WO2018157750A1 - 一种利福霉素-硝基咪唑偶联分子的新应用 - Google Patents

一种利福霉素-硝基咪唑偶联分子的新应用 Download PDF

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WO2018157750A1
WO2018157750A1 PCT/CN2018/076969 CN2018076969W WO2018157750A1 WO 2018157750 A1 WO2018157750 A1 WO 2018157750A1 CN 2018076969 W CN2018076969 W CN 2018076969W WO 2018157750 A1 WO2018157750 A1 WO 2018157750A1
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rifamycin
anaerobic
administration
bacteroides
nitroimidazole
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PCT/CN2018/076969
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English (en)
French (fr)
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马振坤
袁鹰
刘宇
王晓梅
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丹诺医药(苏州）有限公司
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Priority to US16/489,348 priority Critical patent/US20200093807A1/en
Priority to EP18760546.4A priority patent/EP3574900B1/en
Priority to JP2019546356A priority patent/JP2020510654A/ja
Priority to CA3054831A priority patent/CA3054831A1/en
Publication of WO2018157750A1 publication Critical patent/WO2018157750A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • 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
    • A61K31/438—The ring being spiro-condensed with carbocyclic or heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/552—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being an antibiotic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4858—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of medical chemistry, and in particular relates to a new application of a rifamycin-nitroimidazole coupling molecule.
  • Anaerobic bacteria are bacteria that grow on the surface of solid media that are grown under anaerobic conditions and are not able to grow in air (18% oxygen) and/or 10% carbon dioxide. . Such bacteria lack a complete metabolic enzyme system and their energy metabolism proceeds in an anaerobic fermentation.
  • Anaerobic bacteria include the following categories:
  • GPAC Gram-positive anaerobic cocci
  • Including Peptostreptococcus, Finegoldia, Anaerococcus, Peptoniphilus, Veillonella, etc. is the human mouth, upper respiratory tract, gastrointestinal tract and Part of the female genitourinary microflora, a conditional pathogen, can cause various degrees of infection in human parts (25-30% of all anaerobic infections), including mild skin suppuration or severe brain and dura mater External abscess, bacteremia, endocarditis, necrotizing pneumonia and toxic abortion. GPAC causes up to 40% of pleural/pulmonary infections, and the mortality rate can be as high as 75% in the severe wound infections it causes. GAPC can also cause eye, facial features and head, neck infection, meningitis, pericarditis, femoral and joint (including artificial joint) infection, breast abscess, urinary tract infection.
  • GNPC is one of the normal flora of human oral cavity, genitourinary tract, respiratory tract and intestinal tract. The proportion of clinical samples is very low, but there is an increasing trend.
  • Clostridium can cause Clostridium bacteremia, food poisoning, necrotic enteritis, iatrogenic diarrhea (CDI), antibiotic-associated pseudomembranous colitis, skin and soft tissue infections.
  • CDI iatrogenic diarrhea
  • BV Bacterial Vaginosis
  • Gardnerella Vaginalis is one of the main causes of BV. Metronidazole is still the first line of clinical treatment for BV, although the short-term cure rate for BV can reach 70% to 80%. The recurrence rate within 3 months can be as high as 58%. The presence of GV-resistant metronidazole-resistant strains and biofilm formation may be important causes of BV recurrence and treatment failure. Studies have shown that the biofilm's response to antibacterial drugs is significantly different from its planktonic growth pattern, which may be related to the special growth state of the bacteria itself in the biofilm and the biofilm's reduced penetration of antibacterial drugs. Therefore, the discovery of the biofilm structure produced by Gardnerella is a new hotspot in BV recurrence and drug resistance research.
  • the object of the present invention is to propose a novel application of a rifamycin-nitroimidazole coupling molecule, which can effectively inhibit anaerobic flora and can be used for treating bacterial vaginosis.
  • the anaerobic flora comprises Actinomyces naeslundii, Anaerococcus prevotii, Atopobium vaginae, Bacteroides fragilis ( Bacteroides fragilis) (including metronidazole-resistant), Bacteroides thetaiotaomicron (including metronidazole-resistant), Bacteroides gracilis, Bacteroides uniformis, common Bacteroides vulgatus, Bacteroides ovatus (including metronidazole-resistant), Bifidobacterium breve, Bifidobacterium longum, Clostridium Sporogenes), Clostridium perfringens (including metronidazole-resistant), Eubacterium rectale, Fusobacterium nucleatum, Gardnerella Vaginalis), Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensen Ii), Mobiluncus
  • curtisii Mobiluncus mulieris
  • Peptococcus Peptoniphilus asaccharolyticus (including metronidazole resistance) Type)
  • Peptostreptococcus Peptostreptococcus anaerobius
  • Prevotella bivia including metronidazole-resistant
  • Prevotella A combination of one or more of disiens
  • Prevotella intermedia Treponema denticola, and Veionella parvula.
  • the present invention also provides the use of the above rifamycin-nitroimidazole-coupled molecule as a medicament for the preparation of a Bacterial Vaginosis (BV) caused by anaerobic dysregulation.
  • BV Bacterial Vaginosis
  • the effective dose of the rifamycin-nitroimidazole coupling molecule is 10 mg-10 g per day, and the treatment period is 1-15 days.
  • the administration mode employed by the application includes one or a combination of injection administration, oral administration, intraluminal administration, enteral administration, and transdermal absorption.
  • the administration form used in the application includes one or a combination of an injection, a suppository, a tablet, a capsule, a patch, and a sustained release agent.
  • the rifamycin-nitroimidazole coupling molecule of the formula I of the present invention has broad-spectrum antibacterial activity, including activity against anaerobic bacteria such as most vaginal pathogens, and its antibacterial activity in vitro.
  • the activity is stronger than anti-BV drugs such as metronidazole and clindamycin, and has application prospects for the prevention and treatment of anaerobic genus, bacterial vaginosis caused by species, and other anaerobic infections mentioned above.
  • This embodiment provides the use of the rifamycin-nitroimidazole coupling molecule of Formula I for inhibiting anaerobic bacteria
  • anaerobic bacteria include Actinomyces faecalis, Plasmodium anaerobacter, Atopo vaginal bacteria, Bacteroides fragilis (including metronidazole-resistant type), Bacteroides variabilis (including metronidazole-resistant type) ), Bacteroides, Bacteroides, Bacteroides, Bacteroides ovum (including metronidazole-resistant), Bifidobacterium breve, Bifidobacterium longum, Clostridium sporogenes, gas capsule Clostridium (including metronidazole-resistant), Escherichia coli, Clostridium nucleatum, Gardnerella vaginalis, Lactobacillus crispus, Lactobacillus garerii, Lactobacillus johnsonii, C.
  • the clinical isolates tested were either reference bacteria from the American Type Culture Collection (ATCC, Manassas, VA). After the strains were received, they were separately inoculated onto a suitable agar plate and placed under optimized conditions for growth. The grown clone was made into a bacterial suspension in a culture medium containing a cryoprotectant, and stored in a frozen form at -80 ° C after dispensing. Prior to testing, the frozen bacteria were inoculated into a suitable agar dish and grown for growth. Anaerobic bacteria were grown for 48 hours at 35 degrees C in a Bactron II anaerobic cabinet (Shel Lab, Cornelius, OR) prior to testing.
  • the medium for anaerobic agar dilution susceptibility testing is supplemented with Brucella agar (SBA), containing 5 ⁇ g/ml of hemin (BD/BBL; Cat. No. 5300551), 1 ⁇ g/ml of Vitamin K1 (Sigma).
  • MIC Minimum Inhibitory Concentration
  • the MIC value of all microorganisms except Haemophilus was determined using the agar dilution method (1-2) in CLSI. Drug dilution and preparation of drug-containing agar plates were performed manually according to the CLSI guidelines (1-2). To dry the agar surface, the multiwell plate was allowed to stand at room temperature for 1 hour. The agar plates used for testing under anaerobic conditions were pre-set in an oxygen free cabinet for about 1 hour. Each isolate was adjusted to a 0.5 McFarland turbidity standard in a suitable medium using a nephelometer (Dade Behring MicroScan, Wet Sacramento, CA). Each bacterial suspension was then transferred to the wells of the assay plate using a stainless steel replicator.
  • the rifamycin-nitroimidazole coupling molecule (Formula I) is in vitro bacteriostatic to most anaerobic bacteria except for the activity of a few strains and the most commonly used anti-anaerobic drug metronidazole.
  • the activity (MIC) is 100 to 1000 times higher than the nitrate.
  • MIC is 0.03-0.0005 ⁇ g / ml.
  • the broad-spectrum anti-anaerobic activity of the fumycin-nitroimidazole-coupled molecule (Formula I) can be predicted to have a good therapeutic effect on infections caused by other species of bacteria being tested.
  • the effective dose for the bacterial vaginosis is estimated to be 1/100 of the effective dose of metronidazole, equivalent to 10 mg per day, in order to reach For good efficacy, the dose of the rifamycin-nitroimidazole-coupled molecule (Formula I) can continue to increase to 10 grams and has reached its highest effective dose.
  • This example provides a formulation and method of preparation of a rapid release oral formulation of a rifamycin-nitroimidazole coupling molecule of Formula I.
  • the rifamycin-nitroimidazole coupling molecules and excipients of the formula I are weighed according to the above prescribed amounts.
  • Povidone K30 (PVP K30) and sodium dodecyl sulfate (SDS) were dissolved in purified water, stirred for 1 hour, and used as a binder; the rifamycin-nitroimidazole represented by Formula I was coupled.
  • Molecules, mannitol and sodium carboxymethyl starch (DST) were passed through a 30 mesh sieve, added to a granulator, premixed, and the impeller agitation speed was 700 rpm for about 15 minutes.
  • a peristaltic pump to add a proper amount of purified water and binder to the granulator mixture at a fixed speed (145-165 g/min).
  • the granulator impeller stirring speed is 400 rpm for about 1-2 minutes.
  • the binder is added, Continue mixing for 0.5 to 1 minute; dry the wet particles with a fluidized bed, set the inlet air temperature to 60 ° C, and the inlet air volume to 40 m 3 /h; add silica and hard according to the weight of the dried dry particulate material.
  • Magnesium citrate first mixed with silica and dry pellet hopper mixer, mixing time 15 minutes; rotation speed 20 rpm; then add magnesium stearate, mixing time 6 minutes, mixing speed 20 rpm, take the total mixing
  • the material was measured by filling the No. 0 capsule with a capsule filling machine, that is, the rifamycin-nitroimidazole coupled molecular hard capsule represented by the formula I was obtained.
  • the total mixed material is tableted by a tableting machine to obtain a rifamycin-nitroimidazole coupled molecular tablet of the formula I.
  • This example provides an injection preparation method of the rifamycin-nitroimidazole coupling molecule of Formula I.
  • This example provides a method for preparing a topical gelling agent of the rifamycin-nitroimidazole coupling molecule of Formula I.
  • the carbomer was wetted with an appropriate amount of ethanol, diluted to about 1600 mL with purified water under stirring, boiled, and further stirred to make it a transparent gel solution, and allowed to cool. Then take the rifamycin-nitroimidazole coupling molecule of formula I, natural borneol and flavor, dissolve it with ethanol and add it to the gel transparent solution together with the surfactant, then dilute to 2000 mL with purified water, and mix well. , placed for more than 12 hours, atmospheric pressure defoaming, after the test, the rifamycin-nitroimidazole coupled molecular external gel as shown in Formula I was obtained.

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Abstract

本发明揭示了一种式（Ⅰ）所示的利福霉素-硝基咪唑偶联分子在抑制厌氧菌群中的应用。本发明式（Ⅰ）的利福霉素-硝基咪唑偶联分子对厌氧菌具有广谱抗菌活性，包括对绝大多数与细菌性阴道病相关的致病菌具有活性，其体外抗菌活性强于甲硝唑和克林霉素等抗细菌性阴道病药物，对厌氧菌引起的细菌性阴道病或其它相关疾病的预防与治疗方面具有很好的应用前景。

Description

一种利福霉素-硝基咪唑偶联分子的新应用 技术领域
本发明属于医药化学领域，尤其涉及一种利福霉素-硝基咪唑偶联分子的新应用。
背景技术
厌氧菌(anaerobic bacteria)是一类在无氧条件下比在有氧环境中生长好，而不能在空气(18％氧气)和(或)10％二氧化碳浓度下的固体培养基表面生长的细菌。这类细菌缺乏完整的代谢酶体系，其能量代谢以无氧发酵的方式进行。
厌氧菌包括以下几类：
1、革兰氏阳性厌氧球菌(Gram-positive anaerobic cocci，GPAC)
包括消化链球菌(Peptostreptococcus)，微单胞菌(Finegoldia)，厌氧球菌(Anaerococcus)，嗜胨菌(Peptoniphilus)，韦永氏球菌(Veillonella)等，是人口腔、上呼吸道、胃肠道和女性生殖泌尿系统微生物菌群的一部分，为条件致病菌，可造成人体各部分不同程度的感染(占全部厌氧菌感染的25-30％)，包括轻微皮肤化脓或严重的脑及硬膜外脓肿，菌血症，心内膜炎，坏死性肺炎和浓毒性流产。GPAC造成的的胸膜/肺部感染达40％，在其造成的严重伤口感染中，死亡率可高达75％。GAPC还可造成眼、五官及头，颈感染，脑膜炎，心周炎，股和关节(包括人工关节)感染，乳房脓肿，尿道感染等。
2、革兰氏阴性厌氧球菌(Gram-negative anaerobic cocci，GNPC)
GNPC是人口腔，生殖泌尿道，呼吸道及肠道正常菌群之一，临床样本中比重很低，但有增加趋势。
3、非孢子形成革兰氏阳性厌氧杆菌(Non-spore-forming anaerobic Gram-positive rods)
包括丙酸杆菌(Propionibacterium)，乳酸杆菌(Lactobacillus)，放线菌(Actinomyces)，真细菌(Eubacterium)，埃格特菌(Eggerthella)，阿托波菌(Atopobium)，双歧杆菌(Bifidobactrium)，动弯杆菌(Mobiluncus)等。多为与粘膜相关的复合型感染，口腔和粪便是主要感染源。是术后死亡率和发病率的重要原因。
4、革兰氏阴性厌氧杆菌(Anaerobic Gram-negative rods)
包括拟杆菌(Bacteroides)，卟啉单胞菌(Porphyromonas)，普雷沃菌(Prevotella)，梭形杆菌(Fusobacterium)等，主要寄生于人口腔、胃肠道及阴道粘膜，感染一般由于粘膜受损引起，例如阴道炎和牙周炎。
5、孢子形成革兰氏阳性厌氧杆菌(Spore-forming Gram-positive rods)
包括梭状芽孢杆菌属(Clostridium)，可造成梭状芽胞杆菌菌血症，食物中毒，坏死性肠炎，医源性腹泻(CDI)，抗菌素相关伪膜性结肠炎，皮肤及软组织感染等。
细菌性阴道病(Bacterial Vaginosis，BV)是育龄期女性常见的下生殖道感染性疾病，是由于正常阴道微生态菌群构成发生变化引起的一种无阴道粘膜炎症表现的综合征。全世界每年约有数百万女性罹患细菌性阴道病，严重影响健康。BV可引发不良妊娠结局，如自然流产、早产、羊水感染、产后子宫内膜炎和剖腹产切口感染及围产儿并发症等。此外，BV的复发和持续感染还能增加滴虫阴道炎、外阴阴道假丝酵母菌病、宫颈癌和人类免疫缺陷病毒(human immunodeficiency virus，HIV)感染的风险。
阴道加德纳菌(Gardnerella Vaginalis，GV)是BV的主要致病原因之一，甲硝唑仍是目前BV临床常规治疗的一线用药，虽然对BV的短期治愈率可以到达70％～80％，而3个月内的复发率却可高达58％。GV的甲硝唑耐药菌株的存在和生物膜的形成可能是BV复发与治疗失败的重要原因。研究表明生物膜中的细菌对抗菌药物的反应与其浮游生长方式存在显著差异，可能与细菌本身在生物膜中的特殊生长状态和生物膜降低了抗菌药物的穿透力有关。因此，加德纳菌生成的生物膜结构的发现是BV复发与耐药研究的新热点。
在美国专利US 7,678,791B2中公开了化合物4-脱氧-3,4-[2-螺环-[2-(2-甲基-5-硝基-咪唑-1-基)乙基]-哌啶-4-基]]-(1氢)-咪唑并-(2,5-二氢)利福霉素S，该化合物对大肠埃希杆菌等几种细菌具有抗微生物活性，但并没有文献记载其对厌氧菌群具有抗菌活性。
发明内容
鉴于上述现有技术存在的缺陷，本发明的目的是提出一种利福霉素-硝基咪唑偶联分子的新应用，能够有效抑制厌氧菌群，能够用于治疗细菌性阴道病。
本发明的目的将通过以下技术方案得以实现：
一种式Ⅰ所示的利福霉素-硝基咪唑偶联分子在抑制厌氧菌群中的应用；
Figure PCTCN2018076969-appb-000001
优选的，上述的应用中，所述厌氧菌群包括内氏放线菌(Actinomyces naeslundii)、普氏厌氧球菌(Anaerococcus prevotii)、阿托波阴道菌(Atopobium vaginae)、脆弱类拟杆菌(Bacteroides fragilis)(包括甲硝唑耐药型)、多形拟杆菌(Bacteroides thetaiotaomicron)(包括甲硝唑耐药型)、纤维拟杆菌(Bacteroides gracilis)、单形拟杆菌(Bacteroides uniformis)、普通拟杆菌(Bacteroides vulgatus)、卵形拟杆菌(Bacteroides ovatus)(包括甲硝唑耐药型)、短双歧杆菌(Bifidobacterium breve)、长双歧杆菌(Bifidobacterium longum)、产芽胞梭状芽胞杆菌(Clostridium sporogenes)、产气荚膜梭状芽胞杆菌(Clostridium perfringens)(包括甲硝唑耐药型)、直肠真杆菌(Eubacterium rectale)、核粒梭形杆菌(Fusobacterium nucleatum)、阴道加德纳菌(Gardnerella vaginalis)、卷曲乳杆菌(Lactobacillus crispatus)、加氏乳杆菌(Lactobacillus gasseri)、詹氏乳杆菌(Lactobacillus jensenii)、柯氏动弯杆菌(Mobiluncus(curtisii subsp.curtisii))、羞怯动弯杆菌(Mobiluncus mulieris)、消化球菌(Peptococcus)、不解糖嗜胨菌(Peptoniphilus asaccharolyticus)(包括甲硝唑耐药型)、大消化链球菌(Peptostreptococcus)、厌氧消化链球菌(Peptostreptococcus anaerobius)、二路普雷沃菌(Prevotella bivia)(包括甲硝唑耐药型)、解糖胨普雷沃菌(Prevotella disiens)、中间普雷沃菌(Prevotella intermedia)、齿垢密螺旋体(Treponema denticola)和小韦永氏球菌(Veionella parvula)中的一种或多种的组合。
本发明还提供上述的利福霉素-硝基咪唑偶联分子在作为制备治疗由厌氧菌群失调引起的细菌性阴道病(Bacterial Vaginosis，BV)的药物中的应用。
优选的，上述的应用中，所述利福霉素-硝基咪唑偶联分子的人体有效剂量为每日10mg-10g，治疗周期为1-15天。
优选的，上述的应用中，所述应用采用的给药方式包括注射给药、口服给药、腔内给药、肠内给药和透皮吸收中的一种或几种的组合。
优选的，上述的应用中，所述应用所采用的给药剂型包括注射剂、栓剂、片剂、胶囊剂、贴剂和缓释剂中的一种或几种的组合。
本发明的突出效果为：本发明式Ⅰ的利福霉素-硝基咪唑偶联分子具有广谱抗菌活性，包括对绝大多数阴道致病菌在内的厌氧菌具有活性，其体外抗菌活性强于甲硝唑和克林霉素等抗BV药物，对被测定厌氧菌属、种引起的细菌性阴道病和以上所述其它厌氧菌感染的预防与治疗方面具有应用前景。
以下便结合实施例对本发明的具体实施方式作进一步的详述，以使本发明技术方案更易于理解、掌握。
具体实施方式
下面通过具体实施例对本发明的方法进行说明，但本发明并不局限于此。下述实施例中所述实验方法，如无特殊说明，均为常规方法；所述试剂和材料，如无特殊说明，均可从商业途径获得。
实施例1
本实施例提供式Ⅰ所示的利福霉素-硝基咪唑偶联分子在抑制厌氧菌中的应用；
Figure PCTCN2018076969-appb-000002
其中，厌氧菌包括内氏放线菌、普氏厌氧球菌、阿托波阴道菌、脆弱类拟杆菌(包括甲硝唑耐药型)、多形拟杆菌(包括甲硝唑耐药型)、纤维拟杆菌、单形拟杆菌、普通拟杆菌、卵形拟杆菌(包括甲硝唑耐药型)、短双歧杆菌、长双歧杆菌、产芽胞梭状芽胞杆菌、产气荚膜梭状芽胞杆菌(包括甲硝唑耐药型)、直肠真杆菌、核粒梭形杆菌、阴道加德纳菌、卷曲乳杆菌、加氏乳杆菌、詹氏乳杆菌、柯氏动弯杆菌、羞怯动弯杆菌、消化球菌(尼日尔)、不解糖嗜胨菌(包括甲硝唑耐药型)、大消化链球菌、厌氧消化链球菌、二路普雷沃菌(包括甲硝唑耐药型)、解糖胨普雷沃菌、中间普雷沃菌、齿垢密螺旋体和小韦永氏球菌中的一种或多种的组合。
本实施例中，式Ⅰ所示的利福霉素-硝基咪唑偶联分子对细菌性阴道病相关的病原菌的所有测试均使用与临床和实验室标准研究所(Clinical and Laboratory Standards Institute,CLSI；1-3)的指南一致的琼脂稀释法。测试均在无氧条件下进行。对照组化合物为甲硝唑，利褔平和克林霉素。
材料和方法
测试化合物
由丹诺医药有限公司提供，检测前在-20摄氏度保存。三种对照药物由Sigma提供。所有母液在自动除菌前均放置至少1小时。
测试菌株
检测的临床分离株或为来自美国标准菌种保藏中心(American Type Culture Collection，ATCC，Manassas，VA)的参照菌。收到菌株后，将它们分别接种与合适的琼脂板上并置于优化的条件下生长。将长出的克隆在含有冷冻保护剂的培养液中制成细菌悬浮液，分装后在-80摄氏度冷冻保存。测试前，将冷冻的细菌接种到合适的琼脂皿并培养生长。厌氧菌于在测试前于Bactron II无氧柜(Shel Lab，Cornelius，OR)中35摄氏度生长48小时。
测试培养基
用于厌氧琼脂稀释法药敏检测的培养基是补充Brucella琼脂(SBA)，由含有5微克/毫升的血晶素(BD/BBL；货号：5300551)，1微克/毫升的维生素K1(Sigma,St.Louis,MO；货号SLBC4685V)和5％的色淀绵羊血(Cleveland Scientific,Bath,OH,货号291958)的Brucella琼脂。
以上所有培养基的制备和储存均按照CLSI(1-3)进行。
琼脂稀释法测定最低抑菌浓度(Minimum Inhibitory Concentrations,MIC)
应用CLSI中的琼脂稀释法(1-2)确定除嗜血杆菌属外的所有微生物的MIC值。按照CLSI指南(1-2)手工进行药物稀释和制备含药物的琼脂板。为使琼脂表面干燥，将多孔板在室温放置1小时。将用于厌氧条件下测试的琼脂板在无氧柜中预置约1小时。用比浊计(Dade Behring MicroScan，Wet Sacramento，CA)把每一分离株在合适的培养基中调节至0.5麦克法兰氏浊度标准。然后用不锈钢复制器将每个细菌悬液转移到检测板的孔中。大约含10(5)/1-2微升的细菌接种到每孔的琼脂表面，干燥后将药物板和无药对照板放入无氧柜中厌氧环境35摄氏度哺育42-48小时。培养后按照CLSI指南确定MIC(1-2)。
测试结果如下表1所示。
表1
Figure PCTCN2018076969-appb-000003
Figure PCTCN2018076969-appb-000004
Figure PCTCN2018076969-appb-000005
由上述测试结果可见，利福霉素-硝基咪唑偶联分子(式Ⅰ)除对少数菌株与目前最常用抗厌氧菌药物甲硝唑的活性相当外，对绝大部分厌氧菌的体外抑菌活性(MIC)高于硝唑100至1000倍。对于常见BV优势菌如阴道加德纳菌，普雷沃菌，动弯杆菌，阿托波阴道菌和消化链球菌均有极强的抑菌活性(MIC为0.03-0.0005微克/毫升)。利福霉素-硝基咪唑偶联分子(式I)对于主要BV菌加德纳菌的抑菌作用(MIC＝0.004微克/毫升)大大强于其两个母体抗菌素，甲硝唑(MIC＝4微克/毫升)和利福平(MIC＝0.5微克/毫升)，显示出化合物I结构中两个共价偶联的功能团之间有很强的协同作用。从福霉素-硝基咪唑偶联分子(式I)的广谱抗厌氧菌活性可以预测其对被检测属的其它种细菌造成的感染也会有好的治疗作用。
根据利福霉素-硝基咪唑偶联分子(式I)的MIC值预测其针对细菌性阴道病的有效剂量为甲硝唑的有效剂量的1/100，相当于每天10毫克，为达到更好的药效，利福霉素-硝基咪唑偶联分子(式I)的剂量可以继续增加到10克已达到其最高有效剂量。
实施例2
本实施例提供式Ⅰ所示的利福霉素-硝基咪唑偶联分子的一种快速释放口服制剂的处方和制备方法。
Figure PCTCN2018076969-appb-000006
按上述处方量称取式Ⅰ所示的利福霉素-硝基咪唑偶联分子和辅料。将聚维酮K30(PVP K30)和十二烷基硫酸钠(SDS)，溶解于纯化水中，搅拌1小时，作为黏合剂备用；将式Ⅰ所示的利福霉素-硝基咪唑偶联分子、甘露醇和羧甲淀粉钠(DST)过30目筛，加入制粒机中，预混，叶轮搅拌速度700rpm，时间约15分钟。再用蠕动泵以固定的速度(145-165g/分钟)加适量的纯化水和黏合剂到制粒机混合物中，制粒机叶轮搅拌速度400rpm，时间约1～2分钟，黏合剂加入完毕后，继续混合0.5～1分钟；采用流化床对湿颗粒进行干燥，设进风温度为60℃，进风量40m 3/h；根据干燥后的干颗粒物料的重量计算应添加二氧化硅和硬脂酸镁，先将二氧化硅与干颗粒置料斗混合器中进行混合，混合时间15分钟；转速20rpm；再加入硬脂酸镁，混合时间6分钟，混合速度为20rpm，取总混后的物料测采用胶囊灌装机充填0号胶囊，即得式Ⅰ所示的利福霉素-硝基咪唑偶联分子硬胶囊剂。
将总混后的物料采用压片机压片，即得式Ⅰ所示的利福霉素-硝基咪唑偶联分子片剂。
实施例3
本实施例提供式Ⅰ所示的利福霉素-硝基咪唑偶联分子的一种注射剂制备方法。
Figure PCTCN2018076969-appb-000007
Figure PCTCN2018076969-appb-000008
在氮气保护下将甘露醇、乙醛次硫酸钠、吐温-80加入适量注射用水中，加入式Ⅰ所示的利福霉素-硝基咪唑偶联分子，中速搅拌10-15分钟，湿润式Ⅰ所示的利福霉素-硝基咪唑偶联分子，1N NaOH缓慢滴加，耗时约175分钟(前快后慢)，至式Ⅰ所示的利福霉素-硝基咪唑偶联分子全部溶解，0.45+0.22μm两道微孔滤膜过滤，滤液灌装至10mL玻璃瓶中，每瓶装3.5mL，玻璃瓶转移至冻干机中进行冻干，扎盖后即得式Ⅰ所示的利福霉素-硝基咪唑偶联分子的冻干粉针剂。
实施例4
本实施例提供式Ⅰ所示的利福霉素-硝基咪唑偶联分子的一种外用凝胶剂制备方法。
Figure PCTCN2018076969-appb-000009
将卡波姆用适量乙醇润湿，搅拌下用纯化水稀释到大约1600mL，煮沸，继续搅拌使其呈透明状凝胶液，放凉。再取式Ⅰ所示的利福霉素-硝基咪唑偶联分子、天然冰片和香精，用乙醇溶解与表面活性剂一起加入凝胶透明液中，再用纯化水定容至2000mL，搅拌均匀，放置12小时以上，常压脱泡，经检验后分装即得式Ⅰ所示的利福霉素-硝基咪唑偶联分子外用凝胶剂。

Claims (6)

  1. 一种式Ⅰ所示的利福霉素-硝基咪唑偶联分子在抑制厌氧菌群中的应用；
    Figure PCTCN2018076969-appb-100001
  2. 根据权利要求1所述的应用，其特征在于，所述厌氧菌群包括内氏放线菌、普氏厌氧球菌、阿托波阴道菌、脆弱类拟杆菌、多形拟杆菌、纤维拟杆菌、单形拟杆菌、普通拟杆菌、卵形拟杆菌、短双歧杆菌、长双歧杆菌、产芽胞梭状芽胞杆菌、产气荚膜梭状芽胞杆菌、直肠真杆菌、核粒梭形杆菌、阴道加德纳菌、卷曲乳杆菌、加氏乳杆菌、詹氏乳杆菌、柯氏动弯杆菌、羞怯动弯杆菌、消化球菌、不解糖嗜胨菌、大消化链球菌、厌氧消化链球菌、二路普雷沃菌、解糖胨普雷沃菌、中间普雷沃菌、齿垢密螺旋体和小韦永氏球菌中的一种或多种的组合。
  3. 权利要求1所述的利福霉素-硝基咪唑偶联分子在作为制备治疗由厌氧菌群失调引起的细菌性阴道病的药物中的应用。
  4. 根据权利要求3所述的应用，其特征在于，所述利福霉素-硝基咪唑偶联分子的人体有效剂量为每日10mg-10g，治疗周期为1-15天。
  5. 根据权利要求3所述的应用，其特征在于，所述应用采用的给药方式包括注射给药、口服给药、腔内给药、肠内给药和透皮吸收中的一种或几种的组合。
  6. 根据权利要求3所述的应用，其特征在于，所述应用所采用的给药剂型包括注射剂、栓剂、片剂、胶囊剂、贴剂和缓释剂中的一种或几种的组合。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106860451A (zh) * 2017-02-28 2017-06-20 丹诺医药(苏州）有限公司 一种利福霉素‑硝基咪唑偶联分子的新应用
CN108047250B (zh) * 2018-02-12 2020-08-14 丹诺医药(苏州）有限公司 一种利福霉素-硝基咪唑偶联分子的应用
CN109464673A (zh) * 2019-01-08 2019-03-15 丹诺医药(苏州）有限公司 利福霉素-喹嗪酮偶联分子及其盐的应用和制剂
WO2024036578A1 (en) * 2022-08-18 2024-02-22 Tennor Therapeutics (Suzhou) Limited Methods for preventing or treating h. pylori infection
CN117535208B (zh) * 2024-01-04 2024-03-29 四川厌氧生物科技有限责任公司 一种卷曲乳杆菌及其在女性生殖道健康中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008008480A2 (en) * 2006-07-12 2008-01-17 Cumbre Pharmaceuticals Inc. Nitroheteroaryl-containing rifamycin derivatives
CN104971061A (zh) * 2015-06-09 2015-10-14 丹诺医药(苏州）有限公司 一种利福霉素-硝基咪唑偶联分子的新用途
CN105037389A (zh) * 2015-06-09 2015-11-11 丹诺医药(苏州）有限公司 一种利福霉素-硝基咪唑偶联分子的制备方法
CN106860451A (zh) * 2017-02-28 2017-06-20 丹诺医药(苏州）有限公司 一种利福霉素‑硝基咪唑偶联分子的新应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ITBO20110461A1 (it) * 2011-07-29 2013-01-30 Alfa Wassermann Spa Composizioni farmaceutiche comprendenti rifaximina, processi per la loro preparazione e loro uso nel trattamento di infezioni vaginali.
MX2015011802A (es) * 2013-03-15 2016-01-08 Alfa Wassermann Spa Rifaximina para usarse en el tratamiento de infecciones vaginales.

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008008480A2 (en) * 2006-07-12 2008-01-17 Cumbre Pharmaceuticals Inc. Nitroheteroaryl-containing rifamycin derivatives
US7678791B2 (en) 2006-07-12 2010-03-16 Cumbre Ip Ventures, L.P. Nitroheteroaryl-containing rifamycin derivatives
CN104971061A (zh) * 2015-06-09 2015-10-14 丹诺医药(苏州）有限公司 一种利福霉素-硝基咪唑偶联分子的新用途
CN105037389A (zh) * 2015-06-09 2015-11-11 丹诺医药(苏州）有限公司 一种利福霉素-硝基咪唑偶联分子的制备方法
CN106860451A (zh) * 2017-02-28 2017-06-20 丹诺医药(苏州）有限公司 一种利福霉素‑硝基咪唑偶联分子的新应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3574900A4

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3868378A4 (en) * 2018-10-16 2021-11-17 Tennor Therapeutics Limited SOLID DISPERSION OF RIFAMYCIN NITROIMIDAZOLE COUPLING MOLECULES AND THEIR USES
CN117535207A (zh) * 2024-01-04 2024-02-09 四川厌氧生物科技有限责任公司 一种格氏乳杆菌及其应用

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