Application of JMJD6 in preparation of medicine for treating myocardial hypertrophy

Abstract

The invention discloses an application of JMJD6 in preparation of a medicine for treating myocardial hypertrophy. A JMJD6 myocardial specific knockout mouse is utilized, and it is found that a myocardial specific deletion JMJD6 mouse shows a phenomenon similar to that of a myocardial hypertrophy animal model; a mouse myocardial hypertrophy model is further prepared by adopting an aortic arch constriction (TAC) operation, meanwhile, a JMJD6 overexpression mouse model is constructed through an adeno-associated virus (AAV9), and it is found that overexpression of JMJD6 can improve the heart function of the mouse damaged by the TAC operation, reduce the myocardial cell area of the mouse and relieve myocardial remodeling, prompting that JMJD6 can improve myocardial hypertrophy; the invention discovers that the histone arginine demethylase JMJD6 is associated with myocardial hypertrophy for the first time, and proves that the myocardial hypertrophy can be improved by increasing the level of the JMJD6. Therefore, JMJD6 is expected to be used as a target spot for screening and preparing medicines for treating myocardial hypertrophy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application of JMJD6 in preparing a drug for treating myocardial hypertrophy. Background Art

[0002] Heart failure is the terminal stage of heart disease, with a high mortality rate and extremely poor prognosis. The incidence of heart failure in my country is 275 per 100,000 person-years, with approximately 3 million new cases each year. Myocardial hypertrophy is the recognized pathological basis of heart failure. Pathological myocardial hypertrophy is a common pathophysiological manifestation of many end-stage cardiovascular diseases such as hypertension, coronary heart disease, and valvular disease, manifested as enlargement and thickening of the myocardial wall under various stresses, injuries, or stimuli. Currently, clinical treatment for pathological myocardial hypertrophy is still mostly focused on improving cardiac function and alleviating clinical symptoms. Therefore, in-depth research on the pathogenesis of pathological myocardial hypertrophy and the discovery of new targets for intervention in pathological myocardial hypertrophy and heart failure are of great significance for preventing the transformation of myocardial hypertrophy to cardiovascular adverse events such as heart failure and prolonging the life of patients with cardiovascular diseases.

[0003] The role of epigenetic modifications in cardiac hypertrophy has garnered increasing attention. Previous studies have shown that histone methylation modifications, such as histone H3K4me3 and H3K27me3, can regulate cardiac hypertrophy, angiogenesis, and subsequent heart failure. Histone demethylases, which regulate gene transcription by altering histone methylation levels, are potential therapeutic targets for cardiac hypertrophy. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. To this end, the main purpose of the present invention is to provide a use of JMJD6 in the preparation of a drug for treating myocardial hypertrophy.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] Application of JMJD6 in the preparation of drugs for treating myocardial hypertrophy.

[0007] Use of a JMJD6 activator in the preparation of a drug for treating myocardial hypertrophy.

[0008] Application of JMJD6 recombinant vector in the preparation of drugs for treating myocardial hypertrophy.

[0009] Use of JMJD6 overexpressing adenovirus or adeno-associated virus in the preparation of a drug for treating myocardial hypertrophy.

[0010] A pharmaceutical composition for treating myocardial hypertrophy, which is selected from one of JMJD6, a JMJD6 activator, a JMJD6 recombinant vector, an MJD6 overexpressing adenovirus or an adeno-associated virus.

[0011] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, such carriers including (but not limited to): diluents, buffers, suspensions, emulsions, granules, encapsulations, excipients, fillers, adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents or adsorption carriers.

[0012] The pharmaceutical composition of the present invention can be prepared into various dosage forms as needed, including but not limited to tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalants or sprays.

[0013] The administration route of the pharmaceutical composition of the present invention is not limited, as long as it can exert the desired therapeutic or preventive effect, including but not limited to oral administration, intravenous injection, intramuscular injection, subcutaneous injection, sublingual dissolution, rectal irrigation, nasal spray, oral spray, local skin or systemic transdermal administration.

[0014] The pharmaceutical composition of the present invention can also be used in combination with other drugs for treating myocardial hypertrophy. The combined use of multiple drugs can greatly improve the success rate of treatment.

[0015] About the definition content:

[0016] The term "expression level" refers to the amount of a biomarker in a biological sample." Expression" generally refers to the process by which information (e.g., genetically encoded and / or epigenetic) is converted into a structure present and operating in a cell. Therefore, as used herein, "expression" can refer to transcription into polynucleotides, translation into polypeptides, or even polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of polypeptides). Fragments of transcribed polynucleotides, translated polypeptides, or polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of polypeptides) should also be considered as expressed, whether or not they are derived from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of polypeptides, such as by proteolysis.

[0017] As used herein, "treatment" refers to clinical intervention intended to alter the natural course of the individual being treated, and may be performed during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating the disease state, and eliminating or improving prognosis.

[0018] The sequence of JMJD6 of the present invention can be found in public databases, and the specific sequence is shown as SEQ ID NO.1;

[0019] mnhkskkrir eakrsarpel kdsldwtrhn yyesyplnpa avpdnverad alqlsvkefveryerpykpv vllnaqegws aqekwtlerl krkyrnqkfk cgedndgysv kmkmkyyiey mestrddsplyifdssygeh pkrrklledy kvpkfftddl fqyagekrrp pyrwfvmgpp rsgtgihidp lgtsawnalvqghkrwclfp tntprelikv treeggnqqd eaitwfnviy prtqlptwpp efkpleilqk pgetvfvpggwwhvvlnldt tiaitqnfas stnfpvvwhk tiqhgqrqck pppatpchcv ptltwrdqls gilkqehpelavladavdlq estgiasdss sdssssssss ssdsdseces gsegdgtthr rkkrrtcsmv gngdttsqddcvskersssr

[0020] Compared with the prior art, the present invention has at least the following advantages:

[0021] 1) The present invention provides a method for preparing a drug for treating myocardial hypertrophy using JMJD6 myocardial-specific knockout mice. The method found that mice with a myocardial-specific JMJD6 deficiency exhibited similar phenomena to animal models of myocardial hypertrophy. Furthermore, a myocardial hypertrophy model was established in mice using transaortic arch constriction (TAC) surgery. A JMJD6 myocardial-specific overexpression mouse model was also constructed using adeno-associated virus (AAV9). The method found that overexpression of JMJD6 in myocardial cells improved cardiac function in mice impaired by TAC surgery, reduced myocardial cell area in mice, and alleviated myocardial remodeling, suggesting that JMJD6 can improve myocardial hypertrophy.

[0022] 2) The present invention discovered the mechanism of action of JMJD6 in treating cardiac hypertrophy. Specifically, through enzyme active site mutation experiments, it was found that JMJD6's protective effect against cardiac hypertrophy is dependent on its demethylase activity. By acting as a demethylase, JMJD6 reduces the enrichment of the H3R2me2a modification in the promoter region of the circadian clock gene Per2, thereby increasing Per2 expression and inhibiting glycolysis, ultimately effectively improving cardiac hypertrophy.

[0023] 3) This study, for the first time, discovered a correlation between the histone arginine demethylase JMJD6 and cardiac hypertrophy, demonstrating that increasing JMJD6 levels can improve cardiac hypertrophy. Therefore, JMJD6 is a promising target for screening and developing drugs to treat cardiac hypertrophy. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the specific embodiments of the present invention, the following briefly introduces the drawings required for the specific embodiments or the description of the prior art.

[0025] Figure 1 The expression of JMJD6 mRNA in the hearts of mice with cardiac hypertrophy induced by TAC surgery ( Figure 1 A) and protein ( Figure 1 B) level.

[0026] Figure 2 The effects of myocardial-specific deletion of JMJD6 on the size of cardiomyocytes and the levels of cardiac hypertrophy markers in mice (A: mouse heart body ratio; B: WGA staining to detect the area of ​​mouse cardiomyocytes; C: qPCR detection of the expression of hypertrophy markers in mouse heart tissue).

[0027] Figure 3 The effects of myocardial-specific overexpression of JMJD6 on the size of cardiomyocytes and the levels of cardiac hypertrophy markers in mice (A: mouse heart body ratio; B: WGA staining to detect the area of ​​mouse cardiomyocytes; C: qPCR detection of the expression of hypertrophy markers in mouse heart tissue).

[0028] Figure 4 The role of JMJD6 histone demethylase activity in cardiac hypertrophy (A: mouse cardiac body ratio; B: WGA staining to detect mouse cardiomyocyte area).

[0029] Figure 5 To screen the targets of JMJD6 histone demethylation (WB detection of histone methylation modification levels in the heart tissue of JMJD6 myocardium-specific knockout mice).

[0030] Figure 6 Screening of downstream molecules regulated by JMJD6 (A: KEGG enrichment analysis; B: circadian rhythm pathway gene heat map; C: CUT&Tag-PCR detection of the effect of JMJD6 on the enrichment of H3R2me2a on the Per2 promoter; D: Immunofluorescence staining to observe the effect of interfering with Per on cardiomyocyte size; E: qPCR detection of the effect of Per2 on the transcription level of hypertrophy marker genes; F: qPCR detection of the effect of Per2 on the expression of glycolysis-related genes). DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to the accompanying drawings and embodiments, but it should not be understood as limiting the scope of protection of the present invention. Any formal equivalent transformation based on the concept of the present invention should be considered as the scope of the present invention.

[0032] This document provides general and / or specific descriptions of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods. All reagents and instruments used, unless the manufacturer is specified, are commercially available, conventional products and were prepared or used using conventional methods.

[0033] Example 1: Expression of JMJD6 in hypertrophic myocardium

[0034] This example uses an animal model of cardiac hypertrophy to study changes in JMJD6 expression and clarify the role of JMJD6 in cardiac hypertrophy;

[0035] Materials and methods:

[0036] 1) Establishment of a mouse model of pathological cardiac hypertrophy:

[0037] A mouse model of pathological myocardial hypertrophy was induced using transverse aortic constriction (TAC). After anesthesia, the mice underwent routine skin preparation from the neck to the anterior chest. After endotracheal intubation, the chest was opened midway, the muscles were bluntly separated, the incision was opened with a spreader, the thymus was separated, and the aortic arch was exposed. The innominate artery, left common carotid artery, and left subclavian artery were identified along the aortic arch. A suture was threaded between the right innominate artery and the left common carotid artery. A 26G constriction needle was placed parallel to the aortic arch and tied. After the suture was tightened, the constriction needle was slowly withdrawn. The chest was closed and sutured layer by layer. After the mouse recovered spontaneous breathing, the tube was removed and the mouse was returned to the cage for further observation.

[0038] 6-8 week-old C57BL / 6J mice were selected and randomly divided into a sham operation group (Sham) and a transcatheter artery thrombosis (TAC) group. Mice in the TAC group underwent TAC surgery. Four weeks after the surgery, heart tissues were obtained from the mice, and the expression of JMJD6 was detected by qPCR and immunoblotting.

[0039] 2) qRT-PCR fluorescence quantitative PCR experiment

[0040] Total RNA was extracted from mouse heart tissue using Trizol (TAKARA, Japan). RNA was reverse transcribed into cDNA using a reverse transcription kit (TAKARA, Japan). JMJD6 expression was detected using fluorescent quantitative PCR (TAKARA, Japan). Primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. and have the following sequences:

[0041]

[0042] 3) Western blot

[0043] Total protein was extracted from mouse heart tissue using Western blotting and IP lysis buffer (Beyond the Sun, Shanghai). Loading buffer (Quanshi Gold, Beijing) was added and then heat-denatured. The membrane was separated by SDS-PAGE gel and transferred to NC membrane (PALL, USA). The membrane was incubated with JMJD6 primary antibody (Proteintech, Wuhan) diluted 1:100 at 4°C and developed the next day with immunofluorescence secondary antibody (Invitrogen, USA) at room temperature.

[0044] Results and discussion: The experimental results showed that the expression of JMJD6 mRNA ( Figure 1 A) and protein ( Figure 1 B) The level of JMJD6 decreased significantly, demonstrating that JMJD6 plays an important role in cardiac hypertrophy.

[0045] Example 2: Effect of JMJD6 cardiomyocyte-specific deletion on cardiac hypertrophy

[0046] 1) JMJD6 cardiomyocyte-specific knockout mice (Jmjd6 f / f ;Myh6-Cre ERT2 ) construction and induction:

[0047] Beijing Biocytogen was commissioned to design and construct a JMJD6 conditional knockout gene (Jmjd6 f / f ) model mice, using CRISPR technology to construct targeting vectors and identify mouse genotypes until Jmjd6 f / f Homozygous mice were then crossed with inducible myocardial-specific knockout mice (Myh6-Cre ERT2 ) were bred until JMJD6 myocardium-specific knockout mice (Jmjd6 f / f ;Myh6-Cre ERT2 ).

[0048] At 4 weeks of age, JMJD6 cardiomyocyte-specific knockout mice were intraperitoneally injected with 50 mg / kg tamoxifen (Sigma, USA) in a 9:1 corn oil:anhydrous ethanol solution (100 μl per mouse). After 4 weeks of feeding, mice were sacrificed by cervical dislocation, and body weight, heart weight, and left tibia length were recorded.

[0049] 2) WGA staining:

[0050] Mouse heart tissue was fixed with 4% paraformaldehyde (Biyuntian, Shanghai), dehydrated, and embedded as usual, preparing 3-4 μm tissue sections. After dewaxing, 10 μl of 5 μg / ml WGA stain was added to each section of heart tissue and incubated at 37°C in the dark for 20 minutes. Cardiomyocyte size was observed under a fluorescence microscope, and photographs were taken for statistical analysis.

[0051] 3) qRT-PCR fluorescence quantitative PCR experiment

[0052] Referring to Example 1, the expression of hypertrophy marker genes ANP and BNP in the heart tissue of JMJD6 myocardium-specific knockout mice was detected. ANP and BNP primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. and the sequences are as follows:

[0053]

[0054] Results and Discussion: Compared with wild-type (WT) mice, JMJD6 cardiomyocyte-specific knockout mice (Jmjd6 f / f ;Myh6-Cre ERT2 ) Heart volume and weight increased, heart-to-body ratio (HW / BW) and heart weight to left tibia length ratio (HW / TL) increased ( Figure 2 A). WGA staining showed an increase in the cross-sectional area of ​​cardiomyocytes ( Figure 2 B). Increased expression of hypertrophy marker genes ANP and BNP in cardiac tissue ( Figure 2 C). This suggests that myocardial JMJD6 deficiency can induce cardiac hypertrophy.

[0055] Example 3: Effect of myocardial-specific overexpression of JMJD6 on myocardial hypertrophy

[0056] The purpose of this example is to study the protective effect of JMJD6 on myocardial hypertrophy, specifically:

[0057] Materials and methods: Four-week-old C57BL / 6J mice were randomly divided into a control group (AAV9-Control) and an overexpression group (AAV9-JMJD6). The control group mice were injected with a control adeno-associated virus via the tail vein, while the overexpression group mice were injected with a JMJD6 overexpression adeno-associated virus via the tail vein. JMJD6 overexpression adeno-associated virus (AAV9-JMJD6) or control adeno-associated virus (AAV9-Control) were purchased from Shanghai Hanheng Biotechnology Co., Ltd. with a viral titer of 5×10 11vg, 100 μl per mouse. Four weeks after viral infection, the mice were randomly divided into a sham-operated group (sham group) and a TAC-operated group (TAC group), with or without TAC surgery. Four weeks after surgery, the mice were killed by cervical dislocation, and heart and body weights were recorded. Hearts were removed and the area of ​​myocardial cells was observed using WGA staining. qPCR was used to measure the expression of the hypertrophy marker genes ANP and BNP.

[0058] Results and Discussion: Compared with wild-type (WT) mice, myocardium-specific overexpression of JMJD6 in mice after TAC surgery showed a decrease in the heart-to-body ratio (HW / BW) and the ratio of heart weight to left tibia length (HW / TL). Figure 3 A), the cross-sectional area of ​​myocardial cells decreases ( Figure 3 B), expression of cardiac hypertrophy marker genes decreased ( Figure 3 C) These results demonstrate that overexpression of JMJD6 in the myocardium can ameliorate TAC-induced cardiac hypertrophy.

[0059] Example 4: Role of JMJD6 histone demethylase activity in cardiac hypertrophy

[0060] This example studies whether the protective effect of JMJD6 on cardiac hypertrophy depends on its histone demethylase activity, specifically:

[0061] Materials and methods: Four-week-old C57 / BL6J mice were randomly divided into blank group (AAV9-control+Sham), control group (AAV9-control+TAC), overexpression group (AAV9-JMJD6+TAC) and mutant group (AAV9-JMJD6mut+TAC). The mice were injected with control adeno-associated virus (AAV9-Control), JMJD6 overexpression adeno-associated virus (AAV9-JMJD6) and JMJD6 enzyme active site mutant virus (AAV9-JMJD6mut) (purchased from Shanghai Hanheng Biotechnology Co., Ltd.) through the tail vein, respectively. The injected virus titer was 5×10 11 vg, 100 μl per mouse. Four weeks after infection, mice underwent TAC surgery. Four weeks after surgery, mice were killed by cervical dislocation and heart and body weights were recorded. Hearts were removed and WGA staining was used to observe the area of ​​myocardial cells.

[0062] Results and Discussion: Compared with JMJD6 cardiomyocyte-specific overexpression mice, the heart-body ratio (HW / BW) and heart weight to left tibia length ratio (HW / TL) of the cardiomyocyte-specific overexpression JMJD6 enzyme active site mutant mice were increased after TAC surgery ( Figure 4 A), the cross-sectional area of ​​myocardial cells increases ( Figure 4 B). This study demonstrates that JMJD6 ameliorates TAC-induced cardiac hypertrophy through its histone demethylase activity.

[0063] Example 5: Histone modification targets of JMJD6 regulating cardiac hypertrophy

[0064] This example studies the histone modification targets of JMJD6 demethylation, specifically:

[0065] Materials and methods: JMJD6 cardiomyocyte-specific knockout mice (Jmjd6 f / f ;Myh6-Cre ERT2 ), the construction method was the same as in Example 2, mouse heart tissue was obtained, and the modification targets of JMJD6 were screened by immunoblotting experiments.

[0066] Results and Discussion: Compared with wild-type (WT) mice, the level of histone methylation modification H3R2me2a was significantly increased in the heart tissue of JMJD6 cardiomyocyte-specific knockout mice ( Figure 5 ), suggesting that JMJD6 plays a role in cardiac hypertrophy by regulating H3R2me2a modification.

[0067] Example 6: Downstream molecules regulated by JMJD6

[0068] This example studies the downstream molecular mechanism of JMJD6 regulated by H3R2me2a, specifically:

[0069] Materials and methods:

[0070] 1) Transcriptome sequencing of cardiac tissue from JMJD6 cardiomyocyte-specific knockout mice:

[0071] Four replicate samples of the hearts of tamoxifen-induced myocardial-specific JMJD6 knockout mice and the hearts of the control group (WT wild-type) mice were sent to Guangzhou Kidio Biotechnology Co., Ltd. for transcriptome sequencing. The brief steps are as follows: RNA extraction and quality inspection, mRNA enrichment, purification and fragmentation, first-strand cDNA synthesis, second-strand cDNA synthesis, adapter ligation, ligation product purification, library amplification, magnetic bead purification of amplified products, data quality control, alignment to the reference genome, and analysis of differentially expressed genes.

[0072] 2) Isolation of primary rat cardiomyocytes:

[0073] Newborn SD rats aged 1-3 days were obtained. The chest cavity was opened, the heart was removed, and the heart was placed in pre-chilled PBS, minced, and washed to remove residual blood. 0.08% type II collagenase (purchased from Washington, USA) was used for batch digestion for 30 minutes. The digestion fluid was collected and serum was added to terminate the digestion. High and low density Percoll buffers (Cytiva, Sweden) were prepared and used for density gradient centrifugation to isolate cardiomyocytes. The cells were resuspended in complete culture medium and transferred to a cell culture incubator for culture.

[0074] 3) CUT&Tag-PCR experiment:

[0075] The CUT&Tag-PCR kit was purchased from Nanjing Novozymes. The specific steps are briefly summarized as follows: cell collection, primary antibody incubation, secondary antibody incubation, transposon binding, fragmentation, DNA extraction, and qPCR. qPCR was used to verify the enrichment of H3R2me2a in the Per2 promoter region.

[0076] 4) Primary cardiomyocytes transfected with siRNA:

[0077] The small interfering RNA (siRNA) used in this example was synthesized by Shanghai Sangon Biotechnology Co., Ltd. The sequence of rat Per2 siRNA is as follows:

[0078] Sense: 5'-GGAAGCCUCUGUUGCAGAATT (SEQ ID NO.10);

[0079] Anti-sense: 5'-UUCUGCAACAGAGGCUUCCTT (SEQ ID NO.11);

[0080] 100 nM siRNA was prepared with Opti-MEM (Gibco, USA) and added to primary cardiomyocytes for 48 hours to interfere with the expression of Per2.

[0081] Results and Discussion: Transcriptome sequencing of cardiac tissues from JMJD6 myocardium-specific knockout mice showed that differentially expressed genes were significantly enriched in circadian rhythm pathways ( Figure 6 A) Among all rhythm-related differentially expressed genes enriched in the transcriptome, the change in cyclin 2-Per2 was the most significant ( Figure 6 B), and CUT&Tag-PCR results showed that JMJD6 could reduce the enrichment of H3R2me2a in the Per2 promoter region ( Figure 6 C), these results suggest that JMJD6 regulates cardiac hypertrophy by affecting Per2. Further interference with Per2 expression in primary cardiomyocytes revealed an increase in cardiomyocyte area ( Figure 6 D), expression of cardiac hypertrophy markers was upregulated ( Figure 6 E), while the expression of glycolysis-related genes increased ( Figure 6 F), indicating that Per2 protects against cardiac hypertrophy by inhibiting glycolysis.

[0082] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. Application of JMJD6 in the preparation of drugs for the treatment of myocardial hypertrophy.

2. Application of JMJD6 activator in the preparation of drugs for the treatment of myocardial hypertrophy.

3. Application of JMJD6 recombinant vector in the preparation of drugs for the treatment of myocardial hypertrophy.

4. Application of JMJD6 overexpressing adenovirus or adeno-associated virus in the preparation of drugs for the treatment of myocardial hypertrophy.

5. A pharmaceutical composition for treating myocardial hypertrophy, characterized in that: It is selected from one of JMJD6, a JMJD6 activator, a JMJD6 recombinant vector, a MJD6 overexpression adenovirus or an adeno-associated virus.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition further comprises pharmaceutically acceptable excipients.

7. The pharmaceutical composition according to claim 5, characterized in that The auxiliary materials include one or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers and lubricants.

8. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition is in an oral dosage form or a non-oral dosage form.

9. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition is in the form of tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalants or sprays.

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