论文总字数：35062字

摘 要

达托霉素在体外具有广谱抗革兰氏阳性菌的作用。有耐甲氧西林的金葡菌，糖肽类敏感的金葡菌，耐青霉素的肺炎链球菌和凝固酶阴性的葡萄球菌。目前，临床上对于这些耐药菌可选择的抗生素很少，而达托霉素在临床上有着明显的优势，其临床耐药菌株的出现率非常低，其对抗耐药菌的优势在未来一段时间内会愈加显著，应用前景也非常广阔。因此，开发该产品有着极其重要的临床意义。我国CFDA于2009年已批准同意该品种进口，商品名为克必信，规格为500mg，临床主要用于治疗感染性内膜炎、复杂性皮肤及皮肤软组织感染（CSSSI）。但达托霉素对温度、pH值特别敏感，其制剂仿制难度较高，本课题在仿制国外进口品种克必信（CUBICIN）的基础上研制开发了注射用达托霉素。

本课题根据达托霉素的理化特性，通过前期文献调研，对达托霉素注射剂的处方及制备工艺进行了系统的筛选，得到了符合USP标准的注射用达托霉素。具体研究内容如下：

质量标准研究：为控制注射用达托霉素的质量，根据达托霉素的理化性质，参照其原料的质量标准以及中国药典2010年版二部附录I A对注射剂的要求，按照新药（化学药品）研究技术指导原则的要求，对注射用达托霉素进行了水分、酸度、有关物质检查及含量测定等质量研究工作。采用高效液相色谱法，选用梯度洗脱方式对本品进行有关物质的检查。根据本品检出主要强制降解物的UV吸收情况，确定其检测波长为214nm。达托霉素对温度、pH均较敏感，主要降解产物有脱水达托霉素、β-异构体、内酯水解物、杂质1、杂质2、杂质3、杂质4。最终确定有关物质检测标准：内酯水解物（相对保留时间约为0.73）不得超过1.5%，脱水达托霉素（相对保留时间约为1.33）不得超过3.5 %，β异构体（相对保留时间约为0.86）不得超过2.0%，杂质1（相对保留时间约为0.58）不得超过0.5%，杂质2（相对保留时间约为0.90）不得超过1.5%，杂质3（相对保留时间约为1.15）不得超过1.0%，杂质4（相对保留时间约为1.33）不得超过0.5%，其他最大单杂不得过0.15%，总杂不得超过8.0%；采用高效液相色谱法测定本品含量，检测波长214nm，按无水物计，含达托霉素（C₇₂H₁₀₁N₁₇O₂₆）应为87.0％～103.0％；按

平均装量计，含达托霉素（C₇₂H₁₀₁N₁₇O₂₆）应为标示量的95.0％～115.0％。经方法学验证，本品的有关物质检查方法与含量测定方法灵敏、准确、可靠、重复性高、专属性强，能有效控制产品的质量。

关键词 注射用达托霉素 质量标准研究 分析方法验证

ABSTRACT

Daptomycin can be resistant to gram positive bacteria in vitro．Such as Methicillin-resi-stant staphylococcus aureus，glycopeptide-sensitive Staphylococcus aureus，penicillin-resistant Streptococcus pneumoniae and oagculase negative Staphylococcus ．At present，few antibiotics are available for these resistant strains in clinic．Daptomycin for injection has obvious advantages in clinic，the occurrence rate of clinical resistance strains is very low，the advantage of fighting against drug-resistant bacteria will become more significant in the next period of time，and have wide prospect of application．This topic researches and developments of daptomycin for injection on the basis of generic imported drug Cubicin (CUBICIN)．Daptomycin for injection for the treatment of infective endocarditis complicated skin、skin and soft tissue infection (cSSSI)，The development of this product has extremely important clinical significance．But Daptomycin is particularly sensitive to temperature and pH, so it is difficult to copy into generic drug product , According to physical and chemical properties of Daptomycin, through the early literature research, we systematically screen the prescription and preparation process for Daptomycin for injection, finally we obtain Daptomycin for injection which meets the criteria.

The following research has been done in this paper:

Prescription selection and process study: using related substances of daptomycin to determine its optimal formulation and preparation process. Daptomycin has poor thermal stability , with reference to the original research data (EMEA 《the scientific discussion 》), it’s prescription has no excipient. The raw materials from the drug itself has a good framework; this product mainly take aseptic manipulation combined with filtration sterilization sterile protection process.Raw material is daptomycin freeze-dried powder， the production process comprises the following steps：liquid distribution，pH and concentration adjustment，aseptic filtration，filling，freeze-drying ，nitrogen filling pressure plug，light inspection and packaging. In order

to avoid the degradation of daptomycin in the production process，we use low temperature while liquid　distribution，andnitrogen filled protection in finished product．In-process product should control　pH from 4.5 to 5.0．When filling in excess of 5% in order to ensure sufficient dosage　after　rehydrationin of clinical medication．Compare the quality inspection index of the middle experiment Daptomycin for injection with the foreign commercial products (CUBICIN) such as water, acidity, related substances and determination of the quality of samples etc., they were basically the same.The research results show that the medication design is legitimate, the preparation process is steady, and the quality can be commanded. 24 month long term stability test results show that the product in 2~8 C under the conditions of preservation, the physical and chemical properties of stability.

Quality standard study: To control the quality of Daptomycin for injection,the moisture,acidity,determination and related substances has been detected based on the physicochemical properties of daptomycin , the quality standard of its raw materials and injection requirements in Appendix IA of the Chinese Pharmacopoeia 2010 edition and the new drug (chemical drug) technical research guidelines. Chemical method and HPLC method for identification of the goods; water content was determined according to the first method in Appendix Ⅷ M in Chinese Pharmacopoeia 2010 edition; acidity was tested by referring to Appendix Ⅵ H in Chinese Pharmacopoeia 2010 edition; related substances was determined by HPLC and the choice of gradient elution. Based on UV absorption of the detected main degradation product, its wavelength was determined at 214nm. Daptomycin was sensitive to temperature and pH，the main degradation products was dehydration of daptomycin，β-isomer，lactone hydrolysate, impurity 1, impurity 2, impurity 3, impurity 4．Lactone hydrolysate (relative retention time is about 0.73) was not more than 1.5%, the dehydration of daptomycin (relative retention time is about 1.33) was not more than 3.5%, β-isomer (relative retention time is about 0.86) was not more than 2%, impurity 1 (relative retention time is about 0.58) was not more than 0.5%, impurity 2( the relative retention time is about 0.90) was not more than 1.5%, impurity 3(relative retention time is about 1.15) was not more than 1%, impurity4 (relative retention time is about 1.33) was not more than 0.5%, any other individual impurity was not more than 0.15%, total impurities was not more than 8%; the content of daptomycin forinjection was determined by HPLC on 214nm, daptomycin (C₇₂H₁₀₁N₁₇O₂₆) contained not less than 87% and not mre than 103%,calculated on the anhydrous basis, according to the average weight, daptomycin for injection contained not not less than 95% and not more than 115% of stated amount of daptomycin( C₇₂H₁₀₁N₁₇O₂₆).

The determination method for related substances and assay were sensitive, accurate, reliable, reproducible, and specific, and can effectively control the quality of the products by method validation.

KEYWORDS: Daptomycin for Injection；FormuLation and processing；Quality standard；Validation of analytical method

目 录

摘 要 I

ABSTRACT i

第一章 文献综述 1

1.1 达托霉素的研究概况 1

1.1.1 达托霉素国内外研究情况 1

1.1.2 达托霉素作用机制和药理毒理 2

1.1.3 达托霉素临床研究情况 3

1.2 立题依据及研究思路 6

第二章 注射用达托霉素的质量控制的含量测定方法学 7

2.1 供试材料及试剂 8

2.2 仪器 8

2.3 含量测定方法学 9

2.3.1 含量测定背景介绍 9

2.3.2 含量测定方法学主要验证结果 10

2.3.3 中试三批含量测定结果 11

第三章 注射用达托霉素的质量控制的有关物质方法学………………………………………………………12 3.1 供试材料及试剂 ……………………………………………………………………12

3.2 仪器 13

3.3 有关物质方法学 14

3.3.1 有关物质确立的背景介绍 14

3.3.2 有关物质确立的专属性试验 18

3.3.3 有关物质测定杂质干扰amp;定位试验 19

3.3.4 有关物质测定波长选择 20

3.3.5 有关物质测定系统适用性 20

3.3.6 有关物质测定强制降解试验 21

3.3.7 有关物质测定检测限与定量限 23

3.3.8 有关物质测定线性与范围 24

3.3.9 有关物质测定精密度试验 25

3.3.10 有关物质测定溶液稳定性 27

3.3.11 有关物质测定准确度试验 28

3.3.12 工艺验证三批有关物质测定结果 29

第四章 总结与讨论 31

参考文献 33

第一章 文献综述

1.1 达托霉素的研究概况

1.1.1 达托霉素国内外研究情况

自20世纪70年代以来，细菌对抗生素产生耐药性已成为一个不断升级的问题。在过去的25年，革兰阳性菌引起的感染问题^[1,2]，特别是耐甲氧西林葡萄球菌、青霉素和红霉素耐药肺炎球菌、万古霉素耐药性肠球菌的耐药性问题已经越来越严重。随着高致病耐药菌的不断出现，临床上对新抗生素的需求变得十分迫切^[3]。达托霉素对以上耐药菌均有很好的杀菌效果，且制剂用药方便，毒副作用小^[4]。它的上市为临床医生提供了一种新的治疗方案，成为“病原菌最后一道防线——万古霉素”的最佳替代品^[5]；更具意义的是，达托霉素作为环脂肽类抗生素家族的第一个产品^[6-8]，其化学结构和作用机制不同于已有所有类别的抗生素，是近四十年来继噁唑烷酮类抗生素后，应用到临床的唯一新结构类别抗生素。

此外，由于其作用机制与现已上市的各类抗菌药物都不相同，达托霉素作为应用到临床的首个环脂肽类抗生素，至今尚未有交叉耐药菌的报道，临床耐药菌株的出现几率极低（lt;0.2）^[9,10]。这在医院院内感染严重、高致病耐药菌肆虐的今天，显得尤为重要，2015年其全球的市场份额达到11亿美元^[11-16]。

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