| ObjectiveThe objective of this study is to build a method for local pharmacokinetics research of subcutaneous tumor by microdialysis technique. Hydroxycamptothecin as a model drug, Kunming mouse H22hepatoma subcutaneous tumors as an animal mode, microdialysis was used to study the PK process of HCPT magnetic thermosensitive liposomes in local tumor, evaluating tumor aggregation and release of magnetic thermosensitive liposomes. Based on systematic studies, a method to study local tumor pharmacokinetic with microdialysis technique was built, making up for the scarcity of classical pharmacokinetics in tumor targeting agents, and providing foundation for future research in this area.MethodsThe study contains four parts as follows1Establishment of analytical method of HCPT samplesHPLC-UV was appled to detect the content of ring-opened HCPT(O-HCPT) and ring-closed HCPT(C-HCPT), relative chromatographic conditions were investigated, including the mobile phase optimization, standard curves, linear range, precision test the quantification limit and detection limit.2Establishment of HCPT microdialysis parameterThe influence of perfusion rates, media concentrations and temperatures on the recovery of C-HCPT and O-HCPT in vitro were investigated. The stability of HCPT recovery was also investigated, including vitro and vivo stability of O-HCPT and C-HCPT recovery, observation of recovery stability after O-HCPT and C-HCPT were mixed. The feasibility of using internal standard method for the determination of HCPT recovery was studied.3Blood pharmacokinetics study of HCPTHCPT plasma protein binding rate was detected microdialysis in vitro. The pharmacokinetics process of HCPT injection was compared through tail vein and intraperitoneal injecting, vivo recovery was detected with retrodialysis. The pharmacokinetics process of HCPT magnetic thermosensitive liposomes in rats was studied.4Local pharmacokinetics study of HCPT magnetic thermosensitive liposomes in tumorKunming H22cells subcutaneous tumor model was built. Local pharmacokinetics process was sudied by implanting concentric probe into the subcutaneous tumor. Animals were divided into3groups:Group A, the HCPT magnetic thermosensitive liposomes with magnetic field in vitro; Group B, the HCPT magnetic thermal liposomes without magnetic field:Group C, HCPT injection magnetic field with magnetic field in vitro. Flowing rate was2μL-min-1, vivo recovery was detected with retrodialysis. Data was processed by DAS2.1.1, and SPASS18.0.Result1Establishment of analytical method of HCPTWe developed the method of detecting C-HCPT and O-HCPT by HPLC-UV method. Analytical method is as follows:Waters HPLC system:Waters515pump,2487dual wavelength UV detector, Zhejiang University N3000chromatography workstation; column:Phenomenex Luna C18(12)(4.6mm x250mm,5μm) column; guard column:Phenomenex guard column; mobile phase: acetonitrile-methanol-O.O5mol.L-1KH2PO4(pH6.4)(60:490:450); flowing rate:0.8mL-min"1; detecting wavelength:384nm.; column temperature:25°C; column pressure:120barUnder the chromatographic condition, C-HCPT and O-HCPT had symmetrical peaks with tR4.48,7.62min, and limit of quantification and detection limit with the experimental requirements. The linear range, limit of quantification and the limit of detection could met the requirement.2Establishment of microdialysis sampling method for HCPTMicrodialysis sampling method for HCPT was as follows:perfusion rate2μL-min-1sampling volume12μL. sampling interval6min, recovery was detected with retrodialysis.The recovery changes of mixing C-HCPT and O-HCPT showed that:The recovery detected by increment method was the same as by decrement method. Both recoveries were independent of concentrations in the medium. C-HCPT recovery reduced obviously.while O-HCPT recovery did not reduce.The recovery of C-HCPT and O-HCPT could keep stable within8h in vitro. RSD5.12%,5.99%. The recovery of C-HCPT and O-HCPT could keep stable basically within8h in vivo, RSD7.88%,8.68%.Vitro and vivo studies showed that the proportion of the recovery (or delivery) of the HCPT to that of internal standard was not stable, thus drug concentration around the probe could not be accurately calculated. The vivo recovery of HCPT determined with internal standard method was not feasible.3Blood pharmacokinetics study of HCPTHCPT plasma protein binding rate was72%detected by retrodialysis in vitro.Blood microdialysis studies showed that:pharmacokinetic parameters of commercial HCPT injection after administrated through tail vein and intraperitoneal in a lOmg/kg dose were in line with the two-compartment open model. There was a short-term absorption process through intraperitoneal; Tmax,ti/2a,ti/2βand MRT(O-oo) through tail vein were much smaller than those through intraperitonea, which showed that HCPT eliminated and distributed quickly administrated through tail vein; Cmax of tail vein group was13.3times as much as that of the intraperitoneal group, AUC(O-co)4.3times, indicating administrating through the tail vein could significantly improve the HCPT plasma concentration and bioavailability.The HCPT magnetic thermosensitive liposomes administrated through tail vein greatly increased the C-HCPT plasma concentration. Pharmacokinetic parameters of HCPT magnetic thermosensitive liposomes administrated through tail vein in a lOmg/kg dose were in line with the two-compartment open model. t1/2a,t1/2β,Tmax,and MRT(O-oo) of magnetic thermosensitive liposomes werell.1、22、4、3.5times as much as those of commercial HCPT injection, which showed that elimination and distribution of HCPT magnetic thermosensitive liposomes were slower with a longer retention time; Cmax of magnetic thermosensitive liposomes was0.6times as much as that of HCPT injection, AUC(O-oo)4.3times, indicating magnetic thermosensitive liposomes could improve bioavailability,decrease Cmax and reduce the toxicity.4Local pharmacokinetics study of HCPT magnetic thermosensitive liposomes in tumorPharmacokinetic parameters of Group A, B and C were in line with the two-compartment open model. Tmax of Group A, B and C were6,12,18min, indicating that compared with Group B and C, HCPT magnetic thermosensitive liposomes could qickly assemble and release drug in tumor with magnetic field in vitro. Cmax and AUC(O-oo) had statistical difference between any two of three groups(Sig.<0.05), which showed that magnetic thermosensitive liposomes could improve drug concentration in tomor, highly targeted aggregation and releasing drug.Conclusion1HPLC-UV method could separate C-HCPT and O-HCPT, and the minimum detection limit could meet the minimum concentration. 2C-HCPT recovery would reduce,while O-HCPTwould not when mix both together, however dialysis characteristics of both did not change.3Due to the specialty of HCPT lactone ring structure, it is hard to find a suitable internal standard, vivo recovery of HCPT determined with internal standard method was not feasible.4Rat plasma protein binding rate of HCPT in vitro with retrodialysis is in line with those studied by the traditional method, verifying the feasibility of study HCPT vivo pharmacokinetics with microdialysis.5Blood pharmacokinetics studies of HCPT magnetic thermosensitive liposomes showed that magnetic thermosensitive liposomes could protect HCPT lactone ring structure and increase the proportion of C-HCPT in plasma.6Kunming mouse H22hepatoma cells subcutaneous tumor model has a short cycle, a high rate of tumor formation, suitable for the microdialysis study.7HCPT magnetic thermosensitive liposomes could assemble and release drug in tumor quickly with magnetic field in vitro and a high tumor temperature(≥43℃),high tumor target.8.The subject established a general method for researching tumor local pharmacokinetic study of anticancer drugs targeted delivery system based on microdialysis technique, providing foundation for future tumor local pharmacokinetic study. |
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