Interconvention Of Lactone And Carboxylate 9-notrocamptothecin And Research Of Its Antitumor Effect

Camptothecin and its derivatives play an important role in the treatment of malignancies.It is recognized at home and abroad that the intact lactone ring is the critical structural feature with respect to the antitumor activity.Carboxylate camptothecin has only one-tenth the antitumor potency of the lactone camptothecin.Therefore,in order to administratered with 100%lactone ratio of it,the design principle of camptothecin and its derivatives is to protect the stability of lactone form as much as possible.9-Nitrocamptothecin(9-NC),a new derivative of camptothecin,has demonstrated potent anti-tumor effects.The lactone ring is the critical structural feature with respect to the antitumor activity,but the lactone ring is easy to be affected by many foctors,such as pH,plasma albumin binding.The efficacy will be seriously affected.The interconvention of lactone and carboxylate 9-notrocamptothecin in vivo is the key point to the development of a novel formulation.But the lactone/carboxylate balance in vivo has not been reperted.The purpose of this article is to determine the optimal lactone proportion of 9-NC by investigating the pharmacokinetics,tissue distribution,toxicity and pharmacodynamics.Therefore,100%,75%,50%,25%,0%lactone ratios of 9-NC solutions were prepared by adjusting the pH,which is the important factor to affect the interconversion of lactone and carboxylate 9-notrocamptothecin.An HPLC method was established for the simultaneous determination of 9-NC lactone and total forms in rat plasma.The mobile phase condition is pH6.5.The method is simple,fast,high accuracy,and can meet the needs of drug analysis in vivo.The equilibrium of different lactone ratios of 9-NC solutions between lactone form and carboxylate form in pH7.4 buffer solutions,rats plasma and whole blood has been measured.The results showed that the speed of lactone/carboxylate interconversion were different,while the final equilibrium ratio has no correlation with the original lactone ratio of 9-NC.In addition,it was also found that the effects on lactone/carboxylate equilibrium were in the following order:blood cells>plasma albumin>pH.After iv.administration different lactone ratios of 9-NC solutions to rats,the concentrations of lactone and total forms were measured.The results showed that the behavior of total 9-NC is one-room model.The lactone concentrations in plasma were changed with the lactone ratios of the injection,while the total concentrations had no correlation with the lactone ratios of the injection.The AUC values of lactone and total forms were similar between the 75%and 100%lactone ratio groups.But the measured lactone concentrations were reduced in the lower lactone ration(50%,25%and 0%)groups.For a more comprehensive understanding of different lactone ratios of 9-NC in vivo process,the apparent partition coefficients(P)of 9-NC lactone and carboxylate forms were determined by shake-flask method.It was found that the P values of lactone and carboxylate forms were 145.5±2.96 and 0.034±0.26,respectively.HPLC method were established for the simultaneous determination of 9-NC lactone and total forms in heart,liver,spleen,lung,kidney,stomach,small intestine,plasma and tumor in S180 tumor-bearing mice.After iv.administration of different lactone ratios of 9-NC to S180 tumor-bearing mice,the concentrations of lactone and total forms were measured.It can be seen that the lactone has a larger apparent distribution volume compared to the carboxylate,suggesting higher tissue accessibility and binding.It was found that the lactone ratio of 9-NC injection had a significant effect on the lactone concentration in tissues.The measured lactone concentrations in plasma were changed with the lactone ratios of the injection,while the total form concentrations had no correlation with the lactone ratios of the injection.The AUC values of lactone and total forms in tumor were similar between the 75%and 100%lactone ratio groups.But the measured lactone and total forms concentrations were reduced in the lower lactone ration(50%,25%and 0%)groups.There was no significant correlation in kidney between the measured and administrated lactone form,even a negative correlation in liver and small was observed.It might be associated with the fact that carboxylate form was mainly excreted by liver and kidney route and the influence by enterohepatic circulation.The excretion of total 9-NC in urine and bile following intravenous administration of different ratios of 9-NC solutions to rats also were investigated.The results showed that renal excretion is the main way of 9-NC to excrete.The measured 9-NC total concentrations were increased with the reduce of 9-NC lactone ration of the injection.The excretion of total 9-NC in urine following administration of carboxylate form was significantly higher than that of lactone form.The carboxylate form of 9-NC is easier to excrete due to its high polarity.To improve the results,the long-term safety of the five different lactone ratios of 9-NC solutions and its solvent were investigated.The results showed that the mortality is rare in the whole process.However,the results suggested that higher lactone ration presented higher toxicity based on the lower weight and other immune performance.Subsequently,the anti-tumor effect to S180 tumor-bearing mice of the five different ratios of 9-NC were investigated.The results showed that the inhibitory rateof tumor were similar between the 75%and 100%lactone ratio groups.But the inhibitory rate of tumor were reduced in the lower lactone ration(50%,25%and 0%)groups.Based on the immue organ index,liver function index,liver and kidney biopsy,it was found that the toxicity was reduced with the decrease of lactone ration of the injection.lt is sufficient to prove that if the administration lactone proportion between75%to 100%,it can be achieved not only can obtain the same antitumor effect but also can reduce the toxicity.