Studies On Oral Drug Delivery Systems Of Low Molecular Weight Heparin

With the advent of biotechnology, particularly the advances in recombinant protein technology, macromolecular drugs have received mach attech for their therapeutic roles, especially for their good effects and little side effects. However, Because of their poor biopharmaceutic properties of oral absorption, the overwhelming majority could only be administered by injection with poor-compliace. We choose low molecular weight heparin(LMWH) which prevents and treats the venous thromboembolism as a model drug to explore how to improvement the oral bioavailability of macromolecuars. on the basis of sorting and grouping of former research on oral absorption of LMWH and screening of permeation enhances of LMWH, we developed two noval drug delivery systems for LMWH—nanoemulsion and intestinal adhesive patch. First the first time we developed fully digestible nanoemulsion in favor of intestinal absorption of LMWH based on digestion principle and with phase diagram study. The nanoemulsion showed the good results through in-vivo tests. We firstly used the minitablet to prepare the intestinal patch of LMWH. The in-vitro studies showed good permeability of LMWH across jejunum mucosae in the presence of intestinal patch. This research was to be a foundation to develop oral preparations of LMWH and an extension of nanoemulsion and intestinal patch which were applied on the oral of macromolecuars.Firstly we established the method of determination of LMWH in intestinal permeated solution using Azure A by the spectrophotometric method. The intestinal transport of LMWH was performed by applying everted intestinal sacs method. The absorption of LMWH was passive diffusion and the order of permeation of different gut sacs was duodenum>jejunum>ileum>colon. The order of permeation of various absorption enhancers was 33mMSNAC>0.5% Imwitor742> 10mM NaGC> 13mM NaCap>15mM NaTC>15mM NaOA>1%Tween 80. The intestines membrane did not have obvious damage under the concentration of various enhancers.Under the guidance of 10% water content in water-in-oil and relative low concentration of surfactant, nanoemulsion pseudo-ternary phase diagrams were constructed to evaluate the formulation effect. The results showed that middle-chain triglycerides were more easily to become nanoemulsion than long-chain triglycerides; Under the condition of Miglycol 812N/Surfactant/Imwitor742 and K_m being 1, the requirement of water content can be achieved under the oil content of 50% or 60% in the presence of Tween 80,Cremophor EL or TPGS as surfactants. Under the conditionof Miglycol 812N/Tween 80/Cosurfactant and K_m being 1, the requirement of water content can be achieved under the oil content of 50% or 60% in the presence of PEG400,propranol or Imwitor 742; Under the condition of Miglycol 812N/Tween 80/Imwitor 742 and K_m being 1 or 0.5, the requirement of water content can be achieved under the oil content of 50% or 60%. These results showed that It was hard to effectively screen the formulation of nanoemulsion on the basis of water content in phase diagrams. Generally, the digestion products of middle-chain triglycerides were contributed to permeation of LMWH. So it is necessary to screen the nanoemulsion formulation from digestion point.The progress of in-vitro lipid digrstion was monitored indirectly by pH titration. Effect of bile salts micelles, pH of digestion buffer, concentration of pancreatic lipases and calcium ions on the digestion rate and extent of Miglycol 812N during the 30 minutes digestion. The established in-vitro lipolysis model were 50mM Trizma maleate, 150mM NaCl and 5mM Ca²⁺; pH was adjusted to 7.5; the mixed micelles consisting of 5mM NaTDC and 1.25mM PC; and the concentration of pancreatic lipase were 200 U/ml. The above in-vitro digestion condition was moderate and helped to differentiate the formulation factors which affects the digestion of nanoemulsion.Effect of formulation factor on digestion of nanoemulsion was studied using in-vitro lipolysis model. The studies indicated that when middle-chain glycerides were lipolysed fatty acids released per minute were 5-7 times than long-chain glycerides. The lipolysis of middle-chain triglycerides was inhibited in varying degree in the presence of surfactants, of which the degree of inhibition was Cremophor EL>TPGS >Tween80> Labrasol; and the extent of inhibition were increased with the ratio of Tween80/Miglycol812N added. Eeffect of cosurfactants on lipolysis of Miglycol812N behaved differently. Ethanol and PEG400 inhibited the lipolysis of middle-chain glycerides. Transcutol P and Imwitor 742 speeded up the digestion of middle-chain glycerides. However, only Imwitor742 can rescued the digestion of Tween80/Miglycol812N(1/1), and the recovery of digestion of Tween80/Miglycol812N(1/1) was improved with the ratio of Imwitor742/Tween80 increased, the most efficient when the ratio being 1 and the rate of lipolysis exceeded Miglycol 812N itself. Ratio of Imwitor742/Tween80 (1/1) kept constant, varying ratio of Tween80/Miglycol812N did not influence on the rate of digestion, but the degree of digestion was lowered when the ratio was relatively high.Through results of above study, we concluded the rate and degree of various phase diagrams in the presence of Tween 80. Only under the condition of Miglycol812N/ Tween80/Imwitor742 and K_m being lor 0.5, the rate of digestion of nanoemulsion containing more 10% water content could exceed Miglycol 812N itself. With more oil content in nanoemulsion, the extent of digestion is more complete, and the released fatty acids were more, which lead to more absorption of macromolecuars. This research firstly used digestion law combined with phase diagrams to guid the screening of water-in-oil nanoemlsions, which helped to design digestible or non-digestible nanoemulsion.We designed oral digestible(NE-D) and non-digestible(NE-ND) nanoemulsions, The digestion rate of NE-D was more 7 times than NE-ND duiring 30-minute in-vitro digestion experiments. The particle size of the two nanoemulsion were 25-30nm, and became 200nm upon dilution using bile salt micelles solution that phase inversion to O/W emulsion. The digestion products of NE-D were mixed micelles formed with particle size of 15.1nm. The dissolution test showed 90% LMWH was released in 3 minutes from the two LMWH nanoemulsionsVarious dilution of digestion products of NE-D enhanced more permeation of LMWH through jejunum mucosae than that of NE-ND by applying everted intestinal sacs method, which indicated that mixed micelles played a main role in promotion of drug permeation. The fact that the permeation promotion was lowered with dilution increased indicated that dilution effects of intestinal will affect the absorption of nanoemulsions. LMWH formulation was administered to the rat jejunum at a dose of 50mg/kg. The pharmacologic availability of solution, solution containing 35mg SNAC, NE-D and NE-ND of LMWH was 1.02%,13.24%,15.24% and 4.91%; and the maximum APTT were 27 s, 108.2 s, 96.2 s and 48.4 s; when above LMWH formulations following intragastric administered, the pharmacologic availability(PA) was 0.74%,4.40%, 8.89% and 2.01%; and the maximum APTT were 25.3 s, 55.2s, 74 s and 35.8 s; The PA after jejunal and intragastric administration of various LMWH formulations was followed: digestible nanoemulsin>SNAC agent> non-digestible nanoemulsion>solution. PA and maximum APTT of intragastric administration was more than that of jejunal administration, which showed dilution effects affected the absorption of LMWH formulations. PA of NE-D was 3 times than that of NE-ND, which indicated that digestion of nanoemulsion played an important role in promotion absorption of LMWH. It is concluded that the digestion products ofNE-D — caprylate/caprate sodium and monocaprylin monocaprin, with which bile salts/phosphatidylcholine and Tween80 constituted complex mixed micelles. It is concluded that the mixed micelles promoted more permeation of LMWH than the emulsion drops and agent SNAC.The results of Caco-2 cell test indicated that sodium caprate/monocaprin micelles could open tight junctions to some extent and enhanced transport of FD4. Bile salts micelles itself did not affect TEER and transport of FD4, but with which caprate/monocaprin micelles could open tight junctions and enhanced transport of FD4 to more extent. Tween 80 did not affect the above action.Oral intestinal adhesive patch of LMWH was based on minitablet made up of adhesive materials and constituted of core tablet, impermeable layer and enteric layer , which was one of intestinal bioadhesive drug delivery system. Firstly, Carbomer 934P with the best adhesive strength and CMC-Na 2000cp with the best shear stress were chosen as adhesive materials through screening. After single factor study the following formulation and parameters of preparation was: ratio of Carbomer 934P and CMC-Na2000cp being 1, thickness beinglmm, diameter being 3mm and hardness being 4kg. The backing layer made up of EC membrane could effectively prevent the release of LMWH. The enteric-coated layer constituted of Eudragit L-55 could lead the patch to adhere to small intestinal. The core tablet containing less 10% sodium glycocholate did not affect adhesive performance and release rate. The in-vitro permeation test showed that the intestinal patch could promotion LMWH permeation10.4 times than LMWH solution, and that containing 10% glycocholate enhanced15.5 times. The intestinal patch was promising of promotion the oral absorption of LMWH.