Berberine Hydrochloride Microemulsion For Enhanced Oral Bioavailability:Preparation And In Vitro And In Vivo Evaluation

OBJECTIVESThe study was aim to prepare an O/W microemulsion system of Berberine Hydrochloride (BBR ME),and establish the quality evaluating system of it in vitro; and the pharmacokinetics of BBR ME was studied in rats; transport of BBR ME through the Caco-2cell monolayer as well as the effect on it by P-glycoprotein (P-gp) were studied, to provide new thought for study on the dosage forms of BBR, which was the P-gp substrate with poor solubility and membrane permeability; lay the theoretical foundation for mechanism of absorption enhancement of microemulsion.METHODS1. Preformulation studyDeterminated the dissolubility of BBR in different oils, surfactants and cosurfactants and the apparent oil-water partition coefficient of BBR in n-octanol-water and n-octanol-buffer system with serial pH value.2. Preparation of oil-in-water BBR ME for oral deliveryChose Isopropyl myristate, Caster oil, Ethyl oleate, Labrafac Lipophile wl1349, Labrafil M1944CS as oils, Tween80, Tween20, Labrasol, Glycerol trioleate, Polyoxyethylenated castor oil-35, Ethoxylated hydrogenated castor oil-40as surfactants, PEG-400,1,2-propanediol, Glycerin,1,3-butanediol, DehydratedAlcohol as cosurfactants, to work on compatibility research. Then chose the excipients with high solubility of BBR to study the formulation of it. Draw pseudo-ternary phase diagrams, then screen the broad microemulsion area adjuvant system. Screen the final BBR ME prescription with centrifugal and short-term (2weeks) laid stability test, comparison test of average droplet size、polydipersity index (PDI) and the drug-loading quantity.3. Quality assessment of BBR ME IN VITROEstablish HPLC method to assay the content of BBR in the orally BBR ME; assess BBR ME’s appearance and morphology with visual method and inverted microscope; identify the type of BBR blank ME with centrifugation method, dilution method and conductimetric method; assess BBR ME and blank ME’s average droplet size and particle size distribution with NANO-ZS90nano-granulometer; measure BBR ME and blank ME’s viscosity and conductivity with NDJ-7type rotation viscometer and DDS-11A type conductivitymeter respectively; determine BBRME’s stability with long-term (6months) laid stability test.4. Initial Pharmacokinetics study of BBR MEMale Sprague-Dawley rats were equally divided into2groups by randomized design, and orally administrated BBR suspension and BBR ME, respectively. After oral administration medicine, blood samples (～0.5mL) were withdrawn from the fossa orbitalis of rats at predetermined time intervals. BBR in rat plasma was determined by HPLC, and the data were analyzed by DAS2.1.1software, to obtain pharmacokinetic parameters.5. Transport study through Caco-2cell monolayer of BBR MEHPLC method for determination of BBR in transport solution was developed and validated.In order to investigate the two-way transmembrane transport, Caco-2cell monolayer model was established to compare BBR ME with BBR solution in the same concentration, study the effect of P-gp inhibitor Verapamil on the transport of BBR ME through Caco-2cell monolayer. The amount of transportation of BBR was analysed with HPLC, and the permeability coefficient (Papp) and excretion ratio (ER) were abtained. RESULTS1. Preformulation studyThe solution of BBR in caster oil and Labrafil M1944CS as oils were0.2850mg·mL-1、0.1573mg·mL-1, respectively. BBR dissolubility was larger in surfactants and cosurfactants.The apparent oil-water partition coefficient of berberine hydrochloride in n-octanol-water system was0.084(lgPapp=-1.08) in water at37.0℃. When the pH value is equal to1.0,2.0,11.0,12.0, the apparent oil-water partition coefficients were1.74,1.22,1.91,15.17, resfpectively; when the pH values of the buffer solution were from3.0to10.0, the apparent oil-water partition coefficients were all very low (lgPapp<0) with little change.2. Formulation of oil-in-water BBR METhe blank ME was screened with the effects of influence factor on pseudo-ternary phase diagrams test, and the final blank ME prescription was Labrafil M1944CS/Cremophor RH40/Glycerin/Aqueous phase=0.50:3.39:1.13:13.50(m/m/m/m).3. Quality assessment of orally BBR ME in vitroOrally BBR blank ME is clear and transparent and uniform golden O/W type microemulsion, contains2.32mg·mL-1BBR, with18.98nm droplet size and0.106PDI, whose viscosity and conductivity is mPa-s and177.4μs·cm-1respectively. Long-term laid stability test showed the appearance, drug-loading quantity, droplet size and particle size distribution were stable.4. Initial Pharmacokinetics study of BBR MEAfter oral administration BBR suspension and BBR ME, the pharmacokinetic parameters as follows, Cmax respectively were (0.259±0.179) mg·L-1、(0.404±0.13) mg·L-1; AUC0-24respectively were (1.681±0.558) mg-L-1·h、(4.963±2.952) mg·L-1·h; AUC0-∞respectively were (2.223±0.810) mg-L·1·h、(9.790±8.410) mg·L-1·h;T1/2z respectively were (9.761±6.611)h、(10.737±7.027) h.5. Transport study through Caco-2cell monolayer of BBR MECaco-2cell monolayer transport results showed that, compared with the BBR group, the permeability coefficient of BBR ME group was much more larger in the direction A→B; the transport of three group of BBR sample solution from direction B→A was greater than the transport from A→B direction; compared with the BBR group, BBR ME group was significantly decreased efflux; BBR ME+Ver group significantly decreased efflux, compared to BBR ME group.CONCLUSIONS1. The apparent oil-water partition coefficients of Berberine Hydrochloride in water and phosphate buffer with pH1.0-8.0was both less than1, indicating that Berberine Hydrochloride can’t be absorbed in the gastrointestinal tract easily. Additionally, apparent oil-water partition coefficient is influenced by pH value to some extent.2. The final blank ME prescription for BBR was Labrafil M1944CS/Cremophor RH-40/Glycerin/Aqueous phase=0.50:3.39:1.13:13.50(m/m/m/m).3. Short-term (2weeks) and Long-term (6months) laid stability test showed the appearance, drug-loading quantity, droplet size and particle size distribution were in good situation.4. Pharmacokinetics study showed that compared with the group administrated BBR suspension, the oral bioavailability of the group administrated BBR ME was significantly increased, relative bioavailability was440.40%. The results showed that BBR ME could improve the absorption of BBR in vivo.5. It could be concluded that BBR’s membrane permeability could be greatly improved by the O/W ME, which may be the mechanism of bioavailability improvement of BBR ME in vivo; the main mechanism of BBR ME in intestinal absorption could be passive transport from the direction of A→B; and could be active transport from the direction of B→A, involved by P-gp.