The Study Of TID-101 Self Micro-emulsifying Drug Delivery Systems

"Stroke" is also known as "cerebrovascular accident", which belongs to the acute cerebrovascular disease. In recent years, the incidence of stroke has an upward and youngertrend. In our country, ischemic stroke accounts for about 80% of all stroke,which is due to local brain tissue blood circulation disorders, ischemia, hypoxia induced necrosis, timely and effective treatment is the decisive factor for the disease prognosis.As derivative of propofol analogs bisphenol, TID-101 could fight against ischemic stroke damage in many mechanisms, more importantly, it does not lead to loss of consciousness, so it has important clinical value in the treatment of a variety of ischemic stroke symptoms. However, since the compound is highly lipophilic and poor solubility of water, dissolution is its rate limiting process in absorption. And the further research and development of TID-101 is limited by its poor bioavailability.Currently the main method to solve this problem is lipid delivery system, where the self（micro） emulsion drug delivery system [SEDDS or SMEDDS] is composed of oil phase, surfactant and co-surfactant, forming a homogeneous mixture. With in vitro gentle shake or in vivo gastrointestinal motility, it could immediately form drug oil-in-water micro-emulsion, particle size is less than 150 nm, and could effectively improve the drug dissolution, enhancing drug oral absorption through a variety of mechanisms.In this study, firstly, we carried out the former prescription research, including physicochemical properties, analysis and determination methods of TID-101.Then we prepared nine prescriptions of TID-101 self-microemulsion（SEDDS） containing triglyceride with different chain lengths（short-chain, medium-chain and long-chain）and different content（low, medium, high, nine species） as oil phase. Next, we studied TID-SEDDS preparation process, in vitro dispersion, in vitro lipolytic activity, rats in vivobiological availability and absorption through lymphatic transport.Part One We established HPLC content analysis method of TID-101. Take Agilent Eclipse XDB-C18（4.6 × 150 mm, 5μm） as column; mobile phase was methanol-water with gradient elution; flow rate was 1mL·min^-1; column temperature was 30 ℃ and the detection wavelength was 265 nm.Within 0.1¹00 μg·mL^-1concentration range, TID-101 had good linear relationship with regression equation“A=45.55C-16.16,r2=0.9993”. The detection limit was 40ng·mL^-1, while the lowest limit of quantification was 80ng·mL^-1. Results showed that the selected chromatographic conditions for determination of TID-101 content had good shape,excellent specificity, high sensitivity, stable and accurate determination results.Part Two We measured the basic physical and chemical properties of TID-101,including solubility, dissociation constant pKa, partition coefficient lg P, and so on.The results showed that TID-101 was poor water-soluble（5.57 ± 0.91）μg· mL^-1and good fat-soluble, pKa was（10.12 ± 0.63）, lg P was 7.37. It had considerable solubility in various candidate materials of micro-emulsion as well as good compatibility, meeting all the conditions in SEDDS preparation.Part Three Take TID-101 as model drug, we studied formulation and preparation process of TID-101 SEDDS systematically. We chose Cremophor RH40 to be emulsifier, 1,2-propanediol as co-surfactant, and propyl gallate as anti-oxidants,investigating the influences of different triglycerides（C18 long-chain, C8-C10medium-chain and C2 short-chain） on self emulsifying region of SEDDS. And then we prepared TID-101 SEDDS of these three triglycerides respectively, each of them had low, medium and high content of TID-101.Take particle size distribution,compatibility and emulsifying efficiency as the main evaluation index, we further evaluatedthe in vitro characteristics of the prescriptions composed of different ingredients.Part Four We used in vitro lipolytic dynamic model to evaluate the digestion rate of nine kinds of preparations and drug solubilization capacity changes（precipitation percentage） during fat digestion. During the digestive experiments in vitro, with chain-length and content of triglyceride increase, the digestion rate of SEDDS slowed down. After the end of the in vitro digestion process, the final solubilization capacity of SEDDS for TID-101 enhanced with the increase of triglyceride chain-length and content. In high content and long-chain triglyceride group, the TID-101 content in aqueous dispersion phase was（84.63±13.40）%,precipitation percentage was（12.13±7.30）%; while in low content and short-chaintriglyceride group, the TID-101 content in aqueous dispersion phase was（51.30 ± 7.31）%, precipitation percentage was（43.02±3.42）%, the difference between them was significant（p <0.001）.Part Five We established HPLC method for TID-101 concentration in the lymph,and LC-MS/MS determination method for drug concentration in rats plasma samples.The plasma samples were treated with methanol for precipitating proteins. The chromatographic separation was achieved with a acetonitrile- water mobile phase.Detection of TID-101 and the internal standard（IS） Dexamethasone Acetate were achieved by electrospray ionization（ESI） source in the negative ion mode at m/z353.4�'323.2 and m/z 433.4�'353.4. The method was applied for pharmacokinetics study of TID-101 SEDDS in rats. The method was linear over TID-101 concentration range from 10¹20000 ng·mL^-1 with the correlation coefficients（r2） of 0.9996. The intra-run and inter-run relative standard deviations（RSD） were less than 15% and the average recovery values were 83.89%⁸6.69%. The validated method was applied to a pharmacokinetic study in rats after intravenous administration of TID-101 fat emulsion injection and oral administration of TID-101 suspension and SEDDS at a single dose of 20 mg·kg^-1.The rats were given nine different types（chain-length） and oil content SEDDS prescription(SCT–Low, SCT–Mid, SCT–Hig; MCT–Low, MCT–Mid, MCT–Hig; LCT–Low, LCT–Mid, LCT–Higthrough intragastric administration,then we collected the plasma samples for LC-MS/MS measurement to evaluate bioavailability. We established mesenteric lymph duct and duodenum double cannula model on anesthetized rats, and administrated SEDDS and TID-101 suspension though duodenum, collecting lymph for HPLC measurement and evaluating drug lymphatic transport of TID-101 SEDDS, as well as influence of lymphatic transport on rats oral bioavailability. The results show that in the nine SEDDS prescription, the chain-length of triglycerides in SEDDS is a key factor affecting TID-101 lymphatic transport, and the order of the amount for different chain-length oil carrier lymphatic transportation was LCT（C18）, MCT（C8-C10）, and SCT（C2）, exhibiting significant dose dependence. For the control prescription without oil phase, TID-101 absorption was almost not based on lymphatic transportation. When triglycerides content was low in prescription, lymphatic transportation was also not obvious. While in the high oil dose prescription, lymphatic transportation made big contribution to improve the oral bioavailability.The results of this study showed that, compared with oral drug suspension,SEDDS could improve rat oral bioavailability of TID-101 significantly. The chain length and content of triglycerides in the oil phase of SEDDS greatly affects bioavailability. Based on the ensurement of micro-emulsion system stability, the application of high content long-chain triglycerides（C18） couldeffectively reduce the digestion and precipitation of the drug in the gastrointestinal tract, improve the absorption of the drug through lymphatic transport, then avoid hepatic first-pass effect. According to the physical and chemical properties of candidate compound（TID-101）, the development of TID-101 SEDDS has definitive value.