CN106309415A - 卷柏素类化合物在作为抗真菌药物增效剂中的应用 - Google Patents
卷柏素类化合物在作为抗真菌药物增效剂中的应用 Download PDFInfo
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- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
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- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
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Abstract
本发明公开了selaginellin、selaginellin A等卷柏素类化合物作为抗真菌增效剂的用途。实验结果表明，卷柏素selaginellin、selaginellin A与氟康唑、酮康唑等抗真菌药物合用时，可达到协同增效的作用，可降低唑类药物的用药剂量，从而减轻药物的毒副作用。并且通过实时荧光定量PCR(qRT‑PCR)实验表明，卷柏素selaginellin、selaginellin A可显著逆转唑类药物耐药基因的上调，有逆转唑类药物耐药的作用，具有重要的临床应用价值。
Description
技术领域
本发明涉及一种抗真菌增效剂，具体涉及selaginellin或selaginellin A等卷柏素类化合物在作为抗真菌药物增效剂中的应用，属医药技术领域。
背景技术
近年来，临床真菌感染的发病率和死亡率逐年上升。念珠菌是人体重要的条件致病性真菌，是引起医院感染性疾病主要病原菌之一。唑类药物是临床上常用的能有效治疗深部及浅表部真菌感染的抗真菌药，如咪唑类中的酮康唑；三唑类中的氟康唑等。但是，随着药物长期、大量使用，使得真菌对唑类药物的敏感性降低，且耐药性显著增高，导致不得不加大药物剂量，某些抗真菌药物甚至加大用药量也无效。目前通行的做法为、加快新药研发、联合用药。加大药物剂量的危害是显而易见的，比如副作用增大、加速真菌耐药、
增加社会经济负担等等；如酮康唑等对肝脏或心脏有毒副作用的药物，从而限制了这些药物在临床上的应用。寻找唑类药物的增效剂日益受到重视，已成为抗真菌治疗的主要发展方向之一。所谓增效剂是指对受试生物无药效或药效很低，但与某种药物混合使用时，能明显提高该药物效果的辅助化合物
卷柏素为从卷柏属植物中提取的系列有效成分。具有抗微生、抗氧化、抗衰老，及神经保护等活性，但未见其作为抗真菌药物增效剂与抗菌药物联合使用治疗真菌病的报道。
其中代表性成分selaginellin及selaginellin A的结构式如下；
发明内容
发明目的：本发明所要解决的技术问题是克服现有技术的不足，对selaginellin及selaginellin A的活性进行深入研究，提供selaginellin或selaginellin A卷柏素类化合物在作为抗真菌药物增效剂中的应用。
技术方案：为解决以上技术问题，本发明采取如下技术方案：
卷柏素类化合物在作为抗真菌药物增效剂中的应用
selaginellin或selaginellin A在作为抗真菌药物增效剂中的应用。
作为优选方案，selaginellin或selaginellin A与唑类抗真菌药物合用在制备抗真菌药物中的应用。
selaginellin或selaginellin A与唑类抗真菌药物合用在制备抗真菌药物中的应用，作为优选方案，所述的selaginellin或selaginellin A在总药物中的浓度为0.5～256μg/mL。
selaginellin或selaginellin A与唑类抗真菌药物合用在制备抗真菌药物中的应用，作为优选方案，所述的selaginellin或selaginellin A在总药物中的浓度为2～64μg/mL。
本发明所述的selaginellin或selaginellin A与唑类抗真菌药物合用在制备抗真菌药物中的应用，所述的唑类抗真菌药物为咪唑类或三唑类抗真菌药物。如酮康唑或氟康唑。所述的真菌为念珠菌。
唑类药物发挥抗真菌作用主要是抑制了与细胞膜麦角甾醇合成有关的酶，如:由ERG11编码的14α-羊毛甾醇-去甲基化酶(14-DM)和由ERG5编码的Δ222去饱和酶。ERG11、ERG5发生突变或过表达可以降低真菌对唑类药物的敏感性，并产生耐药。真菌外排作用的增加，导致药物在细胞内蓄积减少，从而降低了菌株对药物的敏感性，因此编码外排蛋白的基因过度表达是真菌对唑类药物耐药的主要机制之一。目前已知与耐药有关的外排蛋白包括含ABC转运蛋白超家族(ABCT)的CDR1、CDR2，多药耐药转运蛋白MDR1，以及易化扩散超家族转运蛋白FLU1。本研究表明白念珠菌暴露于FLC后，ERG11、ERG5、CDR1、CDR2、MDR1与FLU1表达上调，而卷柏素与氟康唑合用可以显著逆转这一趋势，说明两药联用，有可能逆转耐药。
有益效果：本发明和现有技术相比具有如下优点：
本发明通过大量实验深入研究selaginellin或selaginellin A的活性，实验结果表明白念珠菌暴露于氟康唑后，ERG11、ERG5、CDR1、CDR2、MDR1与FLU1表达上调，而卷柏素selaginellin或selaginellin A与氟康唑合用可以显著逆转这一趋势，表明两药联用，可逆转耐药。selaginellin和selaginellin A可用作抗真菌药物的增效剂，从而可减轻药物的毒副作用，特别是氟康唑、酮康唑；并有可能逆转真菌耐药，有效治疗真菌感染，具有重要的临床应用价值。
附图说明
图1为selaginellin或selaginellin A联合氟康唑对白念株菌相关基因表达相对变化差异的结果图。
图2为selaginellin或selaginellin A联合氟康唑对白念株菌相关基因表达水平变化趋势图。
具体实施方式
下面结合具体实施例，进一步阐明本发明，应理解这些实施例仅用于说明本发明而不用于限制本发明的范围，在阅读了本发明之后，本领域技术人员对本发明的各种等价形式的修改均落于本申请所附权利要求所限定的范围。
实施例1 Selaginellin和氟康唑合用对不同临床真菌株的作用
一、材料和方法
试药：
selaginellin及selaginellin A卷柏素类化合物：实验室自制，使用1H-NMR,13C-NMR及ESI-MS对其进行了对其进行结构解析，与文献对照确定其化学结构。经HPLC-UV面积归一化法计算，纯度均在98％以上。氟康唑(FLC)，酮康唑(KEC)：辉瑞制药有限公司。
菌株：
分别为美国模式培养物集存库(American Type Culture Collection,ATCC)标准菌株白念珠菌(Candida albicans)ATCC90028，近平滑念珠菌(C.parapsilosis)ATCC22019，白念珠菌临床株(62342)。。
培养液：
RMPI-1640培养基：RMPI-1640(Gibco公司)10.0g，吗啡啉丙磺酸(Sigma)34.5g，加双蒸水900mL溶解，定容置1000mL，滤过消毒，-20℃保存，使用前检查培养基是否无菌。
SDA培养基：蛋白胨10g，琼脂15g，加双蒸水1000mL，高压灭菌，4℃备用。
YEPD培养液：酵母浸膏10g，蛋白胨20g，加双蒸水1000mL，高压灭菌，4℃备用。
菌液制备：
念珠菌菌液：从4℃保存的SDA培养基上挑取菌少量，接种至1mL YEPD培养液，于30℃以100r/min振荡培养活化，使真菌处于指数生长期。吸取菌液至适量无菌生理盐水中，经血球计数板计数，孢子数约为1.0×106～5.0×106cfu/ml。用倍量RMPI-1640液体培养基稀释成菌1.0×103cfu/ml。
药液的配制：
氟康唑以灭菌蒸馏水配成2560μg·mL-1，selaginellin、selaginellinA以DMSO配成药物原液(浓度为2560μg·mL-1)，置于-20℃冰箱冻存。实验前再用灭菌蒸馏水依次进行倍比稀释。联合药敏孔内药物终浓度范围如下，FLC:16～0.03μg·mL-1，KEC：4～0.008μg·mL-1，selaginellin：128～2μg·mL-1；selaginellinA：64～1μg·mL-1。96孔平板孔内DMSO终浓度≤1％，对菌株生长的影响可忽略不计。于96孔板中每孔加入100μL不同浓度的药液备用，注意避光。
MIC值判定：
100％MIC：以溶媒对照为参照，肉眼观察药基孔中无菌生长为最低抑菌浓度，100％MIC值。
联合药敏试验：
参照M27-A3方案。将制备完毕的平板置于恒温培养箱中，35℃培养48h。(n＝3)。
以FICI法评价唑类药物与selaginellin、selaginellinA的体外联合效果，其公式为∑FIC＝FICA+FICB＝MICAB/MICA+MICBA/MICB，式中的MICA和MICB分别表示A和B单用时的MIC值，MICAB和MICBA分别表示两种药物联用时各自的MIC值。FICI≤0.5为协同作用，FICI＞4为拮抗作用，0.5＜FICI≤4为无关作用。
实验分组：
qRT-PCR和Western Blot实验各指标检测均为4个组别：空白对照组，FLC单用组(0.25μg·mL-1)，selaginellin、selaginellinA单用组(64μg·mL-1)，两药联用组(0.25μg·mL-1 FLC+64μg·mL-1selaginellin)。
qRT-PCR检测相关基因的表达水平
提取总RNA：将上述菌体于-80℃冻干过夜。用1mL Total RNA Extractor重悬，震荡混匀，在4℃12000r/min×10min离心；上清液加入200μL氯仿，震荡15s，离心；抽取上层无色水相加入等体积异丙醇，混匀，离心；去除上清，加入1ml 70％的乙醇，离心；吸尽上清，加入30μL的无RNase水溶解RNA沉淀，待完全溶解后于-70℃保存。
引物设计与合成：从NCBI基因库中查得所需基因序列，并用Primer 5软件设计所需引物，委托南京金斯瑞科技有限公司合成引物。
逆转录：每个组份轻轻混匀，然后2000rpm离心20s；取灭过菌且无核酸酶的0.2mlPCR管，依次加入如下组份：RNA(2μg)1μL，Oligo dT(18)(50μM)2μL，无核酸酶的双蒸水至总体积12.5μL；65℃保温5min，冰浴5min；依次加入如下组份：RNase抑制剂(40u/μL)0.5μL，5×Reaction Buffer 4.0μL，dNTPs(10mM)2.0uL，M-MuLV 1.0μL；混匀后，2000rpm离心20s；42℃保温1h，70℃保温10min，置冰上5min。
qRT-PCR检测目的基因表达：反应程序：95℃×15s(变性)，60℃×20s(退火)，72℃×15s(延伸)。以GAPDH作为内参标准，一个样本基因做3个复孔，结果应用软件GraphpadPrism 5.0分析，基因水平采用倍数变化来表示(2(△△Ct)法)。
实验结果：
1)以棋盘法检测两类药物合用后对C.albicans ATCC90028和临床分离株62342，以及C.parapsilosis ATCC22019的MIC值(见表1、2)，计算对应的FICI指数。结果显示，selaginellin、selaginellin A与FLC、KEC对C.albicans ATCC90028和临床株62342，C.parapsilosis ATCC22019显示出较好的协同增效作用。
表1.棋盘法检测selaginellin与FLC、KEC联用对C.albicans、C.parapsilosis的MIC(μg·mL-1)
SEL表示selaginellin化合物。
表2.棋盘法检测selaginellin A与FLC、KEC联用对C.albicans、C.parapsilosis的MIC(μg·mL-1)
SELA表示selaginellin A化合物。
2)qRT-PCR考察SEL和FLC联用后耐药相关基因的表达差异
采用qRT-PCR技术检测了真菌细胞膜麦角固醇生物合成的关键基因ERG5、ERG11，外排泵基因CDR1、CDR2、MDR1、FLU1的表达水平。结果显示，单用FLC显著上调ERG5、ERG11、CDR1、CDR2、MDR1、FLU1的表达，单用selaginellin、selaginellinA也有一定的上调作用，但氟康唑(FLC)与selaginellin、selaginellinA合用后，selaginellin、selaginellinA可逆转氟康唑(FLC)引起的上述基因的表达上调，分别下调52％、86％、71％、62％、67％、56％、36％(如图1和图2)。从基因表达方面证实selaginellin、selaginellinA可降低真菌对氟康唑的耐药性，取得了非常好的预料不到的技术效果。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (6)
1.卷柏素类化合物在作为抗真菌药物增效剂中的应用。
2.selaginellin或selaginellin A在作为抗真菌药物增效剂中的应用。
3.selaginellin或selaginellin A与唑类抗真菌药物合用在制备抗真菌药物中的应用。
4.根据权利要求2所述的selaginellin或selaginellin A与唑类抗真菌药物合用在制备抗真菌药物中的应用，其特征在于，selaginellin或selaginellin A在总药物中的浓度为0.5～256μg/mL。
5.根据权利要求4所述的selaginellin或selaginellin A与唑类抗真菌药物合用在制备抗真菌药物中的应用，其特征在于，selaginellin或selaginellin A在总药物中的浓度为2～64μg/mL。
6.根据权利要求3所述的selaginellin或selaginellin A与唑类抗真菌药物合用在制备抗真菌药物中的应用，其特征在于，所述的唑类抗真菌药物为咪唑类或三唑类抗真菌药物。
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| CN114814057B (zh) * | 2022-04-28 | 2024-01-16 | 湖南省药品检验检测研究院 | 一种非靶向代谢组学区分卷柏品种真伪的方法及应用 |
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