阅读说明：本技术 榄香素在制备用于治疗溶酶体贮积病的药物中的应用 (Use of elemene in preparing medicine for treating lysosome storage disease ) 是由 李欣然 冯新华 向聪 于 2020-05-20 设计创作，主要内容包括：本发明公开了小分子化合物榄香素在制备用于治疗溶酶体贮积病的药物中的应用。本发明通过对一个天然萃取物的小分子库的筛选,从850种天然小分子中筛选到小分子化合物榄香素能够有效抑制溶酶体贮积病的表型,比如能够抑制溶酶体体积的增加、抑制胆固醇贮积、显著恢复被抑制的溶酶体管成、显著恢复对于重复饥饿的耐受并降低细胞死亡率,从而达到减轻损伤与抑制病情发展的目的。榄香素具有开发为广谱性治疗溶酶体贮积病药物的前景。(The invention discloses an application of a small molecular compound elemene in preparing a medicament for treating lysosomal storage disease. The invention screens a small molecule library of a natural extract, and the small molecule compound elemene screened from 850 natural small molecules can effectively inhibit the phenotype of the lysosome storage disease, such as inhibiting the increase of the volume of lysosome, inhibiting cholesterol storage, remarkably recovering the inhibited lysosome tube, remarkably recovering the tolerance to repeated hunger and reducing the cell death rate, thereby achieving the purposes of reducing injury and inhibiting the disease development. Elemene has the prospect of being developed into a broad-spectrum drug for treating lysosomal storage diseases.)

1. Use of elemene in the manufacture of a medicament for the treatment of lysosomal storage diseases.

2. The use of claim 1, wherein the lysosomal storage disease is GM2 gangliosidosis and the primary storage substance is a glycolipid or polysaccharide containing a galactose chain.

3. The use of claim 1, wherein the lysosomal storage disease is Fabry's disease and the primary storage agent is GM2 ganglioside.

4. The use of claim 1, wherein the lysosomal storage disease is niemann pick C and the primary storage substance is cholesterol.

5. A medicine for treating lysosome storage disease is characterized in that the effective component is elemene.

6. The medicament of claim 5, wherein the lysosomal storage disease is GM2 gangliosidosis and the primary storage substance is a glycolipid or polysaccharide containing a galactose chain.

7. The medicament of claim 5, wherein the lysosomal storage disease is Fabry's disease and the primary storage agent is GM2 ganglioside.

8. The medicament of claim 5, wherein the lysosomal storage disease is niemann pick C and the primary storage substance is cholesterol.

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of elemene in preparing a medicine for treating lysosomal storage diseases.

Background

Lysosomal Storage Disease (LSD) is a general term for a class of inherited diseases caused by lysosome-associated single gene mutations. There are approximately 50 lysosomal storage diseases that have been found in humans, with an average incidence of less than one ten-thousandth of each, but with a total incidence of between about 1/10000 and 1/5000.

The pathogenesis of LSD varies depending on the mutated gene. The mutated gene may be an enzyme responsible for degradation within the lysosome, a protein responsible for transport of substances on the lysosome membrane, a protein associated with lysosomal membrane trafficking, and an enzyme responsible for post-translational modification of lysosomal associated proteins, among others. Although the pathogenesis varies, the onset of all LSDs is primarily accompanied by the accumulation of some lysosomal associated substance. LSDs can be generally classified into the following broad categories according to their primary storage materials:

1. lipid storage diseases, which are further classified into sphingolipid storage, ganglioside storage, leukodystrophy, and the like;

2. mucopolysaccharide storage disease;

3. glycoprotein storage disease;

4. mucolipidosis.

In addition, there are LSDs such as glycogen storage diseases such as Pompe Disease and cystine storage diseases, which do not belong to the above groups. Because the mutual complex influence of the substances stored in lysosomes and the transport and function of lysosome membranes, the various LSDs can finally cause the secondary storage of various lysosome related metabolites, and a similar metabolite storage effect is caused.

Due to similar material accumulation, LSDs are similar in their main pathological phenotype, both at the cellular level and at the human level, except for the phenotype directly related to the function of the mutated gene itself. Among them, the phenotype in the human body is mainly caused by the damage of the terminal differentiated cells (mainly neurons and muscle fibers) with long life, including developmental delay, muscle spasm, insufficient muscle power, mental retardation, visual and auditory injuries, etc. At the cellular level, phenotypes include enlarged lysosomes, accumulation of various lysosome-associated metabolites (cholesterol, sphingomyelin, ceramides, various polysaccharides, etc.), reduced lysosomal degradation and transport functions, hindered lysosomal regeneration, and the like. The phenotypic severity of LSD also varies depending on the severity of the causative mutation. Even relatively mild mutations can cause morbidity during the juvenile period and death usually before the third decade, and severe mutations can cause patients to develop morbidity and death within a few years from infancy to early childhood.

Because LSD is caused by mutation on the gene level, the current biological and medical technology can not achieve radical cure. Although the CRISPR gene editing technology that has appeared in recent years can directly and accurately edit target gene sequences in living cells and higher animals, it has not been applied to clinical experiments and treatments in a short time because of its unresolved off-target effects and ethical problems. By far, treatment regimens directed against LSD are mainly aimed at reducing the uptake of relevant storage substances in order to alleviate the phenotype. Such regimens and their efforts are directed to the patient and family members, and are only suitable for use where the primary stored substance is an externally ingested substance or a direct metabolite thereof. LSDs such as glycoprotein storage disease, mucopolysaccharidosis and the like caused by storage of substances synthesized in vivo cannot be treated by adjusting the manner of intake of substances.

In addition, the treatment modalities reported at present also include gene therapy and protein replacement therapy, but the average number is rare. The former treatment regimen is to deliver the wild type of the mutant gene into the patient via viral encapsulation and express it in place of the mutant gene to perform its function. The latter treatment is to inject normal protein after synthesis into the patient to directly replace the diseased protein to perform the function. The gene therapy is limited by the transfection efficiency and the expression efficiency of the virus, and the insertion of the virus into the DNA fragment of the chromosome may cause the mutation of other genes, the instability of the chromosome and other problems, easily cause other diseases and increase the canceration risk. Protein replacement therapy is generally only applicable to LSD due to enzymatic mutations within lysosomes, and is extremely ineffective against brain cells due to the presence of the blood brain barrier. And protein replacement therapy requires periodic injections of synthetic proteins, which is expensive.

In addition to the above-mentioned disadvantages, there is another limitation in the current treatment modalities for LSD, that is, they are directed to LSD of single or same kind of pathogenesis and cannot be used as universal therapy for most LSDs. However, the incidence of LSD alone is extremely low, which results in extremely low efficiency and minimal benefit for further optimization of the above treatment modalities.

Disclosure of Invention

Based on the above problems currently faced with treatment of LSD, the present application provides a drug that can treat LSD over a broad spectrum and at a relatively low cost. The application provides the use of elemene in the manufacture of a medicament for the treatment of lysosomal storage diseases.

The molecular structure of Elemicin (Elemicin) is shown as formula I.

At present, no medicinal record of elemene exists, but some researches show that elemene can participate in processes of antioxidation, antibiosis and the like, but metabolites of elemene also show certain toxicity in murine experiments. Elemene is abundantly present in some edible and medicinal plants, including nutmeg, celery, etc.

Toxicity studies of elemene have shown that elemene toxicity is mainly caused by metabolism in the liver to 1 '-hydroxyelemene (1' -hydroxyeleemicin) (Wang, Y.K., et al, Role of Metabolic activity element-Induced Cellular activity. J Agric Food Chem,2019.67(29): p.8243-8252.). Toxicity includes mainly the Inhibition of Stearoyl-CoA Desaturase 1(Stearoyl-CoA Desaturase 1) (Yang, X.N., et al, Metabolic Activation of Elericin Leads to the Inhibition of Stearoyl-CoA Desaturase 1.Chem Res Toxicol,2019.32(10): p.1965-1976.), the binding to cysteine/N-ethylcysteine in cells (Wang, Y.K., et al, le of Metabolic Activation-Induced Cellular sensitivity. J.agricultural Food, 2019.67(29): p.8243-8252.), and the binding ability to DNA (van den Berg, S.J., chemical nutritional basic) and certain binding ability to DNA (chemical amplification of protein 52. 12. J.J.). However, in Cellular experiments (Wang, Y.K., et al, Role of Cellular Activation in electric-Induced Cellular susceptibility. J.Agric Food Chem,2019.67(29): p.8243-8252.) and in mouse experiments (van den Berg, S.J., et al, physiology based kinetic models for the alkenyl benzene electric in vitro and human and Cellular inflammations for the isk evaluation. Chem toxin, 2012.25(11): p.2-67.), concentrations of > 250. mu.M and 2.5mM, respectively, which resulted in decreased Cellular activity/mouse liver injury, were well above the 10 micromolar concentration used in this application.

The present application provides, in a first aspect, the use of elemene in the manufacture of a medicament for the treatment of a lysosomal storage disease. Preferably, the lysosomal storage disease is: (1) GM2 gangliosidosis (GM2 gangliosidosis), primary storage substance is glycolipid or polysaccharide containing galactose chain; (2) fabry's Disease, primary storage substance is GM2 ganglioside; (3) niemann Pick type C disease (Niemann-Pick type C), the primary storage material is cholesterol.

The application also provides a medicament for treating lysosomal storage diseases, the active ingredient of which is elemene. Preferably, the lysosomal storage disease is: (1) GM2 gangliosidosis, the primary storage substance is glycolipid or polysaccharide containing galactose chain; (2) fabry's disease, the primary storage substance is GM2 ganglioside; (3) niemann pick C type disease, the primary storage substance is cholesterol.

Because of the unique pathogenesis of LSD, the main pathological manifestations of LSD are highly similar, all appearing at the cellular level as storage of various substances in the lysosome, obstruction of lysosomal membrane transport and regeneration, and degradation of lysosomal degradation functions. Accumulation of harmful substances in nerve and muscle cells due to the impairment of lysosomal function and lysosomal storage is a major cause of impairment of physiological functions and death of patients. Therefore, at the stage that the pathogeny cannot be directly eradicated by the gene editing means at present, the aims of reducing the injury and inhibiting the disease development can be achieved by inhibiting the pathological phenotype at the cell level for a long time.

The invention screens a small molecule library of a natural extract, and the small molecule compound elemene screened from 850 natural small molecules can effectively inhibit the phenotype of the lysosome storage disease, such as inhibiting the increase of the volume of lysosome, inhibiting cholesterol storage, remarkably recovering the inhibited lysosome tube, remarkably recovering the tolerance to repeated hunger and reducing the cell death rate, thereby achieving the purposes of reducing injury and inhibiting the disease development. Elemene has the prospect of being developed into a broad-spectrum drug for treating lysosomal storage diseases.

Drawings

Fig. 1 is a graph of the results of lysosome total volume detection in three LSD model cell lines, where a: GLAKO model, B: hex ako model, C: NPC1KO model lysosomes were labeled with lysotracker in Wild Type (WT) HeLa cells and GLAKO, HEXAKO, NPC1KO HeLa cells, the total intracellular volume of lysosomes was quantified by flow cytometry and normalized against WT. ***: p-value < 0.001.

Fig. 2 is a graph of the results of the detection of total lysosomal cholesterol in three LSD model cell lines, where a: GLAKO model, B: hex ako model, C: NPC1KO model, cholesterol in Wild Type (WT) HeLa cells and GLAKO, HEXAKO, NPC1KO HeLa cells were stained with filipin (cholesterol in cells accumulated in lysosomes), and quantitative analysis was performed by fluorescence confocal irradiation against lysosomal cholesterol. ***: p-value < 0.001.

Fig. 3 is a graph of results of the detection of endolysin tube formation of three LSD model cell lines, wherein a: GLAKO model, B: hex ako model, C: the NPC1KO model was starved for a long time (6 hours) for wild-type (WT) HeLa cells and GLAKO, HEXAKO, NPC1KO HeLa cells, and the number of tubular structures (lysosome vascularization) appearing on lysosomes in the cells was observed and quantified by live cell imaging. The lysosome tube becomes an essential step for lysosome regeneration. ***: p-value < 0.001.

Fig. 4 is a graph of mortality results for three LSD model cell lines under repeated starvation treatments, where a: GLAKO model, B: hex ako model, C: NPC1KO model, cell mortality was quantified by PI staining by repeated starvation (12 hours recovery of normal medium after 12 hours serum starvation, three replicates) of Wild Type (WT) HeLa cells and GLAKO, HEXAKO, NPC1KO HeLa cells. The data show that the mortality of LSD cells was significantly increased compared to wild-type cells. ***: p-value < 0.001.

Fig. 5 is a graph of the results of phenotypic measurements of elemene inhibition of lysosomal volume increase in LSD model cell lines: p-value < 0.001.

Fig. 6 is a graph of the results of phenotypic measurements of cholesterol storage in the elemene-inhibited LSD model cell line: p-value < 0.001.

Fig. 7 is a graph of the results of lysosome formation assays in the elemene-restored LSD model cell line: p-value <0.01,.: p-value < 0.001.

Fig. 8 is a graph of the results of tolerance measurements of the element-restored LSD model cell line to repeated starvation: p-value <0.01,.: p-value < 0.001.

Detailed Description