Study On Pharmacokinetics Of 9-nitrocamptothecin-20-O-Propionate And Apoptotic-induced Mechanisms Of 9-Nitrocamptothecin-20-O-Propionate

Camptothecin(CPT)is a natural alkaloid isolated from Camptotheca acuminata.CPT has been widespread concern due to its strong anti-tumor activity.9-nitrocamptothecin-20-O-propionate(CZ112)and 9-nitrocamptothecin(9NC)are bioactive derivatives of CPT.CZ112 is the esterified product of 9NC,by stabilizing the structure of the lactone ring to reduce its toxicity.The purpose of this study is to investigate CZ112 and 9NC induced apoptosis in NCI-H520 cells and their pharmacokinetics.9NC was identified as the major metabolite of CZ112 in rat plasma by HPLC/photodiode array detection(PDA)and LC-MS/MS analysis.An LC-MS/MS method was developed to quantitatively determine CZ112 and 9NC in rat plasma,tissues and different excretion.This method was also applied to the pharmacokinetic study of CZ112 and its metabolite 9NC in rat after intravenous(i.v.)and intragastric(i.g.)administration.After i.v.administration,CZ112 and 9NC were metabolized rapidly in rat plasma.After i.g.administration,the T1/2 and MRT of CZ112 were shorter than 9NC,indicating that metabolism of CZ112 was slightly faster than 9NC,and the retention time of 9NC in the plasma was longer.Oral bioavailability of CZ112 was low,presumably due to its poor water solubility caused.The tissue distribution of CZ112 and 9NC were determined for rats after i.v.and i.g.administration.After i.v.administration,CZ112 was detected in all tissues tested,for 9NC,only in the heart,spleen and lung tissue can be detected.After i.g.administration,other tissues except brain tissue were able to detect CZ112,for 9NC,only in the heart and lung tissue can be detected.CZ112 and 9NC had a significant accumulation in the lung tissue of rats,suggesting that they had lung targeting,whether it was i.v.or i.g.administration.The accumulation of CZ112 and 9NC in excretion pathway was investigated.After i.v.administration,cumulative excretion of CZ112 and 9NC was 24.17%of the dose in the bile,0.22%in the urine and 0.06%in the feces.After i.g.administration,cumulative excretion of CZ112 and 9NC was 0.05%of the dose in the bile,0.02%in the urine and 6.13%in the feces.In both modes of administration,the excretion of CZ112 and 9NC was small,indicating that most of the drugs were excluded in other forms or by other means.In this study,NCI-H520 cells were used to study the mechanism of CZ112 and 9NC-induced apoptosis of lung squamous cell carcinoma.MTT results showed that CZ112 and 9NC had a significant inhibitory effect on the growth of NCI-H520 cells.Annexin V-FITC and PI double staining showed that CZ112 and 9NC could induce apoptosis of NCI-H520 cells.PI staining showed that the percentage of G0/G1 phase in CZ112 and 9NC treatment group increased significantly,and the percentage of G2/M phase decreased significantly,and both were concentration-dependent.Western blot analysis showed that CZ112 and 9NC could downregulate the expression of CDK2,CDK4,CDK6,Cyclin Dl,PI3K(p110),PI3K(p85)and p-Akt protein in different degree.Therefore,the mechanism of CZ112 and 9NC induced apoptosis in NCI-H520 cells may be as follow:CZ112 and 9NC activated PI3K/Akt signaling pathway,and then PI3K/Akt activated its downstream cell cycle,which induces G0/G1 arrest in NCI-H520 cells,eventually leading to apoptosis.