGUAGUCUGCACAUUGGUUA-3'

[0035] hsa-miR-221-3p sequence (SEQ ID No. 3): 5'-AGCUACAUUGUCUGCUGGGUUUC-3'

[0036] hsa-miR-146a-5p (SEQ ID No.4): 5'-UGAGAACUGAAUUCCAUGGGUU-3'

[0037] hsa-miR-100-5p (SEQ ID No.5): 5'-AACCCGUAGAUCCGAACUUGUG-3'

[0038] hsa-miR-320a (SEQ ID No.6): 5'-AAAAGCUGGGUUGAGAGGGCGA-3'

[0039] hsa-miR-92a-3p (SEQ ID No.7): 5'-UAUUGCACUUGUCCCGGCCUGU-3'

[0040] hsa-miR-423-5p (SEQ ID No.8): 5'-UGAGGGGCAGAGAGCGAGACUUU-3'

[0041] hsa-miR-199a-5p (SEQ ID No.9): 5'-CCCAGUGUUCAGACUACCUGUUC-3'

[0042] hsa-miR-29a-3p (SEQ ID No.10): 5'-UAGCACCAUCUGAAAUCGGUUA-3'

[0043] The expression of exosomal marker proteins was quantitatively analyzed using proteomics (contracted to Hong Kong Chengqi Medical Biotechnology Co., Ltd.): A2 macroglobulin (A2M, Alpha-2-macroglobulin), complement C3 (C3, ComplementC3), immunoglobulin δ-1 heavy chain (Immunoglobulin gamma-1 heavy chain), immunoglobulin λ-1 light chain (Immunoglobulin lambda-1 light chain), immunoglobulin heavy constant μ (IGHM, Immunoglobulin heavy constant mu(IGHM)), immunoglobulin heavy constant α1 (IGHA1, Immunoglobulin heavyconstant alpha 1(IGHA1)), immunoglobulin λ constant 2 (IGLC2, Immunoglobulin lambda constant 2(IGLC2)), complement factor H (CFH, Complement factor H), haptoglobin (HP, Haptoglobin), and complement C4-A (C4-A, Complement C4-A). The expression level of C4-A and above proteins accounts for no less than 60% of the total protein expression level in exosomes, preferably no less than 65%, and more preferably no less than 67.09%.

[0044] The sequence of A2 macroglobulin (A2M, Alpha-2-macroglobulin) is shown in SEQ ID No. 11.

[0045] The protein sequence of complement C3 (C3) is shown in SEQ ID No. 12.

[0046] The sequence of the immunoglobulin δ-1 heavy chain is shown in SEQ ID No. 13.

[0047] The sequence of the immunoglobulin λ-1 light chain is shown in SEQ ID No. 14.

[0048] The immunoglobulin weight constant μ (IGHM) sequence is shown in SEQ ID No. 15.

[0049] The sequence of immunoglobulin weight constant α1 (IGHA1) is shown in SEQ ID No. 16.

[0050] The sequence of immunoglobulin λ constant 2 (IGLC2) is shown in SEQ ID No. 17.

[0051] The protein sequence of complement factor H (CFH) is shown in SEQ ID No. 18.

[0052] The sequence of haptoglobin (HP) is shown in SEQ ID No. 19.

[0053] The protein sequence of complement C4-A (C4-A) is shown in SEQ ID No. 20.

[0054] Example 2: Mesenchymal exosomes with high expression of marker miRNAs and marker proteins improve ovarian function in POF mice.

[0055] Forty female C57BL / 6 mice aged 6 to 8 weeks were divided into 5 groups: (1) normal mice, blank control group without modeling or any treatment; (2) premature ovarian failure (POF) group (model group); (3) POF + low-dose exosome group (low-dose group); (4) POF + medium-dose exosome group (medium-dose group); (5) POF + high-dose exosome group (high-dose group), with 8 mice in each group.

[0056] POF was induced by cyclophosphamide / busulin treatment. Mice were intraperitoneally injected with 120 mg / kg cyclophosphamide and 12 mg / kg busulin for 4 weeks. The control group was injected with the same volume of saline for the same period as the experimental group.

[0057] Subsequently, mice were given weekly intravenous injections of the exosomes obtained in Example 1 for 8 weeks. After the experiment, the mice were euthanized, and ovarian tissue and blood samples were collected. The efficacy of the exosomes was evaluated through histological analysis and hormone assays. Low-dose exosomes: 0.5 μg / 0.5 ml per dose; medium-dose exosomes: 1 μg / 0.5 ml per dose; high-dose exosomes: 2 μg / 0.5 ml per dose.

[0058] Blood samples from sacrificed mice were used for hormone assays, including FSH (follicle-stimulating hormone, purchased from ABclonal, catalog number RK04237), LH (luteinizing hormone, purchased from ELK, catalog number ELK2368), E2 (estradiol, purchased from ELK, catalog number ELK1208), and AMH (anti-müllerian hormone, purchased from ABclonal, catalog number RK09261), with n=8 in each group. Hormone assays were performed using ELISA kits. Results are as follows: Figure 2 As shown in Table 1, compared with the control group, the serum E2 and AMH levels of mice in the POF group were significantly decreased, while the FSH and LH levels were significantly increased. After receiving high-dose exosome treatment, the decrease in E2 and AMH levels and the increase in LH levels were partially reversed.

[0059] Table 1 shows the effects of exosome treatment in Example 2 of this invention on serum (A) E2, (B) FSH, (C) LH, and (D) AMH levels in mice, n = 8, analyzed by one-way ANOVA. Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0060] Table 1. Analysis of Serum Hormone Indicators in Mice

[0061]

[0062] Ki67 (Antigen Kiel 67, cell proliferation nuclear antigen 67) is a proliferation-associated nucleoprotein recruited to condensed chromosomes during mitosis and is typically expressed only in proliferating cells. MVH (Mouse Vasa Homolog, mouse vasa gene homolog) is a germ cell-specific marker; MVH positivity indicates germline stem cells. After sectioning mouse ovarian tissue, paraffin sections are dewaxed with xylene, rinsed with water, and then subjected to a heat retrieval method to expose the antigen. Non-specific binding is then blocked with a blocking solution, followed by incubation with specific primary antibodies (anti-Ki67 and anti-MVH) at 4°C overnight. After incubation, sections are washed with PBS buffer to remove unbound primary antibodies, and then incubated with appropriate secondary antibodies for 30 minutes to 1 hour. After completion, sections are washed again, and a colorimetric reaction is performed. The substrate solution reacts with the enzyme to form a precipitate, revealing the antigen location. Subsequently, sections are contrast-stained with hematoxylin, dehydrated, and cleared, and finally mounted with neutral resin. Figure 3 and Figure 4Compared with the control group, the number of Ki67 and MVH positive cells (appearing brown or light brown) in the ovarian tissue of POF mice was significantly reduced, indicating a decrease in the number of proliferating germ cells and germline stem cells. High-dose exosome treatment could significantly increase the number of Ki67 positive cells, while slightly increasing the number of cells expressing MVH.

[0063] Example 3: Safety evaluation of exosomes (effects on blood biochemistry in POF mice and normal mice)

[0064] Six to eight-week-old female C57BL / 6 mice were divided into six groups: (1) normal mice, a blank control group (no modeling or treatment); (2) premature ovarian failure (POF) group (model group); (3) POF + low-dose exosome group (low-dose group); (4) POF + medium-dose exosome group (medium-dose group); (5) POF + high-dose exosome group (high-dose group); and (6) normal age-matched mice receiving high-dose exosome injections to assess the effects of exosomes on normal mice (exosome group). Each group consisted of 8-10 mice. POF was induced by cyclophosphamide / busulin treatment. Mice were intraperitoneally injected with 120 mg / kg cyclophosphamide and 12 mg / kg busulin for 4 weeks. The control group was injected with the same dose of saline for the same period as the experimental group. Subsequently, mice were given intravenous injections of exosomes once a week for 8 weeks, with low-dose injections of 0.5 μg / 0.5 ml, medium-dose injections of 1 μg / 0.5 ml, and high-dose injections of 2 μg / 0.5 ml. After the experiment, the mice were euthanized, and blood samples were collected for biochemical analysis. Plasma or serum samples were analyzed for sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein and albumin, phosphate, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, direct bilirubin, and total bilirubin.

[0065] Compared with the control group, POF mice showed significantly lower levels of glucose, cholesterol, creatinine, total protein, albumin, and total bilirubin, while ALT and the ALT / AST ratio were significantly higher in POF mice. However, high-dose exosomes could reverse these abnormalities. Furthermore, the results showed that these hematological parameters did not significantly change after normal mice received weekly high-dose exosome injections for 8 weeks, thus confirming the high safety profile of the exosomes (Table 2). Figure 5 ).

[0066] Table 2 shows the biochemical index analysis of mouse blood samples in Example 3 of this invention. One-way ANOVA was performed, n = 8-10. Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0067] Table 2. Analysis of Biochemical Indicators in Mouse Blood Samples

[0068]

[0069]

[0070] The above results show that mesenchymal stem cell exosomes highly expressed by hsa-miR-21-5p, hsa-miR-199a-3p, hsa-miR-221-3p, hsa-miR-146a-5p, hsa-miR-100-5p, hsa-miR-320a, hsa-miR-92a-3p, hsa-miR-423-5p, hsa-miR-199a-5p and hsa-miR-29a-3p, or mesenchymal stem cell exosomes highly expressed by hsa-miR-21-5p, hsa-miR-199a-5p and hsa-miR-29a-3p, or A2 macroglobulin, complement C3, immunoglobulin δ-1 heavy chain, immunoglobulin λ-1 light chain, immunoglobulin weight constant μ, immunoglobulin weight constant α1, immunoglobulin λ constant 2, complement factor H, haptoglobulin, and complement C4-A have reversal and / or protective and / or repairing effects on premature ovarian failure.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A mesenchymal stem cell exosome, characterized in that... The exosome highly expresses the following microRNAs: hsa-miR-21-5p, hsa-miR-199a-3p, hsa-miR-221-3p, hsa-miR-146a-5p, hsa-miR-100-5p, hsa-miR-320a, hsa-miR-92a-3p, hsa-miR-423-5p, hsa-miR-199a-5p, hsa-miR-29a-3p, said microRNAs having the sequences shown in SEQ ID Nos. 1 to 10; or proteins: A2 macroglobulin, complement C3, immunoglobulin δ-1 heavy chain, immunoglobulin λ-1 light chain, immunoglobulin weight constant μ, immunoglobulin weight constant α1, immunoglobulin λ constant 2, complement factor H, haptoglobulin, complement C4-A, said proteins having the sequences shown in SEQ ID Nos. 11 to 20.

2. The mesenchymal stem cell exosomes according to claim 1, characterized in that... The microRNA accounts for no less than 50% of the total RNA expression in the exosome, preferably no less than 54%.

3. The mesenchymal stem cell exosomes according to claim 1, characterized in that... The protein accounts for no less than 60% of the total protein expression in the exosome, preferably no less than 65%, and more preferably no less than 67%.

4. The use of the mesenchymal stem cell exosomes according to any one of claims 1 to 3 in the preparation of a medicament for treating premature ovarian failure.

5. The application according to claim 4, wherein the premature ovarian failure is premature ovarian failure caused by chemotherapy.

6. A composition, characterized in that... It includes mesenchymal stem cell exosomes as described in any one of claims 1 to 3.

7. The composition according to claim 6, characterized in that... The composition contains at least 1.5 x 10^6 active exosomes. 6 per μg.

8. The composition according to claim 6 or 7, characterized in that... The composition is an injectable dosage form.

9. The composition according to claim 8, characterized in that... Administered by injection.

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