WO2022160323A1 - Application of nucleoside analogue in preparation of drugs for preventing or treating gastrointestinal diseases - Google Patents

Application of nucleoside analogue in preparation of drugs for preventing or treating gastrointestinal diseases Download PDF

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WO2022160323A1
WO2022160323A1 PCT/CN2021/074575 CN2021074575W WO2022160323A1 WO 2022160323 A1 WO2022160323 A1 WO 2022160323A1 CN 2021074575 W CN2021074575 W CN 2021074575W WO 2022160323 A1 WO2022160323 A1 WO 2022160323A1
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ulcer
analogs
group
mice
dipivoxil
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PCT/CN2021/074575
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French (fr)
Chinese (zh)
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陈新平
刘菁菁
赵文阳
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兰州大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants

Definitions

  • the invention relates to the technical field of medicine, in particular to the application of nucleoside analogs in the preparation of medicines for preventing or treating gastrointestinal diseases.
  • Gastrointestinal diseases cover diseases of the esophagus, stomach, small intestine, colon and rectum. Common main symptoms include rhythm, periodic epigastric pain, diarrhea, hungry abdominal pain, acid regurgitation, fever, black stool, gastrointestinal bleeding and intestinal obstruction, etc. Gastrointestinal diseases are among the most common diseases in humans, the most common of which include dysphagia, gastric ulcer, peptic ulcer, gastroparesis, delayed gastric emptying, irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Peptic ulcers mainly include gastric ulcers, duodenal ulcers and complex ulcers.
  • gastric ulcers mainly focus on the weakening of the gastric mucosal barrier and the increase of gastrin secretion, while the formation of duodenal ulcers Factors are more focused on the increase in the total volume of parietal cells.
  • exogenous factors such as excessive drinking, irregular eating, long-term mental stress and long-term use of non-steroidal anti-inflammatory drugs (such as aspirin), glucocorticoids, clopidogrel and other drugs are related to the formation of peptic ulcer disease.
  • Inflammatory bowel disease can be caused by bacteria, fungi, viruses, parasites, protozoa and other organisms, as well as by allergic reactions and physical and chemical factors.
  • UC ulcerative colitis
  • IBD Inflammatory bowel disease
  • the main types of western medicines for the treatment of gastrointestinal diseases include gastrointestinal motility drugs, antispasmodics, antiemetics, peptic ulcer drugs, gastric mucosal protective agents, digestive aids, probiotics, etc.
  • the drugs for the treatment of peptic ulcer mainly include proton pump inhibitors, H2-receptor antagonists, bismuth preparations and prostaglandins, etc., which are mainly used to neutralize gastric acid through raw materials containing calcium. It is difficult to completely cure, the curative effect is unsatisfactory, and it is easy to recur.
  • Inflammatory bowel disease is listed as one of the modern intractable diseases by the World Health Organization, and the incidence rate is on the rise in the world.
  • Nucleoside analogs have some special effects, and 2'-deoxynucleoside drugs can specifically infect virus replication.
  • nucleoside compounds are generally used as inhibitors of enzymes in the process of virus replication, blocking the infection of target cells by the virus.
  • Common nucleoside antiviral compounds mainly include Lamivudine (3-TC), Telbivudine (LDT), Zidovudine (AZT), Famciclovir (FCV), Telofovir (PMPA), Adefovir dipivoxil (PMEA) and other drugs.
  • Telbivudine for adult patients with chronic hepatitis B with evidence of viral replication and persistent elevation of serum transaminases (ALT or AST) or evidence of active liver disease; zidovudine, alias azidodeoxythymidine, For the treatment of AIDS or AIDS-related syndrome patients and immunodeficiency virus (HIV) infection, zidovudine is the first anti-AIDS drug approved by the US FDA in the world.
  • ALT or AST serum transaminases
  • zidovudine alias azidodeoxythymidine
  • Acyclovir is the most effective and representative nucleoside analog in clinical application; acyclovir is a highly effective, low-toxic, broad-spectrum antiviral drug, and is currently the drug of choice for the treatment of herpes; Famciclovir, the first oral drug approved in the United States for recurrent herpes simplex virus infection in AIDS patients, not only has a high absorption rate, but also has a long drug duration, and is the only effective drug for reducing postherpetic neuralgia; adefovir , Adenine nucleoside derivatives with broad-spectrum antiviral activity can effectively inhibit the replication and expression of retroviral genes.
  • nucleoside antitumor drugs there are dozens of nucleoside antitumor drugs currently used in clinical and research, their main role is to interfere with the DNA synthesis of tumors, or affect the transcription process of nucleic acid, inhibit the synthesis of proteins, so as to achieve the effect of tumor treatment.
  • cytarabine a pyrimidine antimetabolite that mainly acts on the cell S proliferation phase, interferes with the proliferation of cells by inhibiting the synthesis of cell DNA, and is mainly used for acute leukemia. Nuclear cell leukemia and acute lymphoblastic leukemia are also effective.
  • Gemcitabine a new cytosine nucleoside derivative, has the same mechanism of action as cytarabine. Its main metabolite is incorporated into DNA in cells and mainly acts on G1/S phase. Clinically, gemcitabine and cytarabine have different anti-tumor profiles and are effective against a variety of solid tumors.
  • Metastatic non-small cell lung cancer has been used as a first-line application. Recent data indicate that this product also has palliative effects on ovarian cancer, breast cancer, bladder cancer, cervical cancer, liver cancer, biliary tract cancer, nasopharyngeal cancer, testicular tumor, lymphoma and head and neck cancer.
  • patent CN201880077576.4 discloses nucleotide analogs The preparation method and its application in nucleic acid sequence determination, etc.
  • patent CN201780039312.5 discloses a method for nucleic acid detection using reversible blocking nucleoside analogs
  • patent CN201810489585.6 discloses a skeleton-integrated nucleoside analog drug Functional nucleic acids and derivatives thereof and preparation methods thereof
  • Patent CN201410169585.X discloses that phospho N-fatty acids modified with cyclophosphyl groups and connected to fatty chains are used for the treatment of viral hepatitis and liver cancer
  • Patent CN201910892040.4 discloses Drug aptamers constructed by nucleoside analog drug molecules, preparation methods and applications, etc.
  • patent CN201810077674.X discloses the use of 1,4-disubstituted 1,2,3-
  • the present invention discloses nucleoside analogs or tautomers, mesomers, racemates, enantiomers, diastereomers and mixtures thereof, and The application of a pharmaceutically acceptable salt in the preparation of a medicament for preventing and/or treating gastrointestinal diseases, said gastrointestinal diseases excluding gastric cancer.
  • the nucleoside analogs are selected from any one of cytosine analogs, thymidine analogs, adenosine analogs and guanosine analogs.
  • the cytosine nucleoside analogs are selected from lamivudine, deoxycytidine, gemcitabine hydrochloride, cytarabine hydrochloride, cidofovir, etricitapine, efcitabine, emtrexine Azacitabine, Acadisin, Azacitidine, Decitabine, Thiarabine, Ethynylcytidine, Capecitabine, Escilarabine, Amcitabine, 4-S- ⁇ -D-Cytarabine , 5-aza-4-thio-2-deoxycytidine, cis-1-[4-(hydroxy-methyl)-cyclopent-2-enyl]-5-I-iodocytosine and cis- -1-[4-(Hydroxy-methyl)-cyclopent-2-enyl]-5-(2-I-iodovinyl) cytosine; the thymidine analogs Selected from zidovudine,
  • the cytidine analog is selected from lamivudine or gemcitabine hydrochloride; the thymidine analog is selected from zidovudine or telbivudine; the adenosine The analog is selected from famciclovir, telofovir dipivoxil or adefovir dipivoxil; the guanine nucleoside analog is selected from acyclovir or 6-thioguanine.
  • the gastrointestinal disease is peptic ulcer.
  • the peptic ulcer is one or more of gastric ulcer, duodenal ulcer, retrobulbar ulcer, pyloric ulcer, compound ulcer and kiss ulcer.
  • the peptic ulcers are gastric ulcers and duodenal ulcers.
  • the gastrointestinal disease is inflammatory bowel disease.
  • the inflammatory bowel disease is ulcerative colitis and Crohn's disease.
  • nucleoside analogs or tautomers, mesomers, racemates, enantiomers, diastereomers and mixtures thereof, and pharmaceutically acceptable salts thereof and one or more pharmaceutically acceptable carriers to form a pharmaceutical composition
  • the dosage form of the pharmaceutical composition is injection, tablet, capsule, granule and pill.
  • the invention provides the application of nucleoside analogs in the preparation of medicines for preventing or treating gastrointestinal diseases, and specifically provides some classic nucleoside analogs lamivudine, telbivudine, famciclovir , telofovir dipivoxil, adefovir dipivoxil, acyclovir, gemcitabine hydrochloride, zidovudine and 6-thioguanine, DAI score and colonic colitis mice under different feeding conditions Morphometry, the results showed lamivudine, telbivudine, famciclovir, telofovir dipivoxil, adefovir dipivoxil, acyclovir, gemcitabine hydrochloride, zidovudine and 6-thioguanine and other drugs Effective for ulcerative colitis.
  • Drugs such as purine are effective for both ulcerative colitis and gastric ulcer at the same time, and the therapeutic effect is 40 times higher than that of existing clinical drugs, and has a good clinical application prospect.
  • Figure 1 The effect of lamivudine on body weight and DAI score of C57BL/6N colitis mice
  • Fig. 3 The effect of lamivudine on gastric tissue morphology in gastric ulcer mice
  • Figure 4 The effect of lamivudine on gastric ulcer index, gastric ulcer area and gastric ulcer inhibition rate
  • Figure 5 Effects of adefovir dipivoxil, telofovir dipivoxil, famciclovir and telbivudine on body weight, DAI score, colon morphology and colon length in KM colitis mice;
  • Figure 6 Effects of acyclovir, gemcitabine hydrochloride, zidovudine and 6-thioguanine on body weight, DAI score, colon morphology and colon length in KM colitis mice;
  • Figure 7 Effects of telbivudine, gemcitabine hydrochloride and zidovudine on gastric tissue morphology, ulcer index, ulcer area and ulcer inhibition rate in KM gastric ulcer mice;
  • Figure 8 Effects of adefovir dipivoxil, telofovir dipivoxil, acyclovir, famciclovir and 6-thioguanine on gastric tissue morphology, ulcer index, ulcer area and ulcer inhibition rate in KM gastric ulcer mice;
  • protection scope of the present invention will be described in detail below with reference to specific examples, but it should be noted that the protection scope of the present invention is not limited to the following examples, and a class of compounds that protect nucleoside analogs in all gastrointestinal diseases, especially Therapeutic effects in peptic ulcer disease and inflammatory bowel disease, including different dosage forms, doses, and combination drugs. All technical solutions obtained by those skilled in the art through logical analysis, inference and experimentation in the prior art according to the idea of the present invention belong to the scope of the claimed protection of the present invention.
  • the C57BL/6N mouse described in the present invention is one of the most widely used strains in mice, and is also most commonly used as the female parent of transgenic or gene knockout mice in genetic engineering.
  • the KM mice of the present invention refer to Kunming mice.
  • the following examples of the present invention use sodium dextran sulfate to model colitis in mice.
  • the indications of sulfasalazine enteric-coated tablets are (1) ulcerative colitis to treat mild to moderate ulcerative colitis; it can be used as an adjuvant therapy in severe ulcerative colitis. It can also be used for maintenance treatment of ulcerative colitis in remission; (2) Crohn's disease is used to treat active Crohn's disease, especially those patients with colon involvement; (3) rheumatoid arthritis is resistant to salicylic acid or other For rheumatoid arthritis and juvenile rheumatoid arthritis (polyarticular type) in which the effect of non-steroidal anti-inflammatory drugs is not significant, sulfasalazine enteric-coated tablets are used as positive drugs in Example 1 of the present invention.
  • Cimetidine in the following embodiments of the present invention is a histamine H2 receptor antagonist, mainly used to inhibit the secretion of gastric acid, can significantly inhibit the secretion of basal and nocturnal gastric acid, and can also inhibit the secretion of gastric acid.
  • Gastric acid secretion caused by stimuli such as histamine, peptide gastrin, insulin and food, and reduce its acidity, prevent and protect corrosive gastritis caused by chemical stimulation Bleeding is also effective.
  • cimetidine is used as a positive drug.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms, which are within the scope of sound medical judgment, suitable for use in contact with human and animal tissue without excessive of toxicity, irritation, allergic reactions or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • composition refers to a biologically active compound optionally in admixture with at least one pharmaceutically acceptable chemical component or agent, which is a “carrier” that facilitates For introducing compounds into cells or tissues, include but are not limited to stabilizers, diluents, suspending agents, thickening agents and/or excipients.
  • salts in the present invention can be mentioned metal salts, ammonium salts, salts formed with organic bases, salts formed with inorganic acids, salts formed with organic acids, salts formed with basic or acidic amino acids, etc. .
  • salts can be synthesized from the parent compound containing acid or base by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.
  • treating and other similar synonyms include alleviating, alleviating or ameliorating the symptoms of a disease or disorder, preventing other symptoms, ameliorating or preventing the underlying metabolic cause of the symptoms, inhibiting the disease or disorder, such as preventing the progression of the disease or disorder, alleviating the disease or Condition, amelioration of a disease or condition, alleviation of symptoms caused by a disease or condition, or cessation of symptoms of a disease or condition, in addition, the term may also encompass prophylactic purposes.
  • the term also includes obtaining a therapeutic effect and/or a prophylactic effect.
  • the therapeutic effect refers to curing or ameliorating the underlying disease being treated.
  • curing or amelioration of one or more physiological symptoms associated with the underlying disease is also a therapeutic effect, eg, an improvement in a patient's condition is observed although the patient may still be affected by the underlying disease.
  • the composition or compound may be administered to a patient at risk of developing a particular disease, or to a patient presenting one or more physiological symptoms of the disease even if no diagnosis of the disease has been made. or compound.
  • Embodiment 1 The therapeutic effect of lamivudine on C57BL/6N colitis mice
  • Lamivudine (Lamivudine, 3-TC, HPLC ⁇ 98%), purchased from McLean Biotechnology Co., Ltd., batch number: C10102050, molecular weight 229.25; Sulfasalazine enteric-coated tablets (Salazosulfapyridine, SASP) purchased from Shanghai Xin Yi Tianping Pharmaceutical Co., Ltd.; Dextran Sulfate Sodium (DSS), MW: 40000, purchased from Aladdin Biotechnology Co., Ltd.
  • mice 40 8-week-old male C57BL/6N mice were randomly divided into 5 groups according to their body weight, with 8 mice in each group.
  • the groups and doses were as follows:
  • mice were given lamivudine group (C+3-TC, the dose was 2mg/kg/day)
  • DSS model control group (DSS, orally administered an equal volume of 0.9% normal saline);
  • SASP Positive drug group
  • the model mice were given lamivudine group (D+3-TC, the dosage was 2 mg/kg/day).
  • a 4% DSS distilled water solution was prepared, and the mice in the DSS model group drank the DSS water solution freely for modeling, and kept regular feed for continuous modeling for 7 days.
  • mice in DSS model control group drank DSS aqueous solution freely for modeling mice in DSS+3-TC group drank DSS aqueous solution freely for modeling, and were given 2 mg/kg/day 3-TC; mice in DSS+SASP group drank DSS aqueous solution freely for modeling The model was given 80mg/kg/day of SASP at the same time; the mice in the blank control group drank distilled water freely, and the mice in the blank control group +3-TC group were given 2 mg/kg/day of 3-TC while drinking distilled water freely. Routine feed was fed for 7 consecutive days. On the seventh day, 2 hours after the last administration, the serum and colon tissue were separated for use.
  • the normal stool score is scored as 0, the occult blood bleeding is scored as 1 (judged by the occult blood test results), the bloody stool is scored as 3 with the naked eye, and the average of the above three items is DAI.
  • mice After the mice were sacrificed, the colons of the mice were separated, and the ileum was cut from the junction of the ileum and the colon, and then at the colon near the anus. to the length of the anus, and photographed to record.
  • the experimental data were statistically analyzed by SPSS23.0 software, and the data were expressed as (x ⁇ s), and the comparison between groups was performed by one-way analysis of variance (One-way ANOVA) and LSD-t method. p ⁇ 0.05 was considered statistically significant.
  • mice were monitored for their body weight, fecal viscosity, blood in the stool and occult blood, and SPSS 23.0 software was used for one-way analysis of variance for data statistics.
  • mice in the above groups were sacrificed, their colon tissues were quickly removed, and their morphological changes were observed, and their lengths were measured at the same time, and statistical analysis was performed on the data of each group. Significant differences in colon length of mice in each group were expressed as *p ⁇ 0.05, **p ⁇ 0.01 and ***p ⁇ 0.001. The colon morphology and colon length monitoring results of each group of UC colitis mice are shown in Figure 2.
  • the colon length of the mice in the blank control + lamivudine administration group did not change significantly, indicating that lamivudine has no toxic and side effects on normal mice; compared with the blank control group, the colon in the DSS model group The length was significantly shortened (###p ⁇ 0.001), indicating that the DSS model was successfully prepared; compared with the DSS model control group, both the sulfasalazine administration group and the lamivudine administration group could significantly improve the colon.
  • There were significant differences in edema and shortening (*p ⁇ 0.05 and ***p ⁇ 0.001), and the efficacy of lamivudine was stronger than that of clinical drug sulfasalazine.
  • Embodiment two the therapeutic effect of lamivudine on KM gastric ulcer mice
  • mice 8-week-old SPF grade Kunming (Kunming, KM) male mice, weighing 18-22 g, without any drugs before the experiment, were purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The experimental animals were acclimatized and raised for a week in an environment with a temperature of 24-26 °C and alternating daytime patterns of 12h/12h.
  • Lamivudine (Lamivudine, 3-TC, HPLC ⁇ 98%), purchased from McLean Biotechnology Co., Ltd.; absolute ethanol, Tianjin Damao Chemical Reagent Co., Ltd.; Cimetidine (CIM), purchased from Shanghai Xinyi Tianping Pharmaceutical Co., Ltd.
  • mice 40 KM male mice (20-25g) were raised in the laboratory animal room of Lanzhou University. After the mice were adaptively reared for one week, they were randomly divided into 5 groups with 8 mice in each group. The grouping and dosage are shown in Table 2. shown;
  • mice in the above groups were stimulated for 2 h to end the experiment, and the serum and gastric tissue were separated to determine various indicators.
  • the experimental data were statistically analyzed by SPSS23.0 software, and the data were expressed as (x ⁇ s), and the comparison between groups was performed by one-way analysis of variance (One-way ANOVA) and LSD-t method. p ⁇ 0.05 was considered statistically significant.
  • ulcer area The above-mentioned ulcer area, ulcer index and inhibition rate, and gastric histomorphological measurements show that lamivudine is effective in preventing and treating gastric ulcer, and the curative effect is significantly stronger than that of the clinical drug cimetidine.
  • Embodiment 3 The therapeutic effect of adefovir dipivoxil, telofovir dipivoxil, famciclovir and telbivudine on KM colitis mice
  • Telofovir dipivoxil (PMPA, HPLC ⁇ 98%), adefovir dipivoxil (PMEA, HPLC ⁇ 98%), famciclocir (Famciclocir, FCV, HPLC ⁇ 98%) and telbivudine (Telbivudine, LDT, HPLC) ⁇ 98%) were purchased from Aladdin Biotechnology Co., Ltd.; Sulfasalazine enteric-coated tablets (Salazosulfapyridine, SASP) were purchased from Shanghai Xinyi Tianping Pharmaceutical Co., Ltd.; Dextran Sulfate Sodium (DSS), MW: 40000, purchased from Aladdin Biotechnology Co., Ltd.
  • mice with standard body weight were randomly divided into 7 groups with 8 mice in each group.
  • the grouping and administration doses were as follows:
  • DSS model control group (DSS, orally administered an equal volume of 0.9% normal saline);
  • SASP Positive drug group
  • telofovir disoproxil group (PMPA, administered at a dose of 2 mg/kg/day, intraperitoneal injection);
  • Adefovir dipivoxil group (PMEA, administered at a dose of 2 mg/kg/day, intraperitoneal injection);
  • FCV Famciclovir group
  • Telbivudine group (LDT, administered at a dose of 2 mg/kg/day, intraperitoneal injection).
  • mice of the above groups were monitored for their body weight, fecal viscosity, blood in the stool and occult blood, colon morphology and length, and SPSS 23.0 software was used for one-way analysis of variance for data statistics.
  • Embodiment 4 Therapeutic effect of acyclovir, gemcitabine hydrochloride, zidovudine and 6-thioguanine on KM colitis mice
  • Gemcitabine hydrochloride (Gemzar, HPLC ⁇ 98%), zidovudine/3-azido-3-deoxythymidine (AZT, HPLC ⁇ 98%) and 6-thioguanine (6-TG, HPLC ⁇ 98%) ) was purchased from Aladdin Biotechnology Co., Ltd.; Acyclovir (ACV, HPLC ⁇ 97%) was purchased from McLean Biotechnology Co., Ltd.; Sulfasalazine Enteric-Coated Tablets (Salazosulfapyridine, SASP) were purchased from Shanghai Xinyi Tianping Pharmaceutical Co., Ltd.; Dextran Sulfate Sodium (DSS), MW: 40000, purchased from Aladdin Biotechnology Co., Ltd.
  • mice with standard body weight were randomly divided into 7 groups with 8 mice in each group.
  • the grouping and administration doses were as follows:
  • DSS model control group (DSS, orally administered an equal volume of 0.9% normal saline);
  • SASP Positive drug group
  • Gemcitabine hydrochloride group (Gemzar, the dosage is 2mg/kg/day, intraperitoneal injection);
  • Zidovudine group (AZT, the dose is 2mg/kg/day, intraperitoneal injection);
  • Acyclovir group (ACV, the dose is 2 mg/kg/day, intraperitoneal injection);
  • 6-thioguanine group (6-TG, administered at a dose of 2 mg/kg/day, intragastric administration).
  • mice of the above groups were monitored for their body weight, fecal viscosity, blood in the stool and occult blood, colon morphology and length, and SPSS 23.0 software was used for one-way analysis of variance for data statistics.
  • each administration group of acyclovir, gemcitabine hydrochloride, zidovudine and 6-thioguanine could significantly increase the DAI score (***p ⁇ 0.001, **p ⁇ 0.01 , *p ⁇ 0.05 and *p ⁇ 0.05), improved colonic edema and shortening (***p ⁇ 0.001, **p ⁇ 0.01, **p ⁇ 0.01 and **p ⁇ 0.01), indicating acyclovir , gemcitabine hydrochloride, zidovudine and 6-thioguanine have therapeutic effects on ulcerative colitis, among which the curative effect of acyclovir is stronger than that of the clinical drug sulfasalazine.
  • the curative effects of each administration group are compared as follows: Acyclovir > sulfasalazine.
  • Embodiment 5 The protective effect of telbivudine, gemcitabine hydrochloride and zidovudine on KM gastric ulcer mice
  • mice 8-week-old SPF grade Kunming (Kunming, KM) male mice, weighing 18-22 g, without any drugs before the experiment, were purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.
  • Gemcitabine hydrochloride (Gemzar, HPLC ⁇ 98%), zidovudine/3-azido-3-deoxythymidine (AZT, HPLC ⁇ 98%) and telbivudine (Telbivudine, LDT, HPLC ⁇ 98%) were purchased from Aladdin Biotechnology Co., Ltd.; anhydrous ethanol, Tianjin Damao Chemical Reagent Co., Ltd.; Cimetidine (CIM) tablets were purchased from Shanghai Xinyi Tianping Pharmaceutical Co., Ltd.
  • mice 48 KM male mice (20-25g) were raised in the laboratory animal room of Lanzhou University. After the mice were adaptively reared for one week, they were randomly divided into 6 groups with 8 mice in each group. The grouping and dosage are shown in Table 3. shown;
  • mice in the above groups were terminated after 2 hours of ethanol stimulation of the mice in the above groups, and the serum and gastric tissue were separated to determine various indicators.
  • the experimental data were statistically analyzed by SPSS23.0 software, and the data were expressed as (x ⁇ s), and the comparison between groups was performed by one-way analysis of variance (One-way ANOVA) and LSD-t method. p ⁇ 0.05 was considered statistically significant.
  • Embodiment 6 Protective effect of adefovir dipivoxil, telofovir dipivoxil, acyclovir, famciclovir and 6-thioguanine on KM gastric ulcer mice
  • mice 8-week-old SPF grade Kunming (Kunming, KM) male mice, weighing 18-22 g, without any drugs before the experiment, were purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.
  • Telofovir dipivoxil (PMPA, HPLC ⁇ 98%), adefovir dipivoxil (PMEA, HPLC ⁇ 98%), famciclocir (Famciclocir, FCV, HPLC ⁇ 98%) and 6-thioguanine (6-TG , HPLC ⁇ 98%) was purchased from Aladdin Biotechnology Co., Ltd.; Acyclovir (ACV, HPLC ⁇ 97%) was purchased from Maclean Biotechnology Co., Ltd.; Anhydrous ethanol, Tianjin Damao Chemical Reagent Co., Ltd.; Cimetidine (CIM) was purchased from Shanghai Xinyi Tianping Pharmaceutical Co., Ltd.
  • mice 64 KM male mice (20-25g) were raised in the laboratory animal room of Lanzhou University. After the mice were adaptively reared for one week, they were randomly divided into 8 groups with 8 mice in each group. The grouping and dosage are shown in Table 4. shown;
  • mice in the above groups were terminated after 2 hours of ethanol stimulation of the mice in the above groups, and the serum and gastric tissue were separated to determine various indicators.
  • the experimental data were statistically analyzed by SPSS23.0 software, and the data were expressed as (x ⁇ s), and the comparison between groups was performed by one-way analysis of variance (One-way ANOVA) and LSD-t method. p ⁇ 0.05 was considered statistically significant.
  • ulcer area The above-mentioned ulcer area, ulcer index and inhibition rate, and gastric histomorphological measurements show that adefovir dipivoxil, telofovir dipivoxil, acyclovir, famciclovir and 6-thioguanine are effective in preventing and treating gastric ulcer.
  • the nucleoside analogs of the present invention lamivudine, telbivudine, famciclovir, telofovir dipivoxil, adefovir dipivoxil, acyclovir, gemcitabine hydrochloride, zidovudine and 6-thioguanine
  • the DAI score and colon morphometry of colitis mice showed that lamivudine, telbivudine, famciclovir, telofovir dipivoxil, adrenalin
  • Drugs such as fovir dipivoxil, acyclovir, gemcitabine hydrochloride, zidovudine, and 6-thioguanine are all effective for ulcerative colitis.
  • nucleoside analogs have the effect of treating gastrointestinal diseases.
  • nucleoside analogs can be further classified into cytosine analogs, thymidine analogs, adenosine analogs and guanosine analogs.
  • the inventors selected thymidine analogs from Zidovudine or Telbivudine; the adenosine analogs are selected from famciclovir, telofovir dipivoxil or adefovir dipivoxil; the guanosine analogs are selected from acyclovir or 6-thioguanine.
  • related experiments on gastrointestinal diseases were carried out.
  • telbivudine famciclovir
  • telofovir dipivoxil adefovir dipivoxil
  • gemcitabine hydrochloride zidovudine and 6-thiol Guanine has a significant effect on the treatment of gastrointestinal diseases.
  • the experimental results are shown in the above-mentioned examples.
  • the inventors have confirmed the above inferences and concluded that all nucleoside analogs have effects in the treatment of gastrointestinal diseases, and can be used with For the preparation of medicines for treating gastrointestinal diseases, a new use is provided for medicines of nucleoside analogs, which can be recognized by those skilled in the art.

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Abstract

An application of a nucleoside analogue in preparation of drugs for preventing or treating gastrointestinal diseases. The nucleoside analogue is selected from any one of a cytosine nucleoside analogue, a thymine nucleoside analogue, an adenine nucleoside analogue, and a guanine nucleoside analogue. Experimental results show that the nucleoside analogue is effective in treatment of peptic ulcer disease and inflammatory bowel disease, has an activity of 40 times higher than that of the existing clinical drugs, can effectively improve the integrity of mucosae, promote the improvement of tissue morphologies and restore the weight of animals, and has a good clinical application prospect.

Description

核苷类似物在制备预防或治疗胃肠道疾病药物中的应用Application of nucleoside analogs in the preparation of drugs for preventing or treating gastrointestinal diseases 技术领域technical field
本发明涉及医药技术领域，具体涉及核苷类似物在制备预防或治疗胃肠道疾病药物中的应用。The invention relates to the technical field of medicine, in particular to the application of nucleoside analogs in the preparation of medicines for preventing or treating gastrointestinal diseases.
背景技术Background technique
胃肠道疾病涵盖食管、胃、小肠、结肠和直肠等疾病，常见的主要症状包括节律性、周期性上腹痛、腹泻、饥饿性腹痛、反酸、发烧、黑便血便、消化道出血和肠梗阻等。胃肠道疾病是人类最常见的疾病之一，其中最常见的包括吞咽障碍、胃溃疡、消化性溃疡、胃轻瘫、胃排空延迟、肠易激综合征(IBS)和炎症性肠病(IBD)。消化性溃疡主要包括胃溃疡、十二指肠溃疡和复合型溃疡等，目前认为胃溃疡的形成因素较多着重于胃粘膜屏障的削弱和胃泌素分泌的增加，而十二直肠溃疡的形成因素则较多着重于壁细胞总体积的增大。此外，过度饮酒、进食无规律、长期精神紧张和长期服用非甾体抗炎药(如阿司匹林)、糖皮质激素、氯吡格雷等药物等外源性因素均与消化性溃疡病的形成有关。炎症性肠病可由细菌、真菌、病毒、寄生虫、原虫等生物引起，亦可由***反应及理化因子引起。根据病因不同，可分为特异性炎性病变和非特异性炎性病变，前者指感染性结肠炎、缺血性结肠炎和伪膜性结肠炎等，后者主要包括溃疡性结肠炎(Ulcerative Colitis，UC)与结肠克罗恩病(Crohn’s Disease，CD)。UC是一种炎症性疾病(Inflammatory bowel disease，IBD)，作为消化内科常见的病症，可引起消化道发生长期炎症和溃疡，以慢性复发性肠道炎症和肠上皮细胞损伤为主要特征，多发于20-30岁，临床症状主要有腹泻、腹痛、血便和肠梗阻等。已有许多研究表明，UC的发病与免疫因素、炎症、环境遗传和压力及感染等因素有关。胃炎由慢性胃炎、急性胃炎等组成。Gastrointestinal diseases cover diseases of the esophagus, stomach, small intestine, colon and rectum. Common main symptoms include rhythm, periodic epigastric pain, diarrhea, hungry abdominal pain, acid regurgitation, fever, black stool, gastrointestinal bleeding and intestinal obstruction, etc. Gastrointestinal diseases are among the most common diseases in humans, the most common of which include dysphagia, gastric ulcer, peptic ulcer, gastroparesis, delayed gastric emptying, irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Peptic ulcers mainly include gastric ulcers, duodenal ulcers and complex ulcers. At present, it is believed that the formation factors of gastric ulcers mainly focus on the weakening of the gastric mucosal barrier and the increase of gastrin secretion, while the formation of duodenal ulcers Factors are more focused on the increase in the total volume of parietal cells. In addition, exogenous factors such as excessive drinking, irregular eating, long-term mental stress and long-term use of non-steroidal anti-inflammatory drugs (such as aspirin), glucocorticoids, clopidogrel and other drugs are related to the formation of peptic ulcer disease. Inflammatory bowel disease can be caused by bacteria, fungi, viruses, parasites, protozoa and other organisms, as well as by allergic reactions and physical and chemical factors. According to different etiologies, it can be divided into specific inflammatory lesions and non-specific inflammatory lesions. The former refers to infectious colitis, ischemic colitis and pseudomembranous colitis, and the latter mainly includes ulcerative colitis. , UC) and colonic Crohn's disease (Crohn's Disease, CD). UC is an inflammatory disease (Inflammatory bowel disease, IBD). As a common disease in gastroenterology, it can cause long-term inflammation and ulcers in the digestive tract. It is mainly characterized by chronic recurrent intestinal inflammation and intestinal epithelial cell damage. 20-30 years old, the main clinical symptoms are diarrhea, abdominal pain, bloody stool and intestinal obstruction. Many studies have shown that the pathogenesis of UC is related to factors such as immune factors, inflammation, environmental genetics, stress and infection. Gastritis consists of chronic gastritis and acute gastritis.
目前主要治疗胃肠道疾病的西药类型有促胃肠动力药、解痉药、止吐药、消化性溃疡药物、胃黏膜保护剂、助消化药、微生态制剂等。其中治疗消化性溃疡的药物主要包括质子泵抑制剂、H2-受体拮抗剂、铋制剂和***素类等，其主要是通过含有钙质的原料来中和胃酸，虽能改善缓解症状，但很难彻底治愈，疗效无法令人满意，易反复发作。炎症性肠病被世界卫生组织列为现代难治病之一，且发病率在全球均呈上升趋势，结肠炎的发病机制尚未完全阐明，临床治疗常用的药物包括氨基水杨酸类制剂、糖皮质激素和免疫抑制剂等，短期使用可控制结肠炎的症状，但治愈率极低，长时间使用可诱发多种不良反应，且停药可复发等问题，严重者可引起癌变，因此，开发出一种治疗胃肠道疾病的新药物是目前亟需解决的技术问题。At present, the main types of western medicines for the treatment of gastrointestinal diseases include gastrointestinal motility drugs, antispasmodics, antiemetics, peptic ulcer drugs, gastric mucosal protective agents, digestive aids, probiotics, etc. Among them, the drugs for the treatment of peptic ulcer mainly include proton pump inhibitors, H2-receptor antagonists, bismuth preparations and prostaglandins, etc., which are mainly used to neutralize gastric acid through raw materials containing calcium. It is difficult to completely cure, the curative effect is unsatisfactory, and it is easy to recur. Inflammatory bowel disease is listed as one of the modern intractable diseases by the World Health Organization, and the incidence rate is on the rise in the world. The pathogenesis of colitis has not been fully elucidated. Commonly used drugs for clinical treatment include aminosalicylic acid preparations, sugar Corticosteroids and immunosuppressants, etc., short-term use can control the symptoms of colitis, but the cure rate is very low, long-term use can induce a variety of adverse reactions, and the withdrawal of drugs can cause recurrence and other problems, and severe cases can cause cancer. Therefore, the development of The development of a new drug for the treatment of gastrointestinal diseases is a technical problem that needs to be solved urgently.
核苷类似物具有一些特殊的作用，2’-脱氧核苷药物能特异性的感染病毒的复制。目前临 床上用于治疗肝炎、艾滋病、疱疹等病毒性疾病的药物有很大一部分都是核苷类化合物，它们一般都是作为病毒复制过程中酶的抑制剂，阻断病毒对于靶细胞的浸染，常见的核苷类抗病毒化合物主要包括拉米夫定(Lamivudine,3-TC)、替比夫定(Telbivudine，LDT)、齐多夫定(Zidovudine，AZT)、泛昔洛韦(Famciclovir，FCV)、替洛福韦(PMPA)、阿德福韦酯(PMEA)等药物。替比夫定，用于有病毒复制证据以及有血清转氨酶(ALT或AST)持续升高或肝组织活动性病变证据的慢性乙型肝炎成人患者；齐多夫定，别名叠氮脱氧胸苷，用于艾滋病或与艾滋病有关的综合征患者及免疫缺陷病毒(HIV)感染的治疗，齐多夫定是世界上第一个获得美国FDA批准生产的抗艾滋病药品，其作用机制主要是与病毒的DNA聚合酶结合，中止DNA链的增长，从而阻止病毒的复制；拉米夫定，抗病毒药物，对病毒DNA链的合成和延长有竞争性抑制作用，其主要用于乙型肝炎和肝胆疾病的治疗，是目前临床应用中疗效最好、最具代表性的核苷类似物；阿昔洛韦，是一种高效、低毒、广谱的抗病毒药物，目前是治疗疱疹的首选药物；泛昔洛韦，是第一个在美国获准用于艾滋病患者复发性单纯疱疹病毒感染的口服药，不仅人体吸收率高，而且药物持续时间长，是减少疱疹后神经痛唯一有效的药物；阿德福韦，具有广谱抗病毒活性的腺嘌呤类核苷衍生物，可以有效地抑制逆转录病毒基因的复制和表达。Nucleoside analogs have some special effects, and 2'-deoxynucleoside drugs can specifically infect virus replication. At present, a large part of the drugs used in the clinical treatment of viral diseases such as hepatitis, AIDS, and herpes are nucleoside compounds. They are generally used as inhibitors of enzymes in the process of virus replication, blocking the infection of target cells by the virus. , Common nucleoside antiviral compounds mainly include Lamivudine (3-TC), Telbivudine (LDT), Zidovudine (AZT), Famciclovir (FCV), Telofovir (PMPA), Adefovir dipivoxil (PMEA) and other drugs. Telbivudine, for adult patients with chronic hepatitis B with evidence of viral replication and persistent elevation of serum transaminases (ALT or AST) or evidence of active liver disease; zidovudine, alias azidodeoxythymidine, For the treatment of AIDS or AIDS-related syndrome patients and immunodeficiency virus (HIV) infection, zidovudine is the first anti-AIDS drug approved by the US FDA in the world. DNA polymerase binds to stop the growth of the DNA chain, thereby preventing the replication of the virus; Lamivudine, an antiviral drug, has a competitive inhibitory effect on the synthesis and extension of the viral DNA chain, which is mainly used for hepatitis B and hepatobiliary diseases Acyclovir is the most effective and representative nucleoside analog in clinical application; acyclovir is a highly effective, low-toxic, broad-spectrum antiviral drug, and is currently the drug of choice for the treatment of herpes; Famciclovir, the first oral drug approved in the United States for recurrent herpes simplex virus infection in AIDS patients, not only has a high absorption rate, but also has a long drug duration, and is the only effective drug for reducing postherpetic neuralgia; adefovir , Adenine nucleoside derivatives with broad-spectrum antiviral activity can effectively inhibit the replication and expression of retroviral genes.
除了常见的抗病毒的核苷类化合物，据报道，一些2’和3’-脱氧核苷类药物能特异性的干扰病毒复制，并且可以选择性的引导病变癌细胞向正常细胞分化。In addition to common antiviral nucleoside compounds, it has been reported that some 2' and 3'-deoxynucleoside drugs can specifically interfere with viral replication and selectively guide the differentiation of diseased cancer cells into normal cells.
目前用于临床和正在研究的核苷类抗肿瘤药物有数十种，他们的主要作用是干扰肿瘤的DNA合成，或者影响核酸的转录过程，抑制蛋白质的合成，从而达到***的效果。例如阿糖胞苷，主要作用于细胞S增殖期的嘧啶类抗代谢药物，通过抑制细胞DNA的合成，干扰细胞的增殖，主要用于急性白血病，对急性粒细胞白细胞疗效最好，对急性单核细胞白血病及急性淋巴细胞白血病也有效。对恶性淋巴瘤、肺癌、消化道癌、头颈部癌有一定的疗效，对病毒性角膜炎及流行性结膜炎等也有一定的疗效。吉西他滨，为一种新的胞嘧啶核苷衍生物，作用机制和阿糖胞苷相同，其主要代谢物在细胞内掺入DNA，主要作用于G1/S期。在临床上，吉西他滨和阿糖胞苷的抗瘤谱不同，对多种实体瘤有效，用于晚期胰腺癌患者在氟尿嘧啶类失败后作为二线用药，能改善患者的生活；其次是对局部晚期和已经有转移的非小细胞肺癌作为一线应用。近有资料说明本品对卵巢癌、乳腺癌、膀胱癌、子***、肝癌、胆道癌、鼻咽癌、睾丸肿瘤、淋巴瘤及头颈部癌也具有姑息性疗效。There are dozens of nucleoside antitumor drugs currently used in clinical and research, their main role is to interfere with the DNA synthesis of tumors, or affect the transcription process of nucleic acid, inhibit the synthesis of proteins, so as to achieve the effect of tumor treatment. For example, cytarabine, a pyrimidine antimetabolite that mainly acts on the cell S proliferation phase, interferes with the proliferation of cells by inhibiting the synthesis of cell DNA, and is mainly used for acute leukemia. Nuclear cell leukemia and acute lymphoblastic leukemia are also effective. It has a certain curative effect on malignant lymphoma, lung cancer, digestive tract cancer, head and neck cancer, and also has a certain curative effect on viral keratitis and epidemic conjunctivitis. Gemcitabine, a new cytosine nucleoside derivative, has the same mechanism of action as cytarabine. Its main metabolite is incorporated into DNA in cells and mainly acts on G1/S phase. Clinically, gemcitabine and cytarabine have different anti-tumor profiles and are effective against a variety of solid tumors. They are used as second-line drugs for patients with advanced pancreatic cancer after the failure of fluorouracils, which can improve the life of patients; Metastatic non-small cell lung cancer has been used as a first-line application. Recent data indicate that this product also has palliative effects on ovarian cancer, breast cancer, bladder cancer, cervical cancer, liver cancer, biliary tract cancer, nasopharyngeal cancer, testicular tumor, lymphoma and head and neck cancer.
目前，研究人员针对核苷及其类似物的研究中，更多的是关于其剂型、检测方法、制备方法、结构修饰以及癌症治疗等方面研究，例如专利CN201880077576.4公开了核苷酸类似物的制备方法及其在核酸序列测定等方面的应用；专利CN201780039312.5公开了使用可逆封闭 核苷类似物进行核酸检测的方法；专利CN201810489585.6公开了一种骨架整合有核苷类似物药物的功能性核酸及其衍生物及其制备方法；专利CN201410169585.X公开了环磷酰化基团修饰后并连接脂肪链的磷酸N-脂肪酸用于病毒性肝炎和肝癌的治疗；专利CN201910892040.4公开了核苷类似物药物分子构建的药物适配体、制备方法及其应用等；专利CN201810077674.X公开了1,4-二取代1,2,3-三氮唑核苷类似物在抗肿瘤药物尤其是胃癌中的应用；然而没有研究者针对核苷类似物治疗胃肠道疾病的相关研究，亦没有相关的文献或者专利公开其具有治疗胃肠道疾病的新用途。发明人在研究过程中意外的发现，核苷类似物对胃肠道疾病具有显著的疗效，在临床上具有广阔的应用前景。At present, the research on nucleosides and their analogs by researchers is more about their formulations, detection methods, preparation methods, structural modifications, and cancer treatment. For example, patent CN201880077576.4 discloses nucleotide analogs The preparation method and its application in nucleic acid sequence determination, etc.; patent CN201780039312.5 discloses a method for nucleic acid detection using reversible blocking nucleoside analogs; patent CN201810489585.6 discloses a skeleton-integrated nucleoside analog drug Functional nucleic acids and derivatives thereof and preparation methods thereof; Patent CN201410169585.X discloses that phospho N-fatty acids modified with cyclophosphyl groups and connected to fatty chains are used for the treatment of viral hepatitis and liver cancer; Patent CN201910892040.4 discloses Drug aptamers constructed by nucleoside analog drug molecules, preparation methods and applications, etc.; patent CN201810077674.X discloses the use of 1,4-disubstituted 1,2,3-ribavirin analogs in antitumor drugs Especially the application in gastric cancer; however, there is no relevant research on nucleoside analogs for the treatment of gastrointestinal diseases, and no relevant literature or patents disclose that they have new uses for the treatment of gastrointestinal diseases. During the research process, the inventor unexpectedly discovered that nucleoside analogs have significant curative effect on gastrointestinal diseases, and have broad application prospects in clinical practice.
发明内容SUMMARY OF THE INVENTION
针对上述技术问题，本发明公开了核苷类似物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体及其混合物形式，及其可药用盐在制备预防和/或治疗胃肠道疾病药物中的应用，所述的胃肠道疾病不包括胃癌。In view of the above technical problems, the present invention discloses nucleoside analogs or tautomers, mesomers, racemates, enantiomers, diastereomers and mixtures thereof, and The application of a pharmaceutically acceptable salt in the preparation of a medicament for preventing and/or treating gastrointestinal diseases, said gastrointestinal diseases excluding gastric cancer.
优选地，所述的核苷类似物选自胞嘧啶核苷类似物、胸腺嘧啶核苷类似物、腺嘌呤核苷类似物和鸟嘌呤核苷类似物中的任一种。Preferably, the nucleoside analogs are selected from any one of cytosine analogs, thymidine analogs, adenosine analogs and guanosine analogs.
优选地，所述的胞嘧啶核苷类似物选自拉米夫定、脱氧胞苷、盐酸吉西他滨、盐酸阿糖胞苷、西多福韦、依曲西他平、艾夫他滨、恩曲他滨、阿卡地新、阿扎胞苷、地西他滨、Thiarabine、Ethynylcytidine、卡培他滨、艾西拉滨、安西他滨、4-S-β-D-阿糖胞嘧啶核苷、5-氮杂-4-硫代-2-脱氧胞苷、顺式-1-[4-(羟基-甲基)-环戊-2-烯基]-5-I-碘胞嘧啶和顺式-1-[4-(羟基-甲基)-环戊-2-烯基]-5-(2-I-碘乙烯基)胞嘧啶中的任一种；所述的胸腺嘧啶核苷类似物选自齐多夫定、替比夫定、索非布韦、5-氟尿嘧啶、脱氧尿苷、克来夫定、司他夫定、氟铁龙、双呋氟尿嘧啶、替加氟、氟尿嘧啶、卡莫氟、4-硫代胸腺嘧啶、5-牛磺酸甲基-2-S-尿苷、2’-氟-5-甲基-β-L-阿糖呋喃尿苷、2，3-胸腺嘧啶二脱氧碳环核苷衍生物、3-苯甲酰胸腺嘧啶和三氟胸苷中的任一种；所述的腺嘌呤核苷类似物选自腺苷、脱氧腺苷、脱氧腺苷酸、泛昔洛韦、替洛福韦、替洛福韦酯、富马酸替洛福韦二吡呋喃、阿德福韦、阿德福韦酯、二磷酸阿德福韦、阿地福韦、氯法拉滨、克拉屈滨、曲沙他滨、Aristeromycin和Neplanocin A中的任一种；所述的鸟嘌呤核苷类似物选自8-羟基-2-脱氧鸟嘌呤核苷、阿昔洛韦、更昔洛韦、恩替卡韦、三磷酸恩替卡韦、LB80380/ANA380、奈拉滨、9-β-D-阿糖呋喃糖鸟嘌呤、Forodesine hydrochloride、巯鸟嘌呤、6-巯基鸟嘌呤、6-硫基鸟嘌呤、6-硫代鸟嘌呤核苷酸、2-脱氧鸟苷一磷酸和5-(2-呋喃基)-2-脱氧鸟苷中的任一种。Preferably, the cytosine nucleoside analogs are selected from lamivudine, deoxycytidine, gemcitabine hydrochloride, cytarabine hydrochloride, cidofovir, etricitapine, efcitabine, emtrexine Azacitabine, Acadisin, Azacitidine, Decitabine, Thiarabine, Ethynylcytidine, Capecitabine, Escilarabine, Amcitabine, 4-S-β-D-Cytarabine , 5-aza-4-thio-2-deoxycytidine, cis-1-[4-(hydroxy-methyl)-cyclopent-2-enyl]-5-I-iodocytosine and cis- -1-[4-(Hydroxy-methyl)-cyclopent-2-enyl]-5-(2-I-iodovinyl) cytosine; the thymidine analogs Selected from zidovudine, telbivudine, sofosbuvir, 5-fluorouracil, deoxyuridine, clavudine, stavudine, flutron, difur-fluorouracil, tegafur, fluorouracil, cardinolone Morpho, 4-thiothymine, 5-taurine methyl-2-S-uridine, 2'-fluoro-5-methyl-β-L-arabinofuranoside, 2,3-thymidine Any one of pyrimidine dideoxycarbocyclic nucleoside derivatives, 3-benzoyl thymidine and trifluorothymidine; the adenosine analogs are selected from adenosine, deoxyadenosine, deoxyadenylic acid , famciclovir, telofovir, telofovir dipivoxil, telofovir disoproxil fumarate, adefovir, adefovir dipivoxil, adefovir diphosphate, adefovir, clofala any one of pyridoxine, cladribine, trosacitabine, Aristeromycin and Neplanocin A; the guanosine analog is selected from 8-hydroxy-2-deoxyguanosine, acyclovir, more Ciclovir, entecavir, entecavir triphosphate, LB80380/ANA380, nerabine, 9-β-D-arabinofuranoguanine, Forodesine hydrochloride, mercaptoguanine, 6-mercaptoguanine, 6-thioguanine , any of 6-thioguanine nucleotides, 2-deoxyguanosine monophosphate and 5-(2-furyl)-2-deoxyguanosine.
优选地，所述的胞嘧啶核苷类似物选自拉米夫定或盐酸吉西他滨；所述的胸腺嘧啶核苷类似物选自齐多夫定或替比夫定；所述的腺嘌呤核苷类似物选自泛昔洛韦、替洛福韦酯或阿 德福韦酯；所述的鸟嘌呤核苷类似物选自阿昔洛韦或6-硫基鸟嘌呤。Preferably, the cytidine analog is selected from lamivudine or gemcitabine hydrochloride; the thymidine analog is selected from zidovudine or telbivudine; the adenosine The analog is selected from famciclovir, telofovir dipivoxil or adefovir dipivoxil; the guanine nucleoside analog is selected from acyclovir or 6-thioguanine.
优选地，所述的胃肠道疾病为消化性溃疡。Preferably, the gastrointestinal disease is peptic ulcer.
优选地，所述的消化性溃疡为胃溃疡、十二指肠溃疡、球后溃疡、幽门管溃疡、复合溃疡和对吻溃疡中的一种或几种。Preferably, the peptic ulcer is one or more of gastric ulcer, duodenal ulcer, retrobulbar ulcer, pyloric ulcer, compound ulcer and kiss ulcer.
优选地，所述的消化性溃疡为胃溃疡和十二指肠溃疡。Preferably, the peptic ulcers are gastric ulcers and duodenal ulcers.
优选地，所述的胃肠道疾病为炎症性肠病。Preferably, the gastrointestinal disease is inflammatory bowel disease.
优选地，所述的炎症性肠病为溃疡性结肠炎和克罗恩病。Preferably, the inflammatory bowel disease is ulcerative colitis and Crohn's disease.
优选地，所述核苷类似物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体及其混合物形式，及其可药用盐和一种或多种药学上可接受的载体组成药物组合物，所述药物组合物的剂型为注射剂、片剂、胶囊剂、颗粒剂、丸剂。Preferably, the nucleoside analogs or tautomers, mesomers, racemates, enantiomers, diastereomers and mixtures thereof, and pharmaceutically acceptable salts thereof and one or more pharmaceutically acceptable carriers to form a pharmaceutical composition, and the dosage form of the pharmaceutical composition is injection, tablet, capsule, granule and pill.
本发明的有益效果是：本发明提供了核苷类似物在制备预防或治疗胃肠道疾病药物中的应用，具体提供了部分经典的核苷类似物拉米夫定、替比夫定、泛昔洛韦、替洛福韦酯、阿德福韦酯、阿昔洛韦、盐酸吉西他滨、齐多夫定和6-硫基鸟嘌呤，在不同饲喂条件下，对结肠炎小鼠进行DAI评分和结肠形态测定，结果显示拉米夫定、替比夫定、泛昔洛韦、替洛福韦酯、阿德福韦酯、阿昔洛韦、盐酸吉西他滨、齐多夫定和6-硫基鸟嘌呤等药物对溃疡性结肠炎均有效。同时，在不同饲喂条件下，对胃溃疡小鼠进行溃疡指数和抑制率及组织形态学检查，实验结果显示拉米夫定、替比夫定、泛昔洛韦、替洛福韦酯、阿德福韦酯、阿昔洛韦、盐酸吉西他滨、齐多夫定和6-硫基鸟嘌呤均具有防治胃溃疡的效果，且大部分药物效果强于现有临床用药西咪替丁的疗效，即本发明所述的核苷类似物拉米夫定、替比夫定、泛昔洛韦、替洛福韦酯、阿德福韦酯、阿昔洛韦、盐酸吉西他滨、齐多夫定和6-硫基鸟嘌呤等药物同时对溃疡性结肠炎和胃溃疡均有效，治疗效果比现有临床药物活性高40倍，具有良好的临床应用前景。The beneficial effects of the invention are as follows: the invention provides the application of nucleoside analogs in the preparation of medicines for preventing or treating gastrointestinal diseases, and specifically provides some classic nucleoside analogs lamivudine, telbivudine, famciclovir , telofovir dipivoxil, adefovir dipivoxil, acyclovir, gemcitabine hydrochloride, zidovudine and 6-thioguanine, DAI score and colonic colitis mice under different feeding conditions Morphometry, the results showed lamivudine, telbivudine, famciclovir, telofovir dipivoxil, adefovir dipivoxil, acyclovir, gemcitabine hydrochloride, zidovudine and 6-thioguanine and other drugs Effective for ulcerative colitis. At the same time, under different feeding conditions, the ulcer index, inhibition rate and histomorphology of gastric ulcer mice were examined. The experimental results showed that lamivudine, telbivudine, famciclovir, telofovir dipivoxil, adefovir Wei Dipivoxil, acyclovir, gemcitabine hydrochloride, zidovudine and 6-thioguanine all have the effect of preventing and treating gastric ulcer, and most of the drug effects are stronger than that of the existing clinical drug cimetidine. Nucleoside analogues lamivudine, telbivudine, famciclovir, telofovir dipivoxil, adefovir dipivoxil, acyclovir, gemcitabine hydrochloride, zidovudine and 6-thioguanidine according to the invention Drugs such as purine are effective for both ulcerative colitis and gastric ulcer at the same time, and the therapeutic effect is 40 times higher than that of existing clinical drugs, and has a good clinical application prospect.
附图说明Description of drawings
图1拉米夫定对C57BL/6N结肠炎小鼠体重和DAI评分的影响；Figure 1 The effect of lamivudine on body weight and DAI score of C57BL/6N colitis mice;
图2拉米夫定对C57BL/6N结肠炎小鼠结肠形态及结肠长度的影响；Figure 2 The effect of lamivudine on colon morphology and colon length in C57BL/6N colitis mice;
###p<0.001 vs.Control组；*p<0.05、**p<0.01和***p<0.001 vs.DSS组。 ### p<0.001 vs. Control group; *p<0.05, **p<0.01 and ***p<0.001 vs. DSS group.
图3拉米夫定对胃溃疡小鼠胃组织形态的影响；Fig. 3 The effect of lamivudine on gastric tissue morphology in gastric ulcer mice;
图4拉米夫定对胃溃疡指数、胃溃疡面积及胃溃疡抑制率的影响；Figure 4 The effect of lamivudine on gastric ulcer index, gastric ulcer area and gastric ulcer inhibition rate;
###p<0.001 vs.Control组；**p<0.01和***p<0.001 vs.Ethanol组。 ### p<0.001 vs. Control group; **p<0.01 and ***p<0.001 vs. Ethanol group.
图5阿德福韦酯、替洛福韦酯、泛昔洛韦和替比夫定对KM结肠炎小鼠体重、DAI评分、结肠形态和结肠长度的影响；Figure 5 Effects of adefovir dipivoxil, telofovir dipivoxil, famciclovir and telbivudine on body weight, DAI score, colon morphology and colon length in KM colitis mice;
###p<0.001 vs.空白对照组；*p<0.05、**p<0.01和***p<0.001 vs.DSS模型组。 ### p<0.001 vs. blank control group; *p<0.05, **p<0.01 and ***p<0.001 vs. DSS model group.
图6阿昔洛韦、盐酸吉西他滨、齐多夫定和6-硫基鸟嘌呤对KM结肠炎小鼠体重、DAI评分、结肠形态和结肠长度的影响；Figure 6 Effects of acyclovir, gemcitabine hydrochloride, zidovudine and 6-thioguanine on body weight, DAI score, colon morphology and colon length in KM colitis mice;
###p<0.001 vs.空白对照组；*p<0.05、**p<0.01和***p<0.001 vs.DSS模型组。 ### p<0.001 vs. blank control group; *p<0.05, **p<0.01 and ***p<0.001 vs. DSS model group.
图7替比夫定、盐酸吉西他滨和齐多夫定对KM胃溃疡小鼠胃组织形态、溃疡指数、溃疡面积和溃疡抑制率的影响；Figure 7 Effects of telbivudine, gemcitabine hydrochloride and zidovudine on gastric tissue morphology, ulcer index, ulcer area and ulcer inhibition rate in KM gastric ulcer mice;
###p<0.001 vs.空白对照组；*p<0.05、**p<0.01和***p<0.001 vs.乙醇模型组。 ### p<0.001 vs. blank control group; *p<0.05, **p<0.01 and ***p<0.001 vs. ethanol model group.
图8阿德福韦酯、替洛福韦酯、阿昔洛韦、泛昔洛韦和6-硫基鸟嘌呤对KM胃溃疡小鼠胃组织形态、溃疡指数、溃疡面积和溃疡抑制率的影响；Figure 8 Effects of adefovir dipivoxil, telofovir dipivoxil, acyclovir, famciclovir and 6-thioguanine on gastric tissue morphology, ulcer index, ulcer area and ulcer inhibition rate in KM gastric ulcer mice;
###p<0.001 vs.空白对照组；*p<0.05、**p<0.01和***p<0.001 vs.乙醇模型组。 ### p<0.001 vs. blank control group; *p<0.05, **p<0.01 and ***p<0.001 vs. ethanol model group.
具体实施方式Detailed ways
以下结合具体实施例对本发明的保护范围进行详细说明，但应当指出的是，本发明的保护范围并不限于以下实施例，同时保护核苷类似物的一类化合物在所有胃肠道疾病尤其是消化性溃疡病和炎症性肠病中的治疗效果，包括不同剂型、剂量、联合用药等。凡本领域技术人员按照本发明的思路在现有技术上通过逻辑分析、推断和实验得出的技术方案，均属于本发明所请求保护的范围。The protection scope of the present invention will be described in detail below with reference to specific examples, but it should be noted that the protection scope of the present invention is not limited to the following examples, and a class of compounds that protect nucleoside analogs in all gastrointestinal diseases, especially Therapeutic effects in peptic ulcer disease and inflammatory bowel disease, including different dosage forms, doses, and combination drugs. All technical solutions obtained by those skilled in the art through logical analysis, inference and experimentation in the prior art according to the idea of the present invention belong to the scope of the claimed protection of the present invention.
应理解，上述简述和下文的详述为示例性且仅用于解释，而不对本文发明主题做任何限制。在本申请中，必须注意，除非文中另有清楚的说明，否则在本说明书和权利要求书中所用的单数形式包括所指事物的复数形式。还应注意，除非另有说明，否则所用“或”、“或者”表示“和/或”。此外，所用术语“包括”以及其他形式，例如“包含”、“含”和“含有”并非限制性。It is to be understood that both the foregoing brief description and the following detailed description are exemplary and explanatory only and are not intended to limit the subject matter herein. In this application, it must be noted that the singular forms used in this specification and the claims include the plural forms of referents unless the context clearly dictates otherwise. It should also be noted that the use of "or" and "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" and other forms such as "comprising", "including" and "comprising" is not intended to be limiting.
本发明所述的C57BL/6N小鼠是小鼠中使用最广泛的品系之一，也是基因工程中最常用作转基因或基因敲除小鼠的母本。The C57BL/6N mouse described in the present invention is one of the most widely used strains in mice, and is also most commonly used as the female parent of transgenic or gene knockout mice in genetic engineering.
本发明所述的KM小鼠是指昆明小鼠。The KM mice of the present invention refer to Kunming mice.
本发明以下实施例使用葡聚糖硫酸钠用于小鼠结肠炎的造模。The following examples of the present invention use sodium dextran sulfate to model colitis in mice.
本发明以下实施例中，柳氮磺吡啶肠溶片的适应症为(1)溃疡性结肠炎治疗轻至中度的溃疡性结肠炎；在重度溃疡性结肠炎中可作为辅助疗法。亦可用于溃疡性结肠炎缓解期的维持治疗；(2)Crohn’s病用于治疗活动期的克隆病，特别是那些累及结肠的患者；(3)类风湿性关节炎对水杨酸类或其他非甾体抗炎药疗效不显著的类风湿性关节炎和幼年类风湿性关节炎(多关节型)，本发明实施例一中将柳氮磺吡啶肠溶片作为阳性药物使用。In the following examples of the present invention, the indications of sulfasalazine enteric-coated tablets are (1) ulcerative colitis to treat mild to moderate ulcerative colitis; it can be used as an adjuvant therapy in severe ulcerative colitis. It can also be used for maintenance treatment of ulcerative colitis in remission; (2) Crohn's disease is used to treat active Crohn's disease, especially those patients with colon involvement; (3) rheumatoid arthritis is resistant to salicylic acid or other For rheumatoid arthritis and juvenile rheumatoid arthritis (polyarticular type) in which the effect of non-steroidal anti-inflammatory drugs is not significant, sulfasalazine enteric-coated tablets are used as positive drugs in Example 1 of the present invention.
本发明以下实施例中的西咪替丁，也称甲氰咪胍，是一种组胺H2受体阻抗剂，主要用 于抑制胃酸的分泌，能明显抑制基础和夜间胃酸分泌，也能抑制由组胺、分肽胃泌素、胰岛素和食物等刺激引起的胃酸分泌，并使其酸度降低，对因化学刺激引起的腐蚀性胃炎有预防和保护作用，对应激性胃溃疡和上消化道出血也有明显疗效。本发明实施例将西咪替丁作为阳性药物使用。Cimetidine in the following embodiments of the present invention, also known as cimetidine, is a histamine H2 receptor antagonist, mainly used to inhibit the secretion of gastric acid, can significantly inhibit the secretion of basal and nocturnal gastric acid, and can also inhibit the secretion of gastric acid. Gastric acid secretion caused by stimuli such as histamine, peptide gastrin, insulin and food, and reduce its acidity, prevent and protect corrosive gastritis caused by chemical stimulation Bleeding is also effective. In the embodiment of the present invention, cimetidine is used as a positive drug.
术语“药学上可接受的”是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms, which are within the scope of sound medical judgment, suitable for use in contact with human and animal tissue without excessive of toxicity, irritation, allergic reactions or other problems or complications commensurate with a reasonable benefit/risk ratio.
术语“药物组合物”是指任选的混合有至少一种药学上可接受的化学成分或试剂的生物活性化合物，所述药学上可接受的化学成分或试剂即为“载体”，其有助于将化合物引入到细胞或组织中，包括但不限于稳定剂、稀释剂、悬浮剂、增稠剂和/或赋形剂。The term "pharmaceutical composition" refers to a biologically active compound optionally in admixture with at least one pharmaceutically acceptable chemical component or agent, which is a "carrier" that facilitates For introducing compounds into cells or tissues, include but are not limited to stabilizers, diluents, suspending agents, thickening agents and/or excipients.
术语“药学上可接受的盐”是指保留了指定化合物的游离酸和游离碱的生物效力，并且在生物学或其它方面上没有不良作用的盐。除特别指示外，本发明中的盐可以提及金属盐、铵盐、与有机碱形成的盐、与无机酸形成的盐、与有机酸形成的盐、与碱性或者酸性氨基酸形成的盐等。The term "pharmaceutically acceptable salts" refers to salts that retain the biological potency of the free acid and free base of the specified compound and are not biologically or otherwise adversely affected. Unless otherwise specified, the salts in the present invention can be mentioned metal salts, ammonium salts, salts formed with organic bases, salts formed with inorganic acids, salts formed with organic acids, salts formed with basic or acidic amino acids, etc. .
药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。一般地，优选醚、乙酸乙酯、乙醇、异丙醇或乙腈等非水介质。Pharmaceutically acceptable salts can be synthesized from the parent compound containing acid or base by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.
术语“治疗”和其它类似的同义词包括缓解、减轻或改善疾病或病症症状，预防其它症状，改善或预防导致症状的潜在代谢原因，抑制疾病或病症，例如阻止疾病或病症的发展，缓解疾病或病症，使疾病或病症好转，缓解由疾病或病症导致的症状，或者中止疾病或病症的症状，此外，该术语还可包含预防的目的。该术语还包括获得治疗效果和/或预防效果。所述治疗效果是指治愈或改善所治疗的潜在疾病。此外，对与潜在疾病相关的一种或多种生理症状的治愈或改善也是治疗效果，例如尽管患者可能仍然受到潜在疾病的影响，但观察到患者情况改善。就预防效果而言，可向具有患特定疾病风险的患者施用所述组合物或化合物，或者即便尚未做出疾病诊断，但向出现该疾病的一个或多个生理症状的患者施用所述组合物或化合物。The term "treating" and other similar synonyms include alleviating, alleviating or ameliorating the symptoms of a disease or disorder, preventing other symptoms, ameliorating or preventing the underlying metabolic cause of the symptoms, inhibiting the disease or disorder, such as preventing the progression of the disease or disorder, alleviating the disease or Condition, amelioration of a disease or condition, alleviation of symptoms caused by a disease or condition, or cessation of symptoms of a disease or condition, in addition, the term may also encompass prophylactic purposes. The term also includes obtaining a therapeutic effect and/or a prophylactic effect. The therapeutic effect refers to curing or ameliorating the underlying disease being treated. In addition, curing or amelioration of one or more physiological symptoms associated with the underlying disease is also a therapeutic effect, eg, an improvement in a patient's condition is observed although the patient may still be affected by the underlying disease. In terms of prophylactic effect, the composition or compound may be administered to a patient at risk of developing a particular disease, or to a patient presenting one or more physiological symptoms of the disease even if no diagnosis of the disease has been made. or compound.
本发明以下实施例中，具体涉及小分子活性化合物如表1所示。In the following examples of the present invention, specific small molecule active compounds are shown in Table 1.
表1实施例中活性小分子一览表List of active small molecules in the examples of Table 1
Figure PCTCN2021074575-appb-000001
Figure PCTCN2021074575-appb-000001
Figure PCTCN2021074575-appb-000002
Figure PCTCN2021074575-appb-000002
Figure PCTCN2021074575-appb-000003
Figure PCTCN2021074575-appb-000003
实施例一、拉米夫定对C57BL/6N结肠炎小鼠的治疗效果Embodiment 1. The therapeutic effect of lamivudine on C57BL/6N colitis mice
1.动物饲喂1. Animal Feeding
SPF级健康雄性C57BL/6N雄性小鼠，体重18-22g，实验前未用过任何药物，由中国农业科学院兰州兽医研究所提供。实验动物于温度为24～26℃，12h/12h的白昼规律交替的环境中适应饲养一周，给予动物饮食并自由饮水，然后分组进行实验。SPF grade healthy male C57BL/6N male mice, weighing 18-22g, had not used any drugs before the experiment, provided by Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The experimental animals were acclimatized and raised for a week in an environment with a temperature of 24-26°C and a 12h/12h alternation of daytime regularity.
2.药物及试剂2. Drugs and reagents
拉米夫定(Lamivudine,3-TC,HPLC≥98％)，购自麦克林生物科技有限公司，批号：C10102050，分子量为229.25；柳氮磺吡啶肠溶片(Salazosulfapyridine，SASP)购自上海信谊天平药业有限公司；葡聚糖硫酸钠(Dextran Sulfate sodium，DSS)，MW:40000，购自阿拉丁生物科技有限公司。Lamivudine (Lamivudine, 3-TC, HPLC≥98%), purchased from McLean Biotechnology Co., Ltd., batch number: C10102050, molecular weight 229.25; Sulfasalazine enteric-coated tablets (Salazosulfapyridine, SASP) purchased from Shanghai Xin Yi Tianping Pharmaceutical Co., Ltd.; Dextran Sulfate Sodium (DSS), MW: 40000, purchased from Aladdin Biotechnology Co., Ltd.
3.实验分组和灌胃给药剂量3. Experimental grouping and intragastric administration dose
取8周龄雄性C57BL/6N小鼠40只，按体重随机分为5组，每组8只，分组和给药剂量如下：40 8-week-old male C57BL/6N mice were randomly divided into 5 groups according to their body weight, with 8 mice in each group. The groups and doses were as follows:
正常对照组(Control，口服给予等体积0.9％生理盐水)；Normal control group (Control, orally administered an equal volume of 0.9% normal saline);
正常鼠给予拉米夫定组(C+3-TC，给药剂量为2mg/kg/day)Normal mice were given lamivudine group (C+3-TC, the dose was 2mg/kg/day)
DSS模型对照组(DSS，口服给予等体积0.9％生理盐水)；DSS model control group (DSS, orally administered an equal volume of 0.9% normal saline);
阳性药组(SASP，口服给予柳氮磺吡啶，80mg/kg/day)；Positive drug group (SASP, oral administration of sulfasalazine, 80 mg/kg/day);
模型鼠给予拉米夫定组(D+3-TC，给药剂量为2mg/kg/day)。The model mice were given lamivudine group (D+3-TC, the dosage was 2 mg/kg/day).
4.溃疡性结肠炎模型的制备4. Preparation of Ulcerative Colitis Model
配制4％的DSS蒸馏水溶液，DSS模型组小鼠自由饮用DSS水溶液造模，并保持常规饲料喂养，连续造模7天。A 4% DSS distilled water solution was prepared, and the mice in the DSS model group drank the DSS water solution freely for modeling, and kept regular feed for continuous modeling for 7 days.
5.给药时间及方法5. Administration time and method
DSS模型对照组小鼠自由饮用DSS水溶液造模、DSS+3-TC组小鼠自由饮用DSS水溶液造模同时给予2mg/kg/day的3-TC；DSS+SASP组小鼠自由饮用DSS水溶液造模同时给予80mg/kg/day的SASP；空白对照组小鼠自由饮用蒸馏水，空白对照组+3-TC组小鼠自由饮用蒸馏水的同时给予2mg/kg/day的3-TC，所有小鼠保持常规饲料喂养，连续给药7天。第七天末次给药2h后，分离血清及结肠组织备用。Mice in DSS model control group drank DSS aqueous solution freely for modeling, mice in DSS+3-TC group drank DSS aqueous solution freely for modeling, and were given 2 mg/kg/day 3-TC; mice in DSS+SASP group drank DSS aqueous solution freely for modeling The model was given 80mg/kg/day of SASP at the same time; the mice in the blank control group drank distilled water freely, and the mice in the blank control group +3-TC group were given 2 mg/kg/day of 3-TC while drinking distilled water freely. Routine feed was fed for 7 consecutive days. On the seventh day, 2 hours after the last administration, the serum and colon tissue were separated for use.
6.溃疡性结肠炎临床指标检测6. Detection of clinical indicators of ulcerative colitis
6.1体重降低情况6.1 Weight loss
按每只鼠降低体重的百分比(％)计算，无体重减轻为0分，体重减轻1％-5％记为1分，体重减轻6％-10％为2分，体重减轻11％-15％记为3分，体重减轻大于15％为4分。Calculated according to the percentage (%) of body weight loss of each mouse, no weight loss is scored as 0, body weight loss of 1%-5% is scored as 1 point, body weight loss of 6%-10% is scored as 2 points, body weight loss is 11%-15% Scored as 3 points, weight loss greater than 15% was scored as 4 points.
6.2大便粘稠度6.2 Stool viscosity
正常大便评分记为0分，松散便记为2分、腹泻记为4分。Normal stool was scored as 0, loose stool as 2, and diarrhea as 4.
6.3便血及隐血情况6.3 Blood in stool and occult blood
正常大便评分记为0分，隐血出血记为1分(按隐血检测结果进行判断)，肉眼可见血便为3分、以上三项均值即为DAI。The normal stool score is scored as 0, the occult blood bleeding is scored as 1 (judged by the occult blood test results), the bloody stool is scored as 3 with the naked eye, and the average of the above three items is DAI.
6.4结肠长度的测量6.4 Measurement of Colon Length
处死小鼠后分离小鼠结肠，从回肠和结肠结合处剪断回肠，再在结肠近***处剪断，分离结肠外的筋膜，使结肠完全伸展开，用直尺测量小鼠结肠自回结肠处至***的长度，并拍照记录。After the mice were sacrificed, the colons of the mice were separated, and the ileum was cut from the junction of the ileum and the colon, and then at the colon near the anus. to the length of the anus, and photographed to record.
7.数据处理7. Data processing
实验数据采用SPSS23.0软件进行统计学分析，数据以(x±s)表示，组间比较采用单因素方差分析(One-way ANOVA)和LSD-t法进行两两比较。p<0.05认为具有统计学意义。The experimental data were statistically analyzed by SPSS23.0 software, and the data were expressed as (x±s), and the comparison between groups was performed by one-way analysis of variance (One-way ANOVA) and LSD-t method. p<0.05 was considered statistically significant.
8.结果分析8. Analysis of results
8.1拉米夫定对C57BL/6N结肠炎小鼠体重和疾病活动指数的影响8.1 Effects of lamivudine on body weight and disease activity index in C57BL/6N colitis mice
上述小鼠在实验过程中，对其体重、粪便粘稠度、便血及隐血情况进行监测，数据统计用SPSS23.0软件进行单因素方差分析。During the experiment, the above-mentioned mice were monitored for their body weight, fecal viscosity, blood in the stool and occult blood, and SPSS 23.0 software was used for one-way analysis of variance for data statistics.
各组小鼠体重差异和DAI评分的显著性差异表示为*p<0.05、**p<0.01和***p<0.001。不同品系UC小鼠的各组小鼠的体重差异和DAI评分检测结果见图1。Significant differences in body weight differences and DAI scores of mice in each group were expressed as *p<0.05, **p<0.01, and ***p<0.001. The body weight difference and DAI score detection results of each group of mice of different strains of UC mice are shown in Figure 1.
由上述数据可见，与空白对照组相比，空白对照+拉米夫定给药组在体重指标和DAI评分指标中均显示无统计学差异，表明拉米夫定对正常小鼠无毒副作用；与空白对照组相比，DSS模型组体重大幅度降低，符合结肠炎患者体重减少的规律，DAI评分呈显著的上升趋势(###p<0.001)，表明DSS模型是制备成功的；与DSS模型对照组相比，柳氮磺吡啶给药组和拉米夫定给药组均能显著的改善体重减低和DAI评分(*p<0.05)，且拉米夫定的的疗效强于临床用药柳氮磺吡啶的疗效。It can be seen from the above data that compared with the blank control group, the blank control + lamivudine administration group showed no statistical difference in body weight index and DAI score index, indicating that lamivudine has no toxic and side effects on normal mice; Compared with the blank control group, the body weight of the DSS model group decreased significantly, which was in line with the law of body weight loss in colitis patients, and the DAI score showed a significant upward trend (###p<0.001), indicating that the DSS model was successfully prepared; Compared with the model control group, both the sulfasalazine administration group and the lamivudine administration group could significantly improve body weight loss and DAI score (*p<0.05), and the efficacy of lamivudine was stronger than that of clinical medication Efficacy of sulfasalazine.
8.2拉米夫定对C57BL/6N结肠炎小鼠结肠形态及结肠长度的影响8.2 Effects of lamivudine on colon morphology and colon length in C57BL/6N colitis mice
上述各组小鼠处死后，迅速摘除其结肠组织，观察其形态改变，同时测量其长度，对各组数据进行统计分析。各组小鼠结肠长度的显著性差异表示为*p<0.05、**p<0.01和***p<0.001。UC结肠炎小鼠的各组小鼠的结肠形态及结肠长度监测结果见图2。After the mice in the above groups were sacrificed, their colon tissues were quickly removed, and their morphological changes were observed, and their lengths were measured at the same time, and statistical analysis was performed on the data of each group. Significant differences in colon length of mice in each group were expressed as *p<0.05, **p<0.01 and ***p<0.001. The colon morphology and colon length monitoring results of each group of UC colitis mice are shown in Figure 2.
与空白对照组相比，空白对照+拉米夫定给药组小鼠结肠长度无明显的变化，表明拉米夫定对正常小鼠无毒副作用；与空白对照组相比，DSS模型组结肠长度显著的缩短(###p<0.001)，表明DSS模型是制备成功的；与DSS模型对照组相比，柳氮磺吡啶给药组和拉米夫定给药组均能显著的改善结肠水肿及缩短情况，并具有显著性差异(*p<0.05和***p<0.001)，且拉米夫定的疗效强于临床用药柳氮磺吡啶的疗效。Compared with the blank control group, the colon length of the mice in the blank control + lamivudine administration group did not change significantly, indicating that lamivudine has no toxic and side effects on normal mice; compared with the blank control group, the colon in the DSS model group The length was significantly shortened (###p<0.001), indicating that the DSS model was successfully prepared; compared with the DSS model control group, both the sulfasalazine administration group and the lamivudine administration group could significantly improve the colon. There were significant differences in edema and shortening (*p<0.05 and ***p<0.001), and the efficacy of lamivudine was stronger than that of clinical drug sulfasalazine.
所述体重差异、DAI评分、结肠形态及结肠长度测定表明，拉米夫定对DSS诱导的结肠炎小鼠有治疗效果，无明显的毒副作用，且疗效显著强于临床用药柳氮磺吡啶的疗效。The measurement of body weight difference, DAI score, colon morphology and colon length showed that lamivudine had a therapeutic effect on DSS-induced colitis mice without obvious toxic and side effects, and the curative effect was significantly stronger than that of clinical drug sulfasalazine. curative effect.
实施例二、拉米夫定对KM胃溃疡小鼠的治疗效果Embodiment two, the therapeutic effect of lamivudine on KM gastric ulcer mice
1.实验动物来源1. Laboratory animal sources
实验动物：8周龄SPF级昆明(Kunming，KM)雄性小鼠，体重为18～22g，实验前未使用任何药物，购自中国农业科学院兰州兽医研究所。实验动物于温度为24～26℃，12h/12h的白昼规律交替的环境中适应饲养一周，给予动物饮食并自由饮用蒸馏水，然后分组进行实验。Experimental animals: 8-week-old SPF grade Kunming (Kunming, KM) male mice, weighing 18-22 g, without any drugs before the experiment, were purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The experimental animals were acclimatized and raised for a week in an environment with a temperature of 24-26 °C and alternating daytime patterns of 12h/12h.
2.药物及试剂2. Drugs and reagents
拉米夫定(Lamivudine,3-TC,HPLC≥98％)，购自麦克林生物科技有限公司；无水乙醇， 天津大茂化学试剂公司；西咪替丁片(Cimetidine，CIM)购自上海信谊天平药业有限公司。Lamivudine (Lamivudine, 3-TC, HPLC≥98%), purchased from McLean Biotechnology Co., Ltd.; absolute ethanol, Tianjin Damao Chemical Reagent Co., Ltd.; Cimetidine (CIM), purchased from Shanghai Xinyi Tianping Pharmaceutical Co., Ltd.
3.KM小鼠酒精性胃溃疡模型的制备3. Preparation of KM mouse model of alcoholic gastric ulcer
40只KM雄性小鼠(20-25g)，饲养于兰州大学实验动物房，小鼠适应性饲养一周后，随机分为5组，每组8只小鼠，分组情况及给药量如表2所示；40 KM male mice (20-25g) were raised in the laboratory animal room of Lanzhou University. After the mice were adaptively reared for one week, they were randomly divided into 5 groups with 8 mice in each group. The grouping and dosage are shown in Table 2. shown;
表2 KM雄性小鼠实验分组及各组的给药剂量Table 2 Experimental grouping of KM male mice and the dosage of each group
组别group 给药剂量Dosage
空白对照组Blank control group 0.3mL/只灌胃0.9％生理盐水0.3mL/only gavage with 0.9% saline
空白+拉米夫定Blank + Lamivudine 2mg/kg/day 3-TC注射给药，2h后0.3mL/只灌胃生理盐水2mg/kg/day 3-TC injection, 0.3mL/dose of normal saline after 2h
乙醇模型组Ethanol model group 0.3mL/只灌胃无水乙醇0.3mL/only anhydrous ethanol by gavage
西咪替丁阳性组Cimetidine positive group 80mg/kg/day西咪替丁灌胃给药，2h后0.3mL/只灌胃无水乙醇80mg/kg/day cimetidine was administered by intragastric administration, and 0.3mL/only anhydrous ethanol was administered by intragastric administration after 2 hours
拉米夫定给药组Lamivudine-administered group 2mg/kg/day 3-TC注射给药，2h后0.3mL/只灌胃无水乙醇2mg/kg/day 3-TC injection, after 2h 0.3mL/dose with absolute ethanol
上述各组小鼠刺激2h后结束实验，分离血清和胃组织测定各项指标。The mice in the above groups were stimulated for 2 h to end the experiment, and the serum and gastric tissue were separated to determine various indicators.
4.溃疡面积、溃疡指数与抑制率4. Ulcer area, ulcer index and inhibition rate
将胃取出，沿胃大弯剪开，冲洗干净内容物，观察胃粘膜溃疡情况，用直尺测量溃疡的横径与纵径，两者乘积为溃疡面积(mm 2)，如公式1所示；然后计算整个胃组织的溃疡面积，从而计算溃疡抑制率(％)，见公式2。同时以各组鼠溃疡点数之和的均值作为溃疡指数(愈合记0分、表浅性黏膜糜烂记1分、深陷性溃疡或透壁性坏死记2分、穿孔或穿透性溃疡记3分) Take out the stomach, cut it along the greater curvature of the stomach, rinse the contents, observe the gastric mucosal ulcer, measure the transverse diameter and longitudinal diameter of the ulcer with a ruler, and the product of the two is the ulcer area (mm 2 ), as shown in formula 1 ; and then calculate the ulcer area of the entire gastric tissue, thereby calculating the ulcer inhibition rate (%), see formula 2. At the same time, the mean of the sum of ulcer points in each group was taken as the ulcer index (0 points for healing, 1 point for superficial mucosal erosion, 2 points for deep ulcers or transmural necrosis, and 3 points for perforation or penetrating ulcers). Minute)
溃疡面积(mm2)＝溃疡最大长径*垂直于最大长径的最大宽径    (1)Ulcer area (mm2) = the maximum length of the ulcer * the maximum width perpendicular to the maximum length (1)
Figure PCTCN2021074575-appb-000004
Figure PCTCN2021074575-appb-000004
5.数据处理5. Data processing
实验数据采用SPSS23.0软件进行统计学分析，数据以(x±s)表示，组间比较采用单因素方差分析(One-way ANOVA)和LSD-t法进行两两比较。p<0.05认为具有统计学意义。The experimental data were statistically analyzed by SPSS23.0 software, and the data were expressed as (x±s), and the comparison between groups was performed by one-way analysis of variance (One-way ANOVA) and LSD-t method. p<0.05 was considered statistically significant.
6.拉米夫定对胃溃疡小鼠胃组织形态、溃疡指数、溃疡面积及溃疡抑制率的影响6. Effects of lamivudine on gastric tissue morphology, ulcer index, ulcer area and ulcer inhibition rate in gastric ulcer mice
上述各组小鼠处死后，迅速取其胃组织，观察其形态改变及溃疡损伤情况，并对其溃疡面积和溃疡指数进行统计分析，实验结果如图3和图4所示。由图3可知，正常组小鼠和正常组给予拉米夫定药物组胃组织宏观形态正常，未出现明显出血性病变，而乙醇模型组小鼠胃组织出血性病变明显，溃疡最为严重，表明乙醇胃溃疡模型制备成功，同时拉米夫定对健康小鼠胃组织无明显的损伤作用；与乙醇模型组相比，拉米夫定和西咪替丁各给药组均能显著 的改善胃粘膜的损伤程度，减少溃疡面积，且拉米夫定的溃疡抑制率(79.2％)高于临床用药西咪替丁(CIM)的溃疡抑制率(73.25％)。以上所述溃疡面积、溃疡指数与抑制率，及胃组织形态学测定表明，拉米夫定防治胃溃疡有效，且疗效显著强于临床用药西咪替丁的疗效。After the mice in the above groups were sacrificed, their stomach tissues were quickly taken to observe their morphological changes and ulcer damage, and statistical analysis was performed on their ulcer area and ulcer index. The experimental results are shown in Figures 3 and 4. It can be seen from Figure 3 that the macroscopic morphology of gastric tissue in the normal group mice and the normal group treated with lamivudine was normal, and no obvious hemorrhagic lesions appeared, while the gastric tissue of the mice in the ethanol model group had obvious hemorrhagic lesions and the most serious ulcers, indicating that The ethanol gastric ulcer model was successfully prepared, and lamivudine had no obvious damage to the gastric tissue of healthy mice; compared with the ethanol model group, each administration group of lamivudine and cimetidine could significantly improve the gastric The degree of mucosal damage was reduced, the ulcer area was reduced, and the ulcer inhibition rate of lamivudine (79.2%) was higher than that of clinical drug cimetidine (CIM) (73.25%). The above-mentioned ulcer area, ulcer index and inhibition rate, and gastric histomorphological measurements show that lamivudine is effective in preventing and treating gastric ulcer, and the curative effect is significantly stronger than that of the clinical drug cimetidine.
实施例三、阿德福韦酯、替洛福韦酯、泛昔洛韦和替比夫定对KM结肠炎小鼠的治疗效果 Embodiment 3. The therapeutic effect of adefovir dipivoxil, telofovir dipivoxil, famciclovir and telbivudine on KM colitis mice
1.药物及试剂1. Drugs and reagents
替洛福韦酯(PMPA，HPLC≥98％)、阿德福韦酯(PMEA，HPLC≥98％)、泛昔洛韦(Famciclocir，FCV，HPLC≥98％)和替比夫定(Telbivudine，LDT，HPLC≥98％)均购自阿拉丁生物科技有限公司；柳氮磺吡啶肠溶片(Salazosulfapyridine，SASP)购自上海信谊天平药业有限公司；葡聚糖硫酸钠(Dextran Sulfate sodium，DSS)，MW:40000，购自阿拉丁生物科技有限公司。Telofovir dipivoxil (PMPA, HPLC≥98%), adefovir dipivoxil (PMEA, HPLC≥98%), famciclocir (Famciclocir, FCV, HPLC≥98%) and telbivudine (Telbivudine, LDT, HPLC) ≥98%) were purchased from Aladdin Biotechnology Co., Ltd.; Sulfasalazine enteric-coated tablets (Salazosulfapyridine, SASP) were purchased from Shanghai Xinyi Tianping Pharmaceutical Co., Ltd.; Dextran Sulfate Sodium (DSS), MW: 40000, purchased from Aladdin Biotechnology Co., Ltd.
2.实验分组和给药剂量：标准体重KM雄性小鼠，随机分为7组，每组8只，分组和给药剂量如下：2. Experimental grouping and administration dose: KM male mice with standard body weight were randomly divided into 7 groups with 8 mice in each group. The grouping and administration doses were as follows:
正常对照组(Control，口服给予等体积0.9％生理盐水)；Normal control group (Control, orally administered an equal volume of 0.9% normal saline);
DSS模型对照组(DSS，口服给予等体积0.9％生理盐水)；DSS model control group (DSS, orally administered an equal volume of 0.9% normal saline);
阳性药组(SASP，口服给予柳氮磺吡啶，80mg/kg/day)；Positive drug group (SASP, oral administration of sulfasalazine, 80 mg/kg/day);
替洛福韦酯组(PMPA，给药剂量为2mg/kg/day，腹腔注射)；Telofovir disoproxil group (PMPA, administered at a dose of 2 mg/kg/day, intraperitoneal injection);
阿德福韦酯组(PMEA，给药剂量为2mg/kg/day，腹腔注射)；Adefovir dipivoxil group (PMEA, administered at a dose of 2 mg/kg/day, intraperitoneal injection);
泛昔洛韦组(FCV，给药剂量为2mg/kg/day，腹腔注射)；Famciclovir group (FCV, the dosage is 2mg/kg/day, intraperitoneal injection);
替比夫定组(LDT，给药剂量为2mg/kg/day，腹腔注射)。Telbivudine group (LDT, administered at a dose of 2 mg/kg/day, intraperitoneal injection).
3.溃疡性结肠炎模型的制备和临床指标检测方法如实施例一中“4-6实验方法”所示。3. The preparation of the ulcerative colitis model and the detection methods of clinical indicators are shown in "4-6 Experimental Methods" in Example 1.
4.结果分析4. Analysis of results
上述各组小鼠在实验过程中，对其体重、粪便粘稠度、便血及隐血情况、结肠形态及长度进行监测，数据统计用SPSS23.0软件进行单因素方差分析。由图5可知，与空白对照组相比，DSS模型组体重大幅度降低，符合结肠炎患者体重减少的规律，DAI评分呈显著的上升趋势(###p<0.001)，结肠长度显著的缩短(###p<0.001)，表明DSS模型是制备成功的；与DSS模型对照组相比，替洛福韦酯、阿德福韦酯、替比夫定和泛昔洛韦给药组均能显著的改善体重减低、DAI评分、结肠水肿和缩短情况，表明替洛福韦酯、阿德福韦酯、替比夫定和泛昔洛韦对溃疡性结肠炎有治疗作用，其中替比夫定的疗效强于临床用药柳氮磺吡啶的疗效。During the experiment, the mice of the above groups were monitored for their body weight, fecal viscosity, blood in the stool and occult blood, colon morphology and length, and SPSS 23.0 software was used for one-way analysis of variance for data statistics. As can be seen from Figure 5, compared with the blank control group, the body weight of the DSS model group was significantly reduced, which was in line with the law of body weight reduction in colitis patients, the DAI score showed a significant upward trend (###p<0.001), and the colon length was significantly shortened (###p<0.001), indicating that the DSS model was successfully prepared; compared with the DSS model control group, the telofovir dipivoxil, adefovir dipivoxil, telbivudine and famciclovir administration groups could significantly increase the Improved weight loss, DAI score, colon edema and shortening, suggesting that telbivudine, adefovir dipivoxil, telbivudine and famciclovir have therapeutic effects on ulcerative colitis, with telbivudine being more effective than Clinical efficacy of sulfasalazine.
实施例四、阿昔洛韦、盐酸吉西他滨、齐多夫定和6-硫基鸟嘌呤对KM结肠炎小鼠的治疗效果Embodiment 4. Therapeutic effect of acyclovir, gemcitabine hydrochloride, zidovudine and 6-thioguanine on KM colitis mice
1.药物及试剂1. Drugs and reagents
盐酸吉西他滨(Gemzar，HPLC≥98％)、齐多夫定/3-叠氮-3-脱氧胸苷(AZT，HPLC≥98％)和6-硫基鸟嘌呤(6-TG，HPLC≥98％)购自阿拉丁生物科技有限公司；阿昔洛韦(Acyclovir，ACV，HPLC≥97％)购自麦克林生物科技有限公司；柳氮磺吡啶肠溶片(Salazosulfapyridine，SASP)购自上海信谊天平药业有限公司；葡聚糖硫酸钠(Dextran Sulfate sodium，DSS)，MW:40000，购自阿拉丁生物科技有限公司。Gemcitabine hydrochloride (Gemzar, HPLC≥98%), zidovudine/3-azido-3-deoxythymidine (AZT, HPLC≥98%) and 6-thioguanine (6-TG, HPLC≥98%) ) was purchased from Aladdin Biotechnology Co., Ltd.; Acyclovir (ACV, HPLC≥97%) was purchased from McLean Biotechnology Co., Ltd.; Sulfasalazine Enteric-Coated Tablets (Salazosulfapyridine, SASP) were purchased from Shanghai Xinyi Tianping Pharmaceutical Co., Ltd.; Dextran Sulfate Sodium (DSS), MW: 40000, purchased from Aladdin Biotechnology Co., Ltd.
2.实验分组和给药剂量：标准体重KM雄性小鼠，随机分为7组，每组8只，分组和给药剂量如下：2. Experimental grouping and administration dose: KM male mice with standard body weight were randomly divided into 7 groups with 8 mice in each group. The grouping and administration doses were as follows:
正常对照组(Control，口服给予等体积0.9％生理盐水)；Normal control group (Control, orally administered an equal volume of 0.9% normal saline);
DSS模型对照组(DSS，口服给予等体积0.9％生理盐水)；DSS model control group (DSS, orally administered an equal volume of 0.9% normal saline);
阳性药组(SASP，口服给予柳氮磺吡啶，80mg/kg/day)；Positive drug group (SASP, oral administration of sulfasalazine, 80 mg/kg/day);
盐酸吉西他滨组(Gemzar，给药剂量为2mg/kg/day，腹腔注射)；Gemcitabine hydrochloride group (Gemzar, the dosage is 2mg/kg/day, intraperitoneal injection);
齐多夫定组(AZT，给药剂量为2mg/kg/day，腹腔注射)；Zidovudine group (AZT, the dose is 2mg/kg/day, intraperitoneal injection);
阿昔洛韦组(ACV，给药剂量为2mg/kg/day，腹腔注射)；Acyclovir group (ACV, the dose is 2 mg/kg/day, intraperitoneal injection);
6-硫基鸟嘌呤组(6-TG，给药剂量为2mg/kg/day，灌胃给药)。6-thioguanine group (6-TG, administered at a dose of 2 mg/kg/day, intragastric administration).
3.溃疡性结肠炎模型的制备和临床指标检测方法如实施例一中“4-6实验方法”所示。3. The preparation of the ulcerative colitis model and the detection methods of clinical indicators are shown in "4-6 Experimental Methods" in Example 1.
4.结果分析4. Analysis of results
上述各组小鼠在实验过程中，对其体重、粪便粘稠度、便血及隐血情况、结肠形态及长度进行监测，数据统计用SPSS23.0软件进行单因素方差分析。During the experiment, the mice of the above groups were monitored for their body weight, fecal viscosity, blood in the stool and occult blood, colon morphology and length, and SPSS 23.0 software was used for one-way analysis of variance for data statistics.
由图6可知，与空白对照组相比，DAI评分呈显著的上升趋势(###p<0.001)，结肠长度明显缩短(###p<0.001)，表明DSS模型是制备成功的；与DSS模型对照组相比，阿昔洛韦、盐酸吉西他滨、齐多夫定和6-硫基鸟嘌呤各给药组均能显著的增加DAI评分(***p<0.001、**p<0.01、*p<0.05和*p<0.05)、改善结肠水肿和缩短情况(***p<0.001、**p<0.01、**p<0.01和**p<0.01)，表明阿昔洛韦、盐酸吉西他滨、齐多夫定和6-硫基鸟嘌呤对溃疡性结肠炎有治疗作用，其中阿昔洛韦的疗效强于临床用药柳氮磺吡啶的疗效，各给药组疗效对比为：阿昔洛韦>柳氮磺吡啶>6-硫基鸟嘌呤>盐酸吉西他滨>齐多夫定。As can be seen from Figure 6, compared with the blank control group, the DAI score showed a significant upward trend (###p<0.001), and the colon length was significantly shortened (###p<0.001), indicating that the DSS model was successfully prepared; Compared with the DSS model control group, each administration group of acyclovir, gemcitabine hydrochloride, zidovudine and 6-thioguanine could significantly increase the DAI score (***p<0.001, **p<0.01 , *p<0.05 and *p<0.05), improved colonic edema and shortening (***p<0.001, **p<0.01, **p<0.01 and **p<0.01), indicating acyclovir , gemcitabine hydrochloride, zidovudine and 6-thioguanine have therapeutic effects on ulcerative colitis, among which the curative effect of acyclovir is stronger than that of the clinical drug sulfasalazine. The curative effects of each administration group are compared as follows: Acyclovir > sulfasalazine > 6-thioguanine > gemcitabine hydrochloride > zidovudine.
实施例五、替比夫定、盐酸吉西他滨和齐多夫定对KM胃溃疡小鼠的保护效果 Embodiment 5. The protective effect of telbivudine, gemcitabine hydrochloride and zidovudine on KM gastric ulcer mice
1.实验动物来源1. Laboratory animal sources
实验动物：8周龄SPF级昆明(Kunming，KM)雄性小鼠，体重为18～22g，实验前未使用任何药物，购自中国农业科学院兰州兽医研究所。Experimental animals: 8-week-old SPF grade Kunming (Kunming, KM) male mice, weighing 18-22 g, without any drugs before the experiment, were purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.
2.药物及试剂2. Drugs and reagents
盐酸吉西他滨(Gemzar，HPLC≥98％)、齐多夫定/3-叠氮-3-脱氧胸苷(AZT，HPLC≥98％) 和替比夫定(Telbivudine，LDT，HPLC≥98％)购自阿拉丁生物科技有限公司；无水乙醇，天津大茂化学试剂公司；西咪替丁片(Cimetidine，CIM)购自上海信谊天平药业有限公司。Gemcitabine hydrochloride (Gemzar, HPLC≥98%), zidovudine/3-azido-3-deoxythymidine (AZT, HPLC≥98%) and telbivudine (Telbivudine, LDT, HPLC≥98%) were purchased from Aladdin Biotechnology Co., Ltd.; anhydrous ethanol, Tianjin Damao Chemical Reagent Co., Ltd.; Cimetidine (CIM) tablets were purchased from Shanghai Xinyi Tianping Pharmaceutical Co., Ltd.
3.KM小鼠酒精性胃溃疡模型的制备3. Preparation of KM mouse model of alcoholic gastric ulcer
48只KM雄性小鼠(20-25g)，饲养于兰州大学实验动物房，小鼠适应性饲养一周后，随机分为6组，每组8只小鼠，分组情况及给药量如表3所示；48 KM male mice (20-25g) were raised in the laboratory animal room of Lanzhou University. After the mice were adaptively reared for one week, they were randomly divided into 6 groups with 8 mice in each group. The grouping and dosage are shown in Table 3. shown;
表3 KM雄性小鼠实验分组及各组的给药剂量Table 3 Experimental grouping of KM male mice and the dosage of each group
组别group 给药剂量Dosage
空白对照组Blank control group 0.25mL/只灌胃0.9％生理盐水0.25mL/gavage of 0.9% saline
乙醇模型组Ethanol model group 0.25mL/只灌胃无水乙醇0.25mL/only anhydrous ethanol by gavage
西咪替丁阳性组Cimetidine positive group 80mg/kg/day西咪替丁灌胃给药，2h后0.25mL/只灌胃无水乙醇80mg/kg/day cimetidine was administered by intragastric administration, 2 hours later, 0.25mL/dose of anhydrous ethanol was administered by intragastric administration
吉西他滨给药组Gemcitabine-administered group 2mg/kg/day Gemzar注射给药，2h后0.25mL/只灌胃无水乙醇2mg/kg/day Gemzar injection, 0.25mL/gavage absolute ethanol after 2h
齐多夫定给药组Zidovudine administration group 2mg/kg/day AZT注射给药，2h后0.25mL/只灌胃无水乙醇2mg/kg/day AZT injection, 2 hours later, 0.25mL/dose with absolute ethanol
替比夫定给药组Telbivudine administration group 2mg/kg/day LDT注射给药，2h后0.25mL/只灌胃无水乙醇2mg/kg/day LDT injection, 0.25mL/dose anhydrous ethanol after 2h
上述各组小鼠乙醇刺激2h后结束实验，分离血清和胃组织测定各项指标。The experiments were terminated after 2 hours of ethanol stimulation of the mice in the above groups, and the serum and gastric tissue were separated to determine various indicators.
4.溃疡面积、溃疡指数与抑制率4. Ulcer area, ulcer index and inhibition rate
将胃取出，沿胃大弯剪开，冲洗干净内容物，观察胃粘膜溃疡情况，用直尺测量溃疡的横径与纵径，两者乘积为溃疡面积(mm 2)，如公式1所示；然后计算整个胃组织的溃疡面积，从而计算溃疡抑制率(％)，见公式2。同时以各组鼠溃疡点数之和的均值作为溃疡指数(愈合记0分、表浅性黏膜糜烂记1分、深陷性溃疡或透壁性坏死记2分、穿孔或穿透性溃疡记3分) Take out the stomach, cut it along the greater curvature of the stomach, rinse the contents, observe the gastric mucosal ulcer, measure the transverse diameter and longitudinal diameter of the ulcer with a ruler, and the product of the two is the ulcer area (mm 2 ), as shown in formula 1 ; and then calculate the ulcer area of the entire gastric tissue, thereby calculating the ulcer inhibition rate (%), see formula 2. At the same time, the mean of the sum of ulcer points in each group was taken as the ulcer index (0 points for healing, 1 point for superficial mucosal erosion, 2 points for deep ulcers or transmural necrosis, and 3 points for perforation or penetrating ulcers). Minute)
溃疡面积(mm2)＝溃疡最大长径*垂直于最大长径的最大宽径  (1)Ulcer area (mm2) = maximum long diameter of ulcer * maximum wide diameter perpendicular to the maximum long diameter (1)
Figure PCTCN2021074575-appb-000005
Figure PCTCN2021074575-appb-000005
5.数据处理5. Data processing
实验数据采用SPSS23.0软件进行统计学分析，数据以(x±s)表示，组间比较采用单因素方差分析(One-way ANOVA)和LSD-t法进行两两比较。p<0.05认为具有统计学意义。The experimental data were statistically analyzed by SPSS23.0 software, and the data were expressed as (x±s), and the comparison between groups was performed by one-way analysis of variance (One-way ANOVA) and LSD-t method. p<0.05 was considered statistically significant.
6.替比夫定、盐酸吉西他滨和齐多夫定对胃溃疡小鼠胃组织形态、溃疡指数、溃疡面积及溃疡抑制率的影响6. Effects of telbivudine, gemcitabine hydrochloride and zidovudine on gastric tissue morphology, ulcer index, ulcer area and ulcer inhibition rate in gastric ulcer mice
上述各组小鼠处死后，迅速取其胃组织，观察其形态改变及溃疡损伤情况，并对其溃疡面积和溃疡指数进行统计分析，实验结果如图7所示。由图7可知，正常组小鼠胃组织宏观形 态正常，未出现明显出血性病变，而乙醇模型组小鼠胃组织出血性病变明显，溃疡最为严重，表明乙醇胃溃疡模型制备成功；与乙醇模型组相比，替比夫定、盐酸吉西他滨、齐多夫定和西咪替丁各给药组均能显著的改善胃粘膜的损伤程度，减少溃疡面积，其中盐酸吉西他滨的溃疡抑制率(91.02％)和齐多夫定的溃疡抑制率(84.47％)高于临床用药西咪替丁(CIM)的溃疡抑制率(65.25％)。以上所述溃疡面积、溃疡指数与抑制率，及胃组织形态学测定表明，替比夫定、盐酸吉西他滨和齐多夫定防治胃溃疡有效。After the mice in the above groups were sacrificed, their gastric tissues were quickly taken to observe their morphological changes and ulcer damage, and the ulcer area and ulcer index were statistically analyzed. The experimental results are shown in Figure 7. As can be seen from Figure 7, the macroscopic morphology of the gastric tissue of the mice in the normal group was normal, and no obvious hemorrhagic lesions appeared, while the gastric tissue of the mice in the ethanol model group had obvious hemorrhagic lesions and the most serious ulcers, indicating that the ethanol gastric ulcer model was successfully prepared; Compared with the other groups, the administration groups of telbivudine, gemcitabine hydrochloride, zidovudine and cimetidine could significantly improve the degree of gastric mucosal damage and reduce the ulcer area, among which gemcitabine hydrochloride had an ulcer inhibition rate (91.02%). ) and zidovudine (84.47%) were higher than that of clinical drug cimetidine (CIM) (65.25%). The above-mentioned ulcer area, ulcer index and inhibition rate, and gastric histomorphological measurements show that telbivudine, gemcitabine hydrochloride and zidovudine are effective in preventing and treating gastric ulcer.
实施例六、阿德福韦酯、替洛福韦酯、阿昔洛韦、泛昔洛韦和6-硫基鸟嘌呤对KM胃溃疡小鼠的保护效果Embodiment 6. Protective effect of adefovir dipivoxil, telofovir dipivoxil, acyclovir, famciclovir and 6-thioguanine on KM gastric ulcer mice
1.实验动物来源1. Laboratory animal sources
实验动物：8周龄SPF级昆明(Kunming，KM)雄性小鼠，体重为18～22g，实验前未使用任何药物，购自中国农业科学院兰州兽医研究所。Experimental animals: 8-week-old SPF grade Kunming (Kunming, KM) male mice, weighing 18-22 g, without any drugs before the experiment, were purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.
2.药物及试剂2. Drugs and reagents
替洛福韦酯(PMPA，HPLC≥98％)、阿德福韦酯(PMEA，HPLC≥98％)、泛昔洛韦(Famciclocir，FCV，HPLC≥98％)和6-硫基鸟嘌呤(6-TG，HPLC≥98％)购自阿拉丁生物科技有限公司；阿昔洛韦(Acyclovir，ACV，HPLC≥97％)购自麦克林生物科技有限公司；无水乙醇，天津大茂化学试剂公司；西咪替丁片(Cimetidine，CIM)购自上海信谊天平药业有限公司。Telofovir dipivoxil (PMPA, HPLC≥98%), adefovir dipivoxil (PMEA, HPLC≥98%), famciclocir (Famciclocir, FCV, HPLC≥98%) and 6-thioguanine (6-TG , HPLC≥98%) was purchased from Aladdin Biotechnology Co., Ltd.; Acyclovir (ACV, HPLC≥97%) was purchased from Maclean Biotechnology Co., Ltd.; Anhydrous ethanol, Tianjin Damao Chemical Reagent Co., Ltd.; Cimetidine (CIM) was purchased from Shanghai Xinyi Tianping Pharmaceutical Co., Ltd.
3.KM小鼠酒精性胃溃疡模型的制备3. Preparation of KM mouse model of alcoholic gastric ulcer
64只KM雄性小鼠(20-25g)，饲养于兰州大学实验动物房，小鼠适应性饲养一周后，随机分为8组，每组8只小鼠，分组情况及给药量如表4所示；64 KM male mice (20-25g) were raised in the laboratory animal room of Lanzhou University. After the mice were adaptively reared for one week, they were randomly divided into 8 groups with 8 mice in each group. The grouping and dosage are shown in Table 4. shown;
表4 KM雄性小鼠实验分组及各组的给药剂量Table 4 Experimental grouping of KM male mice and the dosage of each group
组别group 给药剂量Dosage
空白对照组Blank control group 0.25mL/只灌胃0.9％生理盐水0.25mL/gavage of 0.9% saline
乙醇模型组Ethanol model group 0.25mL/只灌胃无水乙醇0.25mL/only anhydrous ethanol by gavage
西咪替丁阳性组Cimetidine positive group 80mg/kg/day西咪替丁灌胃给药，2h后0.25mL/只灌胃无水乙醇80mg/kg/day cimetidine was administered by intragastric administration, 2 hours later, 0.25mL/dose of anhydrous ethanol was administered by intragastric administration
阿德福韦酯组Adefovir dipivoxil group 2mg/kg/day PMEA注射给药，2h后0.25mL/只灌胃无水乙醇2mg/kg/day PMEA injection, 0.25mL/only anhydrous ethanol after 2h
替洛福韦酯组telofovir dipivoxil group 2mg/kg/day PMPA注射给药，2h后0.25mL/只灌胃无水乙醇2mg/kg/day PMPA injection, 0.25mL/dose of absolute ethanol after 2h
阿昔洛韦组Acyclovir group 2mg/kg/day ACV注射给药，2h后0.25mL/只灌胃无水乙醇2mg/kg/day ACV injection, 2 hours later, 0.25mL/dose with absolute ethanol
泛昔洛韦组Famciclovir group 2mg/kg/day FCV注射给药，2h后0.25mL/只灌胃无水乙醇2mg/kg/day FCV injection, 0.25mL/only anhydrous ethanol after 2h
6-硫基鸟嘌呤组6-thioguanine group 2mg/kg/day 6-TG注射给药，2h后0.25mL/只灌胃无水乙醇2mg/kg/day 6-TG injection, 2h after 0.25mL/dose with absolute ethanol
上述各组小鼠乙醇刺激2h后结束实验，分离血清和胃组织测定各项指标。The experiments were terminated after 2 hours of ethanol stimulation of the mice in the above groups, and the serum and gastric tissue were separated to determine various indicators.
4.溃疡面积、溃疡指数与抑制率4. Ulcer area, ulcer index and inhibition rate
将胃取出，沿胃大弯剪开，冲洗干净内容物，观察胃粘膜溃疡情况，用直尺测量溃疡的横径与纵径，两者乘积为溃疡面积(mm 2)，如公式1所示；然后计算整个胃组织的溃疡面积，从而计算溃疡抑制率(％)，见公式2。同时以各组鼠溃疡点数之和的均值作为溃疡指数(愈合记0分、表浅性黏膜糜烂记1分、深陷性溃疡或透壁性坏死记2分、穿孔或穿透性溃疡记3分) Take out the stomach, cut it along the greater curvature of the stomach, rinse the contents, observe the gastric mucosal ulcer, measure the transverse diameter and longitudinal diameter of the ulcer with a ruler, and the product of the two is the ulcer area (mm 2 ), as shown in formula 1 ; and then calculate the ulcer area of the entire gastric tissue, thereby calculating the ulcer inhibition rate (%), see formula 2. At the same time, the mean of the sum of ulcer points in each group was taken as the ulcer index (0 points for healing, 1 point for superficial mucosal erosion, 2 points for deep ulcers or transmural necrosis, and 3 points for perforation or penetrating ulcers). Minute)
溃疡面积(mm2)＝溃疡最大长径*垂直于最大长径的最大宽径    (1)Ulcer area (mm2) = the maximum length of the ulcer * the maximum width perpendicular to the maximum length (1)
Figure PCTCN2021074575-appb-000006
Figure PCTCN2021074575-appb-000006
5.数据处理5. Data processing
实验数据采用SPSS23.0软件进行统计学分析，数据以(x±s)表示，组间比较采用单因素方差分析(One-way ANOVA)和LSD-t法进行两两比较。p<0.05认为具有统计学意义。The experimental data were statistically analyzed by SPSS23.0 software, and the data were expressed as (x±s), and the comparison between groups was performed by one-way analysis of variance (One-way ANOVA) and LSD-t method. p<0.05 was considered statistically significant.
6.阿德福韦酯、替洛福韦酯、阿昔洛韦、泛昔洛韦和6-硫基鸟嘌呤对胃溃疡小鼠胃组织形态、溃疡指数、溃疡面积及溃疡抑制率的影响6. Effects of adefovir dipivoxil, telofovir dipivoxil, acyclovir, famciclovir and 6-thioguanine on gastric tissue morphology, ulcer index, ulcer area and ulcer inhibition rate in gastric ulcer mice
上述各组小鼠处死后，迅速取其胃组织，观察其形态改变及溃疡损伤情况，并对其溃疡面积和溃疡指数进行统计分析，实验结果如图8所示。由图8可知，正常组小鼠胃组织宏观形态正常，未出现明显出血性病变，而乙醇模型组小鼠胃组织出血性病变明显，溃疡最为严重，表明乙醇胃溃疡模型制备成功；与乙醇模型组相比，阿德福韦酯、替洛福韦酯、阿昔洛韦、泛昔洛韦、6-硫基鸟嘌呤和西咪替丁各给药组均能显著的改善胃粘膜的损伤程度，减少溃疡面积，抑制溃疡程度达50％以上，各药物组与阳性药西咪替丁对比结果为：6-硫基鸟嘌呤>替洛福韦酯>泛昔洛韦>阿昔洛韦>西咪替丁>阿德福韦酯。以上所述溃疡面积、溃疡指数与抑制率，及胃组织形态学测定表明，阿德福韦酯、替洛福韦酯、阿昔洛韦、泛昔洛韦和6-硫基鸟嘌呤防治胃溃疡有效。After the mice in the above groups were sacrificed, their stomach tissues were quickly taken to observe their morphological changes and ulcer damage, and statistical analysis was performed on their ulcer area and ulcer index. The experimental results are shown in Figure 8. It can be seen from Figure 8 that the macroscopic shape of the gastric tissue of the mice in the normal group was normal, and no obvious hemorrhagic lesions appeared, while the gastric tissue of the mice in the ethanol model group had obvious hemorrhagic lesions and the most serious ulcers, indicating that the ethanol gastric ulcer model was successfully prepared; Compared with the control group, the administration groups of adefovir dipivoxil, telofovir dipivoxil, acyclovir, famciclovir, 6-thioguanine and cimetidine can significantly improve the degree of gastric mucosal damage, reduce Ulcer area, inhibited the degree of ulcer by more than 50%, the comparison results of each drug group and the positive drug cimetidine were: 6-thioguanine > telofovir dipivoxil > famciclovir > acyclovir > cimetidine > Adefovir dipivoxil. The above-mentioned ulcer area, ulcer index and inhibition rate, and gastric histomorphological measurements show that adefovir dipivoxil, telofovir dipivoxil, acyclovir, famciclovir and 6-thioguanine are effective in preventing and treating gastric ulcer.
综上所述，本发明所述的核苷类似物拉米夫定、替比夫定、泛昔洛韦、替洛福韦酯、阿德福韦酯、阿昔洛韦、盐酸吉西他滨、齐多夫定和6-硫基鸟嘌呤，在不同饲喂条件下，对结肠炎小鼠进行DAI评分和结肠形态测定，结果显示拉米夫定、替比夫定、泛昔洛韦、替洛福韦酯、阿德福韦酯、阿昔洛韦、盐酸吉西他滨、齐多夫定和6-硫基鸟嘌呤等药物对溃疡性结肠炎均有效。同时，在不同饲喂条件下，对胃溃疡小鼠进行溃疡指数和抑制率及组织形态学检查，实验结果显示拉米夫定、替比夫定、泛昔洛韦、替洛福韦酯、阿德福韦酯、阿昔洛韦、盐酸吉西他滨、齐多夫定和6-硫基鸟嘌呤具有防治胃溃疡的效果，且大部分药物效果强于现 有临床用药西咪替丁的疗效，即本发明所述的核苷类似物拉米夫定、替比夫定、泛昔洛韦、替洛福韦酯、阿德福韦酯、阿昔洛韦、盐酸吉西他滨、齐多夫定和6-硫基鸟嘌呤等药物同时对溃疡性结肠炎和胃溃疡均有效，具有良好的临床应用前景。To sum up, the nucleoside analogs of the present invention, lamivudine, telbivudine, famciclovir, telofovir dipivoxil, adefovir dipivoxil, acyclovir, gemcitabine hydrochloride, zidovudine and 6-thioguanine, under different feeding conditions, the DAI score and colon morphometry of colitis mice showed that lamivudine, telbivudine, famciclovir, telofovir dipivoxil, adrenalin Drugs such as fovir dipivoxil, acyclovir, gemcitabine hydrochloride, zidovudine, and 6-thioguanine are all effective for ulcerative colitis. At the same time, under different feeding conditions, the ulcer index, inhibition rate and histomorphology of gastric ulcer mice were examined. The experimental results showed that lamivudine, telbivudine, famciclovir, telofovir dipivoxil, adefovir Wei dipivoxil, acyclovir, gemcitabine hydrochloride, zidovudine and 6-thioguanine have the effect of preventing and treating gastric ulcer, and most of the drug effects are stronger than the curative effect of the existing clinical drug cimetidine, namely the present invention Described nucleoside analogs lamivudine, telbivudine, famciclovir, telofovir dipivoxil, adefovir dipivoxil, acyclovir, gemcitabine hydrochloride, zidovudine and 6-thioguanine The other drugs are effective for both ulcerative colitis and gastric ulcer, and have good clinical application prospects.
本发明在研究过程中意外的发现，拉米夫定治疗胃肠道疾病具有显著的疗效，实验结果如上述实施例一和实施例二所示，而拉米夫定属于经典的胞嘧啶核苷类似物，因此发明人推断，所有的胞嘧啶核苷类似物均具有治疗胃肠道疾病的疗效，为了验证上述结论，发明人又选取了另外一种胞嘧啶核苷类似物盐酸吉西他滨进行相关实验，实验结果显示，盐酸吉西他滨也具有治疗胃肠道疾病的效果。由此，发明人进一步推断，所有的核苷类似物均具有治疗胃肠道疾病的效果。同时，核苷类似物又可分为胞嘧啶核苷类似物、胸腺嘧啶核苷类似物、腺嘌呤核苷类似物和鸟嘌呤核苷类似物，发明人选取了胸腺嘧啶核苷类似物选自齐多夫定或替比夫定；所述的腺嘌呤核苷类似物选自泛昔洛韦、替洛福韦酯或阿德福韦酯；所述的鸟嘌呤核苷类似物选自阿昔洛韦或6-硫基鸟嘌呤。并进行胃肠道疾病的相关实验，实验结果显示，替比夫定、泛昔洛韦、替洛福韦酯、阿德福韦酯、阿昔洛韦、盐酸吉西他滨、齐多夫定和6-硫基鸟嘌呤均具有显著的治疗胃肠道疾病的效果，实验结果如上述实施例所示，发明人证实了上述推断，并得出所有的核苷类似物治疗胃肠道疾病均具有效果，能够用于制备治疗胃肠道疾病药物，为核苷类似物的药物提供了一种新用途，这是本领域技术人员所能够认可的。During the research process of the present invention, it was unexpectedly found that lamivudine has a significant curative effect in the treatment of gastrointestinal diseases. The experimental results are shown in the above-mentioned Example 1 and Example 2, and lamivudine belongs to the classic cytosine nucleoside. Therefore, the inventors infer that all cytosine nucleoside analogs have curative effects in the treatment of gastrointestinal diseases. In order to verify the above conclusion, the inventor selected another cytosine nucleoside analog, gemcitabine hydrochloride, to conduct related experiments. , the experimental results show that gemcitabine hydrochloride also has the effect of treating gastrointestinal diseases. From this, the inventors further infer that all nucleoside analogs have the effect of treating gastrointestinal diseases. At the same time, nucleoside analogs can be further classified into cytosine analogs, thymidine analogs, adenosine analogs and guanosine analogs. The inventors selected thymidine analogs from Zidovudine or Telbivudine; the adenosine analogs are selected from famciclovir, telofovir dipivoxil or adefovir dipivoxil; the guanosine analogs are selected from acyclovir or 6-thioguanine. And related experiments on gastrointestinal diseases were carried out. The experimental results showed that telbivudine, famciclovir, telofovir dipivoxil, adefovir dipivoxil, acyclovir, gemcitabine hydrochloride, zidovudine and 6-thiol Guanine has a significant effect on the treatment of gastrointestinal diseases. The experimental results are shown in the above-mentioned examples. The inventors have confirmed the above inferences and concluded that all nucleoside analogs have effects in the treatment of gastrointestinal diseases, and can be used with For the preparation of medicines for treating gastrointestinal diseases, a new use is provided for medicines of nucleoside analogs, which can be recognized by those skilled in the art.

Claims (10)

  1. 核苷类似物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体及其混合物形式，及其可药用盐在制备预防和/或治疗胃肠道疾病药物中的应用。Nucleoside analogs or their tautomers, mesomers, racemates, enantiomers, diastereomers and mixtures thereof, and pharmaceutically acceptable salts thereof in the preparation of prophylactic and/or Or the application of drugs for the treatment of gastrointestinal diseases.
  2. 如权利要求1所述的应用，其特征在于，所述的核苷类似物选自胞嘧啶核苷类似物、胸腺嘧啶核苷类似物、腺嘌呤核苷类似物和鸟嘌呤核苷类似物中的任一种。The use according to claim 1, wherein the nucleoside analog is selected from the group consisting of cytosine analogs, thymidine analogs, adenosine analogs and guanosine analogs any of the.
  3. 如权利要求2所述的应用，其特征在于，所述的胞嘧啶核苷类似物选自拉米夫定、脱氧胞苷、盐酸吉西他滨、盐酸阿糖胞苷、西多福韦、依曲西他平、艾夫他滨、恩曲他滨、阿卡地新、阿扎胞苷、地西他滨、Thiarabine、Ethynylcytidine、卡培他滨、艾西拉滨、安西他滨、4-S-β-D-阿糖胞嘧啶核苷、5-氮杂-4-硫代-2-脱氧胞苷、顺式-1-[4-(羟基-甲基)-环戊-2-烯基]-5-I-碘胞嘧啶和顺式-1-[4-(羟基-甲基)-环戊-2-烯基]-5-(2-I-碘乙烯基)胞嘧啶中的任一种；所述的胸腺嘧啶核苷类似物选自齐多夫定、替比夫定、索非布韦、5-氟尿嘧啶、脱氧尿苷、克来夫定、司他夫定、氟铁龙、双呋氟尿嘧啶、替加氟、氟尿嘧啶、卡莫氟、4-硫代胸腺嘧啶、5-牛磺酸甲基-2-S-尿苷、2’-氟-5-甲基-β-L-阿糖呋喃尿苷、2，3-胸腺嘧啶二脱氧碳环核苷衍生物、3-苯甲酰胸腺嘧啶和三氟胸苷中的任一种；所述的腺嘌呤核苷类似物选自腺苷、脱氧腺苷、脱氧腺苷酸、泛昔洛韦、替洛福韦、替洛福韦酯、富马酸替洛福韦二吡呋喃、阿德福韦、阿德福韦酯、二磷酸阿德福韦、阿地福韦、氯法拉滨、克拉屈滨、曲沙他滨、Aristeromycin和Neplanocin A中的任一种；所述的鸟嘌呤核苷类似物选自8-羟基-2-脱氧鸟嘌呤核苷、阿昔洛韦、更昔洛韦、恩替卡韦、三磷酸恩替卡韦、LB80380/ANA380、奈拉滨、9-β-D-阿糖呋喃糖鸟嘌呤、Forodesine hydrochloride、巯鸟嘌呤、6-巯基鸟嘌呤、6-硫基鸟嘌呤、6-硫代鸟嘌呤核苷酸、2-脱氧鸟苷一磷酸和5-(2-呋喃基)-2-脱氧鸟苷中的任一种。The application according to claim 2, wherein the cytosine nucleoside analog is selected from the group consisting of lamivudine, deoxycytidine, gemcitabine hydrochloride, cytarabine hydrochloride, cidofovir, etrexet Tapine, Alfcitabine, Emtricitabine, Acadisine, Azacitidine, Decitabine, Thiarabine, Ethynylcytidine, Capecitabine, Escilarabine, Amcitabine, 4-S- β-D-cytarabine, 5-aza-4-thio-2-deoxycytidine, cis-1-[4-(hydroxy-methyl)-cyclopent-2-enyl] - any of 5-I-iodocytosine and cis-1-[4-(hydroxy-methyl)-cyclopent-2-enyl]-5-(2-1-iodovinyl)cytosine ; Described thymidine analogs are selected from zidovudine, telbivudine, sofosbuvir, 5-fluorouracil, deoxyuridine, clevudine, stavudine, fluorotromidine, dioxyuridine Furflurouracil, Tegafur, Fluorouracil, Carmofur, 4-thiothymine, 5-taurine methyl-2-S-uridine, 2'-fluoro-5-methyl-β-L-alcohol Any one of sugar furanoside, 2,3-thymidine dideoxycarbocyclic nucleoside derivatives, 3-benzoyl thymidine and trifluorothymidine; the adenosine analogs are selected from adenosine glycosides, deoxyadenosine, deoxyadenylate, famciclovir, telofovir, telofovir dipivoxil, telofovir disoproxil fumarate, adefovir, adefovir dipivoxil, adefovir dipivoxil Any one of fovir, adefovir, clofarabine, cladribine, troxacitabine, Aristeromycin and Neplanocin A; the guanosine analog is selected from 8-hydroxy-2-deoxyguanosine Purine nucleosides, acyclovir, ganciclovir, entecavir, entecavir triphosphate, LB80380/ANA380, nerabine, 9-beta-D-arabinofuranoguanine, Forodesine hydrochloride, mercaptoguanine, 6- Any of mercaptoguanine, 6-thioguanine, 6-thioguanine nucleotide, 2-deoxyguanosine monophosphate, and 5-(2-furyl)-2-deoxyguanosine.
  4. 如权利要求3所述的应用，其特征在于，所述的胞嘧啶核苷类似物选自拉米夫定或盐酸吉西他滨；所述的胸腺嘧啶核苷类似物选自齐多夫定或替比夫定；所述的腺嘌呤核苷类似物选自泛昔洛韦、替洛福韦酯或阿德福韦酯；所述的鸟嘌呤核苷类似物选自阿昔洛韦或6-硫基鸟嘌呤。The application according to claim 3, wherein the cytidine analog is selected from lamivudine or gemcitabine hydrochloride; the thymidine analog is selected from zidovudine or tibial Vudine; the adenosine analogs are selected from famciclovir, telofovir dipivoxil or adefovir dipivoxil; the guanosine analogs are selected from acyclovir or 6-thioguanine .
  5. 如权利要求1-4任一项所述的应用，其特征在于，所述的胃肠道疾病为消化性溃疡。The application according to any one of claims 1-4, wherein the gastrointestinal disease is peptic ulcer.
  6. 如权利要求5所述的应用，其特征在于，所述的消化性溃疡为胃溃疡、十二指肠溃疡、球后溃疡、幽门管溃疡、复合溃疡和对吻溃疡中的一种或几种。The application according to claim 5, wherein the peptic ulcer is one or more of gastric ulcer, duodenal ulcer, retrobulbar ulcer, pyloric canal ulcer, compound ulcer and kiss ulcer .
  7. 如权利要求6所述的应用，其特征在于，所述的消化性溃疡为胃溃疡和十二指肠溃疡。The application according to claim 6, wherein the peptic ulcer is gastric ulcer and duodenal ulcer.
  8. 如权利要求1-4任一项所述的应用，其特征在于，所述的胃肠道疾病为炎症性肠病。The use according to any one of claims 1-4, wherein the gastrointestinal disease is inflammatory bowel disease.
  9. 如权利要求8所述的应用，其特征在于，所述的炎症性肠病为溃疡性结肠炎和克罗恩病。The use according to claim 8, wherein the inflammatory bowel disease is ulcerative colitis and Crohn's disease.
  10. 如权利要求1所述的应用，其特征在于，所述核苷类似物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体及其混合物形式，及其可药用盐和一种或多种药学上可 接受的载体组成药物组合物，所述药物组合物的剂型为注射剂、片剂、胶囊剂、颗粒剂或丸剂。The use according to claim 1, wherein the nucleoside analog or its tautomer, meso, racemate, enantiomer, diastereomer and The mixture form, its pharmaceutically acceptable salt and one or more pharmaceutically acceptable carriers constitute a pharmaceutical composition, and the dosage form of the pharmaceutical composition is injection, tablet, capsule, granule or pill.
PCT/CN2021/074575 2021-01-31 2021-01-31 Application of nucleoside analogue in preparation of drugs for preventing or treating gastrointestinal diseases WO2022160323A1 (en)

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