Study On Mechanism Of Intestinal Absorption Of SYS330, A Novel Antitumor Agent

SYS330 is a recently synthesized novel 4-anilinoquinazoline inhibitor of epidermal growth factor receptor tyrosine kinase(EGFR-TK). The main aims of this research were to study the physicochemical properties and the intestinal absorption mechanisms of SYS330, and investigate the factors that affect the absorption of SYS330 for its clinical trials. A high performance liquid chromatographic(HPLC) have been developed and validated for the determination of SYS330. To predict its oral pharmacokinetic behavior and transport nature in the intestine before clinical trials, the absorption and transport of SYS330 in Caco-2(a human colon cancer cell line) cell monolayers and in situ rat Single-Pass Intestinal Perfusion Model(SPIP) were investigated systematically. These researches laid an useful foundation for studying SYS330.1 Establishment of Determination of SYS330A high performance liquid chromatographic(HPLC) method for the determination of SYS330 was developed and validated. The optimum conditions were: column, Hypersil C18 BDS(200mm×4.6mm, 5μm); mobile phase, acetonitrile:0.1mol/L ammonium acetate=40:60, 0.03%TFA; flow rate, 1.0ml/min; wavelength of UV detector, 248nm; temperature of column, 30℃; sensitivity setting, 0.01AUFS; injection volume:10μL. The intra- and inter precision was both below 2%. The accuracy was above 99%. The physicochemical stability of SYS330 was investigated. The results showed that SYS330 was relatively stable in 30 days at the condition that was alkaline and below 4℃. And thrice freeze-thawing was no influence to the stability of SYS330.2 Study on the Cytotoxicity of SYS330 in Caco-2 CellsMethyl thiazolyl tetrazolium chromatometry was applied to study the cytotoxicity of SYS330 in Caco-2 cells. The results showed that compared with the control group, SYS330 solution lower than 30μg/mL had no toxicity to Caco-2 cell. While SYS330 solution higher than 30μg/mL was toxical to Caco-2 cell.3 Establishment and authentication of Caco-2 cell modelCaco-2 cell model was authenticated by determination of TEER of Caco-2 cell monolayers. The results indicated that in our laboratory, Caco-2 cells had reached integrated confluence and formed differentiated monolayers 21 days after seeding. TEER of the cells reached 300Ω·cm² and met the requirement of drug transport. So this model can be an in vitro model that investigated intestinal permeability and transport of SYS330.4 Study on intestinal permeability and transport of SYS330 in Caco-2 cell modelA human colon cancer cell lines, Caco-2 cells, which have been widely used to investigate and predict intestinal permeability and transport of a number of drugs, was used to characterize the permeability and transport of SYS330. The effect of pH, concentration, ATP inhibitor, Na⁺/K⁺ ATPase inhibitor, glucose transporter inhibitor, Surfactant, P-gp inhibitor, MRP1 and MRP2 inhibitor, endocytosis inhibitor on the transport and effiux of SYS330 was investigated. The results indicated that: 1).The transport of SYS330 was passive diffusion as the dominating process. P_app(AP-BL) of SYS330 was (1.061±0.012)×10^-5cm/s. 2) The transport of SYS330 was pH dependant, and the transport was enhanced at weakly acidic pH on the apical side. 3) SYS330 was transported by passive transcellular pathway. Intensive endocytosis decreased its transport.4) SYS330 was probably effiuxed by P-gp, an effiux transporter protein, which inhibited the absorption of SYS330.5 Study on absorption of SYS330 in rat intestine using in situ single-pass perfusion(SPIP) combined with Mesentery Venous Cannula modelIn situ rat Single-Pass Intestinal Perfusion Model(SPIP), commonly used abroad, which was combined with Mesentery Venous Cannula model, was employed to investigate intestinal absorption of SYS330 in rats. The effect of ATP inhibitor, P-gp inhibitor, MRP1å’ŒMRP2 inhibitor on the intestinal absorption of SYS330 was investigated. The results showed that the intestinal absorption of SYS330 was passive diffusion. P_lumen and P_blood of SYS330 in rat intestine were separately (3.100±0.040)×10^-4cm/s and (1.600±0.025)×10^-5 cm/s. The results indicated that SYS330 was probably substrate of P-gp. The intestinal effiux of SYS330 can be inhibited by P-gp inhibitor verapamil, and the intestinal absorption of SYS330 was enhanced.