| In the preliminary study of the project,they had prepared10-hydroxycamptothecine-Tetrandrine complexes liposome?Lp-10-HCPT-TET?by improving the form and combination of the drug to overcome the shortcomings of 10-HCPT,such as poor water solubility,bad stability,poor targeting and easy resistant.And clinical use of hydroxycamptothecin injection?M-HCPT?metabolism is fast.In addition,the preparation process of liposome,preparation method and anti-tumor effect in vivo are studied.In this study,the preparation process of Lp-10-HCPT-TET was further optimized.The pharmacokinetics and tissue distributions of Lp-10-HCPT-TET were studied by high performance liquid chromatography-tandem mass spectrometry?UPLC-MS?.And regulatlons of the absorption,distribution and elimination in vivo were studied.Lp-10-HCPT-TET provides a reference for the safety of clinical medication and drug surveillance.In this paper,we mainly carry out in-depth research on Lp-10-HCPT-TET from the aspects of dosage form optimization,pharmacokinetics and tissue distribution of drugs in vivo.The specific contents are as follows:?1?The analysis method of an efficient and sensitive liquid chromatography tandem triple quadrupole mass spectrometer?LC-QQQ-MS/MS?was established to study pharrmacokinetics of the 10-HCPT and TET in Lp-10-HCPT-TET in vivo.The tail vein of SD rats was injected with Lp-10-HCPT-TET?0.5mg/kg?,and eyelid blood was collected.The samples were pretreated by direct protein precipitation with methanol as the precipitating solvent.The detection of 10-HCPT and TET was performed by Isocratic elution.The C18 ultra high performance liquid chromatography column?2.1 mm x 100 mm,2.1 m?is used.The mobile phase was composed of10mm ammonium acetate solution?A?and acetonitrile?B?,with A ratio of10:90 and A flow rate of 0.5 mL·min-1.Camptothecin was as an internal standard?IS?.Cationic scanning multi-response monitoring mode was adopted,m/z 365.1 321.0 of 10-HCPT,m/z 623.2 280.9 of TET,m/z 349.2305.1 of CPT.The time curves of the mean blood concentration of 10-HCPT and TET in the 10-HCPT-TET group and the 10-HCPT-TET group were calculated,and the relevant pharmacokinetic parameters were calculated.The results show that the established liquid chromatography tandem triple quadrupole mass spectrometry?LC-QQQ-MS/MS?was high sensitivity and specificity.The linear ranges in 10-HCPT and TET were 0.55-309ng·mL-1 and 0.72-194 ng·mL-1,respectively;the LLOQ of 10-HCPT and TET were 0.2 ng·mL-11 and 0.5 ng·mL-1,respectively.The intra-and inter-day precision and accuracy of all QC samples in rat plasma were below 15%.The average recovery of 10-HCPT was 98.12%,and the average recovery of TET was 97.26%.The matrix effect is not obvious.This method was fully validated and successfully applied to compare the pharmacokinetics of the original dosage form and complexes liposome.The results showed that10-HCPT was rapidly eliminated in the original dosage form and could be rapidly absorbed within 1h,and the main pharmacokinetic parameters showed significant differences?P<0.05?.According to the pharmacokinetic parameters of MRT,T1/2 and CL,the Lp-10-HCPT-TET prolongs the retention and circulation time of 10-HCPT in vivo,achieving a good sustained release effect.To our knowledge,the pharmacokinetic properties of Lp-10-HCPT-TET in rats have not been reported yet.Thus,the developed method is providing guidance for pharmacokinetic studies of related drugs.?2?LC-MS method was established for the detection of 10-HCPT and TET in various tissues,and methodological validation was conducted to study the tissue distribution of Lp-10-HCPT-TET in vivo.Lp-10-HCPT-TET,10-HCPT injection and tetrandrine solution were injected into the tail vein at 3 mg/kg respectively.The cervical vertebrae of the mice were dislocated at different time points,and the heart,liver,spleen,lung and kidney were extracted.The tissues were homogenized and pre-treated with a one-step protein precipitation method to prepare a homogenate solution for each tissue.High-efficiency and sensitive liquid chromatograph series triple quadrupole mass spectrometer?LC-QQQ-MS/MS?was used as a detection method.Ultra-high performance liquid chromatography column?2.1mm×100mm,2.1?m?is used.10mM acetic acid ammonium solution and acetonitrile were mobile phases and eluted isocratically.The positive ion scanning mode was used to determine the tissue distribution of 10-HCPT and TET in Lp-10-HCPT-TET.Methodological studies are required to meet relevant requirements such as specificity,standard curve and limit of quantification,precision,accuracy,extraction efficiency and matrix effects.Tissue distribution results showed that the complexes liposome group and injection control group had more 10-hydroxycamptothecin distributed in liver.However,the AUC-and MRT levels of 10-hydroxycamptothecin in the complexes liposome group were significantly higher than those in the injection group?P<0.01?.Compared with the distribution of anti-alkali injection in other organs,the compound liposome group also increased the distribution of liver and spleen,significantly reduced the distribution of kidney and lung,and the AUC-value of liver was significantly higher than those injection group?P<0.01?.The results showed that the compound liposome of 10-hydroxycamptothecin powder and tetrandrine increased the concentration of 10-hydroxycamptothecin in liver tissue,increased the accumulation of drugs,prolonged the action time of drugs,and the alkaline powder in liver tissue.Its distribution is also conducive to giving play to its anti-tumor multi-drug resistance,further improving the efficacy of drug treatment for liver tumors,reducing drug toxicity,delaying renal excretion,and achieving therapeutic effects.This study laid a good foundation for further pharmacodynamic studies of anti-tumor in vivo.?3?On the basis of the previous topic,10-HCPT and TET were encapsulated into the liposomes by membrane dispersion method.Tween 80and oleic acid were added and co-hydrated by the nano particle size analyzer.The encapsulation rate of Lp-10-HCPT-TET was determined by high performance liquid chromatography-high speed centrifugation.The results showed that the addition of 10 mg of oleic acid was most suitable for the particle size of Lp-10-HCPT-TET,with an average particle size of about120 nm and a potential value of-21.5 mV.When Tween was added,the particle size and potential of Lp-10-HCPT-TET did not change much.The encapsulation efficiency of 10-HCPT and TET was 90%and 87%,respectively. |
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