| Chiral drugs, to which scientists pay much attention, are always the focus of the new drugs'discovery. They are both popular subject and challenge to the new drugs'research and development. As the biological macromolecules in the biosystem are chiral, they have ability of recognization of enantiomers of chiral drugs. After chiral drugs enter the organisms, they are recognized differently which will bring about different biological responses and display different pharmacokinetics and pharmacodynamics.Doxazosin is a new drug developed by Pfizer Company, and went to the market in 1988. This drug is long acting, elective antagonist of alpha-1 adrenoceptor, its therapeutic effect for begin prostatic hyperplasia (BPH) has been proved, and obtained generally application in clinical. However, owing to its side effect in cardiovascular system, the dosage of doxazosin is limited to increase for get maximum therapeutic efficacy. Therefore, it is very urgent and important to develop higher elective antagonist of alpha-1 adrenoceptor.Doxazosin is a quinazoline compound, now it has been used as a racemic in clinical. The latest finding by North China Pharmaceutical Group Corporation (NCPC) has make it clear that (-)-doxazosin have high stereoselectivity in cardiovascular system, its side effect is weaker than (+)-doxazosin and (±)-doxazosin obviously. (-)-Doxazosin is showed its superiority to (+)-doxazosin and (±)-doxazosin in treating BPH. By chiral separation, a single enantiomer was obtained. Levodoxazosin mesylate is hope to become a new drug for BPH, which have patent right in the world.Objective: To study on the quality control of Levodoxazosin mesylate, provide data for quality criteria; to establish methods for determination concentration of Levodoxazosin mesylate in biological medium, and to Study the pharmacokinetics of Levodoxazosin mesylate after a single oral dose.Method: 1. Quality control study of Levodoxazosin mesylate: (1) Physical properties:①Solubility: The test was carried out according to the general standard of China Pharmacopoeia 2005 edition Part Two. Several ordinary organic solvents were used for the solubility.②Melting point: According to the appendixⅣA of China Pharmacopoeia 2005 edition part two, the melting point was determinated.③Optical rotation: The optical rotation was determined according to the appendixⅥE of China Pharmacopoeia 2005 edition part two. (2) Identification:①Chemical method: The sample (50mg) and NaOH (0.2g) were dissolved by water. The solution was evaporated to dryness. Then heated until to melt, and continued heating for a few minutes. The reactant was cooled. Water and excess hydrochloric acid were added. The phenomenon was observed.②Infrared method: According to the appendixⅣC of China Pharmacopoeia 2005 edition part two, the sample was tableted with bromatum kalium. The spectrum was obtained and compared with the spectrum of reference substance. (3) Purity test:①General test: According to the appendix of China Pharmacopoeia 2005 edition part two, the water content, loss on drying, residue on ignition and heavy metals were tested.②Related substances test: The related substances were determined by HPLC, and the new method was validated.③Optical purity test: The optical purity of samples was tested by high performance capillary electrophoresis (HPCE), and the new method was validated. (4) Assay method: The content of Levodoxazosin mesylate was determined by HPLC, and the method was investigated.2. Pharmacokinetics study of Levodoxazosin mesylate: (1) The method of Levodoxazosin mesylate's determination:①Chiral separation and detection: The separation was performed on a chiral chromatographic column (ChiralcelOD-R, 4.6mm×250mm, 10μm) with acetonitrile- 0.5mol·L-1 sodium perchlorate aqueous solution (45:55) as the mobile phase, the flow rate was 0.5ml·min-1. Fluorescence detector was used with 270nm as excitation wavelength and 385nm as emission wavelength.②Non-chiral separation and detection: The separation was performed on a ODS column, and the new method was validated. (2) Study on the conversion of enantiomers after a single oral dose of Levodoxazosin mesylate: Select high dosage of 4.0mg·kg-1, the blood samples were collected at 0.5h, 1.0h, 4.0h, the concentration of (-)-doxazosin and (+)-doxazsin were determined by chiral separation method. (3) Pharmacokinetics study of Levodoxazosin mesylate: The oral dosages were high (4mg·kg-1), middle (1mg·kg-1) and low (0.25mg·kg-1). The blood samples were collected at the 0.5h, 1h, 1.5h, 2h, 3h, 6h, 8h and 24h, the concentration of plasmas were determined by HPLC method. (4) The distribution of the drug: Six Wistar male rats were used in this experiment. The dosage was 6mg·kg-1. The male rats were executed respectively at 30min, 120min, 240min. The sample of hearts, livers, spleens, lungs, kidneys, brains, testes, stomachs, small intestines, muscles and fat were collected. The concentrations of the drug in tissues were determined by HPLC. (5) The excretion of the drug: Six Wistar rats were used in this experiment. The dosage was 6mg·kg-1. The rats were put in the metabolism cages. The urines were collected in the range of 0-5h, 5-8h, 8-12h, 12-24h. The feces were collected in the range of 0-12h, 12-24h. Another Six Wistar rats were used in the experiment. The biles were collected in the range of 0-6h, 6-12h, 12-24h. The concentrations of samples were determined. (6) The plasma protein binding of Levodoxazosin mesylate: The equilibrium dialysis method was used. Levodoxazosin mesylate's contents in and out of the bag were determined, and the plasma protein binding rate was calculated. Results: 1. Quality control study of Levodoxazosin mesylate: (1) Physical properties:①Solubility: Levodoxazosin mesylate was sparingly soluble in dimethyl sulfoxide, slightly soluble in methanol, very slightly soluble in water, and insoluble in acetone and chloroform.②Melting point: Levodoxazosin mesylate was melted at 251℃or so, and at the same time it was decomposed.③Optical rotation: The results of optical rotation of samples were all between -67°and -70°. (2) Identification:①Chemical method: The phenomenon is there was SO2 produced.②Infrared method: The infrared spectrum of Levodoxazosin mesylate was corresponded to that of the reference substance. (3) Purity test:①General impurity test: the water content was not more than 1.0%, the ignited residue was less than 0.2%, heavy metals was less than 20ppm, and pH was in the range of 2.54.5.②Related substances: The new HPLC method was established. The separation was performed on a C18 column with water-acetonitrile-glacial acetic acid-triethylamine as the mobile phase (500:200:15:1), the flow rate was 1.0 ml·min-1, the detection was set at 254nm and the column temperature was maintained at 30℃. The limit of detection was 0.16ng.③Optical purity test: The separation HPCE condition was as follow: The phosphate buffer (50mmol·L-1, pH was adjusted to 2.4 by H3PO4) was used as background electrolyte (BGE), 5mmol·L-1 carboxymethyl-β-cyclodextrin (CM-β-CD) was used as chiral resolution agent. The detection wavelength was 254nm. Voltage was 30kV. The limit of detection was 1.0μg·ml-1. (2)Assay: The calibration curve was linear in the concentration range of 0.01-0.4mg·ml-1: y=8.0×107x+285357, r=0.9999. The precision and repeatability were both fine, and the recovery was high. The content of samples were all more than 98.5%.2. Pharmacokinetics study of Levodoxazosin mesylate: (1) The two methods for determination concentration of Levodoxazosin mesylate in biological medium were established. A good linearity was obtained from 2 to 200ng·ml-1 in plasma, brain, kidney, spleen, lung, adipose tissue, muscles and testes, from 2 to 250ng·ml-1 in bile, from 1 to 300ng·ml-1 in urine, from 2 to 500ng·ml-1 in feces. (2) The conversion ratio of enantiomers after a single oral dose of Levodoxazosin mesylate in dogs was less than 5.0%, so the conversion of Levodoxazosin mesylate can be ignored. (3) The pharmacokinetics parameters of Levodoxazosin mesylate were calculated by DAS2.0 pharmacokinetics software, Levodoxazosin mesylate was rapidly absorbed after a single oral dose. The Tmax value was about 1.7hours, the Cmax values of low, middle, and high dosage were 12.44ng·ml-1, 31.05ng·ml-1 and 122.09ng·ml-1 respectively. The plasma time-concentration curve was fitted to two-compartment model. (4) Levodoxazosin mesylate was distributed in all organs after a single dosage of 6mg·kg-1, mainly in stomach and small intestine, least in brain and testes least, the others were in between. (5) At a single dose of 6mg·kg-1, the excretion rates of urine, bile, and feces were 0.02%, 0.03% and 0.3% respectively. (6) The plasma protein binding rate of Levodoxazosin mesylate was high.Conclusion: In this research, the method of identification, purity test and assay for Levodoxazosin mesylate were established. The new methods can be used in quality control; the determination methods of Levodoxazosin mesylate in biological medium were established, and the new methods were in line with the technical requirement of new drugs'preclinical pharmacokinetics research. The study on pharmacokinetics of Levodoxazosin mesylate offered important datas for the clinical and toxicological research of Levodoxazosin mesylate.
|
PDF Full Text Request