Preparation,Quality Control And Pharmacokinetics In Beagle Dogs Of Captopril Delayed-onset, Sustained Release Tablet

The blood pressure of human body displays significant circadian rhythmicity over 24 hours: the blood pressure is lowest during sleep, rising to peak value rapidly in several hours after waking up in early morning. Multiple clinical researches show that risk of many cardiovascular events such as myocardial infarction, myocardial ischemia, sudden cardiac death, cardiac arrest, may be increased in the early morning. According to treating practice of high blood pressure,the best time for antihypertensive drugs to reach effective plasma level is from approximately 2:00 a.m. to 3:00 a.m..Captopril is an angiotensin-converting enzyme (ACE) inhibitor with sulfhydryl-base—a family of drugs used to treat high blood pressure and some types of heart failure. Besides ordinary captopril tablet,capsule and injection, there are sustained-release capsule with twice-daily dosing and sustained-release tablet with once-daily dosing in the market.But there is no formulation of captopril aimed at chronotherapy for hypertension. In this study,we prepared captopril delayed-onset,sustained release tablet with dry-coated technology which was taken at bedtime( 10:00 p.m. ), involving a 4-5 hours lag time in the release of captopril,established its quality controlling methods,and measured the pharmacokinetics parameters and relative bioavailability in Beagle dogs in a randomized cross-over using captopril sustained release tablet as reference preparation.1. Preparation of captopril delayed-onset,sustained release tabletObjective: To prepare captopril delayed-onset,sustained release tablet,that can start releasing after a lag time 5h. Methods: Captopril delayed-onset,sustained release tablets was prepared with dry-coated technology.Central composite design was used to optimize the formulation, artificial neural networks was used to predict the lag time of release,and multiple regression was performed using statistic analysis software (SAS).The observed values of the lag time of captopril delayed-onset,sustained release tablets prepared under the optimum condition were compared with the predicted.The out shell composition was confirmed. The composition of core tablet was optimized using captopril sustained release tablet as reference preparation and the similarity factor^) as the evaluation standard. Then the formulation of captopril dry-coated tablet was finally confirmed.Results: The lag time in vitro of captopril delayed-onset,sustained release tablet prepared under the optimum condition was 5h. The tablet had similar release behavior to reference products(f2=64.06) and the drug release pattern was accorded with the first order kinetics.Conclution: The lag time of this dry-coated tablet was only controlled by the composition of out shell. The sustained release after release starting was controlled by both out shell and core tablet. 2.Quality control of captopril delayed-onset,sustained release tabletObjective : To develop a method for controlling the quality of this pharmaceuticaUncluding assay of captopriK related substances detection and its release in vitro from this dosage form.Methods : The quality of captopril delayed-onset,sustained release tablet was controlled by high performance liquid chromatographic method on a Diamonsil?C|g( 150x4.6mm, 5um)column detected at 220nm with the column temperature 40°C,and the injected volume 20uL The mobile phase was methanol-water-phosphoric acid(35:65:0.03) at a flow rate of l.Oml/min for assay and release of tablet prepared and methanol-water-phosphoric acid(45:55:0.025) at a flow rate of 1.5ml/min for detection of related substances. The release rates of captopril were determined according to the rotating basket method using 750 ml of dissolution medium consisting of 0.1 mol/L hydrochloric acid. The release experiments were carried out at 37±0.5°C at a rotation speed of 100rpm.Results: The specialization of HPLC method developed was excellent. In the determination of captopril, the calibration curve for captopril assay exhibited excellent linear relationship with a correlation coefficient 0.9994 in the concentration range of 26.25-48.75^g/mL,the average recovery of assay was 101.1 %,the intra-day precision(RSD) was 1.0% and the inter-day precision(RSD) was 1.1%. Assay results of 3 batches of sample (030815-K 030902, 030910) were 101.3%,99.96% and 99.26% respectively.The calibration curve for determination of captopril releasing in vitro from delayed-onset,sustained release tablet exhibited excellent linear relationship with a correlation coefficient 0.9995 in the concentration range of 2.5~50Mg/mL,the average recovery of assay was 99.91%,the intra-day precision(RSD) was 0.49% and the inter-day precision(RSD) was 0.65%. The accumulative release percents at 5h of 3 batches of sample (030815-K 030902> 030910) were all about 10%,and the tablet had similar release behavior to ordinary sustained-release tablet after starting release.The calibration curve for captopril disulfide assay exhibited excellent linear relationship with a correlation coefficient 0.9998 in the concentration range of 2.25-13.5Mg/mL,the average recovery of method was 100.9%,the intra-day precision(RSD) was 0.83% and the inter-day precision(RSD) was 1.6%. The limit of detection was 30ng/ml(S/N^3),and the limit of quantitation was 100ng/ml(S/N^10) (RSD=2.2%,n=5). Related substance assay results of 3 batches of sample (030815-K 030902, 030910) were 2.56%,2.53% and 2.53% respectively.Conclution: The methods developed were special > accurate and reproductive,and can be used to control the quality of the tablet tested.3.Development of method for determination of captopril in Beagle dog plasma by pre-column derivation HPLC with solid phase extractionObjective: To develop a method for determination of captopril in Beagle dog plasma.Methods: Captopril in plasma was derivatized with p-bromophenacyl bromide,then the additional products passing through solid phase extraction(SPE) with Waters OASIS? HLB were determined by HPLC.Separation was achieved on a Diamonsil?Ci8 (200x4.6mm, 5um) column and detected at 258nm.The mobile phase consisted of acetonitrile and water(0.25%acetate) (v/v 40:60) at a flow rate of 1ml/min.Results: Under the separation condions,the additional product of captopril derivatized with p-bromophenacyl bromide was separated completely from plasma with resolution>2.0. There were no interferences appeared at the peak postion(tRl6min) of captopril derivation.The captopril linear range was from 10 ng/ml to 500ng/ml,r=0.9999,the limit of detection was 5ng/ml(S/N 2? 3),the limit of quantitation was 10ng/ml(S/N^10) (RSD=7.3%.n=5), and the sensitivity can meet the determination of 1/25 of Cmax. The method recovery was 95.04%~99.59%(n=15),within-day and between-day precisions(RSD) were 1.6%~3.1%(n=15) and 0.70%~2.8%(n=15) respectively.Conclution: We established a method for determination of captopril in Beagle dog plasma. The method is sensitive,rapid and accurate, and is suitable for the pharmacokinetics study. 4.Pharmacokinetics and bioequivalence study of captopril delayed-onset,sustained release tablet in Beagle dogsObjective: To study the pharmacokinetics and relative bioavailability of captopril delayed-onset,sustained release tablet in Beagle dogs. Methods: A single oral dose of tested captopril preparation and referenced captopril preparation were given to 10 Beagle dogs in a randomized cross-over.The plasma concentration of captopril was determined by the developed HPLC method.The pharmacokinetics parameters and relative bioavailability were measured.Results : The main pharmacokinetic parameters of captopril were as follow: AUCo-t were 885.9 + 353.4and 874.9 ± 382.7nghmL'1;AUCo- - were 8882 ± 368.4 and 877.0 ± 392.4nghmL'1;Cmax were 232.5±90.19 and 309.4±1963ngmL'';tmax were 2.5+1.72 and 3.5±1.08h;t m were 2.68 ± 0.67 and 2.26 ± 0.56h for reference and test preparations,respectively.The relative bioavailability of test preparation were Fo-,: 101.5%±32.2% and F0.~:101.5%±33.35%.Conclution: The 90% confidence interval of the ratio of AUCo-t was 82.0%~116.3%,and met the requirement of the Chinese Pharmacopoeia(80%~125%). The 90% confidence interval of the ratio of Cmax was 90.65%~162.1%,and was outside the range of 70%~143% for requirement of the Chinese Pharmacopoeia. Above all, it showed that only the upper limit of Cmax exceeded the value prescribed in the Chinese Pharmacopoeia. Because what theChinese Pharmacopoeia prescribed is only fit for human beings,and animals are of course different from human beings,we conclued that the two tablets were bioequivalence. Compared the captopril plasma concentration-time curves of two preparations,we can see that drug in plasma couldn't be detected until 2h after the the test preparation oral administered,and the plasma concentration was maximal at 3~5h. But drug in plasma couldn't be detected only until 0.5h after the the reference preparation oral administered,and the plasma concentration was maximal at 0.75~3h. We can conclude from the results that the test preparation exhibited delayed-release obviously in Beagle dogs and the lag time was about 2h.