| ObjectiveThrough systematic research, to establish sampling method with microdialysis for gefitinib and study its pharmacokinetic characteristics by microdialysis and traditional blood-drug concentration method jointly. To explore a new idea for the research of anti-tumor drug targeting systems, also, the method provided a new way to research lipophilic and high protein-binding drug by microdialysis.Methods1Established HPLC analytical method for gefitinib.Depending on HPLC-UV, concentration of gefitinib and erlotinib in saline (pH3.22) or methyl alcohol were determined by external standard method. Specificity, standard curve, precision, accuracy, limit of quantification and detection and stability of chromatographic condition were examined.Erlotinib as internal standard, HPLC-UV was used to measure concentration of gefitinib in plasma. Specificity, standard curve, precision, accuracy, limit of quantification and detection and stability of chromatographic condition were confirmed as well as extraction recovery of drug in plasma.2Established retrodialysis method for gefitinib.Adopting concentration difference methods, increment method and decrement method included, we determined the effect of flow rates, pH, medium concentration and temperature on the gefitinib’s related recovery (RR.g). Meanwhile, the stabilities of RR.g and related delivery (RD.g) were researched in vitro and vivo. Feasibility study on pharmacokinetics of gefitinib by retrodialysis method was validated.3Determined plasma protein binding rate of gefitinb in vitro by retrodialysis method.Used physiological saline solution (ph3.22, containing5%dimethyl sulfoxide) as perfusate, putting the microdialysis probe in the drug-containing plasma with gradient concentration, retrodialysis method was adopted to measure plasma protein binding rate of gefitinib in vitro. After reaching balanced state, we collected the dialysate. In the same way, taking the blank plasma as extracellular fluid, perfused drug-containing saline solution (ph3.22, containing5%dimethyl sulfoxide) to detect RD.g.As RD.g to RR.g, combined with the concentration of dialyzate gefitinib’s plasma protein binding rate was calculated.4Established retrodialysis by calibrator method for gefitinib.Taking erlotinib as internal standard, we determined the effect of flow rate, medium concentration and temperature on the relationship of gefitinib’s related recovery (RR.g) and erlotinib’s related delivery (RD.e)-Besides, the stability of RD.g-to-RD.e ratio (P) were researched in vivo with three probe. Possibility of researching pharmacokinetics of gefitinib by retrodialysis by calibrator method was discussed.5Determined pharmacokinetic parameters of gefitinib by blood-drug concentration method.After a single oral or intravenous injection administration of gefitinib to each group of rats, collected blood from the veniplex behind bulbus oculi. Mixing erlotinib as internal standard, HPLC was used to measure the plasma level. WinNonlin4.0.1program software was used for matching the data that reflected the relationship between concentration and time to gain the main pharmacokinetic parameters.6Determined pharmacokinetic parameters of gefitinib by microdialisis and blood-drug concentration method united.Fat emulsion injection containing erlotinib as perfusate, we used retrodialysis by calibrator method to collect free gefitinib from vena jugularis externa, corpus striatum and subcutaneous tissues after a single oral or intravenous injection administration of gefitinib. Concentration of dialyzate was determined by HPLC. RR.g corrected by RD.e and the result of blood-drug concentration method were combined to gain drug level in real time. WinNonlin4.0.1program software was used to fit the data on plasma concentration-time relation and calculated the main pharmacokinetic parameters of gefitinib.7Established method on culturing human lung adenocarcinoma cell.We chose human lung adenocarcinoma cell to be object of study and optimized programe on thawing cells, passage, cryopreservation and transplantation for setting up animal model with localized tumors. Results1HPLC for gefitinib by external standard method was established.Chromatographic column (DIKMA, Platisil ODS,5μm,150×4.6mm) and guard column (Security GuardTM) were used. Mobile phase was composed of methanol and water containing0.2%triethylamine (80:20, v/v). Flow rate was set as1.0mL·min-1. Determine wavelength was330nm Column temperature was remained at25℃. Sample size was10μL.2HPLC for gefitinib and erlotinib by external standard method and internal standard method for gefitinib were established. Chromatographic column (DIKMA, Platisil ODS,5μm,150×4.6mm) and guard column (Security GuardTM) were used. Mobile phase was made up of methanol and ammonium acetate (10mmol·L-1)(70:30, v/v). Flow rate was set as1.0mL·min-1. Determine wavelength was330nm. Column temperature was kept at25℃. Sample size was10μL.3Parameters of retrodialysis method for gefitinib.Taking normal saline (pH3.22) as perfusate, a constant flow velocity was1.0μL·min-1. Sampling interval was15min, so the sample size was15μL. Under velocity gradients, RR,g and RD.g agreed well with each other. RR.g had a reverse correlation with pH value of perfusate, however, temperature of extracellular fluid was exact contrary. Moreover, RR,g maintained a constant even if concentration changed. RR,g and RD.g exhibited a good stability in vitro and vivo.4Plasma protein binding rate of gefitinib in vitro measured by retrodialysis method.In vitro, the plasma protein binding rate of gefitinib was (89.45±1.03)%(RSD1.16%), moreover, the difference among different concentration was not significant (P>0.05).5Parameters of retrodialysis by calibrator method for gefitinib.Taking normal saline containing erlotinib (pH3.22) as perfusate, a constant flow velocity was1.0μL·min-1. Sampling interval was15min, so the sample size was15μL. Under velocity gradients, the RD.g-to-RD.e ratio (P) kept a constant when pH value of perfusate, temperature of extracellular fluid and concentration changed. The concentric probe and linear probe implanted in brain, heart and subcutaneous tissue all kept a constant ratio (P’). Also, it exhibited a good stability.6The main pharmacokinetic parameters of gefitinib measured by blood-drug concentration method.After a single oral or intravenous injection administration of gefitinib to each group of rats, the plasma drug elimination curve was concordant with a two compartment model. Oral bioavailability of gefitinib tablet was59.81%.7The main pharmacokinetic parameters of gefitinib determined by retrodialysis by calibrator method and blood-drug concentration method united.After a single oral or intravenous injection administration of gefitinib to each group of rats, the plasma drug elimination curve was concordant with a two compartment model. Oral bioavailability of gefitinib tablet was63.07%. The main pharmacokinetic parameters agreed well with the date determined by blood-drug concentration method solely. Conclusions1Under the established chromato graphic system, gefitinib and erlotinib all have good linear relationship between concentration and peak area, as well as its ratio. Furthermore, good precision, exclusivity, stability, matrix effects, dilution effect, extraction recovery of drug in plasma all meet the requirements in biological samples. Depending on HPLC-UV, we can gain accurate quantitative analysis for gefitinib and erlotinib.2Within12hours, retrodialysis method reveals a relatively constant for RR.g in vitro and vivo. But, overall internal data shows a downward trend. Simultaneously, the RD.g has a large difference between internal result and the value in vitro. So, the method for sampling gefitinib in vitro is feasible. However, in vitro, if RD.g in the place of RR.g, some certain errors may exist, and we can preliminarily estimate short-time pharmacokinetic characteristics for gefitinib only.3Gefitinib shows high protein-binding rate in vitro, so it may redistribute and cause adverse event or toxicity in clinical application. It is significant to select the proper dose and monitor blood concentration. In addition, developing new preparation to improve the free drug concentration for gefitinib makes perfect sense.4Erlotinib as internal standard substance, RR.g can be calibrated in real time. Nevertheless, because of lipophilic and high protein-binding rate character of gefitinib, conventional perfusate can not collect it in vivo and modified or new form of perfusate is needed to enhance its relative recovery in vivo. Besides, the P’need further correction.5Compared to the case of normal physiological condition, rats absorb and eliminate gefitinib slowly. So, drug accumulation should be taken into account under anesthesia in clinical. To improve bioavailability and cut down poison and sideeffect of gefitinib, developing targeted drug delivery system for gefitinib such as cycbdextrin inclusion compound, lipidosome-CD and magnetic liposomes is significant.6combined retrodialysis by calibrator with blood-drug concentration method, this technique executes real-time correction for RR.g in twice, so more precise and reliable measured data were gained. It can soulve the problem that difficult to determine probe’s relative recovery in vivo. On the other hand, based on protein binding rate in vivo of drug, the concentration of free drug can be calculated accurately. In consequence, the united technique can be popularized. |
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