Study On The Mechanism Of Oral Absorption Of Phospholipid Complex And Its Microemulsion System

Many active compounds can form complexes with phospholipid at a certain ratio with significant changes of the physical and chemical properties, as well as the biological properties. Self-microemulsifying drug delivery system (SMEDDS) consists of a mixture of drug, oil, surfactant and cosurfactant, and the gentle mixing of these ingredients in aqueous media can generate oil-in-water (O/W) microemulsion droplets of solubilized drugs with a mean droplet size<100nm. For SMEDDS based phospholipid complex (PC), PC was formulated to significantly significantly improve the solubility of the drug and drastically facilitate the incorporation of PC into SMEDDS; at the same time, SMEDDS can improve the dispersion and stability of PC. Increased bioavailability of the phospholipid complex over the simpler, non-complexed drug has been demonstrated by pharmacokinetics and activity studies conducted in animals as well as human beings. However, the absorption mechanism of PC and lipid formulation such as SMEDDS based on PC through small intestine remains to be clarified. The main aim of this present work was to study permeability and absorption mechanism using gentiopicroside (GTP, BCS Ⅲ) and silybin (SIB, BCS Ⅱ) as model drugs.1Preparation and evaluation ofphospholipid complex and its SMEDDSThe preparation of silybin-phospholipid complex (SI8-PC) was optimized by investigating the factors affecting the formation of SIB-PC with complexation efficiency and dissolution. SIB and phospholipid were placed in a round-bottom flask and suspended in anhydrous ethanol at a ratio of1:1, and the concentration of SIB was10mg/mL. The mixture was treated with stirring at40℃until the solution becomes clear. After ethanol was evaporated off under vacuum at40℃, the dried residues were gathered as SIB-PC. The complexation efficiency was close to100%. The complex was shown to be amorphous and completely different from simple mixture measured by DSC and XRD analysis. Moreover, SIB-PC could increase the dissolution of SIB. Formulation and preparation of SIB-PC SMEDDS was studied according to the result of SIB-PC solubility in the different oil, surfactant and co-surfactant and pseudo-ternary phase diagram of the formulation. The composition of the best formulation was Castor oil=27.9%, Labrasol=10.2%, Cremophor RH40=40.9%, Transcutol HP=14.0%and SIB-PC=7%. The mixture was stirred at37℃until the solution becomes clear. The particle size of SIB-PC-SMEDDS in water was (92.0±5.3) nm with Gaussian distribution. The zeta potential was (-14.02±7.12) mV. After dilution by distilled water, there was little change of the particle size of SIB-PC-SMEDDS within8h.Gentiopicroside-phospholipid complex (GTP-PC) and GTP-PC self microemulsion drug delivery system (GTP-PC-SMEDDS) were prepared based on our previous study. GTP and phospholipid were placed in a round-bottom flask and suspended in tetrahydrofuran at a molar ratio of1:1. The mixture was treated with stirring at40℃r4h. After tetrahydrofuran was evaporated off under vacuum at40℃, the dried residues were gathered as GTP-phospholipid complex. GTP-PC-SMEDDS was prepared with Maisine35-1: Miglycol(1:2)=30%, Labrasol:Cremorphor EL(1:4)=40%, Transcutol HP=30%as the blank formulation, added with GTP-PC with1:10(g/g), stirred at37℃for Id and placed for2d2Dynamic in Vitro LipolysisA dynamic in vitro digestion model was established. The extent of lipolysis was defined as the percentage of triglycerides digested in vitro lipolysis experiments. It was assumed that percentage of lipolysis was100%at2h. There was no significant difference among the lipolysis curves (lipolysis percentage-time) of GTP-PC, SIB-PC and phospholipid. And GTP-PC-SMEDDS and SIB-PC-SMEDDS were similar with blank SMEDDS. The result indicated that lipolysis of PC and SMEDDS depended on lipid excipients in the preparation.The dissolution of drugs during lipolysis was determined. The drug concentration for SIB-PC and SIB-PC-SMEDDS was significantly higher than SIB in aqueous dispersed phase,148times and247times (Cmax) respectively. And the results of the physical mixture (SIB and PC) were similar to SIB. Precipitation collected after lipolysis was characterized by XRD analysis. Both SIB-PC and SIB-PC-SMEDDS showed no presence characteristic crystalline peaks of SIB in the pellet, suggesting an either amorphous form or a molecular dispersion.3Absorption study by Caco-2cells monolayersThe absorption mechanisms were studied by transport experiments performed across a Caco-2cells culture model. The result showed that phospholipid complex and SMEDDS could enhance the transport of drug significantly. Transport amount of GTP and SIB and their formulations increased with time. The values of SMEDDS/GTP and GTP-PC/GTP decreased with increasing concentration, but there was no significance between Papp of different concentration of GTP, SIB, SIB-PC and SIB-PC-SMEDDS. SIB was a substrate of P-gp, and SIB-PC and SIB-PC-SMEDDS could significantly inhibit P-gp efflux.Cell membrane fluidity and tight junction protein (ZO-1) were analyzed by fluorescence anisotropy using DPH as the fluorescent labels and by Western Blotting, respectively. GTP-PC, SIB-PC and GTP-PC-SMEDDS significantly increase membrane fluidity. GTP-PC-SMEDDS and SIB-PC-SMEDDS could open tight junction protein.Therefore, the possible reasons for PC promoting the transport are inhibiting P-gp efflux and increasing membrane fluidity; for phospholipid complex SMEDDS are inhibiting P-gp efflux, increaseing membrane fluidity and opening cell tight junctions.4Single Pass Intestinal Perfusion StudiesA modified in situ perfusion method in rats was utilized to study absorption characteristics of drug in four different intestinal segments (duodenum, jejunum, ileum and colon).The results indicated that the absorption of GTP-PC-SMEDDS was significantly better than the GTP and GTP-PC. GTP-PC showed better absorption in ileum and colon than GTP. The absorption of GTP-PC-SMEDDS and GTP-PC was colon> ileum>(jejunum and duodenum).The absorption of SIB-PC and SIB-PC-SMEDDS were significantly better than SIB in four intestinal segments. The best segment was duodenum for SIB, but four segments of SIB-PC-SMEDDS was similar. Therefore, PC and its SMEDDS could promote the intestinal absorption of drugs, but there were differences between different intestinal segments-ileum and colon could promote more significantly.5Intestinal lymphatic transport and systemic bioavailability of drugs in ratsThe possibility of intestinal lymphatic transport of drugs was investigated using unconscious rat model with three duct-cannulated. A UPLC method for the determination of GTP and SIB in plasma and lymph was established.After intraduodenal administration of80mg/kg GTP, the total amount of GTP collected in the mesenteric lymph after8h for the GTP, GTP-PC and GTP-PC-SMEDDS was (5.93±1.45)μg,(19.36±5.74) ug and (40.1±6.32)μg, respectively. Compared with GTP, the relative bioavailability GTP-PC and GTP-PC-SEDDS was252.51%and517.50%, respectively.After intraduodenal administration of200mg/kg SIB, The total amount of SIB collected in the mesenteric lymph after8h for the SIB, SIB-PC and SB-PC-SMEDDS was (0.23±0.21) μg,(2.85±1.09) μg and (5.29±1.67) μg, respectively. Compared with SB, the relative bioavailability SIB-PC and SIB-PC-SEDDS was1265.9%and1802.5%, respectively.The PC and its SMEDDS could significantly increase the lymphatic absorption of drugs, and SMEDDS was better than PC. At the same time, the pharmacokinetic results showed that PC and its SMEDDS could also increase the absorption of drugs through the portal vein.Therefore, the PC and its SMEDDS significantly improve the solubility of the drug (to improve the lipophilicity of BCSIII drugs and hydrophilicity of BCSII drugs). The possible" reasons for PC promoting the intestinal absorption are inhibiting P-gp efflux, increasing membrane fluidity and lymphatic absorption; for phospholipid complex SMEDDS are inhibiting P-gp efflux, increaseing membrane fluidity, opening cell tight junctions and enhance lymphatic absorption.This study is meaningful forresearch and development of naturaldrug-phospholipid complex andthe improvementoforal bioavailabilityof phospholipid complex.