CN109966473A - 一种还原型谷肽甘肽联合抗坏血酸和/或卡泊芬净的抗真菌产品及其应用 - Google Patents
一种还原型谷肽甘肽联合抗坏血酸和/或卡泊芬净的抗真菌产品及其应用 Download PDFInfo
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Abstract
本发明公开了一种还原型谷肽甘肽联合抗坏血酸和/或卡泊芬净的抗真菌产品及其应用,所述还原型谷胱甘肽联合抗坏血酸和/或卡泊芬净联合时能够抑制“矛盾现象”的最低有效浓度用量都为1mM,本发明以还原型谷胱甘肽与卡泊芬净的二联,以及还原型谷胱甘肽、抗坏血酸与卡泊芬净的三联对烟曲霉联合抗菌的作用,发现还原型谷胱甘肽、抗坏血酸与卡泊芬净药物联用具有抑制“矛盾现象”的效果,同时具有协同提高杀灭致病真菌烟曲霉常见临床菌株水平的作用,其中三联效果比二联效果更好。谷胱甘肽和抗坏血酸作为抗真菌药卡泊芬净的协同增效杀菌剂,可抑制高卡泊芬净在抗真菌过程中出现的“矛盾效应”,并提高杀灭水平,具有重要的临床应用价值。
Description
技术领域
本发明涉及医药技术领域,具体为一种还原型谷肽甘肽联合抗坏血酸和/或卡泊芬净的抗真菌产品及其应用。
背景技术
近年来,在医学界由于广谱抗生素、抗肿瘤药物、肾上腺皮质激素、免疫抑制剂等的广泛应用甚至滥用,随着器官移植和外科其他介入性治疗的不断开展,导致菌群的失调以致机体对真菌的抵抗力降低,侵袭性真菌感染的发生呈现上升趋势,如假丝酵母(Candida)和曲霉引起的感染,病死率达60%-100%,成为白血病和骨髓移植等重症免疫受损患者死亡的主要原因。
目前治疗深部真菌感染的临床应用最广泛、研究最热门的一类药物是三唑类抗真菌药物,但有毒副作用,已经发现越来越多的临床分离菌对唑类药物产生耐药性。
新型抗真菌药物棘白菌素类(echinocandins)中的卡泊芬净Caspofunginacetate(CS)正在被推荐为侵袭性真菌感染的挽救疗法。卡泊芬净是一种抗真菌类药,其组成为新型多肽类物质,主要作用的部位在真菌细胞壁。真菌细胞壁主要是由β-(1,3)-D-葡聚糖组成的,其主要作用为维持真菌的正常生理功能和细胞生长,通过β-(1,3)-D-葡聚糖合成酶催化转运将β-(1,3)-D-葡聚糖转变成葡萄糖基。卡泊芬净的作用机制为对真菌细胞壁上的β-(1,3)-D-葡聚糖的合成进行非竞争性抑制,将真菌细胞壁上的正常结构破坏,能够有效的对抗真菌活性,因为只有在卡氏肺囊虫及真菌原体上存在β-(1,3)-D-葡聚糖,所以哺乳动物经过卡泊芬净治疗后不会出现类似多烯类和唑类药物的细胞毒性。然而,棘白菌素类抗真菌药物卡泊芬净作为烟曲霉的抑菌剂不能完全杀死标准数量的烟曲霉,且在抗真菌过程中可产生"矛盾现象",即在低药物浓度时念珠菌或曲霉被杀死或抑制,而在高药物浓度时反而刺激出现菌落或菌丝生长的现象。
还原型谷胱甘肽(GSH)是自然界广泛存在的生物活性三肽,是谷氨酸、半胱氨酸和甘氨酸的缩合物。GSH有多种重要生理功能,具有强大的抗氧化能力可有效调节氧自由基的氧化还原循环,因此它在临床治疗、食品、保健和体育等领域的应用非常广泛。现还原型谷胱甘肽运用于肝病的治疗和肝脏的保护,药物性肝肾损害的预防与治疗,甚至作为抗肿瘤治疗辅助用药。但还原型谷胱甘肽很少运用于抗菌治疗。
抗坏血酸(ascorbic acid)即维生素C,是人体不能合成的必需微量元素,参与人体内多种重要的生化反应,具有强大的抗氧化和增强人体免疫力的能力。维生素C现已广泛应用于抗感染辅助用药、过敏性反应、心血管疾病、糖尿病和癌症等临床辅助治疗。
目前尚未发现有将还原型谷胱甘肽、抗坏血酸和抗坏血酸与棘白菌素类药物联合用于抗真菌感染治疗的研究报道和临床应用。
发明内容
本发明的目的在于提供一种还原型谷肽甘肽联合抗坏血酸和/或卡泊芬净的抗真菌产品及其应用以解决上述背景技术中提出的问题。
为实现上述目的,本发明提供如下技术方案:一种还原型谷肽甘肽联合抗坏血酸和/或卡泊芬净的抗真菌产品及其应用。
优选的,还原型谷胱甘肽联合联合抗坏血酸和/或卡泊芬净的抗烟曲霉菌产品中的应用。
优选的,所述产品是以1mM的还原型谷胱甘肽、1mM的抗坏血酸和/或浓度为0-128μg/ml的卡泊芬净为主要活性成分的药品。
优选的,所述还原型谷胱甘肽联合抗坏血酸和/或卡泊芬净能够抑制“矛盾现象”出现时,还原型谷胱甘肽的最低有效浓度为1mM,抗坏血酸的最低有效浓度为1mM,而卡泊芬净的最低有效浓度由其作为抑菌剂单独使用时的无最低抑菌浓度变成最低抑菌浓度为128μg/ml。
优选的,所述的真菌为烟曲霉。
与现有技术相比,本发明的有益效果是:本发明以还原型谷胱甘肽联合抗坏血酸联合卡泊芬净三联时对烟曲霉协同抗菌的作用,发现还原型谷胱甘肽、抗坏血酸与卡泊芬净药物联用具有协同杀灭致病真菌烟曲霉常见临床菌株的作用,谷胱甘肽作为抗真菌药卡泊芬净的协同增效杀菌剂,在抗坏血酸的保护下,既可抑制高浓度的卡泊芬净在抗真菌过程中出现的“矛盾效应”,同时又能提高杀菌率,具有重要的临床应用价值。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例一
还原型谷胱甘肽(GSH)、抗坏血酸(VC)与卡泊芬净(CS)联用对抗烟曲霉的效果测定:
实验材料:
GSH,CS,E-test药敏条(biomerieux),本实施例用到的试剂均为粉末,纯度均>99%;GSH和VC用无菌水配制成50mg/ml并调节pH=6.5-7.0,CS用无菌水配制成2mg/ml储存液;用时根据需要配成所需浓度的工作液。
实验方法:
1.微量液基稀释法:菌株抗菌药物最低有效浓度(MEC)测定:按照美国国家临床实验室标准化委员会(clinical and laboratory standardsinstitute,CLSI)推荐的M38-A2微量液基稀释法,测定临床标准株AF1160对CS的敏感性,每孔中孢子接种量为0.4×104-5×104CFU/ml;抗真菌药物工作浓度:CS为0.03-128μg/ml;96孔板在35℃下孵育48h后读取结果;CS其MEC值判定为抑制生长为小而圆形紧缩的菌丝体时的最低药物浓度。
(1)菌悬液制备:受试菌株在培养基YUU上于37℃培养2天,收孢子,用无菌生理盐水重悬,调整OD值至0.09-0.3(约0.4×106-5×106/ml);将上述悬液用RPMI1640液体培养基进行1:50稀释,即得到工作液(约2倍于终浓度0.4×104-5×104/ml);
(2)加样:将CS药物储存液用RPMI1640液基倍比稀释为0.06-256μg/ml,然后每个CS浓度梯度下分别混合2mM GSH;以及2mM GSH+2mM VC。最终配制成2倍于终浓度的药液。取100μl药液分别加入96孔圆底药敏板第1-10列各孔,第1孔为最高浓度,第10孔为最低浓度,第11列中加入100μl RPMI1640作为生长对照;第12列仅加入200μl RPMI1640培养基作阴性对照。向第1-11孔分别加入100μl菌悬液后,35℃孵育48h判读结果;
(3)重复:复孔3-5个,求均值;先后重复三次实验,同样得均值。将3次均值再求均值及标准差方差分析。
2.E-test实验法:将GSH,GSH+VC分别和100μl 106个孢子/ml测试菌的孢子悬液混合,加入到融化的20ml YUU培养基中使GSH和VC终浓度均为1mM,待培养基凝固并略干,然后用灭菌的镊子夹取CS E-test试条(从下到上药物浓度依次升高,0.002-32μg/ml),轻轻贴于平板中间,注意不要有气泡;35℃培养48h。
实验结果:
1.微量液基稀释法:CS作为霉菌的抑菌剂只有最低有效浓度MEC值(minimumeffective concentration),而无最低抗菌浓度MIC值(minimum inhibitoryconcentration),其MEC值判定为出现小而圆形紧缩呈分支状的菌丝体生长时的最低药物浓度,而“矛盾现象”则判定为出现大而不分枝正常极性生长的菌丝体生长时的最低药物浓度。
本实施例的实验结果为:①CS单独作用时MEC值为0.25μg/ml,菌丝出现“矛盾现象”的对应CS浓度值为≥4μg/ml,当CS+1mM GSH或CS+1mM GSH+1mM VC时MEC值为0.25μg/ml不变,但当CS≥4μg/ml时菌丝不再出现“矛盾现象”,且菌体数量大量减少,其中CS+1mM GSH+1mM VC导致菌体的减少量大于CS+1mM GSH;②当CS单独浓度在128μg/ml时,仍然有菌体生长,当128μg/ml CS+1mM GSH时也有菌生长,但生长量减少,而当128μg/ml CS+1mM GSH+1mMVC时,菌体完全消失,达到杀菌剂效果,即出现了MIC值,MIC=128μg/ml CS+1mM GSH+1mMVC。
2.E-test实验法:该方法可以在固体平板上方便地观察最低有效浓度MEC值,MEC值判定为抑菌圈边缘与E-test药敏条相交处为MEC值。
本实施例的实验结果为CS单独作用MEC值为0.38μg/ml,但抑菌区内出现大量单菌落,即平板中的“矛盾现象”,而联用1mM的GSH时MIC值为0.38μg/ml不变,但是抑菌区内只有少量较小的菌落,即平板中的“矛盾现象”消失,且菌量变少。
尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (5)
1.一种还原型谷胱甘肽联合抗坏血酸和/或卡泊芬净在制备抗真菌药物产品中的应用。
2.根据权利要求1所述的应用,其特征在于:为还原型谷胱甘肽联合抗坏血酸和/或卡泊芬净的抗烟曲霉菌产品中的应用。
3.根据权利要求1或2所述的应用,其特征在于:所述产品是以1mM的还原型谷胱甘肽、1mM的抗坏血酸和/或浓度为0-128μg/ml的卡泊芬净为主要活性成分的药品。
4.根据权利要求1-3任意一项所述的应用,其特征在于:所述还原型谷胱甘肽联合抗坏血酸和/或卡泊芬净能够抑制“矛盾现象”出现时,还原型谷胱甘肽的最低有效浓度为1mM,抗坏血酸的最低有效浓度为1mM,而卡泊芬净的最低有效浓度由其作为抑菌剂单独使用时的无最低抑菌浓度变成最低抑菌浓度为128μg/ml。
5.根据权利要求1或2所述的应用,其特征在于:所述的真菌为烟曲霉。
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