必特螺旋霉素体内代谢与药物动力学研究
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摘要
由中国医学科学院医药生物技术研究所研制的必特螺旋霉素，是由基因工程菌产生的具有异戊酰侧链的螺旋霉素类药物。必特螺旋霉素是多种结构类似成分的混合物，异戊酰螺旋霉素Ⅰ，Ⅱ，Ⅲ是其主要成分，分别占总量的7.4％，22.5％和37.7％，三者之和约占必特螺旋霉素的67.6％。目前作为一类新药候选药物即将进入临床Ⅱ期试验。
螺旋霉素的结构复杂，必特螺旋霉素是具有十余种螺旋霉素衍生物的混合物，其在体内代谢和药物动力学过程研究对于现有的分析方法是一种挑战。本论文目标是利用先进的分析手段，研究必特螺旋霉素这一复杂混合物的体内代谢和药物动力学过程，主要工作如下：
一、必特螺旋霉素体内代谢和质谱研究
在有关文献报道的基础上，首次采用LC／MS~n技术，系统地研究了必特螺旋霉素在大鼠体内的代谢情况。
大鼠灌胃给予40 mg必特螺旋霉素后，分别收集大鼠的尿、粪和胆汁样品进行分析，除10种原形药物外，共发现49种代谢产物。以异戊酰螺旋霉素Ⅲ为例：11个代谢产物分别鉴定为(1)脱去福洛氨糖，生成M1；(2)脱去4″异戊酰侧链，生成M2和M6；(3)醛基发生还原反应，生成M3和M7；(4)发生水解反应，内酯环开环，生成M4和M11；(5)醛基与半胱氨酸结合，生成M5，M8和M10；(6)脱去碳霉糖，生成M9。必特螺旋霉素中其他组分的代谢途径与异戊酰螺旋霉素Ⅲ类似，共分为11个系列。除10种原形药物(M0a-M0i)外，16个代谢产物(M1a-c，M1f，M3a，M5a，M6a-c，M7a-c，M8a-c和M9c)的结构经与对照物质的色谱和质谱行为比较，予以确认。
其中相对含量最大的是螺旋霉素，同时也是发挥抗菌活性的主要活性物质。对于螺旋霉素类衍生物，醛基还原成羟基和脱福洛氨糖是首次在大鼠体内发现的代谢途径，在尿中和胆汁中均未发现醛基还原产物，说明醛基还原成羟基的过程可能是肠道菌群介导的。脱去福洛氨糖后的代谢产物中包括普拉特霉素、交沙霉素、柱晶白霉素和麦迪霉素等几种临床上常见的的十六元环大环内酯类抗生素。
通过LC／MS~n方法，得到必特螺旋霉素及其代谢产物的多级质谱特征丢失碎片，总结质谱裂解规律并作为判断依据，以推测代谢物的结构。
二、定量分析方法的建立
必特螺旋霉素作为多组分混合物，包含有10种主要成分，均为螺旋霉素衍生物。其中异戊酰螺旋霉素是主要有效成分，本文中以异戊酰螺旋霉素作为跟踪原
摘要il
形药物的定量指标，同时以必特螺旋霉素的主要代谢产物螺旋霉素作为跟踪代谢
物的定量指标。
通过对色谱和质谱条件进行优化，建立了同时定量分析异戊酞螺旋霉素I，11，
m或螺旋霉素I，n，m的液相色谱离子阱型质谱联用法(LC/MSn)，对必特螺旋
霉素在大鼠体内药物动力学进行了研究。
在此基础上，为了能满足必特螺旋霉素I期临床药物动力学研究中高灵敏度
和高通量的需要，建立了液相色谱四极杆型质谱联用法(LC舰S舰S)，同时定量
分析沐口血浆和尿样中异戊酸螺旋霉素I，n，m和螺旋霉素I，n，111六种成分.
三、必特螺旋霉素的体外酸稳定性研究
研究了螺旋霉素系列衍生物(大多数为必特螺旋霉素的主要成分)在pH 1 .3
的人工胃液中，37℃下的水解动力学，以LC舰Sn法鉴定了水解产物;用LC乃以sn
法测定了螺旋霉素系列衍生物(螺旋霉素I，n，In，乙酞螺旋霉素In，丙酞螺旋
霉素m，(异)丁酸螺旋霉素m和异戊酞螺旋霉素I，n，m)的降解速率常数，
并进行了比较研究.旨在寻求螺旋霉素系列衍生物体内外酸稳定性的相关性，为
代谢研究及临床用药提供参考数据，同时为研究螺旋霉素衍生物构效关系提供数
据，进而为开发新药莫定基拙.
研究中发现这些化合物在酸环境下易降解。3位羚基酸化后对于螺旋霉素衍
生物的稳定性影响不大，而4’’位夯基酞化后可以提高螺旋霉素衍生物在酸中的稳
定性并且改变降解途径，脱福洛氨糖成为主要降解途径，其产物具有抗菌活性.
四、必特螺旋霉素在大鼠体内的药物动力学研究
吸收动力学研究
分别灌胃或静脉给予大鼠必特螺旋霉素80 mgkg一，后，采用Lc从Sn法测定了
不同时刻血浆中3种主要成分(异戊酞螺旋霉素I，n，m)及其主要活性代谢物
(螺旋霉素I，n，m)的浓度，绘制各自化合物的血浆浓度一时间曲线，计算了
主要的药物动力学参数。
必特螺旋霉素以上述两种方式给药进入体内后，转化为螺旋霉素的程度牙民高.
以母体药物异戊酸螺旋霉素I，n，111和活性代谢物螺旋霉素I，n，111的AUCO一1:h
总和计算的口服绝对生物利用度平均为%.7%，说明以异戊酞螺旋霉素为主的必
特螺旋霉素与螺旋霉素相比具有更好的口服吸收。
组织分布研究
采用LC乃涯S”法，测定了大鼠经灌胃给予必特螺旋霉素后，组织及血浆中必
特螺旋霉素主要成分和主要代谢物在五个不同时间点的浓度.测定大鼠经灌胃给
予等剂量螺旋霉素后，组织及血浆中螺旋霉素在两个不同时间，点的浓度。
嘴周日称111
........侧.......
给予等剂量两种药?
Bitespiramycin (Shengjimycin) was developed by the Institute of Medical Biotechnology, Chinese Academy of Medical Science. It is a group of 4"-acylated spiramycins with 4"-isovalerylspiramycins as the major components, produced by recombinant Streptomyces spiramyceticus F21. The contents of isovalerylspiramycin I, II, III in bitespiramycin are 7.4%, 22.5% and 37.7%, respectively. Minor components in bitespiramycin include about 6 derivatives of spiramycin such as (iso) butanoylspiramycin, propionylspiramycin and acetylspiramycin. Phase II clinical trial of bitespiramycin will be performed soon.
Although spiramycin has been used in clinical therapy for more than 40 years, the research about its metabolism in vivo was limited. This is due in part to difficulties that have been encountered in establishing a sensitive and specific assay for spiramycin and its metabolites. Bitespiramycin is a complex mixture of more than 10 kinds of spiramycin derivatives. The research about its metabolism and pharmacokinetics in vivo was a challenge for the common analytical methods. The aim of this work is to investigate the metabolism and pharmacokinetics of such a complex multicomponents drug in vivo using the advanced LC/MSn and LC/MS/MS method.
1. Identification of the metabolites of bitespramycin in rats by LC/MSn
Metabolites in the urine, bile and feces of 4 rats following a single oral dose of 40 mg bitespiramycin were investigated. A total of 49 metabolites were found in bile, urine and feces by HPLC with ion trap mass spectrometric detection. Using the multi-stage MS (MSn) analysis of bitespiramycin and its metabolites, the characteristic fragment ions were obtained.
The metabolites of isovalerylspiramycin III (MO) were identified as deforosamine derivative of MO (Ml), deisovalery derivative of Ml (M2), reduction derivative of MO (M3), lactone hydrolyzed derivative of MO (M4), cysteine conjugate of MO (M5), deisovalery derivative of MO (M6), reduction derivative of M6 (M7), cysteine conjugate of M6 (M8), demycarose derivative of MO and M6 (M9), cysteine conjugate of M9 (M10), lactone hydrolyzed derivative of M6 (M11). The other components in bitespiramycin have similar metabolic pathways with isovalerylspiramycin III.
The facile procedure led to identification of all the 10 known components of bitespiramycin, in addition to the characterization of at least 49 metabolites including spiramycin I, II, III, platenomycin A1, josamycin, leucomycin A1 and midecamycin A1 which have been used in clinical therapy for decades. Structures of 16 major metabolites (M1a-c, M1f, M3a, M5a, M6a-c, M7a-c, M8a-c, and M9c) were established by chromatographic and mass spectrometric analyses and
comparison with synthesized reference substances. The aldehyde reduction and hydrolysis of the forosamine represent two novel biotransformation pathways for spiramycin derivatives in vivo.
2. The development of the quantitation methods
A sensitive and specific LC/MSn method was developed for the simultaneous determination of major components (isovalerylspiramycin I, II, III) or their major metabolites (spiramycin I, II, III) in biological samples of rats.
A more sensitive and fast LC/MS/MS quantitative method was developed to simultaneously determine six components (isovalerylspiramycin I, II, III and spiramycin I, II, III) in human plasma and urine.
3. Kinetics of acid-catalyzed hydrolysis
The developed LC/MSn method was used to clarify the degradation pathways and validated for monitoring degradation process of spiramycin derivatives at 37C in synthetic gastric fluid to simulate human gastric environment. Spiramycin III, acetylspiramycin III, propionylspiramycin III, (iso)butanoylspiramycin III and isovalerylspiramycin I, II, III were also the major components of bitespiramycin, which was developed as a novel antibiotic. These components were found susceptible to degradation from exposure to acidic condition (pH 1.3). Furthermore, the degradation rate constants (Ke) and half-life (t1/2) of spiramycin derivatives were calculated
螺旋霉素的结构复杂，必特螺旋霉素是具有十余种螺旋霉素衍生物的混合物，其在体内代谢和药物动力学过程研究对于现有的分析方法是一种挑战。本论文目标是利用先进的分析手段，研究必特螺旋霉素这一复杂混合物的体内代谢和药物动力学过程，主要工作如下：
一、必特螺旋霉素体内代谢和质谱研究
在有关文献报道的基础上，首次采用LC／MS~n技术，系统地研究了必特螺旋霉素在大鼠体内的代谢情况。
大鼠灌胃给予40 mg必特螺旋霉素后，分别收集大鼠的尿、粪和胆汁样品进行分析，除10种原形药物外，共发现49种代谢产物。以异戊酰螺旋霉素Ⅲ为例：11个代谢产物分别鉴定为(1)脱去福洛氨糖，生成M1；(2)脱去4″异戊酰侧链，生成M2和M6；(3)醛基发生还原反应，生成M3和M7；(4)发生水解反应，内酯环开环，生成M4和M11；(5)醛基与半胱氨酸结合，生成M5，M8和M10；(6)脱去碳霉糖，生成M9。必特螺旋霉素中其他组分的代谢途径与异戊酰螺旋霉素Ⅲ类似，共分为11个系列。除10种原形药物(M0a-M0i)外，16个代谢产物(M1a-c，M1f，M3a，M5a，M6a-c，M7a-c，M8a-c和M9c)的结构经与对照物质的色谱和质谱行为比较，予以确认。
其中相对含量最大的是螺旋霉素，同时也是发挥抗菌活性的主要活性物质。对于螺旋霉素类衍生物，醛基还原成羟基和脱福洛氨糖是首次在大鼠体内发现的代谢途径，在尿中和胆汁中均未发现醛基还原产物，说明醛基还原成羟基的过程可能是肠道菌群介导的。脱去福洛氨糖后的代谢产物中包括普拉特霉素、交沙霉素、柱晶白霉素和麦迪霉素等几种临床上常见的的十六元环大环内酯类抗生素。
通过LC／MS~n方法，得到必特螺旋霉素及其代谢产物的多级质谱特征丢失碎片，总结质谱裂解规律并作为判断依据，以推测代谢物的结构。
二、定量分析方法的建立
必特螺旋霉素作为多组分混合物，包含有10种主要成分，均为螺旋霉素衍生物。其中异戊酰螺旋霉素是主要有效成分，本文中以异戊酰螺旋霉素作为跟踪原
摘要il
形药物的定量指标，同时以必特螺旋霉素的主要代谢产物螺旋霉素作为跟踪代谢
物的定量指标。
通过对色谱和质谱条件进行优化，建立了同时定量分析异戊酞螺旋霉素I，11，
m或螺旋霉素I，n，m的液相色谱离子阱型质谱联用法(LC/MSn)，对必特螺旋
霉素在大鼠体内药物动力学进行了研究。
在此基础上，为了能满足必特螺旋霉素I期临床药物动力学研究中高灵敏度
和高通量的需要，建立了液相色谱四极杆型质谱联用法(LC舰S舰S)，同时定量
分析沐口血浆和尿样中异戊酸螺旋霉素I，n，m和螺旋霉素I，n，111六种成分.
三、必特螺旋霉素的体外酸稳定性研究
研究了螺旋霉素系列衍生物(大多数为必特螺旋霉素的主要成分)在pH 1 .3
的人工胃液中，37℃下的水解动力学，以LC舰Sn法鉴定了水解产物;用LC乃以sn
法测定了螺旋霉素系列衍生物(螺旋霉素I，n，In，乙酞螺旋霉素In，丙酞螺旋
霉素m，(异)丁酸螺旋霉素m和异戊酞螺旋霉素I，n，m)的降解速率常数，
并进行了比较研究.旨在寻求螺旋霉素系列衍生物体内外酸稳定性的相关性，为
代谢研究及临床用药提供参考数据，同时为研究螺旋霉素衍生物构效关系提供数
据，进而为开发新药莫定基拙.
研究中发现这些化合物在酸环境下易降解。3位羚基酸化后对于螺旋霉素衍
生物的稳定性影响不大，而4’’位夯基酞化后可以提高螺旋霉素衍生物在酸中的稳
定性并且改变降解途径，脱福洛氨糖成为主要降解途径，其产物具有抗菌活性.
四、必特螺旋霉素在大鼠体内的药物动力学研究
吸收动力学研究
分别灌胃或静脉给予大鼠必特螺旋霉素80 mgkg一，后，采用Lc从Sn法测定了
不同时刻血浆中3种主要成分(异戊酞螺旋霉素I，n，m)及其主要活性代谢物
(螺旋霉素I，n，m)的浓度，绘制各自化合物的血浆浓度一时间曲线，计算了
主要的药物动力学参数。
必特螺旋霉素以上述两种方式给药进入体内后，转化为螺旋霉素的程度牙民高.
以母体药物异戊酸螺旋霉素I，n，111和活性代谢物螺旋霉素I，n，111的AUCO一1:h
总和计算的口服绝对生物利用度平均为%.7%，说明以异戊酞螺旋霉素为主的必
特螺旋霉素与螺旋霉素相比具有更好的口服吸收。
组织分布研究
采用LC乃涯S”法，测定了大鼠经灌胃给予必特螺旋霉素后，组织及血浆中必
特螺旋霉素主要成分和主要代谢物在五个不同时间点的浓度.测定大鼠经灌胃给
予等剂量螺旋霉素后，组织及血浆中螺旋霉素在两个不同时间，点的浓度。
嘴周日称111
........侧.......
给予等剂量两种药?
Bitespiramycin (Shengjimycin) was developed by the Institute of Medical Biotechnology, Chinese Academy of Medical Science. It is a group of 4"-acylated spiramycins with 4"-isovalerylspiramycins as the major components, produced by recombinant Streptomyces spiramyceticus F21. The contents of isovalerylspiramycin I, II, III in bitespiramycin are 7.4%, 22.5% and 37.7%, respectively. Minor components in bitespiramycin include about 6 derivatives of spiramycin such as (iso) butanoylspiramycin, propionylspiramycin and acetylspiramycin. Phase II clinical trial of bitespiramycin will be performed soon.
Although spiramycin has been used in clinical therapy for more than 40 years, the research about its metabolism in vivo was limited. This is due in part to difficulties that have been encountered in establishing a sensitive and specific assay for spiramycin and its metabolites. Bitespiramycin is a complex mixture of more than 10 kinds of spiramycin derivatives. The research about its metabolism and pharmacokinetics in vivo was a challenge for the common analytical methods. The aim of this work is to investigate the metabolism and pharmacokinetics of such a complex multicomponents drug in vivo using the advanced LC/MSn and LC/MS/MS method.
1. Identification of the metabolites of bitespramycin in rats by LC/MSn
Metabolites in the urine, bile and feces of 4 rats following a single oral dose of 40 mg bitespiramycin were investigated. A total of 49 metabolites were found in bile, urine and feces by HPLC with ion trap mass spectrometric detection. Using the multi-stage MS (MSn) analysis of bitespiramycin and its metabolites, the characteristic fragment ions were obtained.
The metabolites of isovalerylspiramycin III (MO) were identified as deforosamine derivative of MO (Ml), deisovalery derivative of Ml (M2), reduction derivative of MO (M3), lactone hydrolyzed derivative of MO (M4), cysteine conjugate of MO (M5), deisovalery derivative of MO (M6), reduction derivative of M6 (M7), cysteine conjugate of M6 (M8), demycarose derivative of MO and M6 (M9), cysteine conjugate of M9 (M10), lactone hydrolyzed derivative of M6 (M11). The other components in bitespiramycin have similar metabolic pathways with isovalerylspiramycin III.
The facile procedure led to identification of all the 10 known components of bitespiramycin, in addition to the characterization of at least 49 metabolites including spiramycin I, II, III, platenomycin A1, josamycin, leucomycin A1 and midecamycin A1 which have been used in clinical therapy for decades. Structures of 16 major metabolites (M1a-c, M1f, M3a, M5a, M6a-c, M7a-c, M8a-c, and M9c) were established by chromatographic and mass spectrometric analyses and
comparison with synthesized reference substances. The aldehyde reduction and hydrolysis of the forosamine represent two novel biotransformation pathways for spiramycin derivatives in vivo.
2. The development of the quantitation methods
A sensitive and specific LC/MSn method was developed for the simultaneous determination of major components (isovalerylspiramycin I, II, III) or their major metabolites (spiramycin I, II, III) in biological samples of rats.
A more sensitive and fast LC/MS/MS quantitative method was developed to simultaneously determine six components (isovalerylspiramycin I, II, III and spiramycin I, II, III) in human plasma and urine.
3. Kinetics of acid-catalyzed hydrolysis
The developed LC/MSn method was used to clarify the degradation pathways and validated for monitoring degradation process of spiramycin derivatives at 37C in synthetic gastric fluid to simulate human gastric environment. Spiramycin III, acetylspiramycin III, propionylspiramycin III, (iso)butanoylspiramycin III and isovalerylspiramycin I, II, III were also the major components of bitespiramycin, which was developed as a novel antibiotic. These components were found susceptible to degradation from exposure to acidic condition (pH 1.3). Furthermore, the degradation rate constants (Ke) and half-life (t1/2) of spiramycin derivatives were calculated
引文
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