CN102503994A - Method of treating clostridium difficile-associated diarrhea - Google Patents

Method of treating clostridium difficile-associated diarrhea Download PDF


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CN102503994A CN2011103037682A CN201110303768A CN102503994A CN 102503994 A CN102503994 A CN 102503994A CN 2011103037682 A CN2011103037682 A CN 2011103037682A CN 201110303768 A CN201110303768 A CN 201110303768A CN 102503994 A CN102503994 A CN 102503994A
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A method of treating a disease or disorder caused by the presence of a bacterium selected from the group consisting Clostridium species, Staphylococcus species, Enterococcus species and combinations thereof comprising administering to a patient in need an effective amount of a mixture, which comprises tiacumicin B, lipiarmycin A4, and at least one of other macrocyclic compounds .


治疗艰难梭菌相关的腹泻的方法 Method of treating C. difficile-associated diarrhea

[0001] 本申请是于2006年10月23日提出的国际申请PCT/US2006/041436(国家申请号: 200680047986. 1,发明名称:治疗艰难梭菌相关的腹泻的方法)的分案申请。 [0001] This application is October 23, 2006 proposed international application PCT / US2006 / 041436 (national application number: 200680047986.1, Title: Clostridium difficile-associated diarrhea treatment method) of the divisional application.

[0002] 相关申请 [0002] RELATED APPLICATIONS

[0003] 本申请要求于2005年10月21日提交的美国临时专利申请系列号60/7¾,135,和于2005年12月12日提交的美国临时申请系列号60/749,641的权益。 [0003] This application claims the benefit of US Provisional Patent Application Serial No. 60 on October 21, 2005 submitted / 7¾, 135, and the benefit of US Provisional Application Serial No. 12 December 2005, filed 60 / 749,641 of.

[0004] 上述相关申请的公开内容通过引用完全结合于本文中。 [0004] The relevant disclosure of which is fully incorporated by reference herein.

[0005] 发明背景 [0005] Background of the Invention

[0006] 1.发明领域 [0006] 1. Field of the Invention

[0007] 本发明涉及治疗由选自由梭菌属(Clostridium)物种,葡萄球菌属(Staphylococcus)物种和肠球菌属(Enterococcus)物种及其组合组成的组的细菌的存在引起的疾病,特别是由选自由艰难梭菌(Clostridium difficile, “ C. difficile"), 产气荚膜梭菌(Clostridium perfringens, “ C. perfringens “),金黄色葡萄球菌(Staphylococcus aureus, “ S. aureus")及它们的组合组成的组的细菌的存在引起的疾病,更具体地治疗由艰难梭菌的存在引起的疾病。 [0007] The present invention relates to the presence of bacterial diseases the treatment of selected from the group consisting of Clostridium (Clostridium) species, Staphylococcus (Staphylococcus) and Enterococcus species (of Enterococcus) species, and combinations thereof caused in particular by selected from Clostridium difficile (Clostridium difficile, "C. difficile"), Clostridium perfringens (Clostridium perfringens, "C. perfringens"), Staphylococcus aureus (Staphylococcus aureus, "S. aureus") and their bacterial diseases present the group consisting of a combination of causes, and more particularly to the treatment of diseases caused by the presence of C. difficile. 所述疾病可以是结肠炎,假膜性结肠炎, 或腹泻。 The disease may be colitis, pseudomembranous colitis, or diarrhea.

[0008] 2.相关技术描述 [0008] 2. Description of Related Art

[0009] 抗生素相关的腹泻(AAD)由艰难梭菌,包括甲氧西林抗性的金黄色葡萄球菌(MRSA)的金黄色葡萄球菌,和产气荚膜梭菌(C. perfringens)的产生毒素的菌株导致。 [0009] Antibiotic-associated diarrhea (AAD) of C. difficile, including methicillin-resistant Staphylococcus aureus (MRSA) Staphylococcus aureus, and Clostridium perfringens (C. perfringens) of the toxin strains cause. AAD 是保健系统的主要的经济负担,据保守估计每年只在美国就产生30-60亿美元的额外的医 AAD is a major economic burden on the health care system, according to conservative estimates would only generate additional US $ 30-60 billion in health in the United States each year

院费用。 Hospital costs.

[0010] AAD是医院和长期护理机构以及团体中的严重的问题。 [0010] AAD hospitals and long-term care facilities as well as a serious problem in the community. 艰难梭菌是医院设施中AAD的最常见原因,引起约20%的AAD病例和大部分的抗生素相关的结肠炎(AAC)病例。 Clostridium difficile is the most common cause of hospital facilities in the AAD, causing about 20% of AAD cases of colitis (AAC) and most of the cases of antibiotic-associated. 艰难梭菌相关的腹泻(CDAD)的增加的发生率归因于频繁地给住院患者开广谱抗生素的处方[Wilcox 等,Lancet (柳叶刀)1996,348 :767-8]。 Clostridium difficile-associated diarrhea (CDAD) increased incidence is due to open broad-spectrum antibiotics to hospitalized patients frequently prescribed [Wilcox et, Lancet (Lancet) 1996,348: 767-8].

[0011] 所述疾病的最严重形式是假膜性的结肠炎(PMC),其在组织学上的主要表现是具有粘膜斑的结肠炎,并且在临床上的主要表现是严重的腹泻,腹部绞痛,和全身毒性。 [0011] The most severe form of the disease is pseudomembranous colitis (the PMC), which is mainly in histological colitis with mucosal plaques, and clinically mainly are severe diarrhea, abdominal cramps, and systemic toxicity. 来自CDAD的总的致死率是低的,但是在患有严重结肠炎或系统毒性的患者中却高得多。 The overall mortality rate from CDAD is low, but in patients with severe colitis or systemic toxicity in is much higher. 最近的研究显示,甚至当死亡不是直接归因于艰难梭菌时,与病例相配的对照相比,在CDAD患者中的死亡率也高得多。 Recent studies have shown that, even when death is not directly attributable to C. difficile, compared with matched case-control mortality in CDAD patients are much higher.

[0012] 腹泻和结肠炎由一种或多种艰难梭菌毒素的产生而导致。 Produce toxins [0012] diarrhea and colitis of one or more Clostridium difficile caused. 生物体在已经给药广谱抗生素的患者的结肠中增殖,或较不常见的在癌症化疗的患者的结肠中增殖。 Organisms proliferation in the colon of patients have been administered broad-spectrum antibiotics, or less commonly proliferation in colon cancer chemotherapy patients. 在用所述药剂治疗后,在约20%的患有腹泻的住院患者中诊断出CDAD。 After treatment with the agent, CDAD diagnosed in approximately 20% of patients with diarrhea in hospitalized patients.

[0013] 对于AAD或CDAD的目前的治疗包括所涉及的抗微生物药剂或化疗剂的中止,非特异性支持性测量和用针对艰难梭菌的抗生素进行治疗。 [0013] Current treatments for CDAD or AAD comprises an antimicrobial agent or a chemotherapeutic agent involved suspension, non-specific supportive measurements and treated with antibiotics for C. difficile. 最常见的抗微生物治疗选择包括万古霉素和甲硝唑。 The most common antimicrobial treatment options include vancomycin and metronidazole. 用抗生素治疗CDAD与疾病的临床复发有关。 Clinical treatment of recurrent CDAD and related diseases with antibiotics. 据报道复发的频率是5-50%,其中20-30%的复发率是最常见的引用数字。 It is reported that the frequency of recurrence is 5-50%, 20-30% recurrence rate which is the most common reference numbers. 不管药物,剂量,或用任何上述抗生素的最初治疗的延续时间如何,复发以几乎相等的频率发生。 Regardless of the drug, the dose, or how to, nearly equal to the recurrence frequency of the duration of any initial treatment with the antibiotic. 治疗中的主要挑战在具有多次复发的患者的治疗中,其中抗生素的控制是成问题的。 Treatment of the major challenges in the treatment of patients with relapsed several times, in which control of antibiotics is problematic.

[0014] 两种最常使用的具体治疗是万古霉素和甲硝唑,尽管万古霉素是由FDA为此适应症批准的唯一的药物。 [0014] Two specific therapy is most commonly used vancomycin and metronidazole, though vancomycin is the only drug approved by the FDA for this indication. 然而,万古霉素不是对于CDAD的第一线治疗所推荐的,主要是因为其仅对一些严重的威胁生命的多抗药性细菌具有抗生素活性。 However, vancomycin is not for first-line treatment of CDAD recommended, mainly because of its multi-resistant bacteria only some serious life-threatening antibiotic activity. 因此,在使万古霉素抗性肠球菌(VRE)或万古霉素抗性金黄色葡萄球菌(VRSA)的出现最小化的努力中,除非当绝对必需时,医药团体不鼓励使用该药物。 Therefore, the vancomycin-resistant enterococci (VRE) or appear vancomycin-resistant Staphylococcus aureus (VRSA) minimization efforts, except when absolutely necessary, the pharmaceuticals group does not encourage the use of the drug.

[0015] 出于关于万古霉素抗性消化道菌群,尤其是肠球菌的促进和选择目的,推荐将甲硝唑用作起始治疗。 [0015] For about vancomycin-resistant gastrointestinal flora, especially enterococci promotion and selection purposes, it is recommended to use as a starting metronidazole treatment. 尽管报道艰难梭菌抗性的频率在一些国家可以是>6%,甲硝唑仍然是几乎如万古霉素一样有效,相当地更廉价并且可以用于口服或静脉内使用。 Despite the reported frequency of C. difficile resistance in some countries may be> 6%, metronidazole is still almost as effective as vancomycin, considerably less expensive and can be used orally or intravenously. 甲硝唑与明显的副作用包括恶心,神经病,白细胞减少症,癫痫发作和对乙醇的毒性反应相关。 Metronidazole and significant side effects including nausea, neuropathy, leukopenia, seizures and related to ethanol toxicity. 此外,其用于儿童或怀孕妇女是不安全的。 In addition, for children or pregnant women are not safe.

[0016] 尽管两种药剂在治疗感染中都是有效的,在约20%的开始反应的患者中治疗失败和腹泻复发的增加率是标准疗法的缺陷。 [0016] Although the two agents in the treatment of infections are valid, the increasing rate of treatment failure and relapse in patients with diarrhea for about 20% of the start of the reaction is a defect in the standard therapy. 已经报道用甲硝唑进行的治疗是对于VRE移植(colonization)和感染的重要的危险因子。 It has been reported with metronidazole treatment is carried out for VRE transplant (colonization) and infection of the important risk factors. 此外,目前的治疗方案相当麻烦,在10-14天要求多到500mg —日四次(qid)。 In addition, the current treatment options rather cumbersome, requires more than 10-14 days to 500mg - four times a day (qid). 因此,存在对于CDAD病例以及其它AAD和AAC病例的更好的治疗方法的需求。 Thus, a need exists for CDAD cases and better treatment of other cases of AAD and AAC.

[0017] 因此,需要开发抗菌药物,其具有低的产生抗性的倾向,具有对现存的抗微生物剂减少的没有交叉抗性和/或具有延长的抗生素后效应。 [0017] Therefore, the development of antimicrobial drugs which have a tendency to produce a low resistance, with the existing antimicrobial agent without cross resistance is reduced and / or the effects of prolonged antibiotics.

[0018] 发明概述 [0018] Summary of the Invention

[0019] 本发明提供治疗由选自由梭菌属物种,葡萄球菌属物种,肠球菌属物种,及其组合组成的组的细菌的存在引起的疾病或病症的方法,所述方法包括向需要其的患者施用有效量的混合物。 [0019] The present invention provides a therapeutic selected from the group consisting of Clostridium species, Staphylococcus species, Enterococcus species, and the presence of a disease or disorder of the group consisting of bacteria caused by the combination, in need thereof said method comprising an effective amount of the mixture administered to the patient. 所述混合物包括有效量的台勾霉素B和选自由下列各项组成的组的另外的大环化合物: The mixture includes an effective amount of tiacumicin B and selected from the group consisting of the following additional macrocycle group consisting of:

[0020] [0020]

Figure CN102503994AD00051
Figure CN102503994AD00052

[0023] IV(0P-1415, RT 比率0. 81), [0023] IV (0P-1415, RT ratio of 0.81),

[0024] [0024]

Figure CN102503994AD00061

[0025]V(0P-1417, RT 比率:0. 84), [0025] V (0P-1417, RT ratio: 084),

[0026] [0026]

Figure CN102503994AD00062

[0027]IX(0P-1435, RT 比率:1. 13), [0027] IX (0P-1435, RT ratio: 113),

[0028] [0028]

Figure CN102503994AD00063

[0029]X(0P-1437, RT 比率:1. 19), [0029] X (0P-1437, RT ratio: 119),

[0030] [0030]

Figure CN102503994AD00064

[0031]XI(0P-1402, RT 比率:1. 24), [0031] XI (0P-1402, RT ratio: 124),

[0032] [0032]

Figure CN102503994AD00071

[0033] XII (0P-1433, RT 比率:1. 39), [0033] XII (0P-1433, RT ratio: 139),

[0034] [0034]

Figure CN102503994AD00072

[0035] XIII (0P-1438,RT 比率:1.48), [0035] XIII (0P-1438, RT ratio: 1.48),

[0036] [0036]

Figure CN102503994AD00073

[0037] XIV (闰年霉素A4,0P-1405, RT比率:0. 89),及其组合。 [0037] XIV (lipiarmycin A4,0P-1405, RT ratio: 089), and combinations thereof. 当存在式XIV的化合物时,所述混合物包括约0. 1到约5%的式XIV的化合物。 When the compound of formula XIV is present, said mixture comprises from about 0.1 to about 5% of the formula XIV.

[0038] 优选地,所述混合物包括至少90重量%的台勾霉素B。 [0038] Preferably, the mixture comprises at least 90 wt% of tiacumicin B. 更优选地,所述混合物包括至少95重量%的台勾霉素B。 More preferably, the mixture comprises at least 95 wt% of tiacumicin B.

[0039] 优选地,所述混合物包括至少1重量%,更优选地约2重量%到约5重量%的另外的大环化合物。 [0039] Preferably, the mixture comprises at least 1 wt%, more preferably from about 2% to about 5% by weight of additional macrocycle.

[0040] 优选地,所述混合物包括约0. 1重量%到约5重量%,更优选地0. 3重量%到3重量%,特别地0.3重量%到1.5重量%,尤其是约1重量%的闰年霉素A4。 [0040] Preferably, the mixture comprises from about 0.1% to about 5 wt%, more preferably from 0.3 wt% to 3 wt%, in particular 0.3 wt% to 1.5 wt%, in particular from about 1 wt. % of lipiarmycin A4.

[0041] 优选地,当存在闰年霉素A4时,所述混合物还包含下列化合物的至少一种: [0041] Preferably, when the presence of Lipiarmycin A4, the mixture further comprises at least one of the following compounds:

[0042] [0042]

Figure CN102503994AD00081

[0043] VI (0P-1431,台勾霉素F, RT 比率:0. 92), [0043] VI (0P-1431, tiacumicin F, RT ratio: 092),

[0044] [0044]

Figure CN102503994AD00082

[0045] VII(0p_1432,台勾霉素C,RT 比率:0. 95),和 [0045] VII (0p_1432, tiacumicin C, RT ratio: 095), and

[0046] [0046]

Figure CN102503994AD00083

[0047] VIII(0P-1434,台勾霉素A,RT 比率:1. 10)。 [0047] VIII (0P-1434, tiacumicin A, RT ratio: 110).

[0048] 优选地,所述混合物显示基本在图5中描述的HPLC图谱。 [0048] Preferably, the mixture exhibits HPLC profile substantially depicted in FIG. 5.

[0049] 优选地,根据本发明治疗的疾病或病症与艰难梭菌,产气荚膜梭菌,金黄色葡萄球菌及其组合相关。 [0049] Preferably, the disease or related condition being treated according to the present invention is Clostridium difficile, Clostridium perfringens, Staphylococcus aureus, and combinations thereof. 更优选地,根据本发明治疗的疾病或病症与艰难梭菌相关。 More preferably, depending on the disease or disorder related to the present invention is Clostridium difficile.

[0050] 优选地,根据本发明治疗的疾病是腹泻或结肠炎,特别是腹泻,更具体地是CDAD。 [0050] Preferably, the disease treated in accordance with the present invention is colitis or diarrhea, particularly diarrhea, more particularly CDAD.

[0051] 优选地,根据本发明的混合物通过包括下列步骤的方法制备: [0051] Preferably, preparing the mixture according to the invention by a method comprising the steps of:

[0052] 将微生物培养在营养培养基中以在营养培养基中积累混合物;和 [0052] The microbial culture in a nutrient medium to accumulate the mixture in a nutrient medium; and

[0053] 从营养培养基中分离所述混合物; [0053] separating the mixture from the nutrient medium;

[0054] 所述营养培养基包括吸附所述混合物的吸附剂。 The [0054] nutrient medium comprises an adsorbent to adsorb the mixture.

[0055] 所述营养培养基优选地包括0. 5-15重量%的吸附剂。 The [0055] The nutrient medium preferably comprises 5-15 wt% 0.05 adsorbent. 所述吸附剂优选地是吸附树脂。 The adsorbent is preferably adsorption. 更优选地,所述吸附树脂选自由下列各项组成的组:离子交换树脂(Amberlite® ) XAD16, XAD16HP, XAD2, XAD7HP, XADl 180,XAD1600, IRC50,和Duolite® XAD761。 More preferably, the adsorbent resin is selected from the group consisting of: ion exchange resin (Amberlite®) XAD16, XAD16HP, XAD2, XAD7HP, XADl 180, XAD1600, IRC50, and Duolite® XAD761. 所述微生物优选地是桔橙指孢囊菌(Dactylosporangium aurantiacum)hamdenensis亚禾中。 The microorganism is preferably an alkylene aurantiacus means hamdenensis Wo in cysts (Dactylosporangium aurantiacum) bacteria. 基于重量,营养培养基包括0. 2%到10 %的葡萄糖,0. 02 %到0. 5 %的K2HPO4,0. 02 %到0. 5 %的MgSO4 · 7H20,0. 01%至Ij 0. 3% 的KCl,0. 到2% 的CaCO3,0. 05%到2% 的酪蛋白氨基酸, 0. 05%到2%的酵母提取物,和0. 5%到15%的XAD-16树脂。 Based on the weight of the nutrient medium comprises 0.2% to 10% glucose, 0.02% to 0.5% of K2HPO4,0. 02% to 0.5% of MgSO4 · 7H20,0. 01% to Ij 0 3 percent of KCl, 0. 2% to CaCO3,0. 05% and 2% casamino acid, 0.05% to 2% yeast extract, and 0.5% to 15% of XAD-16 resin. 培养步骤优选地在约25到约350C的温度和从约6. 0到约8. 0的pH进行。 And culturing step is preferably from about pH 6.0 to about 8.0 at a temperature of from about 25 to about 350C.

[0056] 优选地,根据本发明治疗的疾病与抗生素或癌症化学疗法或抗病毒疗法的使用相关。 [0056] Preferably, the disease is associated with an antibiotic or anti-cancer chemotherapy using a viral or related therapy treatment according to the present invention.

[0057] 根据一个优选的实施方案,所述混合物以约50mg到IOOOmg的量,更优选地IOOmg 到400mg的量,特别地200mg的量每日1-3次,更优选地每日一次或两次,特别地每日两次, 在3-15天,特别地约10天内进行施用。 [0057] According to a preferred embodiment, the mixture in an amount of from about 50mg to IOOOmg amount IOOmg to 400mg and more preferably, in particular 1-3 times the amount of 200mg per day, more preferably once a day or two times, in particular twice daily, 3-15 days, in particular administered from about 10 days. 优选口服施用。 Oral administration is preferred.

[0058] 本发明的治疗可以容许有效治疗与艰难梭菌,金黄色葡萄球菌,和产气荚膜梭菌的产肠毒素菌株相关的腹泻疾病,而不会不利于系统抗生素并且不会增加消化道中的万古霉素抗性肠球菌(VRE)。 [0058] The treatments of the present invention may allow an effective treatment of C. difficile, Staphylococcus aureus, and Clostridium gassing enterotoxigenic strains of disease-associated diarrhea without antibiotics and without detrimental increase digestive system tract and vancomycin-resistant enterococci (VRE). 本发明还减少消化道中VRE的存在。 The present invention also reduces the presence of VRE in the gut.

[0059] 从结合附图考虑的下面的详细描述中,本发明的其它目的和特征将变得显而易见。 [0059] from the following detailed description considered in conjunction with the accompanying drawings, other objects and features of the present invention will become apparent. 然而,要理解的是,所述附图只是出于举例说明的目的而设计并不是为了定义本发明的范围,对于本发明的范围应该参考后附的权利要求。 However, it is to be understood that the drawings are only for purposes of illustration and are not designed to define the scope of the present invention, the rights scope of the appended claims of the present invention, reference should be. 还应该理解的是附图不必局限于一定范围,并且除非另外指出,它们仅仅倾向于概念性地举例说明本文所述的结构和方法。 It should also be understood that the drawings are not necessarily limited to a certain range, and unless otherwise indicated, they are merely tend to conceptually illustrate the structures and methods described herein.

[0060] 附图简述 [0060] BRIEF DESCRIPTION

[0061] 在附图中: [0061] In the drawings:

[0062] 图1显示IB-MD阶段给药时间表。 [0062] Figure 1 shows the dosing schedule stage IB-MD.

[0063] 图2显示在治疗后的类菌体数量。 [0063] FIG. 2 shows the number of cells after treatment of class. 配对符号秩次检验(Pairs signed-ranks test),双尾。 Wilcoxon signed rank test (Pairs signed-ranks test), two-tailed. 对于计数<31og 10,使用值2. 9。 For count <31og 10, using the value of 2.9.

[0064] 图3显示万古霉素对脆弱拟杆菌(B. fragilis)群的治疗方法的效果。 [0064] FIG. 3 shows the effect of vancomycin Bacteroides fragilis (B. fragilis) of the treatment group.

[0065] 图4显示在用MCC治疗后,艰难梭菌营养体计数的定量减少。 [0065] Figure 4 shows after treatment with MCC, quantitative vegetative C. difficile counts reduced.

[0066] 图5是混合物的典型HPLC图谱,所述混合物可以用在本发明的方法中。 [0066] FIG. 5 is a typical HPLC profile of the mixture, the mixture may be used in the method of the present invention.

[0067] 本发明的优选实施方案的详细描述 [0067] Detailed description of preferred embodiments of the present invention.

[0068] 将用在本申请中的某些缩写或术语的定义提供如下: [0068] The definitions of certain abbreviations or terms used in the present application are provided below:

[0069] AAD =抗生素相关的腹泻 [0069] AAD = antibiotic associated diarrhea

[0070] ATCC =美国典型培养物保藏中心(American Type Culture Collection) [0070] ATCC = American Type Culture Collection (American Type Culture Collection)

[0071] 1V=碳13 [0071] 1V = C 13

[0072] CO2 = 二氧化碳 [0072] CO2 = carbon dioxide

[0073] N2 =氮 [0073] N2 = nitrogen

[0074] H2 =氢 [0074] H2 = hydrogen

[0075] TAPS = N-三(羟甲基)甲基-3-氨基丙磺酸[0076] MOPS = 3- (N-吗啉代)丙磺酸 [0075] TAPS = N- tris (hydroxymethyl) methyl-3-propionic acid [0076] MOPS = 3- (N- morpholino) propanesulfonic acid

[0077] CDAD =艰难梭菌相关的腹泻 [0077] CDAD = Clostridium difficile-associated diarrhea

[0078] CLSI =临床和实验室标准研究所(Clinical and Laboratory Standards Institute),前NCCLS [0078] CLSI = Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute), the former NCCLS

[0079] ED50 =产生50%反应的有效剂量 [0079] ED50 = effective dose to produce a 50% response

[0080] HPLC=高效液相色谱 [0080] HPLC = High Performance Liquid Chromatography

[0081] 顶=红外光谱法 [0081] Infrared Spectroscopy top =

[0082] LLOQ =定量的下限 [0082] LLOQ = limit of quantification

[0083] MCC =包含大环化合物的组合物[0084] MIC =最小抑制浓度 [0083] MCC = macrocycle containing compositions [0084] MIC = minimum inhibitory concentration

[0085] MIC50 =抑制50%的测试的细菌菌株的最小抑制浓度 [0085] MIC50 = minimum inhibitory concentration inhibiting 50% of the bacterial strains tested

[0086] MIC90 =抑制90%的测试的细菌菌株的最大抑制浓度 [0086] MIC90 = 90% inhibition of the bacterial strains tested maximal inhibitory concentration

[0087] MRSA =甲氧西林-抗性金黄色葡萄球菌 [0087] MRSA = methicillin - resistant Staphylococcus aureus

[0088] NCCLS =国家临床实验室标准委员会,现在的CLSI [0088] NCCLS = National Committee for Clinical Laboratory Standards, now CLSI

[0089] PMC =假膜性结肠炎 [0089] PMC = pseudomembranous colitis

[0090] VRE =万古霉素-抗性肠球菌 [0090] VRE = vancomycin - resistant enterococci

[0091] VRSA =万古霉素-抗性金黄色葡萄球菌 [0091] VRSA = Vancomycin - resistant Staphylococcus aureus

[0092] 术语“抗生素相关的病症”指当抗生素治疗干扰消化道的微生物菌群的平衡,使病原生物如产生肠毒素的艰难梭菌,金黄色葡萄球菌和产气荚膜梭菌菌株繁殖旺盛时而导致的病症。 [0092] The term "antibiotic-associated disorder" refers to antibiotic therapy when the balance of the digestive tract microflora interference, so that pathogenic organisms such as Clostridium difficile enterotoxin produced, S. aureus and C. perfringens strain breeding strong sometimes due to illness. 除了其它症状,这些生物可以引起腹泻,假膜性结肠炎,和结肠炎并且表现为腹泻, 尿急,腹部绞痛,里急后重和发烧。 In addition to other symptoms, these organisms can cause diarrhea, pseudomembranous colitis, and colitis and is manifested as diarrhea, urgency, abdominal cramps, tenesmus and fever. 腹泻,当严重时,导致脱水和与脱水相关的医学并发症。 Diarrhea, when severe, leading to dehydration and the medical complications associated with dehydration.

[0093] 术语“MCC”指对于全部抗生素物质,主要包含台勾霉素B (例如,通过HPLC分析,至少90%,优选地95% -98% )的制剂。 [0093] The term "MCC" refers to the total antibiotic substance, comprising a main tiacumicin B (e.g., by HPLC analysis, at least 90%, preferably 95% -98%) of the formulation. MCC还包括小量(例如,至少1 %,优选地2% -5% ) 的台勾霉素B相关化合物,即闰年霉素A4和至少一种在上面显示的式III-XIV的化合物。 MCC also comprises a small amount (e.g., at least 1%, preferably 2% -5%) of Tiacumicin B related compounds, i.e. compounds of formula Lipiarmycin A4 and at least one III-XIV shown above. PCT申请PCT/USOSAig??,其国际公开号为WO 2004/01^95A2,提供制备包含台勾霉素B的混合物的方法。 PCT Application PCT / USOSAig ??, which is International Publication No. WO 2004/01 ^ 95A2, comprising providing a mixture prepared tiacumicin B. 将该PCT申请的全部内容结合于本文作为参考。 The entire contents of PCT application is incorporated herein by reference. 然而,专门用于非人的MCC 可以包含少于80%的台勾霉素B (针对整个抗生素物质,通过HPLC测定)。 However, the MCC dedicated to non-human may comprise less than 80% of tiacumicin B (for the whole antibiotic substance, by HPLC assay).

[0094] 术语“赋形剂”指被加入药物组合物以进一步有利于施用化合物的惰性物质。 [0094] The term "excipient" refers to an addition of a pharmaceutical composition to further facilitate administration of a compound of an inert substance. 赋形剂的实例包括但不限于,碳酸钙,磷酸钙,各种糖和各种类型的淀粉,纤维素衍生物,明胶, 植物油和聚乙二醇。 Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0095] 术语“卤素”包括F,Cl,Br和I。 [0095] The term "halogen" includes F, Cl, Br and I.

[0096] 术语“大环化合物”指具有通常包含超过10个原子的大环结构的有机分子。 [0096] The term "macrocycle" refers to an organic molecule having a generally comprise more than 10 atoms of the macrocyclic structure.

[0097] 术语“18-元大环化合物”指具有包含18个原子的环结构的有机分子。 [0097] The term "18-membered macrocycles" refers to organic molecules with ring structures containing 18 atoms.

[0098] 术语“元环”可以包括任何环结构,包括如上所述的碳环和杂环。 [0098] The term "membered ring" may include any cyclic structure, including heterocyclic and carbocyclic described above. 术语“元”意指组成环的骨架原子的数量。 The term "membered" is meant the number of skeletal atoms of the ring components. 因此,例如吡啶,吡喃和硫代吡喃是6元环,而吡咯,呋喃和噻吩是5元环。 Thus, for example, pyridine, pyran and thiopyran are 6-membered ring, and pyrrole, furan, and thiophene are 5-membered ring.

[0099] 术语“MIC”或“最小抑制浓度”指在体外抑制细菌分离物的生长所需要的抗生素的最低浓度。 [0099] The term "MIC" or "minimum inhibitory concentration" refers to the lowest concentration of antibiotic inhibiting growth of bacteria needed to isolate in vitro. 确定抗生素的MIC的常用方法是准备一些包含抗生素系列稀释物的管,接着将其用目的细菌分离物进行接种。 The method of MIC determination used antibiotics an antibiotic is to prepare several tubes containing serial dilutions, then it was inoculated with the bacteria was isolated. 在适当的大气压和温度下接种后,抗生素的MIC可以从显示没有浊度(无生长)的最低浓度的管进行确定。 After inoculation at a suitable temperature and atmospheric pressure, MIC lowest antibiotic concentration from the display can no turbidity (no growth) of the tube is determined.

[0100] 术语"MIC5tl”指在给定的细菌物种中抑制50%的测试细菌菌株的生长所需要的抗生素的最低浓度。 [0100] The term "MIC5tl" lowest concentration of the antibiotic which inhibits growth of 50% of the bacterial strains tested within a given desired bacterial species.

[0101] 术语"MIC9tl”指在给定的细菌物种中抑制90%的测试细菌菌株的生长所需要的抗生素的最低浓度。 [0101] The term "MIC9tl" lowest concentration of the antibiotic which inhibits growth of the test bacterial strain of 90% required in a given bacterial species.

[0102] 术语"患者"指需要医学治疗的人或动物。 [0102] The term "patient" means a person or animal in need of medical treatment. 出于本发明的目的,人患者典型地专门在一级医疗保健机构如医院或疗养院受到照料。 For purposes of this invention, typically a human patient in a specialized health care institutions such as hospitals or nursing homes receive care. 然而,在从一级保健机构中出院后,治疗与使用抗生素或癌症化学治疗或抗病毒治疗相关的疾病可以在门诊病人的基础上进行,或可以由独立于一级医疗保健机构的家庭护理的医师开处方。 However, after discharge from the primary care setting, treatment with antibiotics or cancer chemotherapies or antiviral treatment-related disease can be performed on an outpatient basis, or can care for an independent health care institution of the family physician prescription. 需要医学治疗的动物典型地在兽医的护理下。 Need medical treatment under the care of animals typically veterinarian.

[0103] 术语“药用载体”指药用的载体或稀释剂。 [0103] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable carrier or diluent.

[0104] 术语“药用盐”指来自药用无机碱和有机碱的那些。 [0104] The term "pharmaceutically acceptable salts" refers to those derived from pharmaceutically acceptable inorganic bases and organic bases. 来自适合的碱的盐包括碱金属盐(例如,钠盐或钾盐),碱土金属盐(例如,镁盐),铵盐和MC1-C4烷基)4+盐等。 Salts derived from appropriate bases include alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal (e.g., magnesium), ammonium and MC1-C4 alkyl) 4+ salts. 这些中的一些举例说明的实例包括氢氧化钠,氢氧化钾,胆碱氢氧化物,碳酸钠等。 Some of these illustrative examples include sodium hydroxide, potassium hydroxide, choline hydroxide, sodium carbonate and the like.

[0105] 术语"药物组合物"指本文所述的一种或多种台勾霉素,或其生理可接受的盐, 与其它的化学成分如生理可接受的载体和/或赋形剂的混合物。 [0105] The term "pharmaceutical composition" described herein refers to one or more of the tiacumicins, or a physiologically acceptable salt thereof, with other chemical components such as physiologically acceptable carriers and / or excipients mixture. 药物组合物的目的是促进将化合物施用于生物体。 Purpose of a pharmaceutical composition is to facilitate administration of the compound to an organism.

[0106] 术语"生理可接受的载体"指没有导致对生物的明显刺激并且没有消除所施用的化合物的生物活性和性质的载体或稀释剂。 [0106] The term "physiologically acceptable carrier" refers to not cause significant irritation to an organism and does not eliminate carrier or diluent biologically active compound administered and properties.

[0107] 术语"假膜性结肠炎"或"肠炎"指由于小肠和大肠的粘膜的炎症导致形成假膜性物质(即,由血纤蛋白,粘液,坏死上皮细胞和白细胞组成的物质)。 [0107] The term "pseudomembranous colitis" or "colitis" refers to inflammation of the small intestine due to the large intestine mucosa and lead to the formation of pseudomembranous material (i.e., a substance fibrin, mucous, necrotic epithelial cells and leukocytes) is.

[0108] 用于本文时,术语“台勾霉素”指全部包括在下面的式I中显示的18元大环化合 [0108] As used herein, the term "tiacumicin" refers to all including 18-membered macrocyclic compounds shown in the following formula I

物的化合物家族: Family compound thereof:

[0109] [0109]

Figure CN102503994AD00111

[0110]式 I [0110] Formula I

[0111] 用于本文时,术语“台勾霉素B”指在下面的式II中显示的18-元大环化合物: [0111] As used herein, the term "tiacumicin B" refers to a 18-membered macrocyclic compounds of the following formula II shown in:

[0112] [0112]

Figure CN102503994AD00112

[0113]式 II (RT 比率1.0) [0113] Formula II (RT 1.0 ratio)

[0114] 用于本文时,术语闰年霉素A4指在下面的式XIV中显示的18元大环化合物: [0114] As used herein, the term & Lipiarmycin A4 fingers 18-membered macrocyclic compounds of the following formula XIV shown in:

[0115] [0115]

Figure CN102503994AD00121

[0116] 根据本发明的一个实施方案,在口服给药多剂量后,在血浆中检测到低MCC水平, 其大多数在定量界限下。 [0116] According to an embodiment of the present invention, after oral administration in multiple doses, to detect in plasma levels of low MCC, most of which in the limit of quantification. 作为对照,在两项研究中的粪便水平非常高,超过针对艰难梭菌的MIC90 (0. 125 μ g/mL)的10,000 倍。 As a control, the level of fecal two studies is very high, more than 10,000 times for C. difficile MIC90 (0. 125 μ g / mL) of.

[0117] 根据本发明的一个实施方案,在患者中,与艰难梭菌相关的腹泻的复发可以通过以有效抑制艰难梭菌的复发但是对患者的正常消化道菌群没有影响的量和延续时间施用MCC而得到抑制。 [0117] According to an embodiment of the present invention, in a patient, associated diarrhea recurrent C. difficile can be effective to inhibit relapse of C. difficile but do not affect the normal flora in the digestive tract of the patient volume and duration MCC administration be suppressed.

[0118] 根据一个实施方案,对于CDAD的MCC的每日口服剂量将在约50mg到约1. Og的活性剂/天范围内,优选地在从约IOOmg到约600mg/天范围内。 [0118] According to one embodiment, the daily oral dosage of MCC for CDAD will range from about 50mg to about 1. Og active agent / day, preferably in the range from about 600mg / day to range from about IOOmg. 一般而言,治疗将从约3到约15天之间的时间阶段持续。 In general, the treatment time period between about 3 to about 15 days will continue. 根据需要,可以使用更多或更少的量的药物和治疗间隔。 According to need, a greater or lesser amounts of drug and treatment intervals. 例如,根据在下文报道的临床研究结果,在从约10天的过程中,每天给药约100-400毫克的MCC的剂量,证实在治疗CDAD中是有效的,具有最小的临床复发。 For example, according to the results of clinical study hereinafter reported, from about 10 days in the course of daily dosing MCC about 100-400 mg, demonstrated in the treatment of CDAD is effective with minimal clinical relapse.

[0119] 根据本发明的一个实施方案,所述混合物可以通过下列通用方法制备。 [0119] The mixture can be prepared by the general procedure according to one embodiment of the present invention.

[0120] 在从摇瓶到大“批量”发酵器的范围内,在容器中培养产生MCC的细菌。 [0120] in the range from shake flasks to large "batch" fermentors, the culture in the vessel to produce MCC bacteria. 为了产生基本上大量的MCC,在罐中使用深层好氧发酵。 In order to produce a substantially large amount of MCC, use deep aerobic fermentation in tanks. 然而,可以通过摇瓶培养获得小量。 However, a small amount can be obtained by shake-flask culture. 对于罐发酵,优选使用营养体接种物。 For tank fermentation, it is preferable to use vegetative inoculum. 通过用生物的孢子形式,菌丝体片段,或冻干的沉淀物接种小体积的培养基来制备营养体接种物,以获得新鲜,活跃生长的生物体培养物。 By biological spore form of mycelial fragments, or a lyophilized pellet was used to inoculate a small volume of vegetative medium to prepare inoculum, to obtain a fresh, actively growing culture of the organism. 接着,将营养体接种物转移到更大的罐中,其中在经过适合的培养时间后,以大量提高的产量产生MCC 抗生素。 Subsequently, the vegetative inoculum is transferred to a larger tank, where after a suitable incubation time, in order to substantially increase the yield of antibiotic produced MCC. 如果发泡成为问题,那么将小量的消泡剂加入到大规模的发酵培养基中可能是必需的。 If foaming becomes a problem, then a small amount of anti-foaming agent is added to the large-scale fermentation media may be necessary.

[0121] 在对照培养基中用其它的添加剂/成分进行生产以提高产量。 [0121] Other additives / ingredients in a control medium with production to increase yields. 使用液体-浸没、 搅拌培养方法进行MCC的生产。 Liquid - submerged stirring culture method for production of MCC. 发酵在25°C到37°C的温度范围内进行。 Fermentation in the temperature range 25 ° C to 37 ° C is performed. 仔细监测碳源的消耗,并且如果需要加入额外量的碳源。 Carefully monitor the consumption of the carbon source, and if necessary additional amount of carbon source. 发酵的PH优选地维持在约6.0到约8.0。 PH fermentation is preferably maintained at from about 6.0 to about 8.0. 在发酵液接种后,在3-15天之间,生产和积累MCC。 After inoculation the fermentation broth, between 3-15 days, produced and accumulated MCC.

[0122] 发现商购吸附树脂在发酵过程中增加MCC的产量和回收效率。 [0122] found that commercially available adsorption resin to increase production and recovery efficiency of MCC during fermentation. 吸附剂优选地以0. 5-15重量%范围存在。 Adsorbent preferably present in the range of 0. 5-15 wt%. MCC通过树脂吸收以非常好的产量(> 100mg/L培养液)从发酵培养液中回收,并且通过用各种极性的溶剂进行洗涤从树脂和菌丝体上洗脱。 MCC absorbed by the resin is very good yield (> 100mg / L broth) from the fermentation culture was recovered and eluting from the resin and mycelium by washing with solvents of various polarities.

[0123] 首先,在发酵过程中使用吸附树脂如离子交换树脂(Amberlite) (XAD-16)将MCC 从培养液中捕获出来。 [0123] First, the adsorption resin used in the fermentation process such as ion exchange resin (Amberlite) (XAD-16) capture the MCC from the culture broth. 在完成发酵后,将固体块(包括吸附树脂)通过筛分从培养液中分离出来。 After completion of the fermentation, the solid mass (including the adsorbent resin) is separated from the culture solution sieved through. 将固体块用乙酸乙酯洗脱,接着在减压下浓缩。 The solid mass with ethyl acetate, then concentrated under reduced pressure.

[0124] 在发酵结束后,将固体块(包括吸附树脂)通过筛分从培养液中分离出来。 [0124] After the end of the fermentation, the culture broth separated from the sieving the solid mass (including the adsorbent resin) by. 用有机溶剂如乙酸乙酯、甲醇、乙腈或两种或多种有机溶剂的混合物将MCC从树脂上洗脱下来。 The mixture of ethyl acetate, methanol, acetonitrile, or two or more organic solvents MCC is eluted from the resin with an organic solvent. 接着,在减压下将提取物进行浓缩。 Subsequently, the extract under reduced pressure and concentrated. 该残余物通过下列方式来进一步纯化,即,通过用低极性溶剂如己烷,庚烷,甲基环己烷进行研磨,或通过在两相溶剂系统如:乙酸乙酯/水;乙酸乙酯/氯化钠水溶液;甲醇/己烷;乙腈/己烷或其它以各种比率和组合存在的两种或多种溶剂的混合物中分配,或通过用适合的有机溶剂系统进行柱色谱法洗脱。 The residue was further purified by the following manner, i.e., by polishing with a low polar solvent such as hexane, heptane, methylcyclohexane, or by a two-phase solvent system such as: ethyl acetate / water; acetic acid acetate / aqueous sodium chloride solution; methanol / hexanes; acetonitrile / hexane or other in various ratios and combinations of two or more solvents present in the distribution, or washing by column chromatography with a suitable organic solvent system off. 目前MCC的纯化方法基于使用50 : 50 : 1 的CH3CN/H20/Ac0H 或70 : 30 : 1 的Me0H/H20/Ac0H 作为洗脱剂的中压反向(C-18)柱进行。 Currently MCC purification methods based on the use of 50: 1 CH3CN / H20 / Ac0H or 70:: 50 30: 1 in the reverse pressure Me0H / H20 / Ac0H as eluent (C-18) column. 用盐水洗涤包含需要的MCC的级分并且将其进行浓缩。 Washed with brine containing MCC desired fraction was concentrated and subjected. 将残余物溶解在乙酸乙酯中,用水洗涤并且将有机层蒸发到干燥以提供浅黄色泡沫,其又用异丙醇洗涤,并且在减压下干燥以产生白色粉末。 The residue was dissolved in ethyl acetate, washed with water and the organic layer was evaporated to dryness to provide a pale yellow foam, which was washed in turn with isopropanol, and dried under reduced pressure to yield a white powder. 合并纯度>88%的级分。 Fractions with a purity> 88% fraction. 将级分浓缩到起始体积的一半。 The fractions were concentrated to half the initial volume. 过滤沉淀物并用水洗涤滤饼。 The precipitate was filtered and the cake washed with water. 在高真空下将固体干燥过夜以得到白色粉末并且用HPLC分析。 In the solid was dried under high vacuum overnight to give a white powder and analyzed by HPLC. 典型地,混合物包含90% -99%范围的台勾霉素B作为主要成分,闰年霉素A4(0. 和至少一种或多种上述的式III-XIV的大环化合物。 Typically, the mixture comprises from 90% to 99% of Tiacumicin B as a main component, Lipiarmycin A4 (0. Macrocycle and at least one or more compounds of the above formula III-XIV of.

实施例 Example

[0125] 通过描述本发明的具体实施方案而不倾向于以任何方式限制本发明的范围来提供下列实施例。 [0125] By describing particular embodiments of the present invention without in any way we tend to limit the scope of the invention to provide the following examples.

[0126] 根据上述的制备方法制备在下面的实施例中使用的混合物。 [0126] The mixture used in the following examples were prepared according to the method above. 下表显示根据本发明制备的一些示例性混合物的组合物。 The following table shows some exemplary composition mixture prepared according to the present invention.

[0127] [0127]

Figure CN102503994AD00141

[0128] [0128]

Figure CN102503994AD00151

[0133]检测器:230nm。 [0133] Detector: 230nm.

[0134] 流速:约l.OmL/分钟。 [0134] Flow rate: about l.OmL / min.

[0135] 注射体积:约IOiiし [0135] Injection volume: about shi IOii

[0136] 运行时间:约IOiiし [0136] running time: about IOii shi

[0137] 稀释剂:100%乙腈。 [0137] Diluent: 100% acetonitrile.

[0138] [0138]

梯度程序: 时间(分钟) %移动相A Gradient program: Time (min)% Mobile Phase A

移动相B Mobile phase B

0 60 40 06040

3.0 50 50 3.0 5050

14.0 39 61 14.0 3961

14.5 60 40 14.5 60 40

[0139] 注意:混合物的保留时间必须在8-12分钟内。 [0139] Note: The retention time of the mixture must be within 8-12 minutes.

[0140] 标准制备物:精确称取约20mg的混合物到IOOmL容量瓶中,将其用稀释剂溶解并且稀释到一定体积。 [0140] Standard Preparation: Accurately weigh about 20mg of the mixture to a IOOmL volumetric flask, which was dissolved with a diluent and diluted to a certain volume.

[0141] 样品制备物:精确称取约20mg的混合物到IOOmL的容量瓶中。 [0141] Sample Preparation: Accurately weigh about 20mg of the mixture to IOOmL volumetric flask. 加入约60mL稀释 Diluted to about 60mL join

剂并涡旋以溶解。 Agents and vortexed to dissolve. 用稀释剂稀释到一定体积并混合。 It was diluted with a diluent to volume and mixed.

[0142] 系统的适合性:如下述方法所指导,对标准制备物进行色谱法并且记录最大反应。 [0142] System suitability: as directed by a method of preparing the standard was chromatographed and recording the maximum response. 对于五次重复注射的台勾霉素B的峰面积的相对标准偏差是NMT 2.0%,台勾霉素B面积的拖尾因子是NMT 2.0。 The relative standard deviation for five replicate injections tiacumicin B is the peak area NMT 2.0%, tiacumicin B area tailing factor is NMT 2.0.

[0143] 方法:注射约IOii L的稀释剂。 [0143] Method: injection of about IOii L of diluent. 分別注射等体积(约IOii L)的标准和样品制备物,记录色谱并且对于主峰測量检测器反应。 They were injected with equal volumes (about IOii L) of the standard and sample preparation, and record the chromatogram peak measurement detector for the reaction.

[0144] 相对停留时间: [0144] Relative stay:

[0145] [0145]

相关物质 RT比率 RT ratio related substances

式II的化合物(台勾霉素B) 1.0 式III的化合物 0.71 Compound (tiacumicin B) of formula II, formula III 0.71 1.0

[0146] [0146]

Figure CN102503994AD00171

[0147] 计算:使用下列公式计算测定值: [0147] Calculation: measured value using the following formula:

[0148] [0148]

Figure CN102503994AD00172

[0149] 其中:RU =从测定制备物获得的台勾霉素B的峰面积 [0149] wherein: RU = peak area hook amphotericin B was prepared from the obtained measurement station

[0150] Rs =从标准制备物获得的台勾霉素B的峰面积 [0150] Rs = standard preparation obtained from the stage of the peak area of ​​the hook of amphotericin B

[0151] P =参考标准的纯度,包括水因数。 [0151] P = purity of the reference standard, comprising a water factor.

[0152] Wstd =标准重量(mg) [0152] Wstd = Standard weight (mg)

[0153] StdDil =标准稀释(mL). [0153] StdDil = Standard dilution (mL).

[0154] Wsmp =样品重量(mg). [0154] Wsmp = Sample weight (mg).

[0155] WF=样品水因数. [0155] WF = water factor sample.

[0156] 弃去得自稀释剂的峰,并且通过下式计算个体和总的相关物质的w/w百分比: [0156] diluent discarded from a peak, and by w calculated individual and total related substances / w percentages:

[0157] [0157]

个体相关物质(% ) = i Jr點χ m Individual Related Substances (%) = i Jr point χ m

[0158] 其中=Ri =从样品制备物获得的相关物质的峰面积。 [0158] where = Ri = peak area of ​​related substances obtained from the sample preparation.

[0159] Ru =从样品制备物获得的台勾霉素B的峰面积。 [0159] Ru = peak area of ​​the hook of amphotericin B were obtained from the sample preparation station.

[0160] RFi =相关物质反应因子(对于所有的相关物质RFi = 1. 0) [0160] RFi = response factor related substances (for all substances RFi = 1. 0)

[0161] 此外,将根据本发明的混合物的典型HPLC图谱显示在图5中。 [0161] Further, the typical HPLC profile of the mixture according to the present invention is shown in FIG. 5. 在混合物中包含的化合物,例如式II-XIV的化合物可以依据它们的RT比率在HPLC图谱中找到。 Compound contained in the mixture, for example, compounds of formula II-XIV can be found in the HPLC profile depending on their RT ratio. 在图5中的Par-IOl表示RT比率为1. 0的台勾霉素B。 Par-IOl in FIG. 5 indicates the ratio RT of 1.0 units of tiacumicins B.

[0162] 接着,将上述混合物(50mg)与IOOmg Avicel PH 102,FMC(微晶纤维素)在1号大小的胶囊壳(size 1 capsule shell)中混合。 [0162] Next, the above mixture (50mg) and IOOmg Avicel PH 102, FMC (microcrystalline cellulose) were mixed in size No. 1 capsule shell (size 1 capsule shell) in.

[0163] 实施例1.接种物,pH,和阳离子对MCC针对艰难梭菌的体外活性的影响 Example 1. inoculum, pH, and cations impact on MCC in vitro activity against C. difficile [0163] Embodiment

[0164] 已知对于许多抗生素测量的MIC值受到环境变量如pH,二价阳离子如钙和镁的浓度和细菌密度的影响。 [0164] Known such as pH, divalent cations affect the MIC values ​​of antibiotics for many environmental variables such as measured by the concentration of the bacterial density and calcium and magnesium. 抗菌活性对这些因子的依赖性是重要的考虑因素,特别是对于在消化道中靶向细菌的抗生素,其中这些参数可随着饮食和疾病状态而变化较大。 Antibacterial activity dependence on these factors is an important consideration, especially for the target bacterial antibiotic in the digestive tract, where these parameters may vary with diet and disease state changed greatly.

[0165] 当设计用于将来体外测试的方法时,MIC对这些环境变量的敏感性也可以是考虑的重要因素。 [0165] When the in vitro test method for the future of design for, MIC sensitivity to these environmental variables can also be an important factor to consider. 临床和实验室标准研究所,CLSI (前NCCLS)推荐使用补充了维生素K1和氯高铁血红素的Brucella琼脂用于厌氧微生物的最小抑制浓度(MIC)测定。 Clinical and laboratory Standards Institute, CLSI (former NCCLS) recommended supplemented with vitamin K1 and hemin minimum inhibitory concentration of Brucella agar for anaerobes (MIC) assay. 然而,在该培养基中的二价阳离子的水平没有被标准化。 However, levels of divalent cations in the medium is not standardized. 此外,在厌氧手套箱下使用的培养基的pH也可以在不同的气体混合物下变化。 Further, pH of the medium used under anaerobic glove box under a mixture may also vary in different gases. 厌氧微生物典型地在氮气、氢气和二氧化碳的混合物中温育,并且(X)2的存在将酸化培养基并且可以是变异性的重要来源。 Anaerobic microorganisms typically a mixture of nitrogen, hydrogen and carbon dioxide incubated, and (X) in the presence of acidified medium 2 and may be an important source of variability. 考虑到可用于厌氧微生物敏感性测试的气压条件的多样性(H2AX)2产生器,抽真空/替换方法或厌氧室),接种多少也可能难以标准化。 Considering the diversity of atmospheric conditions generator 2 may be used for susceptibility testing of anaerobic microorganisms (H2AX), evacuation / replacement method or anaerobic chamber), may be difficult to standardize the number inoculated. 每个实验室可获得的厌氧条件将确定在工作台面(bench top)操作过程和厌氧平衡中生物体暴露于有氧气氛的持续时间,并因此影响培养物的存活力和实验结果。 Each laboratory will determine the anaerobic conditions obtained in the work surface (bench top) operation of the process and anaerobic organisms balance duration of exposure to oxygen atmosphere, and thus affect the viability of the culture and experimental results thereof.

[0166] 在该研究中,我们检验了二价阳离子钙和镁的水平,PH(从5-8),接种物密度(超过3个数量级)对MIC的影响以及Brucella培养液的不同批次的变异性。 [0166] In this study, we examined the levels of divalent cations calcium and magnesium, the PH (from 5-8), Effects of inoculum density (more than 3 orders of magnitude) as well as different batches of MIC medium is Brucella variability.

[0167] 材料和方法 [0167] Materials and methods

[0168] 细菌菌株: [0168] Bacterial strains:

[0169] 实验室菌株艰难梭菌9689,700057,43255,17857和迟缓真杆菌(Eubacterium lentum) 43055 获自美国典型培养物保藏中心(American Type Culture Collection(ATCC))。 [0169] strain of Clostridium difficile laboratory 9689,700057,43255,17857 slow and Eubacterium (Eubacterium lentum) 43055 were obtained from the American Type Culture Collection (American Type Culture Collection (ATCC)). 所有的菌株被划线接种在brucella琼脂平板上,所述平板补充了氯高铁血红素和维生素K,其来自在使用前在10%甘油中保持在-78°C的冷冻贮液。 All strains were streaked on brucella agar plates, said plates supplemented with hemin and vitamin K, which remains at the 10% glycerol frozen at -78 ° C in the reservoir prior to use.

[0170] MIC 测试: [0170] MIC test:

[0171] 将目前用于厌氧培养液和琼脂稀释的CLSI方法(4)用于进行MIC评估。 [0171] The methods currently used anaerobic CLSI broth and agar dilution (4) for assessment of MIC. 培养液稀释对于梭菌的MIC测试不是一种有效的方法;然而,由于在厌氧室内平衡后测量固体琼脂的PH的潜在的不准确性,对于评估PH效果使用和比较了两种方法。 For the culture of Clostridium dilution MIC testing is not an efficient method; however, due to the potential measurement inaccuracies PH solid agar in an anaerobic chamber after balancing, and for evaluating the effect of using PH compare the two methods.

[0172] 接种物密度对MIC倌的影响: [0172] Effect of inoculum density of MIC groom:

[0173] 使用琼脂稀释方法G),测定了接种物密度对艰难梭菌针对MCC和万古霉素的敏感性的影响。 [0173] using the agar dilution method G), the measured sensitivity of C. difficile inoculum density for MCC and vancomycin. 通过首先制备〜IO8CfuAiL的混悬液来制备接种物,并接着将所述混悬液以10倍因子系列稀释,从而获得在IO5-IO8CfuAiL之间的培养物密度范围以得到IO2-IO5Cfu/ 斑点的斑点密度。 Inoculum was prepared by first preparing a suspension ~IO8CfuAiL, and then the suspension was diluted 10-fold factor series, thereby obtaining a culture density range between IO5-IO8CfuAiL to obtain IO2-IO5Cfu / spot dot density.

[0174] pH对MIC倌的影响: [0174] Effect of pH on the MIC groom:

[0175] 使用琼脂稀释和微量培养液稀释方法,在6-8的PH范围内评价艰难梭菌对MCC的敏感性。 [0175] agar dilution and broth microdilution dilution culture method, evaluation of the sensitivity of C. difficile in the MCC PH range of 6-8.

[0176] 使用琼脂稀释方法,在两个单独的实验中,在6. 2-8.0的pH范围内确定MCC针对艰难梭菌菌株的MIC。 [0176] using the agar dilution method, in two separate experiments, for determining the MIC MCC C. difficile strains in a pH range of 2-8.0 6. 为了获得针对敏感性测试所需要的厌氧性pH,将缓冲液(IOOmM的NaH2PO4或TAPS [N-三(羟甲基)甲基-3-氨基丙磺酸])分别在pH 7和8加入培养基中。 In order to obtain a pH for anaerobic susceptibility testing required, the buffer (IOOmM of NaH2PO4 or TAPS [N- tris (hydroxymethyl) methyl-3-propanesulfonic acid]) were added at pH 7 and 8 medium. 甚至用强缓冲作用,在厌氧气体中平衡后PH稍微变化,并且因此在一些情形中,将培养基在环境空气中滴定至需要的厌氧性pH。 Even with a strong buffer, after equilibration in an anaerobic gas vary somewhat PH, anaerobic pH and thus in some cases, the medium was titrated to ambient air needed. 实际的pH—直在厌氧室内平衡后得到证实。 The actual pH- confirmed straight after the anaerobic indoor balance. 仅在PH 7检测用作对照的万古霉素。 Vancomycin is only used as a control to detect PH 7.

[0177] 使用培养液微量稀释方法,在3个单独的系列中,在6-8的pH范围确定MCC和万古霉素针对艰难梭菌菌株的MIC值。 Liquid microdilution method [0177] using cultured in three separate series, determined the MIC values ​​of MCC and vancomycin for C. difficile strains in a pH range of 6-8. 在第一个系列中,在环境空气中滴定未缓冲的Brucella培养液以获得5-9的pH范围。 In the first series, unbuffered titrated in ambient air Brucella broth to achieve a pH range of 5-9. 然而,在手套箱环境(10^1^/5^(^/85%¾)中,培养基的厌氧平衡降低了培养基的PH,导致在5-7. 5范围内的厌氧pH范围(如使用具有平底pH 探针的便携式PH计测试的,所述探针在手套箱外用缓冲液标准校准,接着被转移到内部)。 对于随后的实验,将缓冲液加入培养基中以对抗由于厌氧平衡导致的PH改变。在第二个系列中,将IOmM 缓冲液[NaH2PO4 ·Η20 pH7.0,M0PS pH 8.0,或TAPS pH 9.0,在环境空气中的PH值)加入pH值大于6的培养基中以在厌氧平衡后获得6-7. 6的pH范围。 However, in a glove box environment (10 ^ 1 ^ / 5 ^ (^ / 85% ¾), the medium anaerobic PH balance of the medium is reduced, leading to anaerobic pH range in the range of 5-7. 5 (e.g., portable test pH meter pH probe having a flat bottom, the probe in a glove box external calibration standard buffer, then transferred to the inside). for subsequent experiments, buffer was added to the medium due to combat pH due to anaerobic balance changes. in the second series, the IOmM buffer [NaH2PO4 · Η20 pH7.0, M0PS pH 8.0, or TAPS pH 9.0, pH value of the ambient air) was added to a pH greater than 6 medium to obtain a pH range of 6-7. 6 after anaerobic balance. 在第三个系列中,对于超过6的pH处理,将缓冲液浓度增加到IOOmM,以获得6-8. 1的厌氧pH范围。 In the third series, the pH of the treatment for more than 6, the buffer concentration was increased IOOmM, to obtain anaerobic pH range of 6-8. 1.

[0178] 二价阳离子浓度对MIC倌的影响: [0178] Effect of divalent cation concentration for the groom MIC:

[0179] 使用琼脂稀释方法确定钙和镁离子浓度对艰难梭菌菌株针对MCC的敏感性的影响。 [0179] using the agar dilution method to determine sensitivity of the calcium and magnesium ion concentrations of MCC against the strains of C. difficile. 通过实验室专家公司(Laboratory Specialists, Inc)确定如生产商所要求的在Brucella培养液中的二价阳离子水平。 Is determined by the corporate expert laboratory (Laboratory Specialists, Inc) as required by the manufacturer divalent cation levels were cultured in the Brucella. 加入额外量的二价阳离子(以氯化钙或氯化镁的形式),从而得到2. 1,3. 0和5. 7mg/dL的钙离子浓度和3. 3,4. 5,和7. 5mg/dL的镁离子浓度。 An additional amount of divalent cations (calcium or magnesium in the form of a), to give 2. 1,3. 0 calcium concentration and 5. 7mg / dL and 3. 3,4. 5, and 7. 5mg / dL of magnesium ion concentration.

[0180] MCC MIC倌关干不同商♦枇次的培养某的再牛 [0180] different providers ♦ loquat times of culture MCC MIC groom off of a dry cow again

[0181] 使用CLSI琼脂稀释方法,还用来自BBL的三个不同的商业批次的Brucella琼脂(批号#30768960,211086,和3167036)检验艰难梭菌针对MCC的敏感性,所述Brucella琼脂补充了不同批次的维生素K(希格玛(Sigma)批号V-3501和0214010)和氯高铁血红素(希格玛批号072K1221 和0;34K7656)。 [0181] Agar dilution method using CLSI, also with three different commercial lots of Brucella agar from the BBL (lot # 30768960,211086, and 3,167,036) testing the sensitivity of MCC against C. difficile, a Brucella agar supplemented with different batches of vitamin K (Sigma (Sigma) lot V-3501, and 0,214,010) and hemin (Sigma lot 072K1221 and 0; 34K7656).

[0182] 结果 [0182] results

[0183] 接种物密度对MIC倌的影响 Effects [0183] MIC of inoculum density of groom

[0184] 表1和2显示接种物密度对MCC和万古霉素针对艰难梭菌的两个菌株(ATCC 9689 和ATCC 700057)的MIC的影响。 [0184] Tables 1 and 2 show the influence inoculum density of MCC and vancomycin against two strains of C. difficile (ATCC 9689 and ATCC 700057) of the MIC. 两个艰难梭菌菌株对MCC的敏感性不受IO5-IO8Cfu/ ml (IO2-IO5CFU/斑点)的接种物浓度的影响,如通过对于所有测试的接种物浓度所获得的相同的MIC值所显示。 Effects of two strains of C. difficile sensitivity of MCC is not IO5-IO8Cfu / ml (IO2-IO5CFU / spot) inoculum concentration, as shown by the same MIC values ​​for all tested concentrations of the inoculum obtained . 然而,万古霉素的MIC随接种物浓度增加而递增,其中与最低接种物密度相比,最高的接种物密度表现出在MIC上四倍的增加。 However, with the MIC of vancomycin inoculum their concentrations, wherein the inoculum compared to the lowest density, highest density inoculum exhibited four-fold increase in the MIC. 这些结果表明接种物密度不是影响艰难梭菌的MCC敏感性测试的结果的重要因素。 These results suggest that inoculum density is not an important factor in the results of MCC susceptibility testing of C. difficile impact.

[0185] 表1. MCC (μ g/mL)对不同接种物密度的艰难梭菌ATCC 9689 (IO2-IO5CFU/斑点) 的体外活性 [0185] Table 1. MCC (μ g / mL) for different C. difficile inoculum density ATCC 9689 (IO2-IO5CFU / spot) activity in vitro

[0186] [0186]

Figure CN102503994AD00191

[0187] 表2. MCC (μ g/mL)对不同接种物密度的艰难梭菌ATCC 700057 (IO2-IO5CFU/斑点) 的体外活性[0188] [0187] Table 2. MCC (μ g / mL) for different C. difficile inoculum density ATCC 700057 (IO2-IO5CFU / spot) activity in vitro [0188]

Figure CN102503994AD00201

[0189] DH对MIC倌的影响 [0189] The MIC groom's influence DH

[0190] 表3描述了不同pH值对艰难梭菌针对MCC的敏感性的效果,其在两个单独的日子,通过琼脂稀释方法所测量。 [0190] Table 3 describes the different pH values ​​for the sensitivity of C. difficile effect of MCC, in which two separate days, as measured by the agar dilution method. 在第一次实验中,对于两个艰难梭菌菌株,与更低的PH处理(pH 6. 2和pH 7. 2)相比,最高的pH处理(pH 7. 9)表现出MIC值的8倍的增加。 In the first experiment, for both strains of C. difficile, as compared to the lower PH treatment (pH 6. 2 and pH 7. 2), the highest pH treatment (pH 7. 9) exhibited MIC values 8-fold increase. 当在最高的pH(pH 8.0)重复进行证实性实验时,MIC值对于两个菌株保持较高。 When repeated experiments demonstrated the highest pH (pH 8.0), MIC values ​​remain high for both strains. 对于任一菌株,在pH 6. 2和pH 7之间,在MCC MIC中没有观察到增加。 For any strain between pH 6. 2 and pH 7, no increase was observed in the MCC MIC.

[0191 ] MIC值随pH的增加不与增加的生长成一致的相关性,因此pH对MIC的影响似乎不仅仅是由于生物体在更高的PH的增加的存活力。 Correlation [0191] MIC value with the increase in pH is not consistent with the increased growth into, so the effect of pH on the MIC of the organism appears not only because of the increase in viability of higher PH. 与pH 6.2和? And pH 6.2 and? !1 7. 9处理相比,pH 7处理具有较不密集的生物体斑点生长。 ! 1 treatment as compared to 7.9, pH 7 treated with less dense spots organism growth.

[0192] 表3. pH对琼脂稀释MIC值的影响(缓冲的培养基) [0192] Table 3. pH Effect on the agar dilution MIC values ​​(buffered medium)

Figure CN102503994AD00202

[0194] 表4,5和6显示在三个单独的日子,用5-8. 1的pH范围进行的培养液微量稀释敏感性方法的MIC数据。 [0194] Tables 4, 5 and 6 show three separate days, the culture was carried out with a pH range of micro-5-8. 1 dilution MIC data of the sensitivity of the method. 在第一个系列中,其中培养基未被缓冲,对于两个艰难梭菌菌株,在pH 7. 5的MCC的MIC比在pH 5. 9的MIC大8倍(表4)。 In the first series, wherein the unbuffered medium, for the two C. difficile strains than in the MIC of the MCC pH 7. 5 times larger in the MIC. 8 pH 5. 9 (Table 4). 在pH 5的MIC不能被确定,因为生物不能在该PH上生长。 It can not be determined in the MIC pH 5 because the organisms can not grow on the PH. 对于艰难梭菌ATCC 9689和ATCC 700057,在MCC MIC上,缓冲的(IOmM) pH 7. 6处理分别比pH 6处理表现出8倍和16倍的增加(表5)。 For C. difficile ATCC 9689, and ATCC 700057, on MCC MIC, buffer (IOmM) pH 7. 6 pH of the treatment process, respectively, than 6 times and 8 exhibited 16-fold increase (Table 5). 在该第三强缓冲(IOOmM)系列中,对于两种生物体观察到类似的结果,其中最高pH处理(pH 8. 1)在MIC上比最低的PH处理(pH 6)表现出16倍增加(表6)。 In this third intensity buffer (IOOmM) family, for both organisms Similar results were observed, the highest pH treatment (pH 8. 1) on the MIC process than the lowest PH (pH. 6) showed a 16-fold increase (table 6). 在所有的三个实验中,万古霉素显示出类似的趋势,其中与最低的PH处理相比,最高的pH处理产生4-8倍更大的MIC。 In all three experiments, vancomycin show similar trends, which compared with the lowest PH treatment, treatment yields the highest pH 4-8 fold greater MIC. [0195]表 [0195] Table

[0196] [0197] 表5.使用弱缓冲的培养基(IOmM)的pH对MIC的影响 Effect of pH [0196] [0197] Table 5. Medium buffered with a weak (IOmM) of the MIC

[0198] [0198]

Figure CN102503994AD00211

[0199] 表6.使用强缓冲的培养基(IOOmM)的pH对MIC的影响 Effect of pH [0199] Table 6. strongly buffered medium (IOOmM) of the MIC

[0200] [0200]

4.使用未缓冲的培养基pH对MIC的影响 4. Effect of pH of the unbuffered medium of MIC

Figure CN102503994AD00212
Figure CN102503994AD00221

[0201] 还在所有的pH处理上对测定平板的总的生长进行视觉检查。 [0201] The total plate growth assay also visually inspected on all pH treatment. 在第一个系列中,其使用未缓冲的培养液,总的培养浊度随PH增加而增加。 In the first series, using unbuffered culture broth, the turbidity increases the total culture PH increases. 除了对于pH 7.2和pH 7.6培养物浊度是相同的之外,在第二个系列中观察到相同的趋势,所述第二个系列使用IOmM的缓冲培养液。 In addition to the pH 7.2 and pH 7.6 cultures than haze are the same, observed in the second series the same tendency, the buffered medium used IOmM the second series. 在第三个系列中,在整个PH处理中,培养物浊度更相等,例外的是pH 7. 5,其是最混浊的。 In the third series, the entire process PH, the more equal the turbidity of the culture, with the exception that the pH 7. 5, which is the most turbid.

[0202] 总而言之,对于敏感性测试的两种方法和各种浓度的缓冲盐,对于艰难梭菌的两个菌株,MCC和万古霉素的MIC值都随pH增加而增加。 [0202] In summary, the sensitivity for both methods buffered saline and tested at various concentrations, for both strains of C. difficile, the MCC and vancomycin MIC values ​​are increased with increasing pH.

[0203] 二价阳离子浓度对MIC倌的影响: [0203] Effect of divalent cation concentration for the groom MIC:

[0204] 在商业Brucella培养液中的钙和镁水平的测量分别显示21和33mg/L的钙和镁离子浓度。 [0204] In commercial calcium measurements Brucella broth and 21 show the levels of magnesium and 33mg / L of calcium and magnesium ion concentrations. 加入各种额外量的二价阳离子,并且在三个不同的钙离子浓度Ol,30和57mg/ L)和三个不同的镁离子浓度(33,45和75mg/L)测试对于艰难梭菌菌株的MCC MIC值。 Adding various additional amount of divalent cations, and three different calcium concentration Ol, and 57mg L) and three different magnesium ion concentration of 30 / (33, 45 and 75mg / L) for test strains of C. difficile the MCC MIC values. 在所有类型的培养基中,MIC值保持相同。 In all types of culture medium, MIC values ​​remain the same. 在具有不同阳离子浓度的培养基中,艰难梭菌9689 具有0. 063 μ g/ml的MIC值,艰难梭菌700057具有0. 125 μ g/ml的MIC值。 Cations in a medium having a different concentration, with C. difficile 9689 0. 063 μ g / MIC values ​​of ml, with C. difficile 700 057 0. 125 μ g / MIC values ​​ml. 测试作为对照的万古霉素,所述测试用补充的Brucella琼脂进行,在实验过程中,其没有添加任何额外的钙或镁作为对照,对于所有的实验证实预期的MIC值为1 μ g/ml (表7和8)。 Vancomycin tested as a control, the test is performed Brucella agar supplemented, during the experiments, it does not add any additional calcium or magnesium as a control for all experiments confirm the expected MIC value of 1 μ g / ml (tables 7 and 8).

[0205] 表7.在补充不同二价阳离子浓度的Brucella琼脂中的MCC的体外活性[0206] [0205] Table 7. In vitro activity of MCC supplemented with different concentrations of divalent cation Brucella agar [0206]

Figure CN102503994AD00231

[0207] 表8.在补充不同二价阳离子浓度的Brucella琼脂中的MCC的体外活性 [0207] Table 8. In vitro activity supplemented with different concentrations of divalent cations in the MCC Brucella agar

[0208] [0208]

Figure CN102503994AD00232

[0209] 关于不同商业批次的培养基的MCC MIC倌: [0209] For the culture of different batches commercial MCC MIC groom:

[0210] 在三个不同的日子使用三种不同批次的补充的Brucella琼脂培养基来比较MCC 针对艰难梭菌菌株的活性。 [0210] using three different batches of complement at different Brucella agar medium three days to compare the activity of MCC against C. difficile strains. 通过测试QC生物体,迟缓真杆菌对在CLSI(NCCU)可接受范围,即0. 06-0. 25 μ g/mL内的克林霉素的活性来控制MIC测定。 QC organisms tested, Eubacterium to slow the CLSI (NCCU) an acceptable range, i.e., 0. 06-0. 25 in the active clindamycin μ g / mL to control the MIC determinations. 进行MIC测定的另一个对照步骤是包括甲硝唑并监测其对艰难梭菌菌株的活性,其在我们实验室中显示出具有范围在0. 25-0. 5 μ g/mL内的MIC值。 Another control is the MIC determination step comprises metronidazole and its monitoring activity C. difficile strains, which exhibit MIC values ​​having a range within 0. 25-0. 5 μ g / mL in our laboratory . 如在表9中所显示,MCC对艰难梭菌的活性没有受到不同批次的补充的Brucella琼脂的影响。 As shown in Table 9, MCC activity is not affected by C. difficile different batches of Brucella agar supplemented. 所有的对照证实在确定范围内的活性。 Activity in determining the scope of all control confirmed.

[0211] 表9.使用三种不同批次的培养基测试的MCC的体外活性 [0211] Table 9. Test using three different batches of culture media in vitro activity of MCC

[0212] [0212]

Figure CN102503994AD00241

[0213] 结论 [0213] Conclusion

[0214] 与万古霉素相反,MCC针对艰难梭菌的活性不受在IO2-IO5Cfu/斑点范围内的接种物浓度的影响。 Effect [0214] In contrast to vancomycin, MCC activity against C. difficile is not within IO2-IO5Cfu / spot inoculum concentration range.

[0215] 艰难梭菌对MCC的敏感性不受阳离子浓度(在2. 1-5. 7mg/dL范围内的钙离子,浓度在3. 3-7. 5mg/dL范围内的镁离子)和不同商业批次的培养基的影响。 [0215] (calcium ions and magnesium ions in the range dL 2. 1-5. 7mg / concentration in 3. 3-7. 5mg / dL range) of C. difficile MCC cation concentration and sensitivity without the impact of different business culture medium batches.

[0216] 在6-8的pH范围内,对于MCC和万古霉素二者的MIC值随pH增加而增加。 [0216] in a pH range of 6-8, the MIC values ​​for both the MCC and vancomycin increases with pH. 在碱性PH上的高MIC值可能是由于在高于它们的pKa时,这两种化合物的酚羟基的去质子形式,在这种情形中,它们形成带电的种类,预期其对细菌细胞具有更少的通透性。 Higher MIC values ​​on basic PH could be due to their having a pKa higher than, deprotonated form of a phenolic hydroxyl group of these two compounds, in this case, they form a charged species, which is expected to have a bacterial cell less permeability. 相反,在低于PKa时(对于MCC,7. 22),抗生素大部分被质子化,并且因此应该更有效地渗透细胞膜。 Conversely, when less than a PKa (for MCC, 7. 22), most of the antibiotic is protonated, and thus should be more efficiently penetrate cell membranes.

[0217] 生物密度通常随增加的pH而增加;在存在缓冲剂时,生长密度,但不是MIC对pH 的依赖性减少。 [0217] Biological density generally increases with increasing pH; in the presence of buffers, growth density, but not MIC reduce dependence on pH. 尽管在缺乏缓冲液时,生物体密度与碱性成正相关,MIC趋势不可能只是pH 对生物密度的影响的结果。 While in the absence of a buffer, with a basic density of the organism was positively correlated, the results of the MIC trend can not just the biological effect of pH on the density. 这是因为在缓冲实验中观察到MIC和pH之间的相同的关系,其中在整个pH处理过程中,生物体密度更加相等,这可能是由于缓冲液类型对生物生长的不同的作用造成的。 This is because the relationship between the observed MIC of the same buffer and pH experiments, wherein the pH throughout the process, the organism is more equal density, which may be due to different type of buffer action caused by the growth of organisms.

[0218] 实施例2.在健康受试者和患有艰难梭菌相关的腹泻(CDAD)的患者中的MCC的安全性、药物代谢动力学和后果 [0218] Example 2. Safety of MCC, pharmacokinetics and consequences of healthy subjects and in patients suffering from Clostridium difficile-associated diarrhea (CDAD) in implementation

[0219] IB-MD 阶段. [0219] IB-MD stage.

[0220] 简述.这是在佛罗里达州迈阿密的迈阿密大学临床药理学部进行的口服、多剂量、双盲、随机、安慰剂作为对照的、剂量逐步上升的研究。 [0220] briefly. This is done orally Clinical Pharmacology at the Faculty of the University of Miami in Miami, Florida, multi-dose, double-blind, randomized, placebo-controlled, dose-escalating. Richard Preston, Μ. D.作为该试验的主要研究者。 Richard Preston, Μ. D. as the principal investigator of the trial. MCC多口服剂量的耐受性和药物代谢动力学在总共M名健康的志愿受试者中进行评估。 MCC Multi tolerability and pharmacokinetics of an oral dose kinetics evaluated in a total of M healthy volunteer subjects. 评估的MCC的口服剂量(在3个组中,每个组8名受试者,其中6名用活性剂处理,2名安慰剂处理)是150,300,和450mg(在包含50mg的研究药物的粉末填充的胶囊中),其在早餐后每日施用,连续进行10天。 MCC oral doses (in three groups, each group of 8 subjects, 6 wherein the active agent used, two placebo-treated) are evaluated 150, 300, and 450 mg of (containing 50mg of the study medication powder filled capsules), which is administered daily after breakfast, for 10 consecutive days. 给药受试者并且在组合的住院病人/门诊患者基础上监测。 Monitoring the subject and is administered in inpatient / outpatient basis on the combination. 在10天的给药阶段中,让受试者在第0天和第9天进入研究单位,并且在每次入院后停留多达48小时。 In the 10-day dosing period, so that subjects at day 0 and day 9 of the unit entered the study, and admission to stay up to 48 hours after each. 在完成计划表安排和程序后,在第2天和第11天让受试者出院。 After completing the schedule arrangements and procedures, in the first 2 days and 11 days for subjects discharged. 在门诊患者阶段,受试者每日向研究单位报告给药情况并且留下观察3小时。 In the outpatient phase, where the subject is administered a daily report to the study unit and leaves was observed for 3 hours.

24[0221] 在多次给药阶段过程中,在不同时间点/间隔收集系列血液,尿液和粪便样品。 24 [0221] In the multiple dose phase, at different points in time / interval series collecting blood, urine and stool samples. 确定MCC的血浆,尿液和粪便浓度,进行药物代谢动力学分析。 Determining the plasma, urine and feces concentration of MCC, pharmacokinetic analysis. 在受试者从研究中被排除之前,在每个研究阶段的第17天安排随访检查的计划表。 Before subjects were excluded from the study, on day 17 of each study period scheduled follow-up examinations schedule. 在整个治疗阶段并且在随访研究中,紧密监测研究受试者的任何副作用的发生或异常实验室测试发现。 Throughout the treatment period and follow-up study, close monitoring of study subjects any side effects or the occurrence of abnormal laboratory tests found. 见,图1,IB-MD阶段给药时间表。 See FIG. 1, stage IB-MD dosing schedule.

[0222] 2A 阶段 [0222] 2A stage

[0223] 简述.这是选择MCC的安全和有效剂量的剂量发现研究。 [0223] briefly. This is the choice of dose safe and effective dose of MCC discovery research. 向受试者随机给药100 (50mg/12 小时),200 (IOOmg/12 小时),或400 (200mg/12 小时)mg/ 天,持续10 天,随后 Random administering to the subject 100 (50mg / 12 hours), 200 (IOOmg / 12 hour), or 400 (200mg / 12 hours) mg / day for 10 days, then

进行临床评估。 Clinical evaluation. 受试者在每日日志卡片上记录了所有的症状。 Subjects recorded all of the symptoms on the daily log card. 对粪便频率和稠度,血液在粪便中的存在和腹部不适给与特别的注意。 Stool frequency and consistency, the presence of blood in the stool and abdominal discomfort given special attention. 在筛选进入和在治疗结束时(第10-12天)或退出时(无论哪一个更早)进行实验室评估。 At screening and entering (10-12 days) or quit at the end of treatment (whichever is earlier) for laboratory evaluation. 在治疗结束(第10-12天)进行临床观察和日志卡片评估。 At the end of treatment (days 10-12) Clinical observations and assessments card log. 在第2天到第9天,第17天和第52天的治疗上进行患者会见。 Day 2 to Day 9, patients undergoing meeting on day 52 and day 17 of treatment. 对于进入参与标准,进行艰难梭菌毒素的测定。 For participate into the standard measurement Clostridium difficile toxin. 对于未对MCC治疗做出反应的受试者和在临床复发的情形中,进行艰难梭菌毒素测定和培养。 For the subject does not respond to treatment and MCC in the case of clinical relapse, for C. difficile toxin assay and culture. 还在未对治疗做出反应的受试者退出时,进行临床、实验室和微生物学评估。 When the subject is still not responding to treatment withdrawal, clinical, laboratory and microbiological assessment. 在给药的第一天和最后一天,在给药前0. 5小时和给药后2小时采取药物代谢动力学血浆样品。 The last day, taken pharmacokinetic plasma samples 0.5 hours prior to dosing and two hours after dosing on the first day of administration.

[0224] 关键参与标准.受试者是患有与艰难梭菌相关的腹泻的患者,其通过下列定义: 1)腹泻(肠习惯的改变,其中在对小时内有3或更多次的未成型的粪便,或在36小时内有超过6次的松散或水样便),和幻在粪便中存在艰难梭菌的毒素A或B。 [0224] The key criteria involved in the subject is a patient suffering from diarrhea associated with Clostridium difficile, which is defined by the following: 1) diarrhea (bowel habits change, of which there are three or more times within hours of unpaired type of feces, or more than 6 times loose or watery stools within 36 hours), and phantom presence of C. difficile toxin a in feces or B.

[0225] 关键排除标准受试者不能患有1)严重或威胁生命的CDAD,2)与CDAD不相关的威胁生命或严重的疾病,3)同时使用:万古霉素,甲硝唑,杆菌肽,或相关药物。 [0225] The key subjects not suffering from exclusion criteria 1) serious or life-threatening CDAD, 2) associated with CDAD are not life-threatening or serious diseases, 3) use: vancomycin, metronidazole, bacitracin or related drugs. (如果在知道粪便毒素的实验室结果前,研究者认为临床有必要开始治疗,那么用甲硝唑和/或万古霉素在多到M小时,但是不超过3次剂量的治疗是容许的);用于治疗CDAD的任何药物;或其它抗生素,4)在过去的三个月里,溃疡性结肠炎或局限性回肠炎和CDAD的多次复发(定位为超过一次复发)的历史。 (If you know the lab results before stool toxin, the researchers believe it is necessary to begin clinical treatment, then with metronidazole and / or vancomycin to a multi-M hours, but no more than three doses of the treatment is tolerated) ; for any drug treatment of CDAD; or other antibiotics, 4) in the past three months, ulcerative colitis or Crohn's disease and multiple recurrences of CDAD (defined as more than one relapse) history. (容许具有一次CDAD的复发的受试者参与)。 (Allowing a subject having a relapse of CDAD involvement).

[0226] 事件的时间计划表 [0226] event time schedule

[0227] 表10.在2A阶段研究中的评估方法的时间计划表 Time schedule Method [0227] Table 10. Evaluation of Phase 2A study

[0228] [0228]

Figure CN102503994AD00261

[0229] a在给药的第一天和最后一天,在施用前0. 5小时和施用后2小时采取用于药物代谢动力学的血液样品 [0229] a last day Blood samples were taken for pharmacokinetics at 2 hours after administration, and 0.5 hours before administration of the first day of administration

[0230] 终点.在治疗结束时,研究者确定受试者是否已经被治愈或失败。 [0230] the end. At the end of treatment, the researchers determine whether the subject has been cured or failure. 此外,将腹泻消退的时间(定义为消退到每天< 3次的松散或水样便)和在治疗的第10天CDAD的症状的完全减轻(完全减轻是消退到每天< 3次总便数,不管是松散还是紧密;并且没有发烧、升高的白血细胞或腹痛)作为初始的终点,而在治疗后6周内的复发(腹泻的复发,定义为每天3次或更多次松散/水样便,具有阳性毒性测试)作为次级终点。 Further, the resolution of diarrhea time (defined as regression to day <3 loose or watery stools) and on day 10 of treatment complete relief of CDAD symptoms (complete amelioration is dissipated to day <3 times the total will number, regardless of loose or tight; and no fever, elevated white blood cells or abdominal pain) as an initial end point, and in the treatment of recurrent after 6 weeks (recurrent diarrhea, defined as three or more loose / watery per day will , having a toxicity test positive) as a secondary endpoint.

[0231] 分析 [0231] Analysis

[0232] 安全群体: [0232] security groups:

[0233] 安全群体包括已经接受研究药物的至少一次给药并且具有已获得安全信息的所有的随机受试者。 [0233] Safety population included has received study drug is administered at least once and have been obtained for all randomized subjects security information.

[0234] 有效群体: [0234] effective population:

[0235] 在按照方法治疗的患者中确定临床成功或失败。 [0235] determine the clinical success or failure in a patient according to the methods of treating. 用于分析腹泻消退和症状完全减轻的时间的群体是改良意向治疗的群体(mITT),其由所有的随机受试者组成,所述受试者接受至少一个剂量的研究药物,具有腹泻史,在M小时内具有3次或更多次的松散便和在基线的阳性艰难梭菌毒素。 Analysis population for complete relief of symptoms and the resolution of diarrhea time is modified intent to treat population (the mITT), which is composed of all randomized subjects, the subjects received at least one dose of study drug, having history of diarrhea, having three or more loose in the M-hour and then at baseline positive C. difficile toxin.

[0236] 腹泻消退的时间定义为从第一次给药研究药物到腹泻消退的时间(以天数表示);在三个治疗组中比较腹泻消退的时间。 [0236] resolution of diarrhea is defined as the time the first dose study medication to a resolution of diarrhea from time (in days); comparison time resolution of diarrhea three treatment groups. 将腹泻停止日定义为在M小时内发生<3次的不成形便(水样或松散)的第一天,并且治疗的持续时间延续到第10天。 Is defined as the date of cessation of diarrhea occur within hours of M <it shapeless (watery or loose) of the first three times a day, and the duration of the treatment is extended to 10 days. 使用本发明的日志记录数据来在第10和第12天评估腹泻的消退。 Using the logging data according to the present invention is to evaluate the regression of diarrhea on days 10 and 12.

[0237] CDAD症状的完全减轻: [0237] CDAD completely alleviate the symptoms:

[0238] 将CDAD的症状的完全减轻定义为到研究的第10天消退到每天彡3次的排便(如在患者日志中所记录的),而没有其它的相关体征/症状如发烧(彡37. 7°C ),腹痛(在日志中没有反应)和升高的WBC (WBC的正常实验范围)。 [0238] The CDAD symptoms completely alleviate defined as day 10 study regression to San three times daily bowel movements (e.g., in a patient log recorded), and no other related signs / symptoms such as fever (San 37 . 7 ° C), abdominal pain (in the log no response) and the normal range of experimental elevated WBC (WBC's). 如果有任何变量未提及,则认为该结果是未知的。 If any variable is not mentioned, it is considered that the outcome is unknown.

[0239] 临床复发率: [0239] Clinical relapse rate:

[0240] 将临床复发率定义为在治疗后6周内> 3次不成形便(松散或水样的)和艰难梭菌毒素A或B的阳性便。 [0240] is defined as the rate of clinical relapses after 6 weeks of treatment> 3 then deformed (loose or watery), and then positive C. difficile toxin A or B.

[0241] 结果 [0241] results

[0242] 参与和人口统计 [0242] participation and demographics

[0243] 下面的部分总结研究群体在IB-MD阶段和2A试验中的参与和人工统计特征。 [0243] The following section summarizes research participation and labor groups in the statistical characteristics of phase IB-MD and 2A trials. 总共M名健康受试者参与IB-MD研究阶段。 A total of M healthy subjects involved in the research phase IB-MD. 交替的男性和女性受试者参与以提供在性别之间的对等。 Alternating male and female subjects to participate to provide between gender. 受试者年龄范围在38-62岁(平均51.6士7.5岁),体重55.5-901^(平均71. 5 士9. 2kg),高度147-183cm (平均164. 8 士10. 8cm)。 Subject age range 38-62 years (mean 51.6 years disabilities 7.5), weighing 55.5-901 ^ (mean 71.5 disabilities 9. 2kg), height 147-183cm (average 164.8 disabilities 10. 8cm).

[0244] 在2B研究阶段,共49名受试者参与。 [0244] In the phase 2B study, a total of 49 subjects were enrolled. 在接受任何研究药物前,一名受试者不同意并且从研究中撤出,因此不能被评估安全性或功效。 Before receiving any study medication, one subject did not agree to withdraw from the study and therefore can not be evaluated for safety or efficacy. 一名受试者GOOmg给药组)在36小时内具有> 6次排便,但是在前M小时< 3次排便,并且不能评估腹泻消退的时间但是可以评估临床反应和安全分析。 One subject GOOmg administration group) within 36 hours of having> 6 bowel movements, but the former M h <3 bowel movements, and can not assess the time resolution of diarrhea but the clinical response can be assessed and safety analysis. 在给药1或2剂量后,三个患者由于不同意(1名受试者,IOOmg 给药组),需要另外的针对肺炎的抗生素(1名受试者,IOOmg给药组)或不能服研究药物(1 名受试者,200mg给药组)而终止。 After administration 1 or 2 doses, because they do not agree to three patients (subjects 1, IOOmg administration group), the need for additional antibiotics pneumonia (1 subject, IOOmg administered group) served or not study drug (1 subject, 200mg administration group) is terminated. 将受试者的人工统计列于表11中。 The subject of artificial statistics are given in Table 11.

[0245] 表11.进行2A阶段研究的简要人工统计;显示在可评估安全性的群体中的48名受试者的人工统计。 [0245] Table 11. Artificial brief Phase 2A study statistics; 48 displays the subject population may be evaluated in the safety manually counting.

[0246] [0246]

27 27

Figure CN102503994AD00281

名受试者中复发CDAD (1/12),在最高剂量给药组中有一名受试者复发CDAD (1/16),总共复 Subjects relapse CDAD (1/12), there is one subject of recurrent CDAD (1/16) in the highest dose group, the total complex

发率为2/41 (5%)0这两例复发都是在治疗结束后约1个月发生。 Hair was 2/41 (5%) 0 are two recurrences occurred in about 1 month after treatment.

[0252] [0252]

Figure CN102503994AD00291

[0253] a在临床成功的患者中评估在治疗后6周内毒素阳性腹泻的复发 [0253] a assessment of relapse after 6 weeks of treatment in patients with diarrhea toxin positive clinical success in

[0254] 将腹泻消退的时间定义为根据患者的日志卡,患者消退到每天少于3次不成形便的时间。 [0254] The resolution of diarrhea is defined as the time of the patient's diary cards, patient subsided to less than three times a shapeless stools every day. 在mITT群体中,减轻的中值时间对于MCC IOOmg/天,200mg/天和400mg/天治疗组分别是5. 5天,3. 5天,和3. 0天。 In mITT population, the median time to reduce the MCC IOOmg / day, 200mg / day and 400mg / day treatment groups were 5.5 days, 3.5 days and 3.0 days. 腹泻消退的平均天数时间在IOOmg/天治疗的受试者中是6. 3士3. 66天,在200mg/天治疗的受试者中是4. 8士3. 56天,在400mg/天治疗的受试者中是3. 6 士2. 03天。 Mean days to resolution of diarrhea is 6.3 in 3.66 days persons subject IOOmg / day treatment, the subject is 4.8 disabilities 200mg / day treatment of 3.56 days, the 400mg / day treated subject is 3.6 persons 2.03 days. 在IOOmg/天和200mg/天治疗组之间和在200mg/天和400mg/天治疗组之间,腹泻消退的时间不存在统计学上显著的差异;然而,在IOOmg/天和400mg/天治疗组之间的差异具有统计学显著性(P = 0. 0506 KaplanMeier估计和ρ = 0. 0503Kruskal-ffallis 检验)。 Between IOOmg / day and 200mg / day treatment groups and between 200mg / day and 400mg / day treatment group, the time resolution of diarrhea no significant differences were present; however, 400mg / day treatment IOOmg / day the difference between the groups was statistically significant (P = 0. 0506 KaplanMeier estimation and ρ = 0. 0503Kruskal-ffallis test).

[0255] 表13.腹泻消退的时间(mITT群体),定义为到每天消退到<3次不成形便的时间(根据患者的日志卡片) [0255] Table 13. The time resolution of diarrhea (the mITT population), defined as regression to a day <3 unformed stool time (according to the patient's card log)

Figure CN102503994AD00301

[0257] a到第10天,其腹泻没有消退到每天< 3次松散便的受试者 [0257] a Day 10 to which no diarrhea per day regression to <3 times subject the loose feces

[0258] bKaplan-Meier 估计 [0258] bKaplan-Meier estimates

[0259]。 [0259]. 获自广义Wilcoxon检验的P值 P values ​​obtained from the generalized Wilcoxon test

[0260] 在表14中显示到治疗结束时CDAD的症状的完全减轻,其被定义为到研究的第10 天,每天< 3次总的排便数(不管成形还是不成形,如在患者的日志卡片中所记载),并且没有发烧、升高的WBC计数或腹痛(根据患者日志卡片的反应)。 [0260] After treatment to show complete relief of symptoms of CDAD, which is defined as the day 10 of the study, day <3 times the total number (whether deformed or shaped bowel, such as log in the patient 14 in Table card described), and no fever, elevated WBC counts or abdominal pain (patient diary card according to the reaction). 37. 5%的IOOmg/天治疗组,50. 0%的400mg/天治疗组和86. 7%的400mg/天治疗组获得完全的减轻。 37.5% of IOOmg / day treatment group, 50.0% of 400mg / day treatment group and 86.7% of 400mg / day treatment group obtained complete relief. 值得注意的是,到第10天没有完全减轻的大多数患者仍然是治疗成功的,其到第17天症状消退并且不需要进一步的治疗。 It is noteworthy that the majority of patients by day 10 is not completely alleviate the still successful treatment, the symptoms subsided to 17 days and does not require further treatment. 从研究中退出的三名患者(一名不同意,一名需要专门的抗生素,一名不能口服药物)也被列在没有完全减轻之列。 Three patients withdrew from the study (one does not agree, need a special antibiotic, an oral medication can not) are also listed in the list is not complete relief.

[0261] 表14.在mITT群体中,到治疗结束时,CDAD症状的完全减轻,其被定义为到研究的第10天,每天< 3次的总排便数(成形或不成形,如在患者的日志卡片上所记载),没有其它的相关体征/症状如发烧、腹痛和升高的WBC [0261] Table 14. In mITT population, to the end of treatment, complete alleviation of the symptoms of CDAD, which is defined as the study day 10, day <3 times the total number of bowel movements (shaped or shapeless, such as a patient log described on the card), no other relevant signs / symptoms such as fever, abdominal pain and elevated WBC

[0262] [0262]

Figure CN102503994AD00311

[0263] 只有2个受试者(在IOOmg/天治疗组中的1名受试者,在400mg/天治疗组中的1名受试者)经历了临床复发。 [0263] Only two subjects (in IOOmg / day treatment group 1 subject, a subject 400mg / day treatment group) experienced a clinical relapse.

[0264] 安全性 [0264] Security

[0265] 在IB-MD阶段研究中,MCC在所有的剂量上被所有的受试者良好耐受。 [0265] In the IB-MD study period, MCC at all doses was well tolerated by all subjects. 报道了14 例不良事件,7例在150mg组中,2例在450mg组中,5例在安慰剂组中。 It reported 14 cases of adverse events in the 150mg group of seven cases, two cases in the 450mg group, 5 cases in the placebo group. 将不良事件总结如下:头痛(2),眩晕⑴,虚弱⑴,疲劳⑴,鼻充血⑴,吞咽困难⑴,咽炎⑴,结膜炎⑴, 上呼吸道感染O),皮疹(1)和瘙痒(1)。 The adverse events are summarized as follows: headache (2), dizziness ⑴, weakness ⑴, fatigue ⑴, nasal congestion ⑴, dysphagia ⑴, pharyngitis ⑴, conjunctivitis ⑴, upper respiratory tract infection O), rash (1) and pruritus (1) . 接受MCC的患者中没有患者具有药物相关的副作用。 MCC in patients receiving no drug-related side effects in patients.

[0266] 在2A阶段研究中,如在表15中所显示,在IOOmg/天治疗组中有4/16 0 % ) 的受试者,在200mg/天治疗组中有4/16Q5. 0% )受试者,在400mg/天治疗组中有1/16(6.3% )受试者在研究中被报道具有至少一个AE。 [0266] Phase 2A study, as shown in Table 15, there 4/16 0%) subjects in IOOmg / day treatment group, there are 4 / 16Q5. 0% in 200mg / day treatment group ) subjects, 1/16 (6.3%) subjects reported at least one AE in the study at 400mg / day treatment groups. 在IOOmg/天治疗组的感染和侵染体系统中报道了AEs的最高频率(3/16 ;18. 8%受试者)。 AEs reported the highest frequency of infection and infection-body system IOOmg / day treated group (3/16;. 188% of subjects). 在IOOmg/天治疗组中有2/16(12.5%)受试者报道患有血管疾病,在200mg/天治疗组中有2/16 (12. 5%)受试者报道患有胃肠疾病。 There 2/16 (12.5%) subjects reported having vascular disease IOOmg / day treatment group, with a 2/16 (12.5%) subjects reported suffering from a gastrointestinal disease 200mg / day treatment group .

[0267] 表15.在2A研究的安全群体中的副作用的发生率,通过系统器官分类和优选的术 [0267] Table 15. The incidence of side effects in the population studied 2A security by system organ class and preferred technique

语总结 Language summary

Figure CN102503994AD00321

[0269] [0269]

Figure CN102503994AD00331

[0270] 注意:百分比是受试者在该类中的比例 [0270] Note: The percentage proportion of subjects in this class

[0271 ] 5名受试者据报道在研究中具有SAEs (表16)。 [0271] 5 subjects reported to have SAEs in the study (Table 16). 在IOOmg/天治疗组中,一名受试者患有中等严重性的腹泻,另一名受试者患有严重恶化的充血性心力衰竭(CHF)。 In IOOmg / day treatment group, one of the subjects with moderate severity of diarrhea, another subject suffering from congestive heart failure (CHF) serious deterioration. 在200mg/ 天治疗组中,一名受试者具有严重的葡萄球菌败血症和严重的脑出血,另一名受试者具有中等严重的胃肠出血,第三名受试者具有中等严重的胸痛。 In the 200mg / day treatment group, one subject had severe staphylococcal sepsis and severe cerebral hemorrhage, other subjects with moderate severe gastrointestinal bleeding, third subjects with moderately severe chest pain . 在MCC 400mg治疗组中没有受试者具有SAE。 No subject in treatment group having MCC 400mg SAE. 认为所有的SAEs与研究药物不相关。 All SAEs considered unrelated to study drug.

[0272] 表16.在2A研究的安全性群体中的严重副作用的发生率 The incidence of [0272] 16. The serious side of the table in the safety population 2A study in

[0273] [0273]

Figure CN102503994AD00341

[0274] a最后一次给药研究药物的日期减去第一次给药研究药物的日期加1。 [0274] a last dose of study drug minus the date of the date of the first administration of study drug plus 1.

[0275] b如下计算研究天数:开始的日期减去研究药物的第一次给药的日期加1。 [0275] Calculation of the number of days following b: date minus the date of the first administration of study drug plus 1.

[0276] e基于研究者的评估。 [0276] e based on investigator assessment.

[0277] d受试者死亡。 [0277] d death of the subject.

[0278] 药物代谢动力学 [0278] The pharmacokinetic

[0279] 血浆浓度数据 [0279] Plasma concentration data

[0280] 在IB-MD阶段研究中,在给与多剂量口服施用后,MCC的血浆浓度通常低于剂量范围的定量的界限。 [0280] In the IB-MD study period, the given dose oral administration, the plasma concentration of MCC is generally below the quantitation limit of the dosage range.

[0281] 仅在来自6名受试者的12个样品中发现可检测的血浆浓度。 [0281] found only detectable plasma concentrations in 12 samples from six subjects.

[0282] 在所述12个可检测的浓度中,仅发现2个明显高于LL0Q,而其它的很少超过5ng/ mL 的LLOQ0 [0282] The concentration of detectably 12, significantly higher than that found only two LL0Q, while others rarely more than 5ng / mL of LLOQ0

[0283] 分别在第1天,第1小时和仅在第10天的第10次剂量前在受试者021中观察到两种浓度(11. 1 和48. Ong/mL)。 [0283] On day 1, respectively, only the first hour and two concentrations was observed (11.1 and 48. Ong / mL) in the subject 021 before the first dose of 10 10 days.

[0284] 应该注意的是,150mg剂量没有产生可检测的浓度。 [0284] It should be noted, 150mg dose did not produce detectable concentrations.

[0285] 由于在浓度范围内的低MCC血浆水平,对于药物代谢动力学分析,没有超过LLOQ 的充分的血浆数据点。 [0285] Because of the low plasma levels of MCC in a range of concentrations for pharmacokinetic analysis, no more than the LLOQ sufficient plasma data points.

[0286] 在2A阶段研究中,在多剂量口服施用后,MCC的血浆浓度大部分低于量化界限,但是在许多样品和许多受试者中具有剂量依赖性的增加,具有可测量的血浆浓度。 [0286] Phase 2A study, after multiple-dose oral administration, the plasma concentration is below the limit of quantification MCC most, but having a dose-dependent increase in the number of samples and many subject having a measurable plasma concentration .

[0287] 在MCC IOOmg/天治疗组中的2/15(13. 3% )受试者中,在MCC200mg/天治疗组中的9/16 (56. 3% )受试者中,在MCC 400mg/天治疗组的13/17(76. 5% )受试者中发现可检测的血浆浓度。 [0287] In 2/15 MCC IOOmg / day treatment group (13.3%) subjects, 9/16 (56.3%) in MCC200mg / day treatment group of subjects, the MCC detectable plasma concentrations of 400mg / 13/17 (76. 5%) of the subject day treatment groups were found.

[0288] 可观察的MCC浓度在MCC IOOmg/天治疗组中范围是9. 45到12. 3ng/mL,在MCC 200mg/ 天治疗组5. 12 到93. 7ng/mL,并且在MCC400mg/ 天治疗组中5. 32 到84. 9ng/mL。 [0288] MCC concentrations observed in MCC IOOmg / day treatment group is in the range of 9.45 to 12. 3ng / mL, the MCC 200mg / day treatment group 5.12 to 93. 7ng / mL, and MCC400mg / day treatment group 5.32 to 84. 9ng / mL.

[0289] 在所有的治疗组的可检测的MCC浓度中,大部分(35/41 ;85. 4% )在21ng/mL以下。 [0289] In all detectable concentration MCC treatment groups, the majority (35/41; 85 4%) at 21ng / mL or less.

[0290] 仅在2名受试者中观察到超过50ng/mL的MCC浓度,在200mg/天给药组中和在400mg/天给药组中各有一名。 [0290] observed only in two subjects over the MCC concentration 50ng / mL in 200mg / day administration group and each in a 400mg / day administration group.

[0291] MCC的泌尿排泄数据 [0291] MCC urinary excretion data

[0292] 在IB-MD阶段研究中尿的MCC水平都低于量化界限(LLOQ = 5ng/mL)。 [0292] Urine is in the IB-MD study period MCC levels were below the limit of quantification (LLOQ = 5ng / mL).

[0293] MCC的粪浓度数据 [0293] MCC density data of fecal

[0294] 表17显示来自IB-MD研究的粪浓度,其被标准化到150mg剂量;粪MCC平均为916. O μ g/g (138. 4-1768. 9 μ g/g)。 [0294] Table 17 shows the concentrations of IB-MD manure from the study, which was normalized to 150mg dose; average fecal MCC 916. O μ g / g (138. 4-1768 9 μ g / g.).

[0295] 表17在IB-MD阶段研究中的MCC粪浓度,其被标准化为150mg剂量 [0295] Table fecal concentration of MCC in the study IB-MD stage 17, which is normalized to 150mg dose

[0296] [0296]

Figure CN102503994AD00351

[0297] 对于2A阶段研究,在MCC IOOmg/天治疗组(n = 11个样品,充足的),在治疗结束时,粪MCC 平均为255. 6 μ g/g (范围:81. 9-558. 3 μ g/g)。 [0297] For Phase 2A study, the MCC IOOmg / day treatment group (n = 11 samples, sufficient), at the end of treatment, mean faecal MCC is 255. 6 μ g / g (range: 819-558 . 3 μ g / g). 在MCC200mg/天治疗组(η = 9 个样品,充足的)中,粪MCC平均为441. 7 μ g/g(范围:11.7-786. 7 μ g/g)。 In MCC200mg / day treatment group (η = 9 samples, sufficient), the average fecal MCC 441. 7 μ g / g (range:. 11.7-786 7 μ g / g). 在MCC 400mg/天治疗组(η = 13个样品,充足的)中,粪MCC平均为1433. 3 μ g/g (范围:389. 0-3974. 8 μ g/ g) ° In MCC 400mg / day treatment group (η = 13 samples, sufficient), the average fecal MCC 1433. 3 μ g / g (range:.. 389 0-3974 8 μ g / g) °

[0298] 表18.在2A阶段研究中治疗结束时,MCC的粪浓度[0299] [0298] Table 18. When the end of treatment in Phase 2A study, MCC fecal concentration of [0299]

Figure CN102503994AD00361

[0300] 结论 [0300] Conclusion

[0301] 总而言之,本研究显示在多到450mg的多次口服剂量后MCC被良好耐受,在作用位点获得高水平,并且在治疗艰难梭菌相关的腹泻中表现出有前景的结果。 [0301] In summary, this study showed that after multiple oral doses of 450mg and more to the MCC was well tolerated, achieve a high level at the site of action, and showed promising results in the treatment of diarrhea associated with C. difficile.

[0302] 本研究还发现1)在任一研究中没有治疗-出现的副作用可能是药物相关的,2) 在多剂量口服施用后,在血浆中检测到低MCC水平,其大部分低于量化界限。 [0302] The study also found 1) no treatment in either study - side effects may be related to the drug, 2) after multiple-dose oral administration, low MCC levels detected in plasma, the majority of which is below the limit of quantification . 由于低血浆浓度,在IB-MD研究收集的尿中没有检测到完整的MCC。 Due to the low plasma concentrations, the IB-MD study was not detected in urine collected to the full MCC. 3)相反,在两个研究中的粪水平非常高,超过针对艰难梭菌的MIC9tl (0. 125yg/mL)的10,000倍,4)在进行完整疗程治疗的45名受试者中,在治疗前或在治疗10天结束时,只有4名受试者被认为是失败,在50-mg ql2hr剂量组中有2名受试者,在100_mg ql2hr剂量组中有2名受试者被认为是失败的。 3) In contrast, fecal levels in both studies is very high, more than 10,000 times for C. difficile MIC9tl (0. 125yg / mL), and 45 subjects 4) during the complete course of treatment, in the pre-treatment or treatment at the end of 10 days, only four subjects were considered a failure, there are 2 subjects in the 50-mg ql2hr dose groups, there is the 100_mg ql2hr dose group were 2 subjects considered a failure. 在200-mg ql2hr剂量中没有失败的记录(0/16),5)在成功治疗后,只有2名受试者观察到复发。 No record failure (0/16) in the 200-mg ql2hr dose, 5) after successful treatment, only 2 subjects relapse was observed. 约在治疗结束后一个月有两名复发,6)尽管不是统计学显著的,腹泻停止的中值时间显示一种趋势,其表明更高的剂量可能更加有效。 About a month after the end of treatment with two recurrence, 6) Although not statistically significant, the median time to stop diarrhea show a trend, which indicates that higher doses might be more effective. 腹泻停止的时间对于50-mg q 12hr剂量组确定是5. 5天,对于100-mg q 12hr剂量组是3. 5天,和对于200-mg q 12hr剂量组是3. 0 天。 Diarrhea stop time determined for 50-mg q 12hr dose group was 5.5 days, for the 100-mg q 12hr dose group was 3.5 days, and 3.0 days for the 200-mg q 12hr dose Yes.

[0303] 实施例3. MCC在体内选择性地针对艰难梭菌有效,并且没有影响厌氧粪菌群的主要成员:这对于更低的复发率是关键的。 [0303] Example 3. MCC selectively effective against C. difficile vivo, and does not affect the fecal flora of anaerobic main members: It is critical for a lower relapse rate.

[0304] 为了测试MCC在体内针对艰难梭菌具有选择性的活性,并且对于正常厌氧粪菌群可以是相对不足(sparing)的假说,在系列粪样品上进行定量粪培养,所述粪样品获自进入MCC的2A阶段剂量范围临床实验(现在称为MCC)的患者。 [0304] To test the MCC selective in vivo activity against C. difficile, for a normal anaerobic fecal flora may be relatively insufficient (sparing) hypothesis quantitatively cultured on a series fecal manure sample, the manure sample obtained from a patient into the MCC's phase 2A clinical trial dose range (now called MCC) is. 艰难梭菌腹泻的优化抗生素治疗应该消除病原体的营养体形式,但是保留(spare)可能负责集群抗性的正常菌群的主要成分。 Optimizing antibiotic treatment of C. difficile diarrhea should eliminate pathogens in the form of nutrition, but retain the (spare) may be responsible for the main component of the cluster resistance of normal flora.

[0305] 方法 [0305] Method

[0306] 患者(n = 32)随机接受一天两次的50,100或200mg的MCC,持续接受10天。 [0306] Patients (n = 32) were randomized to receive MCC 50, 100 or 200mg twice a day, and continued to receive 10 days. M 名患者以前没有接受治疗;8名患者接受1或2个剂量的标准治疗。 M not previously treated patients; 8 patients received standard treatment with one or two doses. 作为生态对照,对7个另外的患者用万古霉素125mg —日四次治疗10天。 Four times a day for 10 days - as an ecological control of seven additional patients treated with vancomycin 125mg. 培养10_2'4'6'8的新鲜粪样品,以获得艰难梭菌营养体和孢子形式;通过细胞测定测试粪滤液的细胞毒素B。 Fresh fecal samples 10_2'4'6'8 culture to obtain a C. difficile vegetative and spore forms; cytotoxic testing fecal filtrate was measured by cell B. 在研究开始时和第10 天,检查稀释10_3'5'7'9的需氧和厌氧粪菌群培养物的主要的菌群变化。 At the beginning of the study and day 10, the major fecal flora aerobic and anaerobic bacteria culture was diluted 10_3'5'7'9 of inspection. 由于类菌体群生物普遍存在并且是可培养的,将该属选择作为微生物菌群完整性的指示物。 Since Bacteroides group ubiquitous organisms and are cultured genus selected as the indicator of the integrity of the microbiota.

[0307] 详细方法显示如下: [0307] Detailed methods shown below:

[0308] 1)在Calgary 健康区聚集区(Calgary Health Region catchment area)中的单中心研究,群体〜1百万 Single-center study [0308] 1) in the Calgary health region gathering area (Calgary Health Region catchment area) in, one million population ~ 1

[0309] 2)随机开放标记,在2A阶段研究范围内给药,每12小时比较口服用于CDAD治疗的50mg,IOOmg 或200mg,持续10 天。 [0309] 2) open label randomized, Phase 2A within the coverage administered every 12 hours 50mg comparing oral treatment for CDAD, IOOmg or 200mg, for 10 days.

[0310] 3)实验募集结束后,用万古霉素125mg—日四次治疗单独的生态对照组的患者, 治疗10天,作为治疗/生态对照,所述患者对于该实验是合格的。 After the [0310] 3) Experimental recruitment, vancomycin 125mg- patients alone four times a day ecological control group treated for 10 days as treatment / control ecological, the patient is eligible for this experiment.

[0311] 4)轻度到中度CDAD :在研究开始时,> 3但是< 12个腹泻样品/¾小时,阳性艰难梭菌毒素测试,发烧< 39°C,WBC < 30,000/mm3,无呕吐,没有严重的腹部不适 [0311] 4) Mild to moderate CDAD: at the start of the study,> 3 but <12 Diarrhea samples / ¾ hr, C. difficile toxin test positive, fever <39 ° C, WBC <30,000 / mm3, no vomiting, no serious abdominal discomfort

[0312] 5)初始CDAD或仅第一次复发发作。 [0312] 5) The initial CDAD or only first relapse episode.

[0313] 6)如果可能,以前没有进行过治疗。 [0313] 6) If possible, have not previously been treated. 该方案容许多到3个在前标准治疗剂量,但是对于该评估,容许最多2个剂量的标准治疗。 The program content to many previous three standard therapeutic dose, but for this evaluation, allowing up to two standard treatment doses. 在该研究群体中二4个患者以前没有进行过治疗。 Not previously been treated four patients in the two study groups.

[0314] 7)没有同时进行对于任何病症的肠胃外抗生素治疗。 [0314] 7) do not simultaneously parenteral antibiotic treatment for any disorder.

[0315] 8)系列粪样品:除了开始的诊断样品,在研究开始时,在研究开始后第4天,第7 天,第10天,第14天,第21天,第观天和第42天获得> 5克(通常是10-30克)的粪的重复收集样品 [0315] 8) Series fecal samples: In addition to diagnostic samples starting at beginning of the study, after the start of the study on day 4, day 7, day 10, day 14, day 21, and day 42 of View days of> 5 g (typically 10-30 g) repeating collected fecal samples

[0316] 9)对于该报道,比较第0天和第10天粪的艰难梭菌计数和主要的正常定居菌群属的数量结果的变化。 [0316] 9) For this report, the result of the comparison changes the number 0 and day 10 fecal counts of C. difficile and primary settled normal flora of the genus.

[0317] 10)用新鲜传代的样品通过HeLa细胞测定确定艰难梭菌定量计数和艰难梭菌细胞毒素B的粪滤液浓度,因为冷冻对于确定艰难梭菌的定量计数是不利的。 [0317] 10) with fresh samples passaged in HeLa cells was measured to determine the concentration of C. difficile fecal filtrate quantitative counts of C. difficile toxin B cells, as freezing is disadvantageous for the quantitative determination by C. difficile counts.

[0318] 11)因为认为类菌体群生物在受试者中普遍存在并且是高计数的,而且可能是赋予“定居抗性”的正常菌群的主要成分之一,将该群用作抑制厌氧粪菌群的指数。 [0318] 11) as considered in the Bacteroides group ubiquitous organisms and are subject to a high count, and may be given to one of the main components of the normal flora "settled resistance", the group serves as the inhibition of anaerobic fecal flora index. 对于在10 天不能显示类菌群群物种的恢复的患者,处理随后的样品以记录该群恢复的时间。 For the recovery of patients can not be displayed based fauna flora of 10 days, subsequent sample processing to record the time of the recovery group. 如果样品没有被立即处理,将等分试样在-80°c冷冻在15%甘油/脑心输注培养液(Brain Heart Infusion Broth)中,用于进行进一步处理。 If the sample is not processed immediately, aliquots frozen at -80 ° c in 15% glycerol / brain heart infusion broth (Brain Heart Infusion Broth) medium, for further processing.

[0319] 12)厌氧菌群培养的培养基和方法基于Wadsworth-KTL厌氧手册,第6版,2002。 [0319] 12) The method of anaerobic culture medium and culture-based group Wadsworth-KTL Anaerobic Manual, 6th edition, 2002. 通过在CCFA琼脂上将样品稀释至10_2'4'6'8/克粪湿重,来确定艰难梭菌计数。 10_2'4'6'8 by dilution to the sample on CCFA agar / gram of wet weight of feces, C. difficile counts determined. 通过用等体积的100%乙醇处理粪的等分试样1小时,离心,洗涤2次并且重悬浮以定量计数来确定孢子数量。 Aliquots of 100% ethanol by treatment with an equal volume of manure for 1 hour, centrifuged, washed twice and resuspended to quantitatively determined by counting the number of spores.

[0320] 13)使用MacConkey,BAP,m_ 肠球菌琼脂,Lab M 厌氧血琼脂,BAP,BBE,KVLB, PEA 琼脂以10_3'5'7'9进行稀释,进行初始检查之前温育48小时,并且再温育7天来量化正常的菌群培养物。 [0320] 13) using MacConkey, BAP, m_ Enterococcus agar, Lab M anaerobic blood agar, BAP, BBE, KVLB, PEA 10_3'5'7'9 agar dilution and incubated before the initial check 48 hours and incubated for 7 days to quantify the normal flora culture.

[0321] 14)对于万古霉素生态对照,使用产气荚膜梭菌作为指示生物,通过生物测定一式三份地确定万古霉素粪滤液浓度。 [0321] 14) vancomycin ecological control, Clostridium perfringens as an indictor, determined three concentrations of vancomycin fecal filtrate through a biometric type.

[0322] 15)使用Wilc0x0n匹配,在Iogltl转化后确定微生物计数中的差异。 [0322] 15) use Wilc0x0n match, determining the difference in the microbial count after Iogltl conversion. 结果 result

[0323] 在研究开始时,艰难梭菌的平均IogltlCFU士SD营养体计数(所有的MCC患者)是6. 8士3. 6,范围2-10. 95 ;在第10天,除了接受50mg的一名患者,所有其他的患者的艰难梭菌定量计数减少<21og1(l/gm粪。万古霉素类似地有效。在研究开始时,在1/3的每组患者中,类菌体群计数是<3,3-8,和8.5-101呢1(^^^!11,其中正常计数是> 11。在类菌体群中的变化显示在表19中。[0324] 表19.类菌体群计数/克粪湿重的IogltlCFU的平均值士SD [0323] In the beginning of the study, the average IogltlCFU persons SD vegetative counts of C. difficile (all patients MCC) is 6.8 3.6 disabilities, range 2-1095; At day 10, in addition to receiving 50mg of one patient, C. difficile counts quantitative reduction of all other patients <21og1 (l / gm feces vancomycin analogously valid at the start of the study, one third of the patients in each group, the group count bacteroids It is <3,3-8, and it 8.5-101 1 (^^^! 11, wherein the normal count is> 11. the change in the class population of cells are shown in table 19. [0324] table 19. bacteria class group body counts / gram of wet weight of feces IogltlCFU average value SD disabilities

[0325] [0325]

Figure CN102503994AD00381

[0326] * 威氏配对符号秩次检验(wilcoxon matched pairs signed-ranks test),双尾; [0326] * Wilcoxon matched-pairs signed rank test (wilcoxon matched pairs signed-ranks test), two-tailed;

[0327] ** 计数< 3Iog10 = 2. 90 [0327] Count ** <3Iog10 = 2. 90

[0328] 下面的附图还举例说明来自本研究的结果。 [0328] The following figures further illustrate the results from the present study.

[0329] 结论 [0329] Conclusion

[0330] 基于定量的类菌体群计数,患有艰难梭菌腹泻的患者在研究开始时,具有可变地受损的正常菌群,其中约1/3在31oglOCFU/gm范围,1/3在4_71oglOCFU计数中,并且剩余的具有更高的计数(没有在正常的ll_121oglOCFU范围内的)。 [0330] Based on quantitative Bacteroides population count, the patient has C. difficile diarrhea at the beginning of the study, have variably impaired normal flora, in which about 1/3 31oglOCFU / gm range of 1/3 in 4_71oglOCFU counts, and the remaining higher count (ll_121oglOCFU not within the normal range). 所有的MCC的剂量似乎减少艰难梭菌的数量,如万古霉素一样。 All MCC dose appears to reduce the number of C. difficile, such as vancomycin. 没有观察到随MCC的剂量增加,类菌体计数的剂量依赖性减少。 MCC was not observed with increasing doses, dose-dependent reduction bacteroids count. 万古霉素在治疗过程中严重地减少类菌体计数,并且尽管大多数患者恢复它们的计数,极少数仍然在延长的时间内缺乏。 Vancomycin in the treatment process severely reduce class cell count, and although most patients recover their count, a very small number still lack an extended period of time.

[0331] 基于显示高响应率和低复发率的这些数据和临床结果,似乎MCC的200mg剂量是进行进一步的临床研究的适合的剂量。 [0331] Based on these data and clinical results show a high response rate and low recurrence rate, it seems 200mg dose of MCC is suitable for further study of the clinical dose.

[0332] 本发明不限于上述的实施方案,所述实施方案仅作为举例的目的而存在,但是其可以在保护范围内以各种方式进行修改,所述保护范围由后附的专利权利要求限定。 [0332] The present invention is not limited to the embodiments described above, the embodiment of example purposes only exists, but it may be modified in various ways within the scope, the scope of protection defined by the appended patent claims .

Claims (10)

1.-种分离的式IX的化合物(0P-1435) 1.- isolates of a compound of formula IX (0P-1435)
Figure CN102503994AC00021
2.药物组合物,其包含权利要求1的化合物。 2. A pharmaceutical composition comprising a compound of claim 1.
3.权利要求2的药物组合物,其还包含式II的化合物(台勾霉素B) The pharmaceutical composition of claim 2, further comprising a compound of formula II (tiacumicin B)
Figure CN102503994AC00022
4.权利要求3的药物组合物,其还包含式XIV的化合物(闰年霉素A4) The pharmaceutical composition of claim 3, further comprising a compound of Formula XIV, (lipiarmycin A4)
Figure CN102503994AC00023
5.权利要求2-4中任一项的组合物用于制备药物的应用,所述药物用于治疗由艰难梭菌(Clostridium difficile)的存在导致的疾病或病症。 2-4 The composition of any one of claim 1 for the preparation of a medicament, the medicament for treating a disease or condition caused by C. difficile (Clostridium difficile) is present.
6.权利要求5的应用,其中所述组合物在3-15天内,以约50mg到约IOOOmg的量,每日1-3次地施用。 Application according to claim 5, wherein said composition is 3-15 days, in an amount of from about 50mg to about IOOOmg, administered 1-3 times a day.
7.权利要求6的应用,其中所述组合物以约IOOmg到约400mg的量每日一次或两次地施用。 Application according to claim 6, wherein said composition is in an amount from about 400mg to about IOOmg administered twice or once daily.
8.权利要求7的应用,其中所述组合物以约200mg的量,每日一次或两次地施用。 Application of claim 7, wherein said composition is in an amount of about 200mg, administered once or twice a day.
9.权利要求5的应用,其中所述组合物以这样的方式施用从而使在患者中,式II的化合物的血浆浓度低于5ng/mL。 9. The use as claimed in claim 5, wherein said composition is administered in a manner such that in a patient, the plasma concentration of the compound of formula II is less than 5ng / mL.
10.权利要求5的应用,其中所述组合物以这样的方式施用从而使在患者中,式II的化合物的尿浓度低于5ng/mL。 10. Application as claimed in claim 5, wherein said composition is administered in a manner such that in a patient, the urine concentration of the compound of formula II is less than 5ng / mL.
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CN105237599A (en) * 2015-10-09 2016-01-13 华北制药集团新药研究开发有限责任公司 Lipiarmycin A4 crystal and preparation method thereof

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TWI523654B (en) 2007-11-27 2016-03-01 默沙東藥廠 Antibiotic macrocycle compounds and methods of manufacture and use thereof
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CN105237599A (en) * 2015-10-09 2016-01-13 华北制药集团新药研究开发有限责任公司 Lipiarmycin A4 crystal and preparation method thereof
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