Design And Evaluation Of Traditional Chinese Medicine Compound Recipe Lianyu Intragastric Floating Osmotic Pump Controlled Release Formulations

Zuojin (ZJ) prescription is a classic prescription with Coptis and Evodia (6:1), the pills and capsules was collected in the2010edition of "Chinese Pharmacopoeia". Pharmacological effects of ZJ prescription are as follows: theinhibition of gastric acid secretion, anti-ulcer, antimicrobial, analgesic and anti-inflammatory. ZJ preseription is used clinically to treat stomach inflammation and ulcer disease.The dynamics of bio-heat, anti-Helicobacter pylori method on the active ingredients of ZJ prescription screening studies were used to prove that the main active substance is Coptis and Evodia alkaloids. The existing pharmacokinetic studies have shown that such ingredients in vivo absorption is poor. Alkaloids only to maintain a certain concentration can play a therapeutic role in the local lesion. The ordinary preparations of ZJ prescription are pills and capsules. Because these agents rapidly into the intestine after oral administration with gastric emptying, so the effective concentration can not be maintained in the stomach. This treatment effect is not ideal.The researcher developed Lianyu intragastric floating osmotic pump controlled release formulations (LIFOPT). This preparation can significantly reduce the dosage of patients, remain in the gastric juice long time, and has a zero-order release characteristics. It enables the effective parts of Coptis and Evodia continuously and quantitatively release in the stomach, and can effectively avoid irritating due to the sudden release of the drug. The active ingredient was directly released to the lesion, so the efficacy and patient compliance can be significantlyimproved. This treatment can successfully cure stomach inflammation and ulcer disease. The traditional framework gastric floating sustained-release formulations has a direct relationship between drug release and solubility. For multi-component drugs, the various componentsis difficult to synchronize the release because of their different solubility. The LIFOPT contains promoting layer and drug layer. Promoting layer contains light porous microspheres, the tablets float in the gastric juice because apparent density of tablets less than water. Ointment-like drug suspension was formed due to water infiltrating into the drug layer, promoting layer absorbed water and expanded, thedrug suspension was introduced from the release hole. The release style can guarantee that each component is synchronously released, The release curve has a zero-order release characteristics.PART1The extraction and purification of Coptis and Evodia Effective partsObjective:To separate and purify the Coptis total alkaloids with macroporous adsorption resin technology and determine the best technology, purify Evodia alkaloids and limonoids with neutral alumina column chromatography and optimize the processes.Methods:Coptis herbs1kg was crushed into coarse powder and reflux extracted3times(each for2h) with12times volume of water. The combined extractand was concentrated to8L and controlled to0.10to0.15g/mL of crude drug mass concentration. AB-8macroporous resin was used to separate alkaloids. The purification process:the impurities were eluted with3BV purified water, alkaloids were eluted with3BV50%ethanol, the extract were derived through vacuum drying after recovery of ethanol.The contents of Coptis alkaloids were determined by HPLC. The separation was carried out on a C18column with a mixture of acetonitrile-0.05mol/L potassium dihydrogen phosphate solution (50:50) as a mobile phase at the flow rate of1.0mL/min and detection wave length was set at345nm.. Evodia herbs was extracted3times(each for2h) with8times volume of70%ethanol. The combined extractand was filtered and the filtrate was evaporated to alcohol-free taste for recycling ethanol. The Concentrate was added24times volume of water and adjusted pH to3with hydrochloric acid. The precipitation was derived thro μ gh4500r/min centrifμgation after Stationary for2h and redissolved with24times volume of95%ethanol. The solution was added into neutral alumina with little methanol after decompression recycling ethanol. The mixture was stired and packed into chromatography column after methanol was evaporated at60℃. The purification process:the effective parts were eluted with6BV ethyl acetate-dichloromethane(70:30), the extract were derived throμgh vacuum drying after recovery of solvent.The contents of Evodia effective parts were determined by HPLC. The separation was carried out on a C18column with a mixture of acetonitrile-water-tetrahydrofuran-acetic acid (41:59:1:0.2) as a mobile phase at the flow rate of1.0mL/min and detection wave length was set at225nm.Results:For the method of Coptis alkaloids determination, there were good linear relationships for coptisine and palmatine within the range of5～100μg/mL and berberine within the range of10.07～201.4μ g/mL. The average recoveries were9947%(RSD=1.29%)、99.73%(RSD=1.16%)、99.41%(RSD=0.99%)The Coptis alkaloids transfer rate were91.72%, the average content of total alkaloids in the extract were81.87%, the average concentration of berberine, palmatine, coptisine, epiberberine was48.54%,13.25%,12.83%,7.26%.For the method of Evodia effective ingredients determination, there were good linear relationships for evodiamine and rutacarpine within the range of10～200μ g/mL and limonin within the range of25.27-505.4μ g/mL. The average recoveries were99.64%(RSD=1.46%)、98.90%(RSD=1.55%).99.00%(RSD=1.10%)The average transfer rate of effective parts of the Evodia was72.04%, the average content of effective parts in the extract were56.45%, the average concentration of evodiamine,rutaecarpine, limonin was8.73%,17.83%,30.18%.Conclusions:The average content of effective parts of the prepared extracts of Coptis and Evodia were more r than50%in line with the requirements of the effective parts.Part2The pharmacodynamics comparison between the refined intermediate and Zuojin pillsObjective:To verify the rationality of the extraction process with the comparison of effect against the experimental gastric ulcer in rats and the gastric acid secretion between refining intermediates and Zuojin pills.Methods:The impact of different dosages of refined intermediate and Zuojin pills on indomethacin gastric ulcer was investigated, as well as ethanol gastric ulcer, the impact of the high dosage group refined intermediate and Zuojin pills on gastric secretion of normal rats was studied.Results:The influence on indomethacin gastric ulcer was as follows:the number of ulcers of the refined intermediate0.096,0.135g/kg groups and Zuojin pills1.05g/kg group had significant difference compared with control group. The influence on ethanol gastric ulcer show that the number of ulcers of the refined intermediate0.135g/kg groups and Zuojin pills1.05g/kg group were significantly less than control group. The influence on gastric secretion of normal rats show that the refined intermediate group and Zuojin pills group can significantly reduce the secretion of gastric juice and gastric acid secretion.Conclusions:After major pharmacodynamic comparison, the efficacy of refining intermediates and Zuojin pills had no significant differences, indicating that the extraction and purification process were reasonable.Part3The preparation of Lianyu intragastric floating osmotic pump controlled release tabletsObjective:To prepare Lianyu intragastric floating osmotic pump controlled release tablets with osmotic pump formulations technology.Methods:The method to prepare LIFOPT:(1) the preparation of light and porous micro spheres:polyacrylate resin RS was dissolved in appropriate amount of ethanol-dichloromethane; mix the solution obtained with0.3%dodecyl sulfate aqueous solution; stir at50-100r/min until the oil droplet diameter increase to0.3-0.8mm; heat the mixture to40℃slowly and solidify for4hours; filter the mixture and pack the microspheres into column, wash with10BV hot water, the light and porous microspheres were derived after drying.(2) the preparation of promoing layer:mix light and porous microspheres, PEO, HPMC, sodium chloride, PVPk30according the prescription; prepare wet mixture with ethanol, prepare granules throμ gh20mesh sieve, dry for12hour at40℃, add magnesium stearate into the granules.(3) the preparation of drug layer: mix the refined intermediate and PEO according the prescription; prepare wet mixture with ethanol, prepare granules through20mesh sieve, dry for12hour at40℃, add magnesium stearate into the granules.4) Pressing tablets and coating:bilayer tablet core was prepared with the above promoing layer and drug layer granules; coat tablets core with the acetone solution of cellulose acetate; an orifice of0.8mm diameter was drilled by micro drilling machine on surface of the drug layer.The main investigated factors were as follows:the influence of different osmotic promoting agent and dosage in promoing layer and drug layer on drug release, the influence of different amount of PEG400in coating solution, the diameter of release hole and hardness of tablet core on drug release, the influence of the suspending agent on flow propertyof suspension including API, the influence of the swelling agent on swelling rate, the elastic modulus and fracture strength of acetyl cellulose film with defferent thickness, the influence of the porous agent on water permeation rate of CA membrane.On the basis of single factor investigation, the orthogonal experiment was designed to optimize formula in which the amount of osmotic agents, swelling agent, PEG400were taken as three influential factors and three different levels were selected to part, each of them was selected refer to the orthogonal design table. The accumulative release percentage of LIFOPT in different osmotic media was determined to verify the influence of the medium osmotic pressure on drug release.Results:According to the results of single factor investigation, the osmotic promoting agent in drug layer was sodium chloride20mg; suspending agents in drug layer was PEO(Mw200,000)(the refined intermediate:PEO=4:1); swelling agent was PEO:HPMC=4:1; the osmotic promoting agent in promoting layer was sodium chloride; the thickness of coating film was0.18-0.20mm. According to the results of orthogonal test, the amount of osmotic agents was20mg; swelling agent was50mg; PEG400in coating solution was10%. The drug release rate decreased significantly in0.1mol/L hydrochloric acid solution (including2mol/L sodium chloride). The phenomenon showed that the driving force of drug release was the osmotic pressure diffefence between the release media and internal preparation.Conclusions:The accumulative ralease percentage within12hours of the osmotic pump tablets prepared according to optimal prescription and process is more than90%, the release curse was in line with the zero-order kinetics equation to meet the design requirements.Part4Studies on dissolution test in vitro of Lianyu intragastric floating osmotic pump controlled release tabletsObjective:To establish an HPLC method for simultaneous determination of berberine, palmatine, evodiamine and rutaecarpine in release medium, investigate the release character of the formulation and set up dissolution test standards. Methods:The contents of berberine, palmatine, evodiamine and rutaecarpine in release medium were determined by HPLC. The separation was carried out on a C18column with a mixture of acetonitrile-0.025mol/L potassium dihydrogen phosphate solution (including20mmol/L sodium dodecyl sulfate, adjust pH to3.5with phosphate)(55:45) as a mobile phase at the flow rate of1.0mL/min and detection wave length was set at225nm.Dissolution test:Medium:0.1mol/L hydrochloric acid solution1000ml; apparatus2:100rpm; times:2、4、6、8、10and12hours; test solution:pass a portion of the solution under test throμgh a suitable0.45-μm filter. The zero-order kinetic equation was taken to fit the release data, the similarity of release curve of the four components were evaluated acording to f2factor.Results:For the method of determination of berberine, palmatine, evodiamine and rutaecarpine in release medium, there were good linear relationships for berberine within the range of500.8-25.04μg/ml, palmatine within the range of1.914-47.85μg/ml, evodiamine and rutacarpine within the range of0.1023-2.046μg/ml. The average recoveries were98.84%(RSD=1.51%),99.17%(RSD=1.12%),97.94%(RSD=1.34%),99.46%(RSD=1.05%). The zero-order kinetic equation was taken to fit the release data in water,0.1mol/L hydrochloric acid solution and phosphate buffer (pH6.8), the r values of regression equation were greater than0.98. The comparison results of release curve of each component showed that the f2factor were greater than50.Conclusions:The HPLC method wasestablished for simultaneous determination of berberine, palmatine, evodiamine and rutaecarpine in release medium. By the method validation, precision, accuracy, and stability meet the requirements of pharmacopoeia. In various release media the release curves of the preparation are in line with the zero-order kinetics equation, the release curve of different composition were similar, indicating that the product can achieve synchronous release of each component at a constant speed. Part5Studies on pharmacokinetics in beagle dogs of Lianyu intragastric floating osmotic pump controlled release tabletsObjective:To establish an HPLC method for simultaneous determination of berberine and palmatine in plasma of beagle dogs, study the pharmacokinetics in beagle dogs of Lianyu intragastric floating osmotic pump controlled release tablets.Methods:Beagle dogs were randomly divided into twogroups, two-period crossover trial were employed to arrange experiment, the test preparation(LIFOPT) and the reference preparation(normal tablets) were taken respectively, the dosage was300mg, crossover study was carried out after two weeks. Sampling time:before administration and1、2、3、4、6、8、10、12、24、36、48、72、96、120hours after administration. Plasma processing: take hindlimb blood5ml, and immediately transfer into heparinized tube, centrif μ ge at10000r/min for10min, separate plasma and store at-20℃The contents of berberine and palmatine in plasma were determined by HPLC. The separation was carried out on a C18column with a mixture of0.1%formic acid and0.1%formic acid in acetonitrile(gradient elution) as a mobile phase at the flow rate of1.0mL/min and detection wave length was set at345nm.. The pharmacokinetic parameters were calculated by DAS software.Results:For the method of determination of berberine and palmatine in plasma, there were good linear relationships for berberine within the range of0.1-5.0μ g/ml, palmatine within the range of0.01-0.5μ g/ml, within-day and between-day RSD were less than15%, the recoveries were84.16%-96.30%and81.05%～93.29%. The main pharmacokinetics parameters were respectively as follows:berberine:tmax (3.890±1.524) and (6.761±2.496) h;cmax (1.4073±0.3804) and (0.8773±0.2395)μ g/ml; t1/2(14.26+4.85) h and (18.33±6.39) h; AUC0～96(35.94±8.86) μg·ml-1·h and (39.27±7.94) μ g·ml-1·h. The relative bioavailability was109.3±22.1%. Palmatine:tmax(4.191±1.527and (8.835±2.906)h; cmax (0.2515±0.0719) and (0.1773±0.0525) μ g/ml; t1/2(11.47±5.63) h and (16.64±5.34) h; AUCo-96(7.48±2.61) μg·ml-1·h and (8.54±2.39) μ g·ml-1·h. The relative bioavailability was114.2±31.9%.Conclusions:The method of determination of berberine and palmatine in plasma had good sensitivity, precision and accuracy, and the sample concentration was within the linear range. On the basis of AUC0～96of berberine and palmatine, the bioavailability had no significant difference between test formulation and reference formulation, tmax significantly extended and Cmax significantly reduced. It could be concluded that the LIFOPT and the normal tablets were bioequivalent, LIFOPT had good effect of sustained-release in vivo.