Anti-Inflammatory Activity Of Fatty Acid-Modified Camptothecin-Amino Acid Prodrugs

Sepsis is a life-threatening organ dysfunction syndrome caused by a dysregulated host response to infection.The cytokine storm in the hyperinflammatory phase of sepsis can lead to multiple organ dysfunction and even body death.At present,the development of drugs for the treatment of sepsis is still facing great challenges.Studies have shown that Camptothecin(CPT)can reduce inflammatory response by inhibiting the activity of topoisomerase I(Top1).However,poor solubility,high toxicity to non-target cells,and low in vivo activity limit the clinical application of CPT.A large number of studies have shown that construction of CPT prodrugs is one of the effective strategies to improve its therapeutic activity.L-type amino acids have good biocompatibility and can greatly improve the solubility of parent drugs,so they are widely used in the study of prodrugs.Octadecanoic acid(octadecanoic acid)is often used to modify drugs to improve their pharmacokinetic characteristics and enhance their therapeutic activity.Therefore,in this study,five L-type amino acids(glutamic acid,arginine,lysine,serine and valine)and octadecanoic acid were coupled to CPT to construct six prodrug compounds-CPTL-7,CPTL-8,CPTL-9,CPTL-10,CPTL-13 and CPTL-14.The anti-inflammatory activity of fatty acid-modified camptothecin-amino acid prodrugs was investigated from three aspects: physical and chemical properties,in vivo and in vitro experiments.After coupling amino acid and octadecanoic acid,the solubility of the analog was improved compared with that of CPT.The solubility of CPTL-9 with lysine and CPTL-10 with serine was increased by 4-and 7-fold,respectively,compared to CPT.MTT assay showed that the cytotoxicity of all the analogues was lower than that of CPT on mouse monocytic macrophage leukemia cells(RAW 264.7).The cytotoxicity of CPTL-7 and CPTL-9 with glutamate was decreased by 8.4 times and 2 times,respectively.In addition,the analogues could effectively inhibit the production of Nitric oxide(NO)in Lipopolysaccharide(LPS)stimulated RAW 264.7.In addition,CPTL-7 and CPTL-9significantly inhibited the expression of IL-1β,IL-6 and TNF-α in LPS-stimulated RAW264.7 cells.The results of the Maximum tolerated dose(MTD)test showed that in addition to the higher toxicity of CPT(MTD of 12 mg/kg)and CPTL-10(MTD of 16mg/kg),The MTD of the other analogues were all higher than 32 mg/kg,indicating that the combined modification of fatty acids and amino acids can reduce the in vivo toxicity of CPT.In the LPS-induced sepsis mouse model,CPTL-7 and CPTL-9 could significantly improve the survival rate of septic mice at a dose of 1 mg/kg,while other analogues did not show significant therapeutic activity.In addition,histopathological sections showed that CPTL-7 and CPTL-9 could significantly reduce the tissue and organ damage induced by LPS in mice.Moreover,CPTL-9 significantly reduced the protein levels of IL-6 and TNF-α in plasma.After optimizing the dose and frequency of administration,although the expected therapeutic effect was not achieved,CPTL-7 and CPTL-9 still significantly improved the survival rate of septic mice.In conclusion,the present study found that the combined modification of fatty acids and amino acids could significantly reduce the in vivo toxicity of CPT.Although all analogs showed some anti-inflammatory activity in vitro,only CPTL-7 and CPTL-9showed significantly increased anti-inflammatory activity in vivo compared with CPT.It is possible that different pharmacokinetic profiles influence the in vivo anti-inflammatory activity of different analogues,which needs to be further demonstrated.Nonetheless,the lower in vivo toxicity and higher in vivo anti-inflammatory activity could make CPTL-7and CPTL-9 potential compounds for the treatment of sepsis.In addition,this study provides a novel modification strategy for the development of novel drugs for the treatment of sepsis.Sepsis is a life-threatening organ dysfunction syndrome caused by a dysregulated host response to infection.The cytokine storm in the hyperinflammatory phase of sepsis can lead to multiple organ dysfunction and even body death.At present,the development of drugs for the treatment of sepsis is still facing great challenges.Studies have shown that Camptothecin(CPT)can reduce inflammatory response by inhibiting the activity of topoisomerase I(Top1).However,poor solubility,high toxicity to non-target cells,and low in vivo activity limit the clinical application of CPT.A large number of studies have shown that construction of CPT prodrugs is one of the effective strategies to improve its therapeutic activity.L-type amino acids have good biocompatibility and can greatly improve the solubility of parent drugs,so they are widely used in the study of prodrugs.Octadecanoic acid(octadecanoic acid)is often used to modify drugs to improve their pharmacokinetic characteristics and enhance their therapeutic activity.Therefore,in this study,five L-type amino acids(glutamic acid,arginine,lysine,serine and valine)and octadecanoic acid were coupled to CPT to construct six prodrug compounds-CPTL-7,CPTL-8,CPTL-9,CPTL-10,CPTL-13 and CPTL-14.The anti-inflammatory activity of fatty acid-modified camptothecin-amino acid prodrugs was investigated from three aspects: physical and chemical properties,in vivo and in vitro experiments.After coupling amino acid and octadecanoic acid,the solubility of the analog was improved compared with that of CPT.The solubility of CPTL-9 with lysine and CPTL-10 with serine was increased by 4-and 7-fold,respectively,compared to CPT.MTT assay showed that the cytotoxicity of all the analogues was lower than that of CPT on mouse monocytic macrophage leukemia cells(RAW 264.7).The cytotoxicity of CPTL-7 and CPTL-9 with glutamate was decreased by 8.4 times and 2 times,respectively.In addition,the analogues could effectively inhibit the production of Nitric oxide(NO)in Lipopolysaccharide(LPS)stimulated RAW 264.7.In addition,CPTL-7 and CPTL-9significantly inhibited the expression of IL-1β,IL-6 and TNF-α in LPS-stimulated RAW264.7 cells.The results of the Maximum tolerated dose(MTD)test showed that in addition to the higher toxicity of CPT(MTD of 12 mg/kg)and CPTL-10(MTD of 16mg/kg),The MTD of the other analogues were all higher than 32 mg/kg,indicating that the combined modification of fatty acids and amino acids can reduce the in vivo toxicity of CPT.In the LPS-induced sepsis mouse model,CPTL-7 and CPTL-9 could significantly improve the survival rate of septic mice at a dose of 1 mg/kg,while other analogues did not show significant therapeutic activity.In addition,histopathological sections showed that CPTL-7 and CPTL-9 could significantly reduce the tissue and organ damage induced by LPS in mice.Moreover,CPTL-9 significantly reduced the protein levels of IL-6 and TNF-α in plasma.After optimizing the dose and frequency of administration,although the expected therapeutic effect was not achieved,CPTL-7 and CPTL-9 still significantly improved the survival rate of septic mice.In conclusion,the present study found that the combined modification of fatty acids and amino acids could significantly reduce the in vivo toxicity of CPT.Although all analogs showed some anti-inflammatory activity in vitro,only CPTL-7 and CPTL-9showed significantly increased anti-inflammatory activity in vivo compared with CPT.It is possible that different pharmacokinetic profiles influence the in vivo anti-inflammatory activity of different analogues,which needs to be further demonstrated.Nonetheless,the lower in vivo toxicity and higher in vivo anti-inflammatory activity could make CPTL-7and CPTL-9 potential compounds for the treatment of sepsis.In addition,this study provides a novel modification strategy for the development of novel drugs for the treatment of sepsis.