WO2020082840A1 - 过氧化氢在制备幽门螺杆菌感染根除治疗药物中的应用及制备的药物 - Google Patents

过氧化氢在制备幽门螺杆菌感染根除治疗药物中的应用及制备的药物 Download PDF

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WO2020082840A1
WO2020082840A1 PCT/CN2019/098129 CN2019098129W WO2020082840A1 WO 2020082840 A1 WO2020082840 A1 WO 2020082840A1 CN 2019098129 W CN2019098129 W CN 2019098129W WO 2020082840 A1 WO2020082840 A1 WO 2020082840A1
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helicobacter pylori
hydrogen peroxide
pylori
drugs
wild
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曹永孝
狄佳
曹蕾
刘静
米燕妮
姚彤
肖雪
黄婷婷
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Xian Jiaotong University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/40—Peroxides
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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  • the invention belongs to the field of medicine, and relates to the application of hydrogen peroxide in the preparation of drugs for eradication of Helicobacter pylori infection and the prepared drugs.
  • Helicobacter pylori is a Gram-negative, micro-aerobic, coryneform coryneform bacterium with stringent requirements on growth conditions. Since the discovery of it by Australian researchers Warren and Marshall in 1983, Helicobacter pylori has been proven to be the main causative agent of chronic gastritis, peptic ulcer and gastric mucosa-associated lymphoma globally, especially in the development of gastric cancer. In 1994, the World Health Organization listed Helicobacter pylori as a class I carcinogen (1. Peleteiro B, Bastos A, Ferro A, et al.
  • Helicobacter pylori vaccine such as recombinant Helicobacter pylori vaccine, Helicobacter pylori whole bacteria Vaccines, etc.
  • Hydrogen peroxide is an oxidizing agent.
  • the aqueous solution is commonly known as hydrogen peroxide. It is a colorless and transparent liquid. Its aqueous solution is widely used for disinfection of medical wounds, food, and the environment.
  • the present invention provides the application of hydrogen peroxide in the preparation of drugs for eradication of Helicobacter pylori infection and the prepared drugs.
  • the H. pylori includes H. pylori international standard strain and H. pylori wild-type strain.
  • the wild-type strains of Helicobacter pylori include Helicobacter pylori wild type strain I, Helicobacter pylori wild type II and Helicobacter pylori wild type III.
  • the site of Helicobacter pylori infection is the digestive tract.
  • the digestive tract includes stomach and duodenum.
  • a medicine for eradicating Helicobacter pylori infection which is obtained by preparing hydrogen peroxide into oral liquid, tablet, capsule, powder or granule.
  • the mass concentration of hydrogen peroxide is 0.2% to 1%.
  • the present invention has the following beneficial technical effects:
  • Helicobacter pylori As an obligate microaerobic bacteria, Helicobacter pylori has the most suitable oxygen concentration in the growth environment of 5% -6%. Increasing the oxygen concentration (such as atmospheric oxygen concentration) will lead to the rapid death of Helicobacter pylori. Therefore, the appropriate concentration of hydrogen peroxide quickly decomposes in the stomach to release a large amount of oxygen, significantly increasing the concentration of oxygen in the stomach, so that Helicobacter pylori infection can be effectively eradicated.
  • Hydrogen peroxide solution is catalyzed by catalase in the stomach to rapidly decompose to generate a large amount of oxygen and water, both of which are harmless to the human body, and due to the low concentration, the oxidation is weak, which is insufficient to cause damage to the human gastric mucosa. Therefore, the present invention adopts the "oxygen environment" strategy to transform the problem of "Helicobacter pylori eradication” into the problem of "deprivation of Helicobacter pylori growth conditions", effectively avoiding the defects of existing therapies, and providing new methods for clinical prevention and treatment of Helicobacter pylori infection Ideas.
  • Liquid culture medium determines the minimum inhibitory concentration of hydrogen peroxide
  • FIG. 1A show that the minimum inhibitory concentration of hydrogen peroxide on the Helicobacter pylori international standard strain ATCC43504 is 0.21%. When the hydrogen peroxide concentration is greater than 0.21%, none of the Helicobacter pylori standard strains grow.
  • Figure 1B show that when the Helicobacter pylori international standard strains ATCC43504 with concentrations of 10 6 , 10 7 , 10 8 , and 10 9 CFU / mL were inoculated into a liquid medium containing 0.21% hydrogen peroxide, the four groups of strains had The growth curve was different. The group with the highest inoculation concentration of 10 9 CFU / mL was the longest, followed by 10 8 , 10 7 , and 10 6 CFU / mL.
  • the Helicobacter pylori international standard strain ATCC43504 was prepared into a 1.0 ⁇ 10 8 CFU / mL bacterial suspension, and the Helicobacter pylori solid medium containing concentration gradient hydrogen peroxide and bismuth was poured respectively. Pipette 0.1 mL of bacterial suspension into H. pylori solid medium containing hydrogen peroxide with a concentration gradient, and spread the sterile inoculation ring evenly. Incubator at 37 °C, incubated for 5 days in microaerophilic environment, all colonies were eluted in sterile saline 2mL determine colony eluate OD 600.
  • the Helicobacter pylori standard strain ATCC43504 was made into a 1.0 ⁇ 10 8 CFU / mL bacterial suspension, and a Helicobacter pylori liquid medium containing a concentration gradient of hydrogen peroxide (concentration higher than the minimum inhibitory concentration), blank Helicobacter pylori was prepared Bacteria solid medium. Add 0.12 mL of bacterial suspension to 11.88 mL of liquid medium containing a concentration gradient of hydrogen peroxide, place in a constant temperature shaking incubator at 37 ° C, and cultivate at 150 r / min in a micro-aerobic environment for 3 days.
  • Table 1 The minimum bactericidal concentration of hydrogen peroxide on Helicobacter pylori ATCC43504
  • Liquid culture medium determines the minimum inhibitory concentration of hydrogen peroxide
  • the wild-type strains of Helicobacter pylori-I, II, and III were prepared into 1.0 ⁇ 10 8 CFU / mL bacterial suspension, and the liquid culture medium of Helicobacter pylori containing concentration gradient hydrogen peroxide was prepared at the same time.
  • Draw 0.12mL of three bacterial suspensions respectively and add 11.88mL of Helicobacter pylori liquid medium containing hydrogen peroxide with a concentration gradient to make the entire liquid medium volume 12mL (bacterial liquid: medium 1: 100).
  • results of A, B, and C in Fig. 3 show that the minimum inhibitory concentrations of wild-type H. pylori strains I, II, and III are 0.08%, 0.16%, and 0.05%, respectively.
  • results of D, E, and F in Figure 3 show that the wild-type H. pylori strains I, II, and III with concentrations of 10 6 , 10 7 , 10 8 , and 10 9 CFU / mL were inoculated with 0.04% and 0.08%, respectively.
  • the liquid medium of 0.04% hydrogen peroxide was used, the growth curves of the strains in each group were different. The highest inoculation concentration was 10 9 CFU / mL group leader, followed by approximately 10 8 , 10 7 , 10 6 CFU / mL group.
  • the wild-type strains of Helicobacter pylori-I, II, and III were prepared into 1.0 ⁇ 10 8 CFU / mL bacterial suspension, respectively, and the solid culture medium of Helicobacter pylori containing concentration gradient hydrogen peroxide and bismuth was poured respectively. Pipette 0.1 mL of bacterial suspension into H. pylori solid medium containing hydrogen peroxide with a concentration gradient, and spread the sterile inoculation ring evenly. Placed in a constant temperature incubator at 37 °C, after 5 days of cultivation in a micro-aerobic environment, all colonies were eluted in 2mL sterile physiological saline, and the colony eluent OD 600 was measured.
  • the Helicobacter pylori wild-type strains-I, II, and III were made into 1.0 ⁇ 10 8 CFU / mL bacterial suspension, and the liquid culture medium of Helicobacter pylori with concentration gradient hydrogen peroxide (concentration higher than the minimum inhibitory concentration) was prepared. 3. Blank Helicobacter pylori solid medium. Add 0.12 mL of bacterial suspension to 11.88 mL of liquid medium containing a concentration gradient of hydrogen peroxide, place in a constant temperature shaking incubator at 37 ° C, and cultivate at 150 r / min in a micro-aerobic environment for 3 days.
  • Table 2 The minimum bactericidal concentration of hydrogen peroxide on three wild-type strains of Helicobacter pylori
  • Each group was administered by intragastric administration at 15 mL / kg, once a day, and was sacrificed after 2 consecutive weeks, stained with Giemsa, and the number of Helicobacter pylori was counted under the microscope.
  • the results in Figure 5 show that compared with the blank control group, the model group was significantly infected with Helicobacter pylori; the hydrogen peroxide treatment group had no significant difference compared with the blank control group, and the 0.4% hydrogen peroxide group had better bactericidal effect The 0.2% hydrogen peroxide group, while the bactericidal effect of the hydrogen peroxide group is better than the triple drug group.
  • mice 81 Kunming mice aged 6-8 weeks, weighing 15-22g, and keep them adaptively for 1 week.
  • the Helicobacter pylori international standard strain ATCC43504 was made into a 1.0 ⁇ 10 8 CFU / mL bacterial suspension, which was intragastrically administered at 20 mL / kg once a day for 10 consecutive days.
  • Kunming mice were divided into groups: 1 blank control group, 2 model group, 3 hydrogen peroxide group, 4 Livzon triple group (bismuth potassium citrate, metronidazole, clarithromycin). Each group was administered by intragastric administration, 20 mL / kg, once a day for 14 consecutive days.
  • One third of the animals were sacrificed on days 4, 7, and 14 respectively, stained with Giemsa, and the number of Helicobacter pylori was counted under the microscope.
  • the results in Figure 6A show that the number of Helicobacter pylori in the stomach of the model group Kunming mice is significantly higher than that of the blank control group, indicating successful modeling; the number of Helicobacter pylori in the stomach of the Kunming mice in the hydrogen peroxide group after 4 days of treatment is no different from the blank control group ; The number of Helicobacter pylori in the stomach of Kunming mice in the triple drug group is still higher than that in the blank control group, and the sterilization effect of the triple drug is weaker than that in the hydrogen peroxide group.
  • the results in Figure 6B show that after 7 days of treatment, the bactericidal effect of the hydrogen peroxide group is better than that of the triple drug group.
  • the results in Figure 6C show that after 14 days of treatment, the bactericidal effect of the hydrogen peroxide group is significantly better than that of the triple drug group.
  • Kunming mice aged 6-8 weeks, weighing 15-22g, were selected and kept adaptively for 1 week.
  • the Helicobacter pylori international standard strain ATCC43504 was made into a 1.0 ⁇ 10 8 CFU / mL bacterial suspension, which was intragastrically administered at 20 mL / kg once a day for 10 consecutive days.
  • Kunming mice were divided into groups: 1 blank control group, 2 model group, 3 hydrogen peroxide group, 4 triple drug group. Each group was administered by intragastric administration, 20 mL / kg, once a day for 10 consecutive days.
  • One-third of animals were killed at 5 and 10 days by intragastric administration, and Helicobacter pylori was detected separately. After 14 days of normal feeding without the administration of the remaining animals, observe whether Helicobacter pylori relapses.
  • the human body weight is calculated by 60kg, and the dosages of the above three drugs are: 7.3, 16.7, 8.3mg / kg.
  • the dose of Kunming mice is calculated as 9 times the human dose.
  • the doses of potassium bismuth citrate, metronidazole and clarithromycin are: 65, 150, 75mg / kg.
  • Figure 7A shows that the number of Helicobacter pylori in the model group is significantly higher than that in the blank control group, indicating successful modeling; the number of Helicobacter pylori in the hydrogen peroxide group after 5 days of treatment is not significantly different from the blank control group The number of Helicobacter pylori in the stomach of Kunming mice in the triple drug group is still higher than that in the blank control group, and the sterilization effect of the triple drug is weaker than that in the hydrogen peroxide group.
  • 7B shows that after 10 days of treatment, there is no difference in the number of H. pylori between the hydrogen peroxide group and the blank control group, while the triple drug group still has H. pylori infection compared with the blank control group.
  • the bactericidal effect is better than the triple drug group.
  • the result of Figure 7C shows that after 14 days of administration, the number of Helicobacter pylori in the triple drug group is significantly increased compared with the blank control group, while the number of Helicobacter pylori in the hydrogen peroxide group is not different from the blank control group, indicating hydrogen peroxide Helicobacter pylori had no recurrence in the group, and Helicobacter pylori in the triple drug group had significant recurrence of infection.
  • hydrogen peroxide is made into oral liquid, powder, tablet, capsule, and the mass concentration of hydrogen peroxide is 0.2% -1%, preferably 0.4%.
  • the oral solution includes the following raw materials: purified water, 30% hydrogen peroxide solution (medicinal), xylitol, and citric acid.
  • the powder includes the following raw materials: starch, solid hydrogen peroxide (2Na 2 CO 3 .3H 2 O 2 ), anhydrous citric acid.
  • the tablet includes the following raw materials: lactose, sodium bicarbonate, magnesium stearate, solid hydrogen peroxide (2Na 2 CO 3 .3H 2 O 2 ), citric acid.
  • the capsule includes the following raw materials: starch, solid hydrogen peroxide (2Na 2 CO 3 .3H 2 O 2 ), anhydrous citric acid.
  • each capsule weighs 0.3g, that is, the finished capsule.
  • the invention relates to a new use of hydrogen peroxide in eradication of Helicobacter pylori.
  • the hydrogen peroxide has a good killing effect on Helicobacter pylori, in particular, it can effectively prevent Helicobacter pylori from producing drug resistance and recurring infection.
  • the minimum inhibitory concentration range of hydrogen peroxide against Helicobacter pylori international standard strains and clinical wild-type strains is 0.05% -0.21%. After 30 generations of H. pylori acting on hydrogen peroxide, the minimum inhibitory concentration range is 0.08% -0.40%.
  • the mouse Helicobacter pylori infection model had no recurrence after treatment with hydrogen oxide.
  • the new use of hydrogen peroxide provides a new option for eradicating Helicobacter pylori drugs, which can be used to treat diseases caused by Helicobacter pylori infection, including chronic gastritis, peptic ulcer, gastric mucosa-associated lymphoma and gastric cancer.

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Abstract

过氧化氢在制备幽门螺杆菌感染根除治疗药物中的应用及制备的药物。适宜浓度的过氧化氢在胃内迅速分解释放大量氧，显著提高胃内氧浓度，使幽门螺杆菌感染得以有效根除。实验证明，适宜浓度的过氧化氢对幽门螺杆菌国际标准菌株和幽门螺杆菌野生型菌株均具有较好的杀灭作用。小鼠幽门螺杆菌感染模型经过氧化氢治疗后无复发。

Description

过氧化氢在制备幽门螺杆菌感染根除治疗药物中的应用及制备的药物 技术领域
本发明属于医药学领域，涉及过氧化氢在制备幽门螺杆菌感染根除治疗药物中的应用及制备的药物。
背景技术
幽门螺杆菌(Helicobacter pylori,Hp)是一种革兰氏阴性、微需氧、对生长条件要求苛刻的弯曲棒状杆菌。从1983年澳大利亚学者Warren和Marshall发现它至今，幽门螺杆菌在全球范围内已被证实是慢性胃炎、消化性溃疡和胃粘膜相关淋巴瘤的主要致病因子，尤其与胃癌的发生关系密切。1994年世界卫生组织将幽门螺杆菌列为I类致癌因子(1.Peleteiro B,Bastos A,Ferro A,et al.Prevalence of Helicobacter pylori infection worldwide:a systematic review of studies with national coverage[J].Dig Dis Sci,2014,59(8):1698-1709；2.Grad YH,Lipsitch M,Aiello AE.Secular trends in Helicobacter pylori seroprevalence in adults in the United States:evidence for sustained race/ethnic disparities[J].Am J Epidemiol,2012,175(1):54-59；3.张万岱，胡伏莲，萧树东，等.中国自然人群幽门螺杆菌感染的流行病学调查[J].现代消化及介入诊疗,2010,15(5):265-270；4.Moeller H,Correa P.Carcinogenicity of some biological agents[J].Cancer Epidemiol Biomarkers Prev,1994,3(7):627)。目前资料显示，全球各个国家和地区均有不同程度的幽门螺杆菌感染，范围超过50％，其中拉丁美洲与拉脱维亚的感染率高达79％。根据中国幽门螺杆菌科研协作组的调查结果(刘力生,王文,姚崇华.中国高血压防治指南(2009)[J].中国高血压杂志,2010,18(1):1-30)，我国幽门螺杆菌自然人群中的平均感染率64％，西藏自治区的平均感染率85％(胡伏莲.幽门螺杆菌研究聚焦和进展[J].胃肠病学,2015(12):705-707)。
上世纪90年代，标准三联方案(质子泵抑制剂+克拉霉素+阿莫西林/ 甲硝唑)是根除幽门螺杆菌最常用的方案，疗效好(Lind T,et al.Eradication of Helicobacter pylori using one-week triple therapies combining omeprazole with two antimicrobials:the MACH I Study[J].Helicobacter,1996；1(3):138-44)，但随着细菌耐药率的显著升高，幽门螺杆菌的根除效果发生了显著变化。美国、欧洲、韩国幽门螺杆菌的根除率低于80％，近年我国报道的根除率低至60％(Malfertheiner P,Bazzoli F,Delchier J C.Helicobacter pylori eradication with a capsule containing bismuth subcitrate potassium,metronidazole,and tetracycline given with omeprazole versus clarithromycin-based triple therapy:a randomised,open-label,non-inferiority,phase 3 trial(vol377,pg 905,2011)[J].LANCET,2011,378(9805):1778-1778)。为提高幽门螺杆菌的根除率，采取的策略主要集中在：三联疗法的改进与加强、中药抗菌与幽门螺杆菌疫苗三个方面。第一，针对三联疗法采取改进与加强措施，将疗程从7天增加到14天，同时增加抗菌药剂量，药物种类由3种增加到4种；第二，采用中药代替抗菌药发挥抗菌作用，避免耐药率升高，例如半夏泻心汤、胃复散、荆花胃康胶丸以及三九胃泰；第三，幽门螺杆菌疫苗，如幽门螺杆菌重组疫苗、幽门螺杆菌全菌疫苗等。根除幽门螺杆菌的方法虽多，但要达到有效根除却并不容易。第一，在三联疗法基础上增加药物种类、延长疗程的缺点：随着抗菌药的长时间大剂量应用，导致幽门螺杆菌的耐药率上升，成为影响幽门螺杆菌根除治疗效果的主要因素；第二，中成药代替抗菌素的缺点：药物成分复杂，抗菌效果不稳定；第三，幽门螺杆菌疫苗的缺点：疫苗的研发尚处在初级阶段，疫苗作用易受佐剂等因素影响，疫苗引发的免疫反应对人体的安全性尚不明确。
过氧化氢是一种氧化剂，水溶液俗称双氧水，为无色透明液体，其水溶液广泛应用于医用伤口、食品、环境等消毒，关于过氧化氢能够治疗幽门螺杆菌感染还未见报道。
发明内容
针对现有技术中存在的问题，本发明提供过氧化氢在制备幽门螺杆菌感染根除治疗药物中的应用及制备的药物。
本发明是通过以下技术方案来实现：
过氧化氢在制备幽门螺杆菌感染根除治疗药物中的应用。
优选的，所述幽门螺杆菌包括幽门螺杆菌国际标准菌株和幽门螺杆菌野生型菌株。
进一步的，所述幽门螺杆菌野生型菌株包括幽门螺杆菌野生型菌株Ⅰ、幽门螺杆菌野生型Ⅱ和幽门螺杆菌野生型Ⅲ。
优选的，所述幽门螺杆菌感染的部位为消化道。
进一步的，所述消化道包括胃和十二指肠。
过氧化氢在制备根除治疗幽门螺杆菌感染性疾病药物中的应用，所述幽门螺杆菌感染性疾病包括慢性胃炎、消化性溃疡、胃粘膜相关淋巴瘤和胃癌。
一种根除治疗幽门螺杆菌感染的药物，将过氧化氢制成口服液、片剂、胶囊剂、粉剂或颗粒剂得到。
优选的，过氧化氢的质量浓度为0.2％～1％。
与现有技术相比，本发明具有以下有益的技术效果：
幽门螺杆菌作为一种专性微需氧菌，其生长环境最适宜的氧浓度为5％-6％，提高氧浓度(如大气环境氧浓度)则导致幽门螺杆菌迅速死亡。因此，适宜浓度的过氧化氢在胃内迅速分解释放大量氧，显著提高胃内氧浓度，使幽门螺杆菌感染得以有效根除。实验证明，适宜浓度的过氧化氢对幽门螺杆菌国际标准菌株和幽门螺杆菌野生型菌株均具有较好的杀灭作用，特别是能够有效防止幽门螺杆菌产生耐药及复发感染。小鼠幽门螺杆菌感染模型经过氧化氢治疗后无复发，能达到根除的作用，为根除幽门螺杆菌药物提供了新选择。过氧化氢溶液受到胃内过氧化氢酶催化迅速分解生成大量氧和水，二 者均对人体无害，而由于浓度低，氧化作用弱，不足以造成人体胃粘膜损伤。因此本发明采用“氧环境”策略，将“幽门螺杆菌根除难题”转化为“剥夺幽门螺杆菌生长必须条件”的问题，有效避免了现有疗法的缺陷，为临床防治幽门螺杆菌感染提供新的思路。
附图说明
图1A为过氧化氢对液体培养幽门螺杆菌国际标准菌株的最小抑菌浓度；均数±标准差，n＝6，与对照组比较*p<0.05，**p<0.01；
图1B为四组不同浓度幽门螺杆菌国际标准菌株的生长曲线；均数±标准差，n＝6，与对照组比较*p<0.05，**p<0.01；
图2为过氧化氢对固体培养幽门螺杆菌国际标准菌株的最小抑菌浓度；CBS为铋剂组，NS为生理盐水；均数±标准差，n＝5，与对照组比较*p<0.05，**p<0.01。
图3A为不同浓度过氧化氢对幽门螺杆菌野生型菌株Ⅰ的作用；均数±标准差，n＝6，与对照组比较*p<0.05，**p<0.01。
图3B为不同浓度过氧化氢对幽门螺杆菌野生型菌株Ⅱ的作用；均数±标准差，n＝6，与对照组比较*p<0.05，**p<0.01。
图3C为不同浓度过氧化氢对幽门螺杆菌野生型菌株Ⅲ的作用；均数±标准差，n＝6，与对照组比较*p<0.05，**p<0.01。
图3D为过氧化氢对不同浓度幽门螺杆菌野生型菌株Ⅰ的作用；均数±标准差，n＝6，与对照组比较*p<0.05，**p<0.01。
图3E为过氧化氢对不同浓度幽门螺杆菌野生型菌株Ⅱ的作用；均数±标准差，n＝6，与对照组比较*p<0.05，**p<0.01。
图3F为过氧化氢对不同浓度幽门螺杆菌野生型菌株Ⅲ的作用；均数± 标准差，n＝6，与对照组比较*p<0.05，**p<0.01。
图4为过氧化氢对固体培养幽门螺杆菌野生型菌株-Ⅰ、Ⅱ、Ⅲ的最小抑菌浓度；均数±标准差，n＝6，与对照组比较*p<0.05，**p<0.01。
图5为过氧化氢给药14天对沙鼠幽门螺杆菌感染的作用；吉姆萨染色，1000×，PBC为枸橼酸铋钾组，均数，n＝8，与对照组比较*p<0.05，**p<0.01。
图6为过氧化氢给药4、7、14天对小鼠幽门螺杆菌感染的作用；吉姆萨染色，1000×，均数，n＝12，与对照组比较*p<0.05，**p<0.01。
图7为过氧化氢清除小鼠幽门螺杆菌感染后的复发情况；吉姆萨染色，1000×，n＝12，与对照组比较*p<0.05，**p<0.01。
具体实施方式
下面结合具体的实施例对本发明做进一步的详细说明，所述是对本发明的解释而不是限定。
实验1：过氧化氢对幽门螺杆菌国际标准菌株的作用
(1)液体培养基测定过氧化氢的最小抑菌浓度
将幽门螺杆菌国际标准菌株ATCC43504配制成为1.0×10 8CFU/mL菌悬液，同时配制含浓度梯度过氧化氢的幽门螺杆菌液体培养基。吸取0.12mL菌悬液加入11.88mL含浓度梯度过氧化氢的幽门螺杆菌液体培养基，使整个液体培养基体积为12mL(菌液:液体培养基＝1:100)。置37℃恒温震荡培养箱，以150r/min在微需氧环境下培养5天，观察幽门螺杆菌生长情况，测定液体培养基OD 600并绘制生长曲线。
将幽门螺杆菌国际标准菌株ATCC43504分别配制成为1.0×10 6CFU/mL、1.0×10 7CFU/mL、1.0×10 8CFU/mL、1.0×10 9CFU/mL菌悬液，分别吸取 0.12mL接种于含0.21％过氧化氢的幽门螺杆菌液体培养基，使培养基体积为12mL(菌液:培养基＝1:100)。置37℃恒温震荡培养箱，以150r/min在微需氧环境下培养5天，观察幽门螺杆菌生长情况，测定培养基OD 600并绘制生长曲线。
图1A结果显示，过氧化氢对幽门螺杆菌国际标准菌株ATCC43504的最小抑菌浓度为0.21％，当过氧化氢浓度大于0.21％时，幽门螺杆菌标准菌株均不生长。图1B结果显示，将浓度分别为10 6、10 7、10 8、10 9CFU/mL的幽门螺杆菌国际标准菌株ATCC43504分别接种于含0.21％过氧化氢的液体培养基时，四组菌株的生长曲线出现差异，接种浓度最高的10 9CFU/mL组率先生长，随后分别为10 8、10 7、10 6CFU/mL组。
(2)固体培养基测定过氧化氢的最小抑菌浓度
将幽门螺杆菌国际标准菌株ATCC43504配制成为1.0×10 8CFU/mL菌悬液，分别浇筑含浓度梯度过氧化氢、铋剂的幽门螺杆菌固体培养基。吸取0.1mL菌悬液加入含浓度梯度过氧化氢的幽门螺杆菌固体培养基，无菌接种环涂抹均匀。置37℃恒温培养箱，在微需氧环境下培养5天后，将全部菌落洗脱于2mL无菌生理盐水中，测定菌落洗脱液OD 600。图2结果显示，与空白对照组相比，幽门螺杆菌国际标准菌株ATCC43504在含0.1％过氧化氢的固体培养基中生长受限，在含0.2％过氧化氢的固体培养基中生长抑制，在含0.8％过氧化氢的固体培养基中生长显著抑制。随着固体培养基中过氧化氢浓度的增加，标准菌株生长量显著降低。
(3)过氧化氢最小杀菌浓度的测定
将幽门螺杆菌际标准菌株ATCC43504制成为1.0×10 8CFU/mL菌悬液，同时配制含浓度梯度过氧化氢的幽门螺杆菌液体培养基(浓度高于最小抑菌 浓度)、空白的幽门螺杆菌固体培养基。将0.12mL菌悬液加入11.88mL含浓度梯度过氧化氢的液体培养基，放置于37℃恒温震荡培养箱，以150r/min在微需氧环境下培养3天。吸取0.1mL培养3天的菌悬液，分别加入空白固体培养基，无菌接种环涂抹均匀。放置于37℃恒温培养箱，在微需氧环境下培养5天后，观察标准菌株的生长情况。表1结果显示，幽门螺杆菌国际标准菌株ATCC43504经含2.28％过氧化氢的液体培养基培养后，转种至空白幽门螺杆菌固体培养基，无菌落生长。
表1过氧化氢对幽门螺杆菌ATCC43504的最小杀菌浓度
Figure PCTCN2019098129-appb-000001
实验2：过氧化氢对幽门螺杆菌野生型菌株的作用
(1)液体培养基测定过氧化氢的最小抑菌浓度
将幽门螺杆菌野生型菌株-Ⅰ、Ⅱ、Ⅲ分别配制成为1.0×10 8CFU/mL菌悬液，同时配制含浓度梯度过氧化氢的幽门螺杆菌液体培养基。分别吸取0.12mL三种菌悬液加入11.88mL含浓度梯度过氧化氢的幽门螺杆菌液体培养基，使整个液体培养基体积为12mL(菌液:培养基＝1:100)。放置于37℃恒温震荡培养箱，以150r/min在微需氧环境下培养5天，观察幽门螺杆菌生长情况，测定整个液体培养基OD 600并绘制生长曲线。
将幽门螺杆菌野生型菌株-Ⅰ、Ⅱ、Ⅲ分别配制成为1.0×10 6、10 7、10 8、10 9CFU/mL菌悬液，分别吸取0.12mL接种于含0.04％、0.08％、0.04％过氧化氢的幽门螺杆菌液体培养基，使培养基体积为12mL(菌液:培养基＝1:100)。放置于37℃恒温震荡培养箱，以150r/min在微需氧环境下培养5天，观察幽门螺杆菌生长情况，测定整个液体培养基OD 600并绘制生长曲线。
图3中A、B、C结果显示，幽门螺杆菌野生型菌株Ⅰ、Ⅱ、Ⅲ的最小抑菌浓度分别为0.08％、0.16％、0.05％。图3中D、E、F结果显示，将浓度分别为10 6、10 7、10 8、10 9CFU/mL的幽门螺杆菌野生型菌株Ⅰ、Ⅱ、Ⅲ分别接种于含0.04％、0.08％、0.04％过氧化氢的液体培养基时，各组菌株的生长曲线出现差异，接种浓度最高的10 9CFU/mL组先生长，随后大致为10 8、10 7、10 6CFU/mL组。
(2)固体培养基测定过氧化氢的最小抑菌浓度
将幽门螺杆菌野生型菌株-Ⅰ、Ⅱ、Ⅲ分别配制成为1.0×10 8CFU/mL菌悬液，分别浇筑含浓度梯度过氧化氢、铋剂的幽门螺杆菌固体培养基。吸取0.1mL菌悬液加入含浓度梯度过氧化氢的幽门螺杆菌固体培养基，无菌接种环涂抹均匀。放置于37℃恒温培养箱，在微需氧环境下培养5天后，将全部菌落洗脱于2mL无菌生理盐水中，测定菌落洗脱液OD 600。图4结果显示，与空白对照组相比，幽门螺杆菌野生型菌株-Ⅰ、Ⅱ、Ⅲ在含1.6％、0.8％、0.2％过氧化氢的固体培养基中生长受限，在含3.2％、1.6％、0.4％过氧化氢的固体培养基中生长显著抑制。随着固体培养基中过氧化氢浓度的增加，三种野生型菌株生长量显著降低。
(3)过氧化氢最小杀菌浓度的测定
将幽门螺杆菌野生型菌株-Ⅰ、Ⅱ、Ⅲ制成为1.0×10 8CFU/mL菌悬液，同时配制含浓度梯度过氧化氢的幽门螺杆菌液体培养基(浓度高于最小抑菌浓度)、空白的幽门螺杆菌固体培养基。将0.12mL菌悬液加入11.88mL含浓度梯度过氧化氢的液体培养基，放置于37℃恒温震荡培养箱，以150r/min在微需氧环境下培养3天。吸取0.1mL培养3天的菌悬液，分别加入空白固体培养基，无菌接种环涂抹均匀。放置于37℃恒温培养箱，在微 需氧环境下培养5天后，观察三种野生型菌株的生长情况。表2结果显示，幽门螺杆菌野生型菌株-Ⅰ、Ⅱ、Ⅲ分别在含有0.24％、0.32％、0.32％过氧化氢的液体培养基培养后，转种至空白幽门螺杆菌固体培养基，无菌落生长。
表2过氧化氢对3种幽门螺杆菌野生型菌株的最小杀菌浓度
菌株 过氧化氢的最小杀菌浓度(％)
菌株Ⅰ 0.24
菌株Ⅱ 0.32
菌株Ⅲ 0.32
实验3：过氧化氢筛选压力下幽门螺杆菌国际标准菌株的最小抑菌浓度变化
上述实验证实，过氧化氢对幽门螺杆菌国际标准菌株的最小抑菌浓度为0.21％，为使幽门螺杆菌国际标准菌株在过氧化氢筛选压力下继续生长，选取低于最小抑菌浓度的过氧化氢浓度0.20％作为幽门螺杆菌国际标准菌株的诱导浓度。配制含0.20％过氧化氢的幽门螺杆菌液体培养基50mL，分别吸取3.6mL培养基于50mL无菌试管，每管等量添加1×10 8CFU/mL幽门螺杆菌国际标准菌株ATCC43504菌液0.4mL，重复5管，每管放置于恒温震荡培养箱，在37℃、150r/min条件下培养24小时记为诱导1次，当分别诱导10、20、30次时，测定过氧化氢诱导后幽门螺杆菌国际标准菌株的MIC 10、MIC 20、MIC 30。表3结果显示，0.20％过氧化氢诱导幽门螺杆菌国际标准菌株10、20、30代后，最小抑菌浓度分别为0.25％、0.29％、0.37％。
表3过氧化氢筛选压力下幽门螺杆菌ATCC 43504的最小抑菌浓度(MIC)变化
诱导数(次) MIC(％)
0 0.21
10 0.25
20 0.29
30 0.37
实验4：过氧化氢筛选压力下幽门螺杆菌野生型菌株的最小抑菌浓度变化
上述实验证实，过氧化氢对幽门螺杆菌野生型菌株-Ⅰ、Ⅱ、Ⅲ的最小抑菌浓度分别为：0.08％、0.16％、0.05％，为使三种野生型菌株在过氧化氢筛选压力下继续生长，因此选取稍低于最小抑菌浓度的过氧化氢浓度：0.04％、0.08％、0.04％分别作为幽门螺杆菌野生型菌株-Ⅰ、Ⅱ、Ⅲ的诱导浓度。表4结果显示，过氧化氢分别诱导幽门螺杆菌野生型菌株-Ⅰ、Ⅱ、Ⅲ生长10、20、30代后，幽门螺杆菌野生型菌株-I的最小抑菌浓度无变化，均为0.08％；幽门螺杆菌野生型菌株-II的最小抑菌浓度增加至0.40％；幽门螺杆菌野生型菌株-Ⅲ的最小抑菌浓度增加至0.30％。
表4过氧化氢筛选压力下幽门螺杆菌野生型菌株-Ⅰ、Ⅱ、Ⅲ的最小抑菌浓度(MIC)变化
Figure PCTCN2019098129-appb-000002
实验5：过氧化氢对沙鼠和小鼠幽门螺杆菌定植的清除作用
1.沙鼠取4周龄蒙古沙鼠80只，体重40-50g，适应性饲养1周。将幽门螺杆菌国际标准菌株ATCC43504制成为1.0×10 8CFU/mL菌悬液，15mL/kg灌胃，每日1次，连续2周。确证幽门螺杆菌模型成功后将蒙古沙鼠分组：①空白对照组，②模型组，③～⑤分别为过氧化氢0.1％、0.2％、0.4％组，⑥丽珠维三联组(枸橼酸铋钾、甲硝唑、克拉霉素)，⑦枸橼酸铋钾组。各组以15mL/kg进行灌胃给药，每日1次，连续2周后处死，进行吉姆萨染色，显微镜下计数幽门螺杆菌数目。图5结果显示，与空白对照组 相比，模型组沙鼠感染幽门螺杆菌明显；过氧化氢治疗组与空白对照组相比，二者无显著差异，0.4％过氧化氢组杀菌效果优于0.2％过氧化氢组，同时过氧化氢组杀菌效果优于三联药物组。
2.小鼠选取6-8周龄昆明小鼠81只，体重15-22g，适应性饲养1周。将幽门螺杆菌国际标准菌株ATCC43504制成为1.0×10 8CFU/mL菌悬液，以20mL/kg进行灌胃，每日1次，连续10天。确证幽门螺杆菌模型成功后将昆明小鼠分组：①空白对照组，②模型组，③过氧化氢组，④丽珠维三联组(枸橼酸铋钾、甲硝唑、克拉霉素)。各组灌胃给药，20mL/kg，每日1次，连续14天。第4、7、14天分别处死三分之一动物，进行吉姆萨染色，显微镜下计数幽门螺杆菌数目。
图6A结果显示，模型组昆明小鼠胃内幽门螺杆菌数目显著高于空白对照组，说明造模成功；过氧化氢组昆明小鼠治疗4天后胃内幽门螺杆菌数目与空白对照组无差别；三联药物组昆明小鼠胃内幽门螺杆菌数目仍高于空白对照组，三联药物杀菌效果弱于过氧化氢组。图6B结果显示，经过7天给药治疗，过氧化氢组的杀菌效果优于三联药物组。图6C结果显示，经过14天给药治疗，过氧化氢组的杀菌效果明显优于三联药物组。
实验6：昆明小鼠幽门螺杆菌清除后复发
选取6-8周龄昆明小鼠105只，体重15-22g，适应性饲养1周。将幽门螺杆菌国际标准菌株ATCC43504制成为1.0×10 8CFU/mL菌悬液，以20mL/kg灌胃，每日1次，连续10天。确证幽门螺杆菌模型成功后将昆明小鼠分组：①空白对照组，②模型组，③过氧化氢组，④三联药物组。各组灌胃给药，20mL/kg，每日1次，连续10天。灌胃给药第5、10天各处死三分之一动物，分别检测幽门螺杆菌。剩余动物不给药正常饲喂14天后，观 察幽门螺杆菌是否复发感染。
给药剂量依据：枸橼酸铋钾、甲硝唑和克拉霉素临床日用量分别为：440、1000、500mg。人体重按60kg计算，上述三种药物的给药剂量分别为：7.3、16.7、8.3mg/kg。昆明小鼠剂量按人剂量9倍计算，枸橼酸铋钾、甲硝唑和克拉霉素的给药剂量分别为：65、150、75mg/kg。
表5幽门螺杆菌感染模型昆明小鼠给药方案
Figure PCTCN2019098129-appb-000003
图7A结果显示，模型组小鼠胃内幽门螺杆菌数目显著高于空白对照组，说明造模成功；过氧化氢组小鼠治疗5天后胃内幽门螺杆菌数目与空白对照组无显著性差别；而三联药物组昆明小鼠胃内幽门螺杆菌数目仍高于空白对照组，三联药物杀菌效果弱于过氧化氢组。图7B结果显示，经过10天给药治疗，过氧化氢组与空白对照组幽门螺杆菌数目无差别，而三联药物组与空白对照组相比，仍存在幽门螺杆菌感染，过氧化氢组的杀菌效果优于三联药物组。图7C结果显示，给药结束后观察14天，三联药物组与空白对照组相比，幽门螺杆菌数目显著增多，而过氧化氢组幽门螺杆菌数目与空白对照组无差别，说明过氧化氢组幽门螺杆菌基本无复发，三联药物组幽门螺杆菌显著复发感染。
实验7：过氧化氢口服剂型的制备
本发明将过氧化氢制成口服液、粉剂、片剂、胶囊剂，过氧化氢质量浓 度均为0.2％-1％，优选0.4％。
所述口服液包括以下原料：纯净水、30％过氧化氢溶液(药用)、木糖醇、柠檬酸。
(1)按照上述配方量取30％过氧化氢溶液13.3mL，称取木糖醇200g，柠檬酸3g，备用；
(2)将上述30％过氧化氢溶液加纯净水溶解至1L，所得0.4％过氧化氢溶液和木糖醇、柠檬酸一同加入至调配罐中，通过搅拌器充分搅拌混合均匀；
(3)经过滤、杀菌、灌装至50mL的瓶中并自动封口，最后经贴标签装箱，即得成品口服液。
与上述口服液制备方法相同，只是将30％过氧化氢溶液的量改为6.5mL，使所得口服液中过氧化氢浓度为0.2％。
所述粉剂包括以下原料：淀粉、固体过氧化氢(2Na 2CO 3·3H 2O 2)、无水柠檬酸。
(1)按照上述配方称取淀粉4g、固体过氧化氢(2Na 2CO 3·3H 2O 2)0.06g，无水柠檬酸0.9g，备用；
(2)将上述原料混合，粉碎至200目
(3)经杀菌后包装至5g的袋中并封口、贴标签，即得成品粉剂。
与上述粉剂制备方法相同，只是将固体过氧化氢的量改为0.15g，使所得粉剂中过氧化氢浓度为1％。
所述片剂包括以下原料：乳糖、碳酸氢钠、硬脂酸镁、固体过氧化氢(2Na 2CO 3·3H 2O 2)、枸橼酸。
(1)按照上述配方称取乳糖73.8g、碳酸氢钠18g、硬脂酸镁5g、固体 过氧化氢(2Na 2CO 3·3H 2O 2)1.2g、枸橼酸2g，备用；
(2)将上述原料混合，粉碎至100目，混匀、杀菌；
(3)20目筛制粒，加硬脂酸镁，压片制成100片，包薄膜衣即得成品片剂。
所述胶囊剂包括以下原料：淀粉、固体过氧化氢(2Na 2CO 3·3H 2O 2)、无水柠檬酸。
(1)按照上述配方称取淀粉96g、固体过氧化氢(2Na 2CO 3·3H 2O 2)1.2g，无水柠檬酸2.8g，备用；
(2)将上述原料混合，粉碎至200目；
(3)经杀菌后灌装胶囊，每粒重0.3g，即得成品胶囊剂。
本发明涉及过氧化氢在根除幽门螺杆菌中的新用途。所述的过氧化氢对幽门螺杆菌有良好杀灭作用，特别是能够有效防止幽门螺杆菌产生耐药及复发感染。过氧化氢对幽门螺杆菌国际标准菌株及临床野生型菌株的最小抑菌浓度范围为0.05％-0.21％。过氧化氢作用幽门螺杆菌30代后，其最小抑菌浓度范围为0.08％-0.40％。小鼠幽门螺杆菌感染模型经过氧化氢治疗后无复发。所述过氧化氢的新用途为根除幽门螺杆菌药物提供了新选择，可以用治疗幽门螺杆菌感染引起的疾病，包括慢性胃炎、消化性溃疡、胃粘膜相关淋巴瘤和胃癌。

Claims (8)

  1. 过氧化氢在制备幽门螺杆菌感染根除治疗药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述幽门螺杆菌包括幽门螺杆菌国际标准菌株和幽门螺杆菌野生型菌株。
  3. 根据权利要求2所述的应用，其特征在于，所述幽门螺杆菌野生型菌株包括幽门螺杆菌野生型菌株Ⅰ、幽门螺杆菌野生型Ⅱ和幽门螺杆菌野生型Ⅲ。
  4. 根据权利要求1所述的应用，其特征在于，所述幽门螺杆菌感染的部位为消化道。
  5. 根据权利要求4所述的应用，其特征在于，所述消化道包括胃和十二指肠。
  6. 过氧化氢在制备根除治疗幽门螺杆菌感染性疾病药物中的应用，其特征在于，所述幽门螺杆菌感染性疾病包括慢性胃炎、消化性溃疡、胃粘膜相关淋巴瘤和胃癌。
  7. 一种根除治疗幽门螺杆菌感染的药物，其特征在于，将过氧化氢制成口服液、片剂、胶囊剂、粉剂或颗粒剂得到。
  8. 根据权利要求7所述的根除治疗幽门螺杆菌感染的药物，其特征在于，过氧化氢的质量浓度为0.2％～1％。
PCT/CN2019/098129 2018-10-22 2019-07-29 过氧化氢在制备幽门螺杆菌感染根除治疗药物中的应用及制备的药物 Ceased WO2020082840A1 (zh)

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