阅读说明：本技术 梓醇在制备降低核苷类药物所产生的线粒体毒性的药物中的应用 (Application of catalpol in preparation of medicine for reducing mitochondrial toxicity generated by nucleoside medicine ) 是由 不公告发明人 于 2019-02-28 设计创作，主要内容包括：本发明主要涉及梓醇在制备降低核苷类药物所产生的线粒体毒性的药物中的应用,特别是在制备降低核苷类药物对肝脏产生的线粒体毒性的药物中的应用。(The invention mainly relates to application of catalpol in preparation of a medicine for reducing mitochondrial toxicity generated by nucleoside drugs, in particular to application of catalpol in preparation of a medicine for reducing mitochondrial toxicity generated by nucleoside drugs to livers.)

1. Catalpol is applied to preparing medicines for reducing mitochondrial toxicity generated by nucleoside medicines.

2. The method of claim 1, wherein: catalpol is applied to preparing medicines for reducing the toxicity of the nucleoside medicines to mitochondria generated by the liver.

3. The method of claim 2, wherein: catalpol is applied to preparing a medicine for reducing the toxicity of zalcitabine to liver mitochondria.

The technical field is as follows:

the invention mainly relates to application of catalpol in preparation of a medicine for reducing mitochondrial toxicity generated by nucleoside drugs, in particular to application of catalpol in preparation of a medicine for reducing mitochondrial toxicity generated by nucleoside drugs to livers.

Background art:

catalpol is an iridoid glucoside compound, and has anticancer, neuroprotective, antiinflammatory, laxative, diuretic, and blood sugar lowering effects. In addition, the research shows that catalpol has obvious neuroprotective function under the condition of transient ischemia, and the catalpol is found to activate cerebral neurons and can treat cranial nerve diseases caused by trauma, metabolic disorder, cerebral ischemia, Parkinson's disease and the like.

Nucleoside drugs are the main drugs clinically used for treating infectious diseases such as AIDS virus, hepatitis B virus, hepatitis C virus, herpes virus and the like and tumors. Nearly 50% of currently used antiviral drugs are nucleoside drugs. Antineoplastic drugs such as zalcitabine, Cytarabine (Cytarabine), Doxifluridine (Doxifluridine) and the like belong to nucleoside drugs. The main application fields of the existing nucleoside drugs mainly comprise:

1. antiviral medicine

Nucleoside antiviral drugs have various varieties and structures, the antiviral mechanism is to destroy virus transcription, interfere or stop synthesis of virus nucleic acid, and the nucleoside antiviral drugs are mainly applied to infectious treatment of DNA and RNA viruses such as herpes viruses, HIV, HBV, influenza, respiratory viruses and the like at present.

2. Antitumor drug

At present, there are dozens of nucleoside antitumor drugs used in clinic and under study, such as cytarabine, doxifluridine, etc., which mainly act to interfere with DNA synthesis of tumor, or affect transcription process of nucleic acid, inhibit synthesis of protein, thereby achieving effect of treating tumor.

3. Antifungal medicine

Nucleoside compounds having this effect have been used in various clinical applications such as griseofulvin and 5 fluorocytosine, and some of them have inhibitory effects on various fungi and are almost non-toxic to mammals.

4. Antidepressant drug

Nucleoside drugs such as efavirenz, paroxetine, efavirenz and the like can be used for treating nervous system diseases, have very strong antidepressant effect, and some drugs can be used as analgesics for treating joint diseases and are also effective on cerebrovascular dysfunction.

However, with the long-term and widespread use of nucleoside analogs, adverse reactions have not been limited to acute liver failure occurring with viral variation and drug withdrawal rebound, and myopathy associated with mitochondrial toxicity, peripheral neuropathy, acute pancreatitis, hyperlactacidosis, lactic acidosis, kidney damage, rhabdomyolysis, and the like can also occur, with fatal patients. The nucleoside drugs approved to enter the market at present comprise lamivudine, adefovir dipivoxil (Hevili), entecavir, telbivudine, tenofovir, acyclovir, ribavirin and zalcitabine, and the nucleoside drugs can have mild and serious adverse reactions after being taken for a long time. These adverse effects include upper respiratory infection-like symptoms: headache, nausea, malaise, general weakness; gastrointestinal tract reaction: mild to moderate gastrointestinal discomfort, common diarrhea, abdominal pain, anorexia, nausea, vomiting, flatulence, pancreatitis; symptoms of the metabolic system: hypophosphatemia (1% incidence); fat accumulation and redistribution: including central obesity, buffalo back, peripheral wasting, enlarged breast, cushing's syndrome, and possibly lactic acidosis, hepatomegaly associated with steatosis, etc.; skin symptoms: drug eruptions.

The action mechanism of the nucleoside drugs is mainly to stop viral DNA or RNA chains by inhibiting the activity of viral DNA polymerase, but simultaneously, due to the similarity between the viral DNA polymerase and human nuclear DNA polymerase beta and between the viral DNA polymerase and human nuclear DNA polymerase gamma, the drugs can cause nuclear DNA defect in a patient body or reduction of intracellular mitochondrial mtDNA while inhibiting viruses, so that adverse reactions related to mitochondrial injury, such as hepatotoxicity, toxic kidney injury, myopathy, lactic acidosis, peripheral neuropathy and the like, can occur.

Through a large number of experiments and researches, the application of catalpol in preparing the medicine for reducing the mitochondrial toxicity generated by the nucleoside medicine, particularly the application of catalpol in preparing the medicine for reducing the mitochondrial toxicity generated by the nucleoside medicine to the liver, is invented by the applicant of the invention.

The invention content is as follows:

the invention aims to provide the application of catalpol in preparing a medicine for reducing the mitochondrial toxicity generated by nucleoside drugs, in particular to the application of catalpol in preparing a medicine for reducing the mitochondrial toxicity generated by nucleoside drugs to liver.

Specifically, the applicant of the invention invents the application of catalpol in preparing a medicament for reducing the mitochondrial toxicity generated by nucleoside medicaments, particularly the application of catalpol in preparing a medicament for reducing the mitochondrial toxicity generated by the nucleoside medicaments to liver on the basis of the prior art through a large number of pharmacological experiments and research analyses.

Specifically, the invention is realized by the following technical scheme.

The mitochondrial toxicity of the traditional Chinese medicine catalpol is evaluated in a HepG2 cell model by taking the marketed nucleoside drug zalcitabine (ddC) as a control.

First, experimental material

1.1 catalpol as test drug

The molecular formula is as follows: C11H15N5O3

Molecular weight: 265.27

Name: catalpol

Batch number: 20161126

Content and properties: white powder, 99.5%

Providing a unit: qinghai Central Zong pharmaceutical Co Ltd

Solubility: about 1g/100ml (water) at 17 ℃.

The preparation method and the storage condition are as follows: solvent dmso (sigma); the concentration of mother liquor is 100 mM; storing at-20 deg.C; it is diluted with culture medium to desired concentration for use.

1.2 Positive drugs

Zalcitabine (ddC)

Name: zalcitabine (2 ', 3' -Dideoxycytidine)

Molecular weight: 211.22

Content and properties: white powder

Solubility: about 1g/100ml (water) at 17 ℃.

Production unit: TOKYO Chemical Industry co., LTD.

The preparation method and the storage condition are as follows: solvent dmso (sigma); the concentration of the mother liquor is 1M; storing at-20 deg.C; it is diluted with culture medium to desired concentration for use.

Zalcitabine is mainly used for AIDS patients and AIDS-related syndromes which cannot tolerate lamivudine treatment clinically, has additive or synergistic effect on HIV when being combined with lamivudine, and can prevent the occurrence of drug-resistant virus strains and reduce toxic reaction. The adverse reaction is mainly peripheral sensory neuropathy, with burning pain on both feet as the prominent symptom, and half of patients involve both hands. The severity of nervous system response is related to the dosage, with larger dosages occurring earlier and with heavier disease conditions and slower recovery. Other adverse reactions include rash, fever, edema of ankles and lips. The product has no obvious blood toxicity, and is occasionally reduced in platelets or neutrophils.

1.3 cell models

Cell lines: human liver cancer HepG2 cell line was purchased from the Wuhan university culture Collection center (batch number: CCTCC, GDC024) and ordered 6, 10 days in 2008.

1.4 cell culture media and reagents related thereto

Low sugar MEM medium purchased from Hyclone; newborn fetal bovine serum, purchased from BI corporation (israel); trypsin 1: 250 from Merk (batch number T0012-25 g); DMSO was purchased from Sigma (batch No. 038K 0710).

1.5 drug concentration settings

Catalpol cytotoxic concentration is 200 μ M, 100 μ M, 50 μ M, 25 μ M, 12.5 μ M; ddC 50. mu.M.

Second, the experimental contents

2.1 cell proliferation inhibition assay

Taking HepG2 cells in logarithmic phase, inoculating the HepG2 cells in 1 ten thousand per well into a disposable 96-well cell culture plate (Corning) for culture, adding drugs with different concentrations after 24 hours, placing the cells into a 37 ℃ and 5% CO2 incubator for culture, adding 20 mu l MTT (thiazole blue) for treatment for 4 hours after 5 days of drug treatment, then removing supernatant, adding a proper amount of DMSO (dimethyl sulfoxide) for detecting the OD (optical density) value of each well, and counting the cell growth percentage of each drug concentration group according to comparison. The results show that catalpol has no obvious cytotoxicity at 5 concentrations of 200 mu M, 100 mu M, 50 mu M, 25 mu M, 12.5 mu M and the like, and IC50 (half inhibition concentration) > 200 mu M.

2.2 Effect of lactic acid production levels

Taking HepG2 cells in logarithmic phase, inoculating the HepG2 cells in 10 ten thousand per hole into a disposable 6-hole cell culture plate for culture, adding drugs with different concentrations, placing the cell culture plate into a 37 ℃ and 5% CO2 incubator for culture for 15 days, changing the culture solution once every 3 days, collecting cell supernatant after 15 days, and directly measuring by using a lactic acid measuring kit. The results are shown in figure 1 in the attached figure of the specification (the effect of catalpol on lactic acid secretion caused by ddC induced mitochondrial toxicity (. p < 0.05)). As can be seen from FIG. 1, the positive drug ddC 50. mu.M significantly promoted the increase in lactic acid levels of HepG2 cells (P < 0.01) compared to the control; the tested drug catalpol 50 mu M has no effect. The level of the cell supernatant lactic acid after the catalpol and ddC combined administration is basically equivalent to that of a normal control group, which shows that the catalpol can obviously reduce the increase of the content of the lactic acid caused by the mitochondrial toxicity induced by ddC.

2.3 comparison of mtDNA content

HepG2 cells in logarithmic growth phase are taken, inoculated into a disposable 6-hole cell culture plate for culture according to 10 ten thousand per hole, added with drugs with different concentrations, placed into a 37 ℃ and 5% CO2 incubator for culture for 15 days, changed with liquid once every 3 days, collected after 15 days, and subjected to total DNA extraction according to a cell tissue DNA extraction kit of Qiagen. The extracted DNA was quantified on a nucleic acid protein analyzer and then frozen at-70 ℃ in a freezer. The mtDNA and nuclear DNA were quantified by fluorescent quantitative PCR, and the difference in the ratio was compared. The results are shown in fig. 2 (catalpol effect on mitochondrial mtDNA of cells) and fig. 3 (catalpol effect on reducing mitochondrial mtDNA by ddC (. P < 0.05,. P < 0.01)) in the figures of the specification. As can be seen from FIG. 2, the content of mitochondrial DNA (mtDNA) in cells after the test catalpol administration groups (12.5-200 μ M) are treated for 15 days is basically equivalent to that of the blank control group, while the content of mtDNA in the positive ddC 50 μ M administration group is obviously reduced and is very different from that of the blank control group (p is less than 0.001). The results show that catalpol has no obvious influence on the content of mitochondrial mtDNA.

The effect of catalpol co-administration with ddC on mtDNA is shown in FIG. 3. As can be seen from FIG. 3, the content of mtDNA in the catalpol and ddC combination administration group was significantly increased compared to the ddC single administration group, and was statistically different from the ddC single administration group (p < 0.01).

Thirdly, conclusion:

in a HepG2 cell model, zalcitabine (ddC) is taken as a positive drug, and the effect of catalpol as a test drug on reducing the mitochondrial toxicity of nucleoside drugs is researched from 3 aspects such as cell proliferation inhibition test, cell supernatant lactic acid level, mitochondrial mtDNA content and the like.

The test result shows that the catalpol has no obvious influence on cell proliferation and cell supernatant lactic acid level compared with a control group under the condition of 200 mu M high dose of the test drug catalpol, and the catalpol has no mitochondrial toxicity under the condition of 200 mu M high dose. Meanwhile, the positive drug ddC 50 mu M compared with the same period shows obvious mitochondrial toxicity in the detection indexes. Catalpol and ddC are jointly administrated, so that the lactic acid level caused by mitochondrial toxicity induced by ddC can be obviously reduced, and the effect of ddC on reducing the mtDNA content of cells can be effectively inhibited. In conclusion, the in vitro test results show that the tested catalpol has the effect of reducing the mitochondrial toxicity of the nucleoside drugs.

Meanwhile, it should be noted that, for those skilled in the art, zalcitabine is only a positive tool in the experiments of catalpol for reducing the efficacy of mitochondrial toxicity, and it cannot be understood narrowly that catalpol is only effective on mitochondrial toxicity caused by zalcitabine, but is effective on the efficacy of nucleoside drugs.

In conclusion, the inventor obtains that catalpol has a remarkable effect of reducing the mitochondrial toxicity caused by nucleoside drugs through a large number of experimental researches and analyses. Catalpol can be prepared into clinically and therapeutically acceptable dosage forms, including tablets, capsules, pills, granules, oral liquid, patches and the like, according to a conventional preparation method in pharmaceutics when being used for preparing a medicine for reducing the mitochondrial toxicity of nucleoside drugs on livers.