药品注册分类包括中药、化学药及生物制品三大类。药物的临床药代动力学研究旨在阐明药物在人体内的吸收、分布、代谢和排泄的动态变化规律，是全面认识人体与药物间相互作用不可或缺的重要组成部分，也是临床制定合理用药方案的依据。由于生物样品一般来自血清、血浆、尿液等临床生物样品，具有取样量少、药物浓度低、干扰物质多以及个体差异大等特点，因此建立灵敏、精确、可靠的生物样品定量分析方法，并对方法进行验证十分重要。

   本论文围绕生物制品药物聚乙二醇化重组人粒细胞刺激因子（PEG-rhG-CSF，商品名：津优力）和化学药物盐酸莎巴比星，分别建立了快速、灵敏的酶联免疫吸附分析法（ELISA）测定人血清中PEG-rhG-CSF及rhG-CSF的药物浓度和超高效液相色谱质谱联用（UPLC-MS/MS）法测定人尿液中盐酸莎巴比星及其醇类代谢物M3的浓度，并对方法的标准曲线范围、精密度与准确度、选择性、稳定性等方面进行完整验证，以期获得可靠和可重复的定量分析方法，为上述药物的药代动力学研究和治疗药物浓度监测提供方法学参考，同时探讨上述药物在肿瘤患者体内的药代动力学特征，为临床安全、有效和合理用药提供科学依据。主要内容分为两个部分：

第一部分：聚乙二醇化重组人粒细胞刺激因子（PEG-rhG-CSF，商品名：津优力）和重组人粒细胞刺激因子（rhG-CSF，商品名：津恤力）的药代动力学比较研究

   聚乙二醇是一类具有良好的生物相容性且无毒可生物降解的多聚体，rhG-CSF经高分子聚乙二醇修饰后，其作用机制仍与G-CSF相同，但极大地延长了G-CSF半衰期，从而避免了其给药频繁、依从性差和治疗周期较长的不足。本研究进行的PEG-rhG-CSF与rhG-CSF的药代动力学研究将进一步考察PEG-rhG-CSF在广泛使用条件下的疗效和安全性，具体研究内容如下：

  第一章：建立人血清中聚乙二醇化重组人粒细胞刺激因子与重组人粒细胞刺激因子的检测方法。采用ELISA方法测定人血清中聚乙二醇化重组人粒细胞刺激因子与重组人粒细胞刺激因子的药物浓度，并对方法学进行了完整验证。结果显示，聚乙二醇化重组人粒细胞刺激因子与重组人粒细胞刺激因子在39 ~2500pg/mL范围内线性均良好，重组人粒细胞刺激因子的批间准确度范围为-9.3% ~ -4.6%，批内精密度范围为0.1% ~ 12.8%；聚乙二醇化重组人粒细胞刺激因子的批间准确度范围为-6.0% ~ -11.7%，批内精密度范围为0.9% ~ 11.8%；方法学的验证结果符合临床药代动力学研究检测要求。

  第二章：聚乙二醇化重组人粒细胞刺激因子与重组人粒细胞刺激因子在实体瘤患者中的临床药代动力学研究。本研究采用单中心、开放、对照临床研究方法，完成试验组（PEG-rhG-CSF组）13例和对照组（rhG-CSF组）6例受试者血清中药物浓度检测数据，血药浓度-时间曲线呈现单峰特征，聚乙二醇化重组人粒细胞刺激因子和重组人粒细胞刺激因子的半衰期分别为22.82±6.17h和2.76±1.36h；平均体内暴露量分别为8982.94±4775.82 ng•h/mL和121.14±57.94 ng•h/mL，结果显示聚乙二醇化重组人粒细胞刺激因子的半衰期明显长于重组人粒细胞刺激因子，且体内药物暴露量明显较高，二者的t_1/2、C_max、AUC_0-t、CL等均具有统计学差异。

  第三章：建立人血清中粒细胞刺激因子免疫原性的检测方法。采用ELISA方法测定人血清中抗粒细胞刺激因子（G-CSF）抗体的浓度，并对方法学进行完整验证，结果显示该方法在12.5 ~ 400 ng/ml范围内线性良好，批间准确度范围为-4.8% ~ -0.4%，批内精密度范围为0.5% ~ 11.5%，不同储存条件下方法均稳定，能够满足临床免疫原性研究评价的需要。

第二部分：盐酸莎巴比星在难治或复发小细胞肺癌的I期临床药代动力学研究

  注射用盐酸莎巴比星作为一种具有新糖基的蒽环类药物，其抗肿瘤作用机制与该类药物相同，但具有更强的药效和更弱的心脏毒性。本研究旨在进一步探讨盐酸莎巴比星在广泛期小细胞肺癌患者体内药代动力学特征、安全性和耐受性，为II期临床推荐给药剂量提供依据，具体研究内容如下：

第一章：建立人尿液中莎巴比星及其代谢物M3的检测方法。采用超高效液相质谱联用（UPLC-MS/MS）法测定人尿液中莎巴比星及其醇类代谢物M3的浓度，并对方法学进行完整验证。结果显示莎巴比星和代谢物M3在2 ~ 200ng/ml范围内线性良好，莎巴比星和代谢物M3的批内精密度范围分别为2.6% ~ 8.2%和7.0% ~ 11.2%，批间准确度的范围分别为-5.6% ~ 3.2%和-1.2% ~ 10.7%，未见明显基质效应，方法学的验证结果达到临床药代动力学研究所需检测要求。

  第二章：盐酸莎巴比星在难治或复发小细胞肺癌患者中的药代动力学研究。采用开放、非随机和剂量递增的研究设计，共获得15例受试者给药后的尿液药物浓度和药代动力学参数，对莎巴比星的药代动力学特征进行分析。

关键词：重组人粒细胞刺激因子；盐酸莎巴比星；超高效液相色谱质谱联用；临床药代动力学

The classification of drug registration include traditional Chinese medicine, chemical medicine and biological products. The research of pharmacokinetic study aims at illuminating the absorption, distribution, metabolism and excretion properties of drugs in human, and it’s an essential part for comprehensive understanding of the interaction between the human and drug. Also it has been playing an important role in guiding clinical rational administration. Most biological samples come from serum, plasma, urine and so on. All these clinical biological samples have a common feature in less sample, low concentration, various interferents and individual differences. Therefore, it is of great importance to develop and evaluate a sensitive, accurate, reliable quantitative method for detecting the drug concentration of biological samples.

In this study, we aim to assess the clinical pharmacokinetic profiles of pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF) and sabarubicin in patients with advanced cancer respectively. We haved developed and evaluated a rapid and sensitive method to determine the concentration of PEG-rhG-CSF and rhG-CSF in human serum using enzyme-linked immune sorbent assay (ELISA), and a method for determination the concentration of sabarubicin and its alchol metabolism M3 in human urine using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Both the methods can supply information for their pharmacokinetic studies and monitoring the drugs concentration, so as to explore the pharmacokinetic characteristics, and provide reference for clinical applications.

Section 1. The comparative pharmacokinetic studies of PEG-rhG-CSF and recombinant human granulocyte colony-stimulating factor (rhG-CSF)

Polyethylene glycol is a flexible, water-soluble and low toxic polymer. The mechanism of PEG-rhG-CSF is the same as rhG-CSF with longer half-life in serum, thus we can avoid frequent intakes of medicines, poor compliance, longer treatment cycles, and so on. The aim of this comparative pharmacokinetic studies between PEG-rhG-CSF and rhG-CSF is to further investigate the efficacy and safety of PEG-rhG-CSF while widely used.

Chapter 1: Established an ELISA method for determination of PEG-rhG-CSF and rhG-CSF. The assay was validated over the concentration range of 39 ~ 2500 pg/mL for both PEG-rhG-CSF and rhG-CSF. The range of intra-day accuracy of the quality control samples is -9.3% ~ -4.6% and -6.0% ~ -11.7% for PEG-rhG-CSF and rhG-CSF respectively. The range of inter-day precision of the quality control samples is 0.1% ~ 12.8% and 0.9% ~ 11.8% respectively. The method was successfully applied to perform serum pharmacokinetic studies of PEG-rhG-CSF and rhG-CSF in cancer patients.

Chapter 2: Clinical pharmacokinetic studies of PEG-rhG-CSF and rhG-CSF in advanced cancer patients. This study was designed as a single-center, open label and comparative study. Now we have finished the concentration detection of 13 patients for the treatment group and 6 patients for the control group. The serum concentration-time curve shows single apex profiles. The half-time of PEG-rhG-CSF was significantly longer than rhG-CSF (22.82±6.17h and 2.76±1.36h, respectively). The drug exposure of PEG-rhG-CSF was 8982.9±4775.8 ng•h/mL, and 121.1±57.9 ng•h/mL for rhG-CSF. And the results also showed significant differences between the two groups in t_1/2, C_max, AUC_0-t and CL.

Chapter 3: Development and validation of an ELISA method for determination the immunogenicity of G-CSF. The assay was validated over the concentration range of 12.5 ~ 400 ng/mL. The range of intra-day accuracy and inter-day precision of the quality control samples is –4.8% ~ -0.4% and 0.5% ~ 11.5%. This method was proved to be stable at several conditions, and able to meet the requirement of clinical immunogenicity accessment.

Section 2. Phase I clinical pharmacokinetic studies of sabarubicin in patients with extensive small cell lung cancer

Sabarubicin is a relative novel anthracyclines with disaccharide, and it has similar anti-tumor effect and mechanism with anthracyclines, but with stronger efficacy and more slightly cardiotoxicity. In this study, we aim to investigate the pharmacokinetics of sabarubicin in extensive stage small cell lung cancer patiens, and then can provide a basis for phase II dosage.

Chapter 1: Development and validation of a UPLC-MS/MS method for determination of sabarubicin and its metabolism M3 in human urine. A seven-point calibration curve exhibited excellent linearity over the range 2 ~ 200 ng/ml for both sabarubicin and M3. The intra- and inter-day precision and accuracy of the quality control samples at low, medium and high concentration levels showed 2.6% ~ 8.2% for sabarubicin and 7.0% ~ 11.2% for M3 relative standard deviation (RSD), and -5.6% ~ 3.2% and -1.2% ~ 10.7% for sabarubicin and M3 relative errors (RE), respectively. The method was successfully applied to perform urine pharmacokinetic studies of sabarubicin in extensive stage SCLC after intravenous infusion.

Chapter 2: Clinical pharmacokinetic studies of sabarubicin in refractory or recurrent SCLC patients. This study was designed as an open-label, non-randomized and dose-escalation study. A total of 15 patients were enrolled and given intravenous infusion sabarubicin, and we have finished analyzing the pharmacokinetic characteristics in this study.

Keywords: rhG-CSF; Sabarubicin; UPLC-MS/MS; Clinical pharmacokinetics