The Synthesis And Pharmacokinetic Study Of Metolazone

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 3.5⁸.9% and 5.7⁸.5% relative standard deviation （RSD）, repectively. The inter-day accuracy of the QC samples was 93.4¹10.8% 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-C₁₈ （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 6.9¹1.7% and6.9¹1.0% relative standard deviation （RSD）, repectively. The inter-day accuracy of the QC samples was 100.3¹08.8% of the nominal values. The extraction recoveries of the assay were 77.9⁸1.7%. 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.