多晶型药物美托拉宗合成及药动学研究
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摘要
同一种元素或化合物在不同条件下生成结构、形态、物性完全不同的晶体的现象称为多晶现象。而药物不同固态晶格中的不同填充、排列方式将影响药物在人体内吸收的速度和程度,进而导致临床药效的差异。晶型是影响药物生物利用度的最主要因素之一,也正因此优化制剂工艺、优化结晶条件,研究药物晶型与生物利用关系成为近年来药物制剂领域研究的热点和焦点。
美托拉宗,一种利尿剂,最早于1974年由美国Pennwalt公司制造在英国和美国上市,随后,在德、意、日、瑞士和阿根廷等许多国家上市,在临床使用已达三十年,被美国药典23、24、25版收载。美托拉宗的商品名为Zaroxolyn?或Diulo? (规格:2.5mg、5mg、10mg);2001年8月10日,由FDA批准Celltech Pharmaceuticals. Inc公司生产Mykrox?(规格:0.5 mg)并上市。两种产品的最主要的区别是新产品使用的美托拉宗原料的晶型与早期生产的原料不同,所以Mykrox吸收快,其生物利用度与Zaroxolyn?和Diulo?相比有明显的提高;Mykrox?达峰浓度时间是2~4 h,而美托拉宗的其他规格达峰浓度时间是8 h。Mykrox?的优点是半衰期、作用时间较长,分别达8 h、12~24 h;而且不抑制碳酸酐酶,从而不影响H+和氨的分泌,血氨降低;对肾功能不全病人,其与吲达帕胺要比其它噻嗪类药物略胜一筹。
正是由于美托拉宗的上述优点,本课题组与西安力邦制药有限公司合作共同开发了美托法宗片(规格:0.5 mg),其小试、放大工艺成熟稳定,且无知识产权限制,产品质量达到美国药典要求,现已获得临床研究批件。本次试验目的是优化美托拉宗生产工艺及结晶条件,考察美托拉宗不同晶型与生物利用度的关系,阐明其在中国人体内吸收、分布、代谢、排泄的规律,为美托法宗II期临床试验的给药方案提供依据。
一、美托拉宗合成及结构鉴定
目的:探索美托拉宗合成路线,并对美托拉宗结构及晶型进行确证。
方法:以2-甲基-5氯苯胺为起始原料,经乙酰化、氯磺化、氨解、氧化、缩醛反应制得美托拉宗。并采用红外光谱(IR)、核磁共振氢谱(NMR)、质谱(MS)、示差扫描量热法等方法对其结构及晶型进行确证。
结果:最后美托拉宗的回收率为84.5%,物料基本平衡。元素分析结果显示:分子中原子个数比C:H:N:S:Cl:O = 16:16:3:1:1:3得美托拉宗最简化式为C_(16)H_(16)N_3SClO_3,式量为365;由MS得美托拉宗分子量为365,故可知美托拉宗最简化式即为分子式。计算分子中不饱和度Ω为10与分子中存在6个双键、3个环、1个羰基相符合。IR分析结果显示:美托拉宗分子结构中主要官能团有苯环、羰基、氨基、磺酰基、氯原子等,IR图谱中各官能团特征吸收明显,与美托拉宗结构相符。NMR分析结果显示:从美托拉宗结构上看,存在1个手性碳原子,加上二氢嘧啶酮环的椅式构型反转造成的构象异构,分子中各C、H原子化学环境复杂,仅从~(13)C-NMR、~1H-NMR图谱不易分解,结合DEPT、HMBC、HMQC等联合进行解析,确认为美托拉宗分子的C、H分布。MS分析结果看MS图谱中得到的美托拉宗分子离子峰[M+]365,主要裂解发生在苯环及二氢嘧啶酮环上,裂解途径与美托拉宗分子结构相符。
结论:本研究室合成的美托拉宗纯度较高,产率较高,具有产业化前景,且IR、NMR、MS对美托拉宗结构进行解析,并与国外对照品进行了比对,可确认结构为7-氯-1,2,3,4-四氢-2-甲基-3-(2-甲基苯基) -4-氧代-6-喹唑磺酰胺。
二、液相色谱-串联质谱法测定血桨中美托拉宗浓度及其药动学的应用
目的:建立快速、灵敏的液相色谱-串联质谱血浆中定性定量检测方法,并基于此方法研究美托拉宗片单次及多次给药后在中国健康人体内的药代动力学特征,为制订美托拉宗II期临床试验的给药方案提供依据。
方法:选用Agilent TC-C_(18) (5μm,4.6×150 mm ID, Aglient)的色谱柱,流动相为甲醇-去离子水(70:30,V/V);流速为0.5 ml·min~(-1),柱温:35℃,三重四极杆质谱以多反应监测方式进行检测。检测离子为:美托拉宗,[M-H]~-离子,m/z 364.1→257,氢氯噻嗪,[M-H]~-离子,m/z 296.2→269.1。30名健康受试者随机分为3组,每组10人,分别参加美托拉宗单次(0.5 mg、1 mg、2 mg),在给药后的0.5、1.0、1.5、2.0、3.0、4.0、5.0、6.0、8.0、12.0、24.0、36.0、48.0 h分别采集静脉血约5ml,离心分离出血浆置无菌试管中,置于-80℃冰箱中保存,用HPLC/MS/MS法测定血浆中美托拉宗的浓度,计算药动学参数。单次给药中剂量组继续参加多次药代动力学(1 mg)研究。
结果:美托拉宗线性范围为0.050-100.0ng·ml~(-1);低、中、高三个浓度质控批内精密度为3.5~8.9%,批间精密度为5.7~8.5%,准确度为93.4~110.8%;提取回收率为70.5~72.6%。口服美托拉宗0.5 mg、1 mg以及2mg后,美托拉宗的消除半衰期t_(1/2)为6.6±2.8h、7.9±1.2h、7.6±1.9h,AUC_(0-48)为32.9±9.2ng·h·ml~(-1)、122.5±36.3 ng·h·ml~(-1)、162.4±26.9 ng·h·ml~(-1),达峰时间T_(max)分别为1.6±0.9h、1.6±0.6 h以及1.5±1.3 h和达峰浓度C_(max)分别为6.9±2.6ng?ml~(-1)、20.6±4.8 ng·ml~(-1)以及36.8±7.1 ng·ml~(-1)。多次口服美托拉宗,稳态平均血药浓度Cssav为9.7±1.8 ng·ml~(-1),稳态血药浓度波动度DF为2.1±0.3,蓄积常数R为74.9±5.2%,消除半衰期t1/2为8.9±1.4 h,MRT为8.5±1.0 h,AUC0-τ为(156.8±31.6) ng?h?ml~(-1),达峰时间T_(max)和达峰浓度C_(max)分别为2.4±1.4 h和22.4±5.0 ng?ml~(-1)。
结论:在0.5~2 mg剂量范围内美托拉宗的AUC(0-48)、C_(max)均与剂量呈一定线性关系。连续7天给药,体内无蓄积。推荐临床剂量为0.5mg,每天1次,如疗效欠佳,增大剂量为0.5mg,每天2次。
三、高效液相色谱荧光法测定尿液中美托拉宗及其药动学的应用
目的:建立快速、灵敏的液相色谱法尿液中美托拉宗定量检测方法,并基于此方法研究美托拉宗片单次给药后在中国健康人体内的药动学特征,为制订美托拉宗II期临床试验的给药方案提供依据。
方法:选用Agilent TC-C_(18) (5μm,4.6×150 mm ID, Aglient)的色谱柱,流动相为甲醇-去离子水(43:57,V/V);流速为1.0 ml·min~(-1),柱温:35℃;荧光检测器检测,美托拉宗:激发波长235nm,发射波长410nm;地西泮:激发波长235nm,发射波长354nm。30名健康受试者随机分为3组,每组10人,分别参加美托拉宗单次(0.5 mg、1 mg、2 mg)研究,收集单次给药前(0h)及服药后0-2、2-4、4-6、6-8、8~(-1)0、10~(-1)2、12~(-1)6、16-24、24-48h各时间段所有尿液,记录各时段尿量后,留取3ml尿样于-80℃避光保存待测。
结果:美托拉宗线性范围为0.2-500.0 ng·ml~(-1);低、中、高三个浓度质控批内精密度为6.9~11.7%,批间精密度为6.9~11.0%,准确度为100.3~108.8%;提取回收率为77.9~81.7%。单次口服美托拉宗0.5mg、1mg、2mg后48小时的美托拉宗平均尿药累积排泄率(%)分别为59.7±16.4%、61.1±24.3%和62.3±14.4%,估算美托拉宗的消除半衰期t1/2为8.6±2.6h、9.2±2.0h、7.4±0.9h。
结论:健康受试者单次口服0.5 mg、1.0 mg、2.0 mg西安力邦制药有限公司研制的美托拉宗后,其在低、中、高三个剂量组的消除半衰期及体内平均驻留时间相近;在0.5 ~ 2 mg剂量范围内美托拉宗的累积排泄率相近,初步表明0.5 ~2 mg剂量范围内药物在体内为线性代谢。
Polymorphism in materials science is the ability of a solid material to exist in more than one form or crystal structure. Polymorphism can potentially be found in any crystalline material including polymers, minerals, and metals, and is related to allotropy, which refers to elemental solids. But the drugs’polymorphism may affect the rate and speed of absorption of drug in human body to result in discrepancy of clinical therapeutic effect. Crystal form is one of the main factors impacting oral bioavailability of drugs, so it is very important to optimize crystallization condition in drug production process. And in recent years, more and more pharmaceutical experts pay more attention to the relationship between drug crystal form and bioavailability.
Metolazone is a diuretic ("water pill") used in the treatment of high blood pressure and fluid accumulation. It works by blocking salt and fluid retention in the kidneys, thereby increasing urinary output of salt and water (diuresis). Although it is not a true thiazide, metolazone is chemically related to the thiazide class of diuretics, and works in a similar manner. In the market, there are two formulations of metolazone, one is the original formulation of metolazone (Zaroxolyn, or Diulo) and the absorption of these two drugs is relatively incomplete; another is an improved formulation of metolazone (Mykrox), which has more complete absorption, longer t1/2 , higher AUC and longer effects. Therefore, less Mykrox needs to be given to have the same effects as a larger dose of Zaroxolyn or Diulo. Mykrox was developed by Celltech Pharmaceuticals Inc, USA and was put into market in many countries in 2001. Recently, this formulation of metolazone has been developed and approved to conduct clinical trial in China. However, there was significant pharmacokinetic different between the original formulation of metolazone (Zaroxolyn, or Diulo) and improved formulation of metolazone (Mykrox) and the pharmacokinetic study of the improved metolazone formulation (Mykrox) has rarely been reported although this drug has been prescribed in clinic for decades. Therefore, it is urgent to investigate the pharmacokinetic properties of metolazone in man. The aim of our study was to assess the pharmacokinetic properties of metolazone in healthy Chinese volunteers.
Part 1 The synthesis and structural appraisement of metolazone Aims: To synthesize metolazone and make structural and crystal form appraisement of metolazone.
Method: To synthesize metolazone with raw material of 2-methyl-5- chloroaniline by the way of acetylation, chloro-sulfonation, ammonolysis, oxidation and acetal. The structural and crystal form appraisement of metolazone has been validated by IR, MS, NMR, EAL and X-ray diffraction.
Results: Recovery rate of metolazone production was 84.5%. Element analysis result showed that the ratio of atoms (C:H:N:S:Cl:O) in the molecule of metolazone was 16:16:3:1:1:3 and the simplest chemical formula of metolazone was C16H16N3SClO3 with chemical formula weight of 365. The degree of unsaturation in the metolazone molecule was ten, which is consistent with the six C=C double bonds, three rings and one C=O Carbonyl. IR result showed that the main functional groups of the metolazone molecular structure include benzene ring, carbonyl, amino, sulfuryl and chlorine. NMR result indicated that there was one chiral carbon atom, and conformational isomerism of chair form configuration reversal of dihydro-pyrimidone ring. MS result indicated that molecular ion of metolazone is [M+] 365. Its splitting occurred in the benzene ring and dihydro-pyrimidone ring.
Conclusions: The molecular structure of the synthesized products is 7-chloro- 1,2,3,4-tetrahydro-2-methyl-4-oxo-3-o-tolyl-6-quinazoline-sulfonamide.
Part 2 An LC-MS/MS method for quantitative determination of metolazone in human plasma and its application to a pharmacokinetic study.
Aims: To establish a LC-MS/MS method to determine metolazone in human plasma and study the pharmacokinetic characteristic of metolazone in Chinese healthy volunteers, and to provide references or recommendations for dosage regimen for phase II clinical trial study of metolazone.
Method: A simple, rapid and accurate liquid chromatography-tandem mass spectrometry method has been developed and validated for the quantitative measurement of metolazone in human plasma. After a liquid-liquid extraction procedure, samples were chromatographed on an agilent TC-C18 (150mm×4.6mm, 5μm) column using an isocratic elution mobile phase composed of methanol and distilled water (70:30, v/v) at a flow rate of 0.5 mL/min. The column oven temperature was set at 35 ?C. Mass spectrometric detection was performed on a Series 6410 Triple Quad LC-MS/MS (Agilent Technologies, USA) in electrospray negative ionization using multiple reaction monitoring (MRM). The mass transition was m/z 364.1→257 for Metolazone, and m/z 296.2→269.1 for IS, respectively. Thirty healthy male and female subjects were enrolled in this study. All subjects were randomly divided into three groups, such as Groups A-C (five males and five females in each group). Groups A-C were administered a single dose of metolazone tablet 0.5 mg, 1.0 mg and 2.0 mg, respectively. Blood samples (4 mL) were collected at 0 h (pre-dose) and 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 24, 36 and 48 h post dose. The samples were transferred to heparinized tube and centrifuged at 3000 g for 10 min. Plasma was separated and stored at -80°C until analysis. In the design of multiple doses, Group B received 1 mg metolazone at 8:00 a.m. for consecutive 6 days. In days 3, 4 and 5, 4 mL of venous blood was drawn to observe minimum value of steady plasma-drug concentration before every dosing at 8:00 a.m. In day 7, the procedure was the same as that of single dose mentioned above.
Results: A simple, rapid and sensitive method was developed for the determination of metolazone in human plasma and was used to compare the pharmacokinetic parameters after single and multiple doses oral administration. Good linearity was obtained over the concentration range of 0.05-100 ng/mL with correlation coefficients r = 0.995. The intra-day and inter-day precision of the quality control (QC) samples was 3.5~8.9% and 5.7~8.5% relative standard deviation (RSD), repectively. The inter-day accuracy of the QC samples was 93.4~110.8% of the nominal values. The extraction recoveries of the assay were 72.6±7.7, 71.3±10.1 and 70.5±4.0% for the low, middle and high concentrations(0.1, 5 and 100 ng/mL), respectively. The pharmacokinetic parameters of single doses of 0.5 mg, 1 mg and 2mg metolazone were as follows: t1/2 were .6±2.8h, 7.9±1.2h and7.6±1.9h; AUC0-48 were 32.9±9.2 ng·h·ml~(-1), 122.5±36.3 ng·h·ml~(-1) and 162.4±26.9 ng·h·ml~(-1); T_(max) were 1.6±0.9h, 1.6±0.6 h and 1.5±1.3 h; C_(max) were 6.9±2.6ng·ml~(-1), 20.6±4.8 ng·ml~(-1), 36.8±7.1 ng·ml~(-1). The pharmacokinetic parameters of multiple dose of 01 mg metolazone were as follows: Cav (9.7±1.8) ng/mL; DF (2.1±0.3); AUCss (116.8±21.1) ng·h/mL; R(74.9 5.2%); T_(max) (2.4±1.4) h and C_(max)( 22.4±5.0) ng·ml~(-1). Conclusions: The t1/2, C_(max) and AUC of multiple-dose were not significantly different from those of single-dose (P>0.05). The t_(1/2) of single dose and multiple doses were (7.9±1.2) and (8.9±1.4) h, respectively; The AUC of single dose and multiple doses were (123.9±36.7 and (156.8 31.6) ng·h/mL, respectively. These findings suggested that there was no accumulation of metolazone in plasma. The recommended dose was 0.5 or 1.0 mg metolazone per day for patients.
Part 3 An improved HPLC method for quantitative determination of metolazone in human urine and its application to a pharmacokinetic study.
Aims: To establish a HPLC method to determine metolazone in human urine and study the pharmacokinetic characteristic of metolazone in Chinese healthy volunteers, and to provide references or recommendations for dosage regimen for phase II clinical trial study of metolazone.
Method: A simple, rapid and accurate liquid chromatography spectrometry method has been developed and validated for the quantitative measurement of metolazone in human urine. After a liquid-liquid extraction procedure, samples were chromatographed on an agilent TC-C_(18) (150mm×4.6mm, 5μm) column using an isocratic elution mobile phase composed of methanol and distilled water (43:57, v/v) at a flow rate of 1.0 mL/min. The column oven temperature was set at 35?C. Fluorescence detector: excitation wave was 235nm and emission wave was 410nm for metolazone, and excitation wave was 235nm and emission wave was 354nm for diazepam. Thirty healthy male and female subjects were enrolled in this study. All subjects were randomly divided into three groups, such as Groups A-C (five males and five females in each group). Groups A-C were administered a single dose of metolazone tablet 0.5 mg, 1.0 mg and 2.0 mg, respectively.Urine samples (3 mL) were collected at 0 h (pre-dose) and 0-2, 2-4, 4-6, 6-8, 8-10, 10-12,12-16, 16-24, 24-48h h post dose. The samples were stored at -80 ?C until analysis.
Results: A simple, rapid and sensitive method was developed for the determination of metolazone in human urine and was used to study the pharmacokinetic parameters of metolazone after single doses oral administration. Good linearity was obtained over the concentration range of 0.2-500 ng/mL with correlation coefficients r = 0.998. The intra-day and inter-day precision of the quality control (QC) samples was 6.9~11.7% and6.9~11.0% relative standard deviation (RSD), repectively. The inter-day accuracy of the QC samples was 100.3~108.8% of the nominal values. The extraction recoveries of the assay were 77.9~81.7%. The pharmacokinetic parameters of single doses of 0.5 mg, 1 mg and 2mg metolazone were as follows: t_(1/2) were 8.6±2.6h, 9.2±2.0h and 7.4±0.9h and The mean cumulative urinary excretion amount of 10 volunteers up to 48h was59.7±16.4%, 61.1±24.3% and 62.3±14.4% for single doses of .5mg, 1mg and 2mg metolazone.
Conclusions: The pharmacokinetic parameters including elimination half life (t1/2) and accumulative excretory rate were similar for a single dose of 0.5 mg, 1.0 mg and 2.0 mg metolazone in Chinese healthy volunteers. It was suggested that the pharmacokinetics of metolazone fitted the linear dynamic feature over the dose range of metolazone studied.
美托拉宗,一种利尿剂,最早于1974年由美国Pennwalt公司制造在英国和美国上市,随后,在德、意、日、瑞士和阿根廷等许多国家上市,在临床使用已达三十年,被美国药典23、24、25版收载。美托拉宗的商品名为Zaroxolyn?或Diulo? (规格:2.5mg、5mg、10mg);2001年8月10日,由FDA批准Celltech Pharmaceuticals. Inc公司生产Mykrox?(规格:0.5 mg)并上市。两种产品的最主要的区别是新产品使用的美托拉宗原料的晶型与早期生产的原料不同,所以Mykrox吸收快,其生物利用度与Zaroxolyn?和Diulo?相比有明显的提高;Mykrox?达峰浓度时间是2~4 h,而美托拉宗的其他规格达峰浓度时间是8 h。Mykrox?的优点是半衰期、作用时间较长,分别达8 h、12~24 h;而且不抑制碳酸酐酶,从而不影响H+和氨的分泌,血氨降低;对肾功能不全病人,其与吲达帕胺要比其它噻嗪类药物略胜一筹。
正是由于美托拉宗的上述优点,本课题组与西安力邦制药有限公司合作共同开发了美托法宗片(规格:0.5 mg),其小试、放大工艺成熟稳定,且无知识产权限制,产品质量达到美国药典要求,现已获得临床研究批件。本次试验目的是优化美托拉宗生产工艺及结晶条件,考察美托拉宗不同晶型与生物利用度的关系,阐明其在中国人体内吸收、分布、代谢、排泄的规律,为美托法宗II期临床试验的给药方案提供依据。
一、美托拉宗合成及结构鉴定
目的:探索美托拉宗合成路线,并对美托拉宗结构及晶型进行确证。
方法:以2-甲基-5氯苯胺为起始原料,经乙酰化、氯磺化、氨解、氧化、缩醛反应制得美托拉宗。并采用红外光谱(IR)、核磁共振氢谱(NMR)、质谱(MS)、示差扫描量热法等方法对其结构及晶型进行确证。
结果:最后美托拉宗的回收率为84.5%,物料基本平衡。元素分析结果显示:分子中原子个数比C:H:N:S:Cl:O = 16:16:3:1:1:3得美托拉宗最简化式为C_(16)H_(16)N_3SClO_3,式量为365;由MS得美托拉宗分子量为365,故可知美托拉宗最简化式即为分子式。计算分子中不饱和度Ω为10与分子中存在6个双键、3个环、1个羰基相符合。IR分析结果显示:美托拉宗分子结构中主要官能团有苯环、羰基、氨基、磺酰基、氯原子等,IR图谱中各官能团特征吸收明显,与美托拉宗结构相符。NMR分析结果显示:从美托拉宗结构上看,存在1个手性碳原子,加上二氢嘧啶酮环的椅式构型反转造成的构象异构,分子中各C、H原子化学环境复杂,仅从~(13)C-NMR、~1H-NMR图谱不易分解,结合DEPT、HMBC、HMQC等联合进行解析,确认为美托拉宗分子的C、H分布。MS分析结果看MS图谱中得到的美托拉宗分子离子峰[M+]365,主要裂解发生在苯环及二氢嘧啶酮环上,裂解途径与美托拉宗分子结构相符。
结论:本研究室合成的美托拉宗纯度较高,产率较高,具有产业化前景,且IR、NMR、MS对美托拉宗结构进行解析,并与国外对照品进行了比对,可确认结构为7-氯-1,2,3,4-四氢-2-甲基-3-(2-甲基苯基) -4-氧代-6-喹唑磺酰胺。
二、液相色谱-串联质谱法测定血桨中美托拉宗浓度及其药动学的应用
目的:建立快速、灵敏的液相色谱-串联质谱血浆中定性定量检测方法,并基于此方法研究美托拉宗片单次及多次给药后在中国健康人体内的药代动力学特征,为制订美托拉宗II期临床试验的给药方案提供依据。
方法:选用Agilent TC-C_(18) (5μm,4.6×150 mm ID, Aglient)的色谱柱,流动相为甲醇-去离子水(70:30,V/V);流速为0.5 ml·min~(-1),柱温:35℃,三重四极杆质谱以多反应监测方式进行检测。检测离子为:美托拉宗,[M-H]~-离子,m/z 364.1→257,氢氯噻嗪,[M-H]~-离子,m/z 296.2→269.1。30名健康受试者随机分为3组,每组10人,分别参加美托拉宗单次(0.5 mg、1 mg、2 mg),在给药后的0.5、1.0、1.5、2.0、3.0、4.0、5.0、6.0、8.0、12.0、24.0、36.0、48.0 h分别采集静脉血约5ml,离心分离出血浆置无菌试管中,置于-80℃冰箱中保存,用HPLC/MS/MS法测定血浆中美托拉宗的浓度,计算药动学参数。单次给药中剂量组继续参加多次药代动力学(1 mg)研究。
结果:美托拉宗线性范围为0.050-100.0ng·ml~(-1);低、中、高三个浓度质控批内精密度为3.5~8.9%,批间精密度为5.7~8.5%,准确度为93.4~110.8%;提取回收率为70.5~72.6%。口服美托拉宗0.5 mg、1 mg以及2mg后,美托拉宗的消除半衰期t_(1/2)为6.6±2.8h、7.9±1.2h、7.6±1.9h,AUC_(0-48)为32.9±9.2ng·h·ml~(-1)、122.5±36.3 ng·h·ml~(-1)、162.4±26.9 ng·h·ml~(-1),达峰时间T_(max)分别为1.6±0.9h、1.6±0.6 h以及1.5±1.3 h和达峰浓度C_(max)分别为6.9±2.6ng?ml~(-1)、20.6±4.8 ng·ml~(-1)以及36.8±7.1 ng·ml~(-1)。多次口服美托拉宗,稳态平均血药浓度Cssav为9.7±1.8 ng·ml~(-1),稳态血药浓度波动度DF为2.1±0.3,蓄积常数R为74.9±5.2%,消除半衰期t1/2为8.9±1.4 h,MRT为8.5±1.0 h,AUC0-τ为(156.8±31.6) ng?h?ml~(-1),达峰时间T_(max)和达峰浓度C_(max)分别为2.4±1.4 h和22.4±5.0 ng?ml~(-1)。
结论:在0.5~2 mg剂量范围内美托拉宗的AUC(0-48)、C_(max)均与剂量呈一定线性关系。连续7天给药,体内无蓄积。推荐临床剂量为0.5mg,每天1次,如疗效欠佳,增大剂量为0.5mg,每天2次。
三、高效液相色谱荧光法测定尿液中美托拉宗及其药动学的应用
目的:建立快速、灵敏的液相色谱法尿液中美托拉宗定量检测方法,并基于此方法研究美托拉宗片单次给药后在中国健康人体内的药动学特征,为制订美托拉宗II期临床试验的给药方案提供依据。
方法:选用Agilent TC-C_(18) (5μm,4.6×150 mm ID, Aglient)的色谱柱,流动相为甲醇-去离子水(43:57,V/V);流速为1.0 ml·min~(-1),柱温:35℃;荧光检测器检测,美托拉宗:激发波长235nm,发射波长410nm;地西泮:激发波长235nm,发射波长354nm。30名健康受试者随机分为3组,每组10人,分别参加美托拉宗单次(0.5 mg、1 mg、2 mg)研究,收集单次给药前(0h)及服药后0-2、2-4、4-6、6-8、8~(-1)0、10~(-1)2、12~(-1)6、16-24、24-48h各时间段所有尿液,记录各时段尿量后,留取3ml尿样于-80℃避光保存待测。
结果:美托拉宗线性范围为0.2-500.0 ng·ml~(-1);低、中、高三个浓度质控批内精密度为6.9~11.7%,批间精密度为6.9~11.0%,准确度为100.3~108.8%;提取回收率为77.9~81.7%。单次口服美托拉宗0.5mg、1mg、2mg后48小时的美托拉宗平均尿药累积排泄率(%)分别为59.7±16.4%、61.1±24.3%和62.3±14.4%,估算美托拉宗的消除半衰期t1/2为8.6±2.6h、9.2±2.0h、7.4±0.9h。
结论:健康受试者单次口服0.5 mg、1.0 mg、2.0 mg西安力邦制药有限公司研制的美托拉宗后,其在低、中、高三个剂量组的消除半衰期及体内平均驻留时间相近;在0.5 ~ 2 mg剂量范围内美托拉宗的累积排泄率相近,初步表明0.5 ~2 mg剂量范围内药物在体内为线性代谢。
Polymorphism in materials science is the ability of a solid material to exist in more than one form or crystal structure. Polymorphism can potentially be found in any crystalline material including polymers, minerals, and metals, and is related to allotropy, which refers to elemental solids. But the drugs’polymorphism may affect the rate and speed of absorption of drug in human body to result in discrepancy of clinical therapeutic effect. Crystal form is one of the main factors impacting oral bioavailability of drugs, so it is very important to optimize crystallization condition in drug production process. And in recent years, more and more pharmaceutical experts pay more attention to the relationship between drug crystal form and bioavailability.
Metolazone is a diuretic ("water pill") used in the treatment of high blood pressure and fluid accumulation. It works by blocking salt and fluid retention in the kidneys, thereby increasing urinary output of salt and water (diuresis). Although it is not a true thiazide, metolazone is chemically related to the thiazide class of diuretics, and works in a similar manner. In the market, there are two formulations of metolazone, one is the original formulation of metolazone (Zaroxolyn, or Diulo) and the absorption of these two drugs is relatively incomplete; another is an improved formulation of metolazone (Mykrox), which has more complete absorption, longer t1/2 , higher AUC and longer effects. Therefore, less Mykrox needs to be given to have the same effects as a larger dose of Zaroxolyn or Diulo. Mykrox was developed by Celltech Pharmaceuticals Inc, USA and was put into market in many countries in 2001. Recently, this formulation of metolazone has been developed and approved to conduct clinical trial in China. However, there was significant pharmacokinetic different between the original formulation of metolazone (Zaroxolyn, or Diulo) and improved formulation of metolazone (Mykrox) and the pharmacokinetic study of the improved metolazone formulation (Mykrox) has rarely been reported although this drug has been prescribed in clinic for decades. Therefore, it is urgent to investigate the pharmacokinetic properties of metolazone in man. The aim of our study was to assess the pharmacokinetic properties of metolazone in healthy Chinese volunteers.
Part 1 The synthesis and structural appraisement of metolazone Aims: To synthesize metolazone and make structural and crystal form appraisement of metolazone.
Method: To synthesize metolazone with raw material of 2-methyl-5- chloroaniline by the way of acetylation, chloro-sulfonation, ammonolysis, oxidation and acetal. The structural and crystal form appraisement of metolazone has been validated by IR, MS, NMR, EAL and X-ray diffraction.
Results: Recovery rate of metolazone production was 84.5%. Element analysis result showed that the ratio of atoms (C:H:N:S:Cl:O) in the molecule of metolazone was 16:16:3:1:1:3 and the simplest chemical formula of metolazone was C16H16N3SClO3 with chemical formula weight of 365. The degree of unsaturation in the metolazone molecule was ten, which is consistent with the six C=C double bonds, three rings and one C=O Carbonyl. IR result showed that the main functional groups of the metolazone molecular structure include benzene ring, carbonyl, amino, sulfuryl and chlorine. NMR result indicated that there was one chiral carbon atom, and conformational isomerism of chair form configuration reversal of dihydro-pyrimidone ring. MS result indicated that molecular ion of metolazone is [M+] 365. Its splitting occurred in the benzene ring and dihydro-pyrimidone ring.
Conclusions: The molecular structure of the synthesized products is 7-chloro- 1,2,3,4-tetrahydro-2-methyl-4-oxo-3-o-tolyl-6-quinazoline-sulfonamide.
Part 2 An LC-MS/MS method for quantitative determination of metolazone in human plasma and its application to a pharmacokinetic study.
Aims: To establish a LC-MS/MS method to determine metolazone in human plasma and study the pharmacokinetic characteristic of metolazone in Chinese healthy volunteers, and to provide references or recommendations for dosage regimen for phase II clinical trial study of metolazone.
Method: A simple, rapid and accurate liquid chromatography-tandem mass spectrometry method has been developed and validated for the quantitative measurement of metolazone in human plasma. After a liquid-liquid extraction procedure, samples were chromatographed on an agilent TC-C18 (150mm×4.6mm, 5μm) column using an isocratic elution mobile phase composed of methanol and distilled water (70:30, v/v) at a flow rate of 0.5 mL/min. The column oven temperature was set at 35 ?C. Mass spectrometric detection was performed on a Series 6410 Triple Quad LC-MS/MS (Agilent Technologies, USA) in electrospray negative ionization using multiple reaction monitoring (MRM). The mass transition was m/z 364.1→257 for Metolazone, and m/z 296.2→269.1 for IS, respectively. Thirty healthy male and female subjects were enrolled in this study. All subjects were randomly divided into three groups, such as Groups A-C (five males and five females in each group). Groups A-C were administered a single dose of metolazone tablet 0.5 mg, 1.0 mg and 2.0 mg, respectively. Blood samples (4 mL) were collected at 0 h (pre-dose) and 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 24, 36 and 48 h post dose. The samples were transferred to heparinized tube and centrifuged at 3000 g for 10 min. Plasma was separated and stored at -80°C until analysis. In the design of multiple doses, Group B received 1 mg metolazone at 8:00 a.m. for consecutive 6 days. In days 3, 4 and 5, 4 mL of venous blood was drawn to observe minimum value of steady plasma-drug concentration before every dosing at 8:00 a.m. In day 7, the procedure was the same as that of single dose mentioned above.
Results: A simple, rapid and sensitive method was developed for the determination of metolazone in human plasma and was used to compare the pharmacokinetic parameters after single and multiple doses oral administration. Good linearity was obtained over the concentration range of 0.05-100 ng/mL with correlation coefficients r = 0.995. The intra-day and inter-day precision of the quality control (QC) samples was 3.5~8.9% and 5.7~8.5% relative standard deviation (RSD), repectively. The inter-day accuracy of the QC samples was 93.4~110.8% of the nominal values. The extraction recoveries of the assay were 72.6±7.7, 71.3±10.1 and 70.5±4.0% for the low, middle and high concentrations(0.1, 5 and 100 ng/mL), respectively. The pharmacokinetic parameters of single doses of 0.5 mg, 1 mg and 2mg metolazone were as follows: t1/2 were .6±2.8h, 7.9±1.2h and7.6±1.9h; AUC0-48 were 32.9±9.2 ng·h·ml~(-1), 122.5±36.3 ng·h·ml~(-1) and 162.4±26.9 ng·h·ml~(-1); T_(max) were 1.6±0.9h, 1.6±0.6 h and 1.5±1.3 h; C_(max) were 6.9±2.6ng·ml~(-1), 20.6±4.8 ng·ml~(-1), 36.8±7.1 ng·ml~(-1). The pharmacokinetic parameters of multiple dose of 01 mg metolazone were as follows: Cav (9.7±1.8) ng/mL; DF (2.1±0.3); AUCss (116.8±21.1) ng·h/mL; R(74.9 5.2%); T_(max) (2.4±1.4) h and C_(max)( 22.4±5.0) ng·ml~(-1). Conclusions: The t1/2, C_(max) and AUC of multiple-dose were not significantly different from those of single-dose (P>0.05). The t_(1/2) of single dose and multiple doses were (7.9±1.2) and (8.9±1.4) h, respectively; The AUC of single dose and multiple doses were (123.9±36.7 and (156.8 31.6) ng·h/mL, respectively. These findings suggested that there was no accumulation of metolazone in plasma. The recommended dose was 0.5 or 1.0 mg metolazone per day for patients.
Part 3 An improved HPLC method for quantitative determination of metolazone in human urine and its application to a pharmacokinetic study.
Aims: To establish a HPLC method to determine metolazone in human urine and study the pharmacokinetic characteristic of metolazone in Chinese healthy volunteers, and to provide references or recommendations for dosage regimen for phase II clinical trial study of metolazone.
Method: A simple, rapid and accurate liquid chromatography spectrometry method has been developed and validated for the quantitative measurement of metolazone in human urine. After a liquid-liquid extraction procedure, samples were chromatographed on an agilent TC-C_(18) (150mm×4.6mm, 5μm) column using an isocratic elution mobile phase composed of methanol and distilled water (43:57, v/v) at a flow rate of 1.0 mL/min. The column oven temperature was set at 35?C. Fluorescence detector: excitation wave was 235nm and emission wave was 410nm for metolazone, and excitation wave was 235nm and emission wave was 354nm for diazepam. Thirty healthy male and female subjects were enrolled in this study. All subjects were randomly divided into three groups, such as Groups A-C (five males and five females in each group). Groups A-C were administered a single dose of metolazone tablet 0.5 mg, 1.0 mg and 2.0 mg, respectively.Urine samples (3 mL) were collected at 0 h (pre-dose) and 0-2, 2-4, 4-6, 6-8, 8-10, 10-12,12-16, 16-24, 24-48h h post dose. The samples were stored at -80 ?C until analysis.
Results: A simple, rapid and sensitive method was developed for the determination of metolazone in human urine and was used to study the pharmacokinetic parameters of metolazone after single doses oral administration. Good linearity was obtained over the concentration range of 0.2-500 ng/mL with correlation coefficients r = 0.998. The intra-day and inter-day precision of the quality control (QC) samples was 6.9~11.7% and6.9~11.0% relative standard deviation (RSD), repectively. The inter-day accuracy of the QC samples was 100.3~108.8% of the nominal values. The extraction recoveries of the assay were 77.9~81.7%. The pharmacokinetic parameters of single doses of 0.5 mg, 1 mg and 2mg metolazone were as follows: t_(1/2) were 8.6±2.6h, 9.2±2.0h and 7.4±0.9h and The mean cumulative urinary excretion amount of 10 volunteers up to 48h was59.7±16.4%, 61.1±24.3% and 62.3±14.4% for single doses of .5mg, 1mg and 2mg metolazone.
Conclusions: The pharmacokinetic parameters including elimination half life (t1/2) and accumulative excretory rate were similar for a single dose of 0.5 mg, 1.0 mg and 2.0 mg metolazone in Chinese healthy volunteers. It was suggested that the pharmacokinetics of metolazone fitted the linear dynamic feature over the dose range of metolazone studied.
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[50]肖淑华,魏广力,刘昌孝等.美托拉宗人体药动学研究.《中国药理学会制药工业专业委员会第十二届学术会议、中国药学会应用药理专业委员会第二届学术会议、2006年国际生物医药及生物技术论坛(香港)会议论文集》2006.