WO2019085828A1 - 一种天冬甜素的新用途 - Google Patents

一种天冬甜素的新用途 Download PDF

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WO2019085828A1
WO2019085828A1 PCT/CN2018/112031 CN2018112031W WO2019085828A1 WO 2019085828 A1 WO2019085828 A1 WO 2019085828A1 CN 2018112031 W CN2018112031 W CN 2018112031W WO 2019085828 A1 WO2019085828 A1 WO 2019085828A1
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compound
bacterial
formula
use according
drug
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French (fr)
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孔韧
朴莲花
许晓双
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江苏理工学院
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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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

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  • the invention belongs to the technical field of medicine and relates to a new use of aspartame. Specifically, aspartame is used as a bacterial beta glucuronidase inhibitor and its application.
  • bacterial beta-glucuronidase Through bacterial ⁇ -glucuronidase, microorganisms in the gastrointestinal tract take glycosylation from the glucuronidation compound for energy supply, resulting in the conversion of various drugs or their metabolites from a non-toxic state to a toxic state, thereby affecting the drug Metabolic and toxic side effects [1, 2] .
  • Symptoms of bacterial beta-glucuronidase include delayed onset diarrhea caused by irinotecan and gastrointestinal ulcers caused by non-steroidal anti-inflammatory drugs [3,4] .
  • Irinotican (CPT-11), trade name Camptosar, is one of the commonly used anti-tumor chemotherapy drugs in the clinic, and its main target is type I DNA topoisomerase. Since its introduction in 1996, irinotecan has been widely used in the treatment of colorectal cancer, lung cancer, brain tumors, and drug-resistant leukemia and lymphoma, and has been listed as a first-line treatment for metastatic colorectal cancer. Many years of clinical application found that enterotoxicity is one of the main dose-limiting side effects of irinotecan, manifested as delayed diarrhea, usually occurs 24 hours after the patient uses the drug, the main features include unpredictability, high morbidity and certain Degree of fatality.
  • irinotecan liposome formulation capsule It was approved by the US FDA in 2015; PEG-SN38 (SN38 linked to polyethylene, Belrose Pharma Inc.) was in the clinical phase III trial. Although these "new irinotecans” increase bioavailability and efficacy, diarrhea is still an insurmountable dose-limiting side effect.
  • CPT-11 The mechanism of diarrhea is closely related to the metabolic pathway of irinotecan.
  • the main metabolic site of CPT-11 is the liver.
  • CPT-11 first removes the dipiperidinyl group by carboxylesterase (CES) to form the active metabolite SN-38; followed by glucuronyltransferase (UDP-glucuronosyl).
  • CES carboxylesterase
  • UGT glucuronyltransferase
  • SN-38 is the main active metabolite of irinotecan.
  • the concentration of toxic compound SN-38 in the intestinal lumen is greatly increased, killing intestinal epithelial cells and causing irreversible damage to the intestinal mucosa, eventually leading to delayed diarrhea.
  • Produce [3,9] the selective inhibition of the activity of the bacterial ⁇ -GUS enzyme in the intestinal tract can directly reduce the local concentration of SN-38 and protect the intestinal tissue, thereby fundamentally preventing and treating delayed diarrhea.
  • the bacterial ⁇ -GUS enzyme exists in the form of a tetramer, and the small molecule compound binds to the catalytic pocket of the enzyme, and has a strong interaction with a loop structure consisting of a 17 amino acid residue unique to the bacterial enzyme. Since the loop region is distinct in the sequence and structure of the corresponding region in the human homologous protein ⁇ -GUS, the combination with the bacterial loop structure makes the series of compounds selectively inhibit ⁇ -GUS in bacteria, for mammals. ⁇ -GUS has no significant effect [3] . Animal experiments showed that the bacterial ⁇ -GUS inhibitor inhibitor 2 can reduce the incidence of diarrhea and prolong the survival time of the animal, further verifying the safety and effectiveness of the target.
  • the invention adopts integrated virtual screening and experimental determination methods to find that aspartame can selectively inhibit bacterial glucuronidase.
  • Asparagus is a food additive that has been used in humans. Its safety and pharmacy properties are clear. It can be quickly entered into clinical trials to relieve diarrhea caused by irinotecan, caused by non-steroidal anti-inflammatory drugs. Gastrointestinal ulcers, or the regulation of other drug effects and side effects via bacterial glucuronidase metabolism, as well as related conditions caused by bacterial glucuronidase.
  • the invention adopts integrated virtual screening and experimental determination methods to find that aspartame can selectively inhibit bacterial glucuronidase.
  • Asparagus is a food additive that has been used in humans. Its safety and pharmacy properties are clear. It can be quickly entered into clinical trials to relieve diarrhea caused by irinotecan, caused by non-steroidal anti-inflammatory drugs. Gastrointestinal ulcers, or the regulation of other drug effects and side effects via bacterial glucuronidase metabolism, as well as related conditions caused by bacterial glucuronidase.
  • the invention provides the use of a compound A for the preparation of a medicament for the treatment of a disease caused by a bacterial glucuronidase, the compound A being a metabolite of a compound of formula I, a compound of formula I or a compound of formula I in pharmaceutically acceptable form salt.
  • the compound A of the present invention is preferably used for diarrhea, gastrointestinal toxicity or other conditions due to bacterial glucuronidase.
  • the diarrhea includes, but is not limited to, caused by a chemotherapeutic agent; the gastrointestinal toxicity is caused by, but not limited to, by a non-steroidal anti-inflammatory drug.
  • the chemotherapeutic drugs include, but are not limited to, irinotecan or its active metabolite 7-ethyl-10 hydroxycamptothecin, or other drugs containing camptothecin-based compounds and derivatives thereof as main active ingredients.
  • the compound A of the present invention may constitute a pharmaceutical composition together with one or more pharmaceutically acceptable carriers.
  • the invention also provides the use of a chemotherapeutic drug and Compound A in the manufacture of a combination for the treatment of a tumor, the compound A being a compound of formula I, a metabolite of a compound of formula I or a pharmaceutically acceptable salt of a compound of formula I.
  • the chemotherapeutic drugs include, but are not limited to, irinotecan or its active metabolite 7-ethyl-10 hydroxycamptothecin, or other drugs containing camptothecin-based compounds and derivatives thereof as main active ingredients.
  • the Compound A and one or more pharmaceutically acceptable carriers constitute a pharmaceutical composition in the form of a tablet, capsule, granule, pill or other form which can be prepared.
  • the tumor of the present invention is preferably, but not limited to, one or more of colorectal cancer, gastric cancer, liver cancer, breast cancer, brain tumor, drug-resistant leukemia, lymphoma, prostate cancer, lung cancer or bladder cancer. .
  • the invention adopts the drug relocation as the basic strategy, adopts a computer simulation method to preliminarily evaluate the effectiveness of the drug molecule, and adopts a biological experiment method to measure the activity, and finds that aspartame is an effective bacterial ⁇ -GUS enzyme selective inhibition.
  • the enzymatic level of IC 50 is 3.6040 ⁇ 0.4755 ⁇ M (Fig. 1); its IC 50 value is 5.3730 ⁇ 1.6070 ⁇ M (Fig. 2); for mammalian-derived ⁇ -GUS enzyme at 100 ⁇ M Significant effect, indicating its good selectivity (Figure 3); at the same time the compound has no significant effect on the growth of E. coli, indicating that the compound has no obvious cytotoxicity, can inhibit its ⁇ -GUS enzyme without killing E. coli Activity ( Figure 4).
  • an appropriate dose of aspartame can be used to selectively inhibit the bacterial ⁇ -GUS enzyme, thereby affecting the efficacy and side effects of the drug metabolized by the bacterial ⁇ -GUS enzyme, and treating the condition caused by the bacterial ⁇ -GUS enzyme.
  • aspartame can be used in combination with CPT-11 or other camptothecin derivatives to reduce intestinal toxicity, relieve side effects of diarrhea, and potentially increase antitumor effects.
  • Asparagus is a food additive that has been used in humans. Its safety and pharmacy properties are clear. It can be quickly entered into clinical trials to relieve diarrhea caused by irinotecan or to regulate other bacterial gluconates. The efficacy and side effects of metabolic drugs, as well as other related conditions caused by bacterial glucuronidase.
  • Figure 1 aspartame at the protein level suppression IC 50 of FIG ability of bacterial ⁇ -GUS enzyme. Aspartame has a good bacterial ⁇ -GUS enzyme inhibitory effect at the protein level with an IC 50 value of 3.6040 ⁇ 0.4755 ⁇ M.
  • Figure 2 aspartame inhibition at the cellular level IC 50 of FIG ability of bacterial ⁇ -GUS enzyme. Aspartame has a good bacterial ⁇ -GUS enzyme inhibitory effect at the cellular level, and its IC 50 value is 5.3730 ⁇ 1.6070 ⁇ M, respectively.
  • FIG. 3 Aspartame has no significant inhibitory activity against mammalian-derived Bovine taurus ⁇ -GUS enzyme at a concentration of 100 ⁇ M.
  • the Bovine taurus ⁇ -GUS enzyme activity against mammalian origin was tested using a concentration of 100 ⁇ M aspartame, and the compound was found to have no significant inhibitory effect on mammalian-derived ⁇ -GUS enzyme.
  • Figure 4 Cytotoxicity test of aspartame on bacteria, in which black, dark gray and light gray columns respectively indicate co-incubation with E. coli in 1% DMSO solution, 100 ⁇ M aspartame and 10 ⁇ M concentration of aspartame, respectively. result. The results showed that the compound had no significant effect on the growth of E. coli. The compound had no obvious cytotoxicity and could inhibit the activity of ⁇ -GUS enzyme without killing E. coli.
  • Protein Preparation Wizard in the software package processes the crystal structure of the bacterial ⁇ -GUS enzyme (PDB: 3LPF), including modifying the bond level, adding hydrogen atoms and partial charges. Remove all crystal water molecules and optimize the entire system based on the OPLS-2005 force field when the RMSD value is reached Time optimization is terminated.
  • Isomers which produce a variety of possible chiral low-energy compound structures for chiral compounds that do not have a chiral center. Under default conditions, up to 32 stereoisomers are produced per molecule, resulting in approximately 50,000 compounds. Conformation.
  • the Glide SP mode is first used for docking and scoring, retaining all small molecule binding patterns.
  • the active molecule basically contains two distinct pharmacophore features: hydrogen bonding with GLU413 and hydrophobic interaction with PHE365. For the first 10,000 small molecule binding modes produced by docking, the pharmacophore characteristics were used as screening conditions, and the binding mode of hydrogen bonding with GLU413 and hydrophobic interaction with PHE365 was selected. Finally, some compounds were selected and purchased for experiment. Activity assay.
  • the specific steps are: 1) adding 20 ⁇ l of the test compound to a 96-well plate (blackboard); 2) adding 40 ⁇ l of GUS enzyme (125 pM); 3) adding 40 ⁇ l of substrate (4MUG, 312.5 ⁇ M); 4) incubating for 30 minutes at room temperature The reaction was terminated by the addition of 40 ⁇ l of 1 M Na2CO3; 5) Fluorescence detection was performed on an EnVision (Perkin Elmer USA) multi-function microplate reader: excitation wavelength 335 nm, emission wavelength 460 nm. The well plate without the compound was used as a positive control, and the plate without the enzyme was used as a negative control.
  • the specific procedure was the same as the bacterial glucuronidase enzymatic level activity test in which the enzyme was replaced with a Bovine taurus ⁇ -GUS enzyme of mammalian origin with a final concentration of 1 nM.
  • the empty plasmid pGex-4T-1 was transformed into E. coli (DH5 ⁇ ), and cultured overnight at 37 ° C in LB (100 ⁇ M ammonia-cillin). Then 1/100 was expanded to OD600 to 0.6, and centrifuged at 8000 rpm for 5 minutes. The precipitate was washed twice with 50 mM HEPES (100 ⁇ M ampicillin, pH 7.4), and the precipitated OD600 was concentrated to 1.0, and the bacterial solution was replaced with the GUS enzyme for experimental detection. The reaction was carried out at 37 ° C for 2 h, and the buffer was 50 mM HEPES (pH 7.4). The other experimental procedures and data processing were the same as the GUS enzyme assay.
  • Cytotoxicity experiments were performed using E. coli bacteria used in GUS cell experiments. Cytotoxicity experiments were performed using two concentrations of the test compound at a final concentration of 100 ⁇ M and 10 ⁇ M (1% DMSO). 20 ⁇ L of the test compound and 80 ⁇ L of the bacterial solution were added to a 96-well plate, 1% DMSO was used as a control group, and reacted at 37 ° C for 2 hours, and then 10 ⁇ L of Cell Counting Kit-8 (Dojindo, Japan) was added to each well, and mixed. The reaction was carried out at 37 ° C for 5 minutes, 30 minutes, and 60 minutes, respectively, and then the absorbance at 490 nm was measured with a multi-function microplate reader (Thermo Scientific, USA).
  • the IC50 curve for inhibiting bacterial ⁇ -GUS activity at the protein level is shown in Figure 1.
  • Aspartame has a good bacterial ⁇ -GUS enzyme inhibitory effect at the protein level with an IC 50 value of 3.6040 ⁇ 0.4755 ⁇ M.
  • the IC50 curve for inhibition of the bacterial ⁇ -GUS enzyme at the cellular level is shown in Figure 2.
  • Aspartame has a good bacterial ⁇ -GUS enzyme inhibitory effect at the cellular level with an IC 50 value of 5.3730 ⁇ 1.6070 ⁇ M.
  • the ⁇ -GUS enzyme activity against mammalian origin was tested using a concentration of 100 ⁇ M aspartame, and it was found that the compound had no significant inhibitory effect on mammalian-derived ⁇ -GUS enzyme ( FIG. 3 ).
  • the CT-26 cell line was used to construct a mouse tumor model. Eighteen female Balb/cJ mice, 6-8 weeks old, were selected and subcutaneously injected with PBS suspension of cells in the posterior dorsal position of the mice. After about 10 days, the tumor of the mouse reached about 500 mm 3 (the tumor volume was calculated by the formula ⁇ /6 ⁇ a 2 ⁇ b, where a is the short axis of the tumor and b is the long axis of the tumor).
  • mice were then randomly divided into three groups for administration: (1) control group, receiving the same volume of intraperitoneal injection of distilled water, and 1% DMSO solution (a total of about 100 ⁇ L twice a day); (2) CPT-11 The drug-administered group was intraperitoneally injected with CPT-11 at a dose of 50 mg/kg and 1% DMSO solution (about 100 ⁇ L in total, twice daily); (3) CPT-11 plus aspartame combination group, intraperitoneal injection CPT-11, a dose of 50 mg/kg, and an aspartame solution (about 100 ⁇ L total, twice daily) at a dose of 50 mg/kg.
  • the CPT-11 administration group and the combination group were continuously injected with CPT-11 for 9 days; the combination group started oral administration of aspartame from the day before CPT-11 injection, and after the end of CPT-11 injection The oral administration was continued for another two days, and then the administration was terminated.
  • the diarrhea status of the control group, the CPT-11 administration group, and the CPT-11 plus aspartame combination group were observed, and the body weight and tumor size of the mice were recorded.
  • the experimental results showed that the incidence of bloody diarrhea in the CPT-11 group was 83.33%, while the incidence of bloody diarrhea in the CPT-11 plus aspartame group was 50%.
  • the combination was more than the single use of CPT-11. Significantly improve the incidence of diarrhea.
  • the weight loss of the combination group was significantly improved compared with the single CPT-11 group (P ⁇ 0.05). At the same time, there was no significant difference in the effect of inhibiting tumor size between the combination group and the single CPT-11 administration group.

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Abstract

本发明公开一种化合物治疗细菌葡萄糖醛酸酶导致的疾病的药物中的用途，该化合物为天冬甜素、及其代谢产物或该化合物在药学上可接受的盐。以上化合物可以用于癌症的治疗、也可以缓解伊立替康引起的腹泻，非甾体类抗炎药导致的胃肠道溃疡，或调节其他经由细菌葡萄糖醛酸酶代谢的药物疗效与副作用，以及由细菌葡萄糖醛酸酶引起的相关病症。

Description

一种天冬甜素的新用途 技术领域
本发明属于医药技术领域，涉及天冬甜素的新用途，具体而言，天冬甜素作为细菌β葡萄糖醛酸酶抑制剂及其应用。
背景技术
通过细菌β-葡萄糖醛酸酶，胃肠道中的微生物从葡萄糖醛酸化的化合物中夺走糖基用于能量供应，导致多种药物或其代谢产物从非毒性状态转化为毒性状态，从而影响药物的代谢和毒副作用 [1,2]。细菌β-葡萄糖醛酸酶影响的症状包括伊立替康导致的迟发性腹泻，以及非甾体类抗炎药导致的胃肠道溃疡等 [3,4]。
伊立替康(Irinotican，CPT-11)，商品名开普拓(Camptosar)，是临床上常用的抗肿瘤化疗药物之一，其主要靶点为I型DNA拓扑异构酶。自1996年上市以来，伊立替康广泛应用于结直肠癌，肺癌，脑肿瘤以及耐药性白血病和淋巴癌的治疗，并被列为治疗转移性结直肠癌的一线治疗药物。多年的临床应用发现，肠毒性是伊立替康主要的剂量限制性副作用之一，表现为迟发性腹泻，通常在病人使用药物24小时以后出现，主要特征包括不可预知性、高发病率及一定程度的致死率。统计数据表明，在接受伊立替康治疗的病人中高达88％的比例出现腹泻症状，其中20-30％的病人出现3-4级严重腹泻(CTCAE分级)，约3.5％的病人因严重腹泻引发的并发症而死亡 [5,6]。相当数目的病人因腹泻必须减少用药剂量甚至停止用药，迄今为止尚无有效治疗手段。
虽然近年来已发展出多种“第二代伊立替康”，如ONIVYDE(又称MM-398,PEP02,或nal-IRI,Merrimack Pharmaceuticals,Inc.)，一种伊 立替康脂质体配方胶囊于2015年被美国FDA批准上市；PEG-SN38(连接聚乙烯的SN38，BelrosePharma Inc.)在临床三期实验阶段。尽管这些“新型伊立替康”提高了生物可利用度和药效，但是腹泻仍是难以克服的剂量限制性副作用。
腹泻发生机制与伊立替康的代谢途径具有密切关系。CPT-11的主要代谢场所为肝脏，CPT-11首先经羧酸酯酶(carboxylesterase，CES)脱去二哌啶基团生成活性代谢产物SN-38；随后在葡萄糖醛酸转移酶(UDP-glucuronosyl-transferase,UGT)的催化下SN-38接受糖基化修饰进一步转化成非活性代谢产物SN-38G [7]。SN-38是伊立替康的主要活性代谢产物，对DNA拓扑异构酶的抑制能力是原药CPT-11及非活性代谢产物SN-38G一百倍以上，对快速***的肿瘤细胞具有极强的杀伤力 [8]。CPT-11及代谢产物部分进入血液循环，其余部分随胆汁排入肠腔。研究发现，肠道中富含的共生细菌将非活性代谢产物SN-38G作为能量来源之一，由细菌的β-葡萄糖苷酸酶(β-glucuronidase，β-GUS)脱去SN-38G的糖基以供给自身的能量，将其重新活化成SN-38，从而导致肠腔中毒性化合物SN-38浓度极大增加，杀伤肠上皮细胞并造成肠粘膜不可逆转的损伤，最终导致了迟发型腹泻的产生 [3,9]。因此选择性抑制肠道内细菌β-GUS酶的活性，可直接降低SN-38的局部浓度并保护肠道组织，从根本上预防和治疗迟发性腹泻。
细菌β-GUS酶以四聚体形式存在，小分子化合物结合在酶的催化口袋，与细菌酶特有的一段17个氨基酸残基组成的loop结构具有较强的相互作用。由于这段loop区域在人的同源蛋白β-GUS中相应区域序列及结构区别明显，与细菌酶特有loop结构的结合使得该系列化合物可选择性抑制细菌中的β-GUS，对哺乳动物的β-GUS则无明 显影响 [3]。动物实验结果表明，细菌β-GUS抑制剂inhibitor 2可降低腹泻的发生率，延长动物的存活时间，进一步验证了该靶点的安全性和有效性。基于酶活性的高通量筛选方法发现了一系列β-GUS抑制剂 [3,10]。但是，由于全新开发的化合物本身的毒理和药学性质尚不清楚，与CPT-11联合使用以后的安全性和药效更加难以评估，因此与临床应用仍存在较大差距。
本发明采用整合虚拟筛选与实验测定的方法发现天冬甜素可选择性抑制细菌葡萄糖醛酸酶。由于天冬甜素是一种食品添加剂，已在人体中使用，其安全性及药学性质明确，可快速进入临床实验，用于缓解伊立替康引起的腹泻，非甾体类抗炎药导致的胃肠道溃疡，或调节其他经由细菌葡萄糖醛酸酶代谢的药物疗效与副作用，以及由细菌葡萄糖醛酸酶引起的相关病症。
发明内容
本发明采用整合虚拟筛选与实验测定的方法发现天冬甜素可选择性抑制细菌葡萄糖醛酸酶。由于天冬甜素是一种食品添加剂，已在人体中使用，其安全性及药学性质明确，可快速进入临床实验，用于缓解伊立替康引起的腹泻，非甾体类抗炎药导致的胃肠道溃疡，或调节其他经由细菌葡萄糖醛酸酶代谢的药物疗效与副作用，以及由细菌葡萄糖醛酸酶引起的相关病症。
本发明提供一种化合物A用于制备治疗细菌葡萄糖醛酸酶导致的疾病的药物中的用途，所述化合物A为式I化合物、式I化合物的代谢产物或式I化合物在药学上可接受的盐。
Figure PCTCN2018112031-appb-000001
本发明所述化合物A优选用于腹泻、胃肠毒性或其他由于细菌葡萄糖醛酸酶所致的病症。所述腹泻包括但不限于是由化疗药物引起；所述胃肠毒性是包括但不限于是由非甾体类抗炎药引起。所述化疗药物包括但不限于伊立替康或其活性代谢物7-乙基-10羟基喜树碱，或其他以喜树碱类化合物及其衍生物为主要有效成分的药物。
本发明化合物A可以和一种或多种药学上可接受的载体组成药物组合物。
本发明还提供一种化疗药物和化合物A在制备***的联合用药中的用途，所述化合物A为式I化合物、式I化合物的代谢产物或式I化合物在药学上可接受的盐。所述化疗药物包括但不限于伊立替康或其活性代谢物7-乙基-10羟基喜树碱，或其他以喜树碱类化合物及其衍生物为主要有效成分的药物。所述化合物A和一种或多种药学上可接受的载体组成药物组合物，该药物组合物的剂型为片剂、胶囊剂、颗粒剂、丸剂或其他可制备的剂型。
本发明所述肿瘤优选为但不限于结直肠癌、胃癌、肝癌、乳腺癌、脑肿瘤、耐药性白血病、淋巴癌、***癌、肺癌或膀胱癌中的一种或多种。。
本发明以药物重定位为基本策略，采用计算机模拟方法对药物分子的有效性进行先期评估，并采用生物学实验方法测活，发现了天冬甜素为有效的细菌β-GUS酶选择性抑制剂，其酶学水平IC 50值为3.6040±0.4755μM(图1)；其细胞水平IC 50值为5.3730±1.6070μM(图2)；对哺乳动物来源的β-GUS酶在100μM的浓度下无明显作用，表明了其良好的选择性(图3)；同时化合物对大肠杆菌的生长无明显影响，表明化合物无明显细胞毒性，可在不杀死大肠杆菌的情况下抑制其β-GUS酶的活性(图4)。
适当剂量的天冬甜素可用于选择性抑制细菌β-GUS酶，从而影响通过细菌β-GUS酶代谢的药物的药效及副作用，治疗由细菌β-GUS酶引发的病症。例如天冬甜素与CPT-11或其他喜树碱类衍生物药物联合使用，可用于降低肠毒性，缓解腹泻副作用，并有可能提高抗肿瘤效果。
由于天冬甜素是一种食品添加剂，已在人体中使用，其安全性及药学性质明确，可快速进入临床实验，用于缓解伊立替康引起的腹泻，或调节其他经由细菌葡萄糖醛酸酶代谢的药物的疗效与副作用，以及其他由细菌葡萄糖醛酸酶引起的相关病症。
附图说明
图1：天冬甜素在蛋白水平上对细菌β-GUS酶抑制能力的IC 50图。天冬甜素在蛋白水平上具有良好的细菌β-GUS酶抑制效果，其IC 50值为3.6040±0.4755μM。
图2：天冬甜素在细胞水平上对细菌β-GUS酶抑制能力的IC 50图。天冬甜素在细胞水平具有良好的细菌β-GUS酶抑制效果，其IC 50值分别为5.3730±1.6070μM。
图3：天冬甜素在100μM浓度下对哺乳动物来源的Bovine taurus β-GUS酶无明显抑制活性。使用100μM浓度的天冬甜素测试其对哺乳动物来源的Bovine taurus β-GUS酶活性，发现化合物对哺乳动物来源的β-GUS酶无明显的抑制效果。
图4：天冬甜素对细菌的细胞毒性实验，其中黑色、深灰色和浅灰色柱状分别表示采用1％DMSO溶液、100μM天冬甜素以及10μM浓度天冬甜素分别与大肠杆菌共孵育的结果。结果表明化合物对大肠杆菌的生长无明显影响，化合物无明显细胞毒性，可在不杀死大肠杆菌的情况下抑制其β-GUS酶的活性。
具体实施例
试验例
一、天冬甜素对细菌葡萄糖醛酸酶抑制活性测定
1.实验方法
1.1基于受体的虚拟筛选
首先采用
Figure PCTCN2018112031-appb-000002
软件包中的蛋白准备模块Protein Preparation Wizard对细菌β-GUS酶的晶体结构(PDB：3LPF)进行处理，包括修正键级，添加氢原子及部分电荷。删除所有的结晶水分子，将整个体系基于OPLS-2005力场进行优化，当RMSD值达到
Figure PCTCN2018112031-appb-000003
Figure PCTCN2018112031-appb-000004
时优化终止。采用
Figure PCTCN2018112031-appb-000005
中的Ligprep 2.5模块对已知药物分子库LOPAC和Microsource Spectrum电子版分子文件进行预处理，包括去重、去除盐离子和无机物，在pH＝7.4条件下产生化合物的可能离子化状态和互变异构体，对于未确定手性中心的手性化合物产生各种可能手性的低能化合物结构，在缺省条件下，每个分子最多产生 32个立体异构体，最后得到大约5万个化合物构象。首先采用Glide SP模式进行对接和打分，保留所有小分子结合模式。在文献报道中，活性分子基本包含两个明显的药效团特征：与GLU413形成氢键，与PHE365形成疏水相互作用。对于对接产生的前1万个小分子结合模式，采取药效团特征作为筛选条件，选出与GLU413形成氢键，并与PHE365形成疏水相互作用的结合模式，最后挑选并购买了部分化合物进行实验活性测定。
1.2细菌葡萄糖醛酸酶酶学水平测活步骤
将4mg/ml细菌β-GUS(300,000×125pM)采用50mM HEPES缓冲液(pH7.4with 0.017％Triton x-100)稀释至1×125pM，底物4MUG采用50mM HEPES(pH7.4)溶解稀释为312.5μM。具体操作步骤为：1)向96孔板(黑板)加入20μl的待测化合物；2)加入40μl GUS酶(125pM)；3)加入40μl底物(4MUG，312.5μM)；4)室温孵育30分钟,加入40μl 1M Na2CO3终止反应；5)在EnVision(Perkin Elmer USA)多功能酶标仪上进行荧光检测：激发波长335nm，发射波长460nm。其中不加化合物的孔板作为阳性对照，不加酶的孔板作为阴性对照。
1.3哺乳动物来源葡萄糖醛酸酶酶学水平测活步骤
具体步骤同细菌葡萄糖醛酸酶酶学水平活性测试实验，其中酶更换为哺乳动物来源的Bovine taurus β-GUS酶，其终浓度为1nM。
1.4细胞水平GUS酶活性测试实验
将空质粒pGex-4T-1转化到E.coli(DH5α)中，37℃，LB(100μM氨比西林)中培养过夜。然后1/100扩大培养到OD600至0.6，8000rpm离心5分钟。用50mM HEPES(100μM氨比西林，pH 7.4)清洗沉 淀两次，浓缩沉淀的OD600至1.0，此菌液将替换GUS酶用于实验检测。反应在37℃进行2h，缓冲液采用50mM HEPES(pH7.4)，其他实验步骤与数据处理均与GUS酶实验相同。
1.5细菌细胞毒性实验
采用GUS细胞实验所用大肠杆菌菌液进行细胞毒性实验。选用待测化合物终浓度100μM和10μM(1％DMSO)两个浓度进行细胞毒性实验。向96孔板中加入待测化合物20μL和菌液80μL，1％DMSO作为对照组，37℃反应2h，然后每孔中加入Cell Counting Kit-8(Dojindo,Japan)10μL，混匀。在37℃分别反应5分钟，30分钟，60分钟，随后用多功能酶标仪(Thermo Scientific,USA)测490nm的吸光度。
2.实验结果
测定了天冬甜素在蛋白水平上针对细菌β-GUS以及哺乳动物来源的β-GUS酶活性，以及化合物在细胞水平上针对细菌β-GUS酶的活性，和化合物的细胞毒性。
在蛋白水平上抑制细菌β-GUS活性的IC50曲线如图1所示。天冬甜素在蛋白水平上具有良好的细菌β-GUS酶抑制效果，其IC 50值为3.6040±0.4755μM。
在细胞水平上抑制细菌β-GUS酶的IC50曲线如图2所示。天冬甜素在细胞水平上具有良好的细菌β-GUS酶抑制效果，其IC 50值为5.3730±1.6070μM。
使用100μM浓度的天冬甜素测试其对哺乳动物来源的β-GUS酶活性，发现该化合物对哺乳动物来源的β-GUS酶无明显的抑制效果(图3)。
使用10μM，100μM浓度的天冬甜素与大肠杆菌共孵育，对大肠 杆菌的生长无明显影响，说明该化合物无明显细胞毒性，可在不杀死大肠杆菌的情况下抑制其β-GUS酶的活性(图4)。
二、天冬甜素缓解伊立替康导致的小鼠腹泻实验
CT-26细胞系被用来构建小鼠肿瘤模型。选取6-8周的雌性Balb/cJ小鼠18只，在小鼠后背部位置皮下注射细胞的PBS悬浮液。10天左右以后，小鼠的肿瘤达到约500mm 3(肿瘤体积用公式π/6×a 2×b计算，其中a为肿瘤的短轴，b为肿瘤的长轴)。然后将小鼠随机分为三组进行给药：(1)对照组，接受同等体积的腹腔注射蒸馏水，以及1％DMSO溶液灌胃(总共约100μL，每天两次)；(2)CPT-11给药组，腹腔注射CPT-11，剂量为50mg/kg,以及1％DMSO溶液灌胃(总共约100μL，每天两次)；(3)CPT-11加天冬甜素联合用药组，腹腔注射CPT-11，剂量为50mg/kg,以及天冬甜素溶液灌胃(总共约100μL，每天两次)，剂量为50mg/kg。为获得小鼠腹泻模型，其中CPT-11给药组以及联合用药组连续注射9天CPT-11；联合用药组从CPT-11注射前一天开始口服天冬甜素，到CPT-11注射结束后，再继续口服给药两天，然后结束给药。分别观察对照组，CPT-11给药组，和CPT-11加天冬甜素联合用药组的腹泻状况，记录小鼠的体重以及肿瘤大小。实验结果显示，CPT-11给药组小鼠血样腹泻发生率为83.33％，而CPT-11加天冬甜素联合用药组血样腹泻的发生率为50％，联合用药比单一使用CPT-11有明显的改善腹泻发生率的作用。此外，联合用药组体重下降情况与单一的CPT-11给药组比也有明显的改善(P<0.05)。同时，联合用药组与单一的CPT-11给药组在抑制肿瘤大 小效果方面无明显差异。

Claims (10)

  1. 一种化合物A用于制备治疗细菌葡萄糖醛酸酶导致的疾病的药物中的用途，其特征在于，所述化合物A为式I化合物、式I化合物的代谢产物或式I化合物在药学上可接受的盐
    Figure PCTCN2018112031-appb-100001
  2. 根据权利要求1所述的用途，其特征在于，所述疾病为腹泻、胃肠毒性或其他由于细菌葡萄糖醛酸酶所致的病症。
  3. 根据权利要求2所述的用途，其特征在于，所述腹泻是由化疗药物引起；所述胃肠毒性是由非甾体类抗炎药引起。
  4. 根据权利要求3所述的用途，其特征在于，所述化疗药物是指伊立替康或其活性代谢物7-乙基-10羟基喜树碱，或其他以喜树碱类化合物及其衍生物为主要有效成分的药物。
  5. 根据权利要求1所述的用途，其特征在于，所述式I化合物和一种或多种药学上可接受的载体组成药物组合物。
  6. 化疗药物和化合物A在制备***的联合用药中的用途，其特征在于，所述化合物A为式I化合物、式I化合物的代谢产物或式I化合物在药学上可接受的盐。
  7. 根据权利要求6所述的用途，其特征在于，所述化疗药物为伊立替康或其活性代谢物7-乙基-10羟基喜树碱，或其他以喜树碱类化合 物及其衍生物为主要有效成分的药物。
  8. 根据权利要求6所述的用途，其特征在于，所述化合物A和一种或多种药学上可接受的载体组成药物组合物。
  9. 根据权利要求8所述的用途，其特征在于，所述药物组合物的剂型为片剂、胶囊剂、颗粒剂、丸剂或其他可制备的剂型。
  10. 根据权利要求6所述的用途，其特征在于，所述肿瘤包括结直肠癌、胃癌、肝癌、乳腺癌、脑肿瘤、耐药性白血病、淋巴癌、***癌、肺癌或膀胱癌中的一种或多种。
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