The Primary Non-clinical Pharmacokinetic Study Of A New Targeting Antitumor Candidate Compound Z-GP-Dox

BACKGROUND & OBJECTIVE:Z-GP-Dox, designed and synthesized by our lab, is a new tumor-targeting candidate compound activated by FAPa enzyme. It is constituted of N-extremity blocked Gly-Pro dipeptide which is specifically hydrolysised by FAPa, and Free amino bond, derived from Dox. The primary study has proved that the Z-GP-Dox could concentrate at the tumor, release Dox, the active metabolite of Z-GP-Dox,and kill tumor cell with less toxicity. So far, the study of Z-GP-Dox supported by National Key Drug Innovation" Fund and "thel lth five-year" Plan Fund has been developed into drugability research stage. As an important part of drugability research, the primary pharmacokinetic research of Z-GP-Dox aims to learn the regularity of the Z-GP-Dox plasma concentration in rats at different time, acquire the main parameters of the Z-GP-Dox pharmacokinetic, observe the characteristic for the distribution of Z-GP-Dox in mouse tissues and plasma protein- binding rate,as well as to illustrate the dynamic changing regulation of Z-GP-Dox in vivo, by which the necessary pharmacokinetics evidence for the drugability evaluation of this new candidate compound can be obtained.METHODS:1. To establish the analytical method of Z-GP-Dox in biological matrixA simple, specific and highly sensitive method by using reversed—Phase liquid Chromatography has been developed for the quantitative analysis of Z-GP-Dox in rat plasma and tissue sample. After comparing the absolutely recovery rate of Z-GP-Dox with different extracting agent such as methol, acetonitrile, ethyl acetate, methylene dichloride and the mixture of them with different ratio, we chose acetonitrile-methylene dichloride (1:4) as the extracting agent. As to the elution, according to the reviews about Dox and our research, we chose acetonitrile -0.1% TFA(2:3) as the elution. After the Three-dimensional fluorescence detector full stereo wavelength scanning,the excitation spectrum is set at 238nm,the emission spectrum is set at 554nm,the flow is set at 1mL/min, the temperature is set at 25℃2. To determine the plasma concentration of Z-GP-Dox and calculate its primary pharmacokinetics parameters in rat in vivo1) Choosing the solvent and dosage for the administration of Z-GP-Dox, and the time points to take blood samplesAfter comparing the solubility of Z-GP-Dox in DMSO, methanol, propylene glycol and the respective toxicity of those above solvent, propylene glycol was chosen as the solvent for administration. Taking consideration of the effective dosage of Z-GP-Dox by the pharmacodynamics studies and the maximum solubility of Z-GP-Dox in propylene glycol as well as the toxicity of propylene glycol, we gave rats at three dosages respectively:20mg/kg, 10mg/kg, and 5mg/kg as high, middle and low dosage. In primary experiment, Z-GP-Dox in rat plasma can no longer be detected in 5h after administration. So we chose 12 time points to collect plasma with drug:3min,6min,10min,15min,20min,30min,40min,1h,1.5h,2h,3h,5h. Basing on the relevant references of Dox, we chose DNR as the internal standard substance for the detection of Z-GP-Dox in this experiment.2) Administration, taking blood samples, and detectionFollowing a single iv administration with different dosage of Z-GP-Dox to rat(5,10,20),the blood samples were collected at 3 min,6 min,10 min,15 min,20 min,30 min,40 min,1 h,1.5h,2h,3h,5h after dosing. All collected blood samples were centrifuged to obtain plasma and the concentration of Z-GP-Dox in plasma were determined by HPLC method described as above. Then the concentration-time curve of Z-GP-Dox was depicted. Andthe primary pharmacokinetic parameters were calculated by the professional pharmacokinetic analysis program:DAS ver1.0 (Drug And Statistics for Windows).3. The study of the tissue distribution features of Z-GP-Dox in miceThe mice were executed at 10min,20min,30min, 1h,2h and 3h after i.v. administration (20mg/kg).Tissues such as heart, liver, spleen, lung and kidney were taken out and washed out the blood and other material by NS, blotted up the NS by filter and abraded them into homogenate by Phosphate buffer saline, determined by HPLC method described as above.4. The study of plasma protein-binding of Z-GP-Dox in vitroThe plasma protein-binding rate of Z-GP-Dox was tested at the plasma concentration of 0.5μg/mL, 1μg/mL,2μg/mL,4μg/mL,8μg/mL with the method of Equilibrium dialysis. The analysis method and detective condition of Z-GP-Dox in dialyzed solution was the same as the plasma samples.RESULTS AND CONCLUSION1. The establishment of analytical method of Z-GP-Dox in biological matrixA simple, specific and highly sensitive method using reversed--Phase liquid chromatography has been developed for the quantltative analysis of Z-GP-Dox in rat plamas and mouse tissues. Z-GP-Dox were extracted by the extraction which is constituted by acetonitrile-methylene dichloride (1:4) from biological samples to eliminate proteins and others substance. Extraction was evaporated by Nitrogen blow instrument. The residue is dissolved by methanol. The sample was chromatographed on a Ultimate AQ-C18 cloumn((4.6×250mm,5μm, Welch.cn).The mobile phase of gradient elution consisted of acetonitrile--0.1%TFA was run at flow rate of lml/min for different biological matrix,the temperature is 25℃, the quantity of injection was 20μL by 717 sample auto injector with 515 pump.2475 detector was used and the excitation spectrum is 238nm,the emission spectrum is 554nm. The internal standard substance is DNR. Methodology validation showed that the recovery rate of the biology sample of Z-GP-Dox is 83% with a precision of 12%. No notable interference was observed in the chromatograms acquired from biological sample in this detection, suggesting that the established analysis method in this experiment is consistent with the requirement by the guideline of New Drug (western drug) Preclinical Research and basically suitable for the non-clinical pharmacokinetic study of Z-GP-Dox.2. The detection of the drug concentration and acquisition of primary pharmacokinetics parameters of Z-GP-Dox in rat in vivoAs what the result showed, within a dosage ranging from 10 to 20mg/kg, Z-GP-Dox was consistent with the linear kinetics characteristic. Following a single i.v. administration with different dosage (20mg/kg,10mg/kg) of Z-GP-Dox to rat, the C3min and AUC0-of Z-GP-Dox in rat blood circulation was increasing as the increase of the dosage. The C3m is 5.63mg/L, 19.56mg/L respectively; AUC0-t is 1.8mg/L×h,7.5mg/L×h respectively; K,Vd,t1/2,MRT made no significant change with the increase of the dosage. K is 0.37,0.43 respectively; Vd is 4.93L/kg,4.1L/kg respectively;t1/2 is 1.9h,1.65h respectively; MRT is 0.84h,1.3h respectively. However, as to the low dosage of 5mg/kg, because of the low blood concentration, we can hardly get the detected points. What's more, because of the great deviation and error at the low concentration, the detected data could not be taken into calculation.Meanwhile, according to the retain time (about 4min) for Dox under the detection condition of this experiment, we conducted analysis on Dox in plasma sample collected at different time point after high, middle and low dosage administration and no Dox peak was observed.3. The tissue distribution features of Z-GP-Dox in miceThis experiment also learned the distribution of Z-GP-Dox in mice tissues such as heart, liver, spleen, lung and kidney. The result showed that Z-GP-Dox could be distributed in those tissues as quickly as possible. Within 10～180min after administration, the quantity of Z-GP-Dox in lung per gram is the most of all tissue detected. Notably, the distribution of Z-GP-Dox in heart, which was the primary target organ of toxicity for Dox, was obviously reduced. Moreover, in this study, Dox could not be detected in during 10-180min respectively in plasma, lung, spleen, kidney and heart, suggesting that Z-GP-Dox was stable in those tissues. Besides, since 1h after the administration, an additional chromatographic peak was observed in both liver and kidney at the retain time of 13min, which required further identification. However, according to the retain time, this peak could not represent Dox (4min), but probably a suspected metabolite in liver and kidney of mice. Basing on this, we speculated that the liver and kidney might be the key metabolic organ of this candidate compound.4. The determination of the rate for plasma protein-binding of Z-GP-DoxUnder the detecting condition of this experiment, the rat plasma protein-binding of Z-GP-Dox is about 85% at the drug plasma concentration of 8μg/mL with the method of Equilibrium dialysis.