Preparation And Application Of Harmine In Situ Gel

Object: Colorectal cancer is a common malignant tumor that seriously endangers human health.Colorectal cancer refers to a cancerous change that occurs in the colon or rectum,most commonly in the sigmoid colon and rectum.Globally,the incidence of colorectal cancer is the third highest in all malignancies,with a higher incidence among male than among female.The lack of specific symptoms in the early stage of colorectal cancer makes it easy for patients to ignore it.Harmine(HM),a uyghur medicine,has been proved to have a significant inhibitory effect on colorectal cancer.However,HM can cause central nervous system toxicity when systemic injection,which limits its clinical application.In this study,we prepared HM in situ thermosensitive gel(HM gel)for local administration of colorectal region,and evaluated its efficacy in the treatment of colorectal cancer in vivo and in vitro.Methods:(1)The content of HM was determined by high-phase liquid chromatography(HPLC).(2)We used a single factor experimental design to investigate the influence of the dosage of HM,benzalkonium bromide,Poloxamer 407(P407)and Poloxamer 188(P188)on the gel temperature.Then the HM gel formula was optimized by central composite design-response surface methodology to select the optimal dosage of P407,P188 and hydroxypropyl methyl cellulose(HPMC).(3)The optimal formulation was determined by detecting the viscosity,rectal retention and the in vitro release of HM gel.(4)The results of in vitro application: the inhibitory effect of HM gel on proliferation of colon cancer CT26 cells was investigated by CCK-8 method;the percentage of apoptotic cells induced by HM gel was determined by Annexin V-FITC /PI double staining;the ability of HM gel to inhibit CT26 cell migration was investigated by scratch method.(5)The results of in vivo application: CT26 cells were injected into the rectum of BALB/c mice through the rectal mucosa to establish a mouse model of colorectal cancer;and the anti-tumor effect of the drug in vivo was evaluated through tumor volume,pathological sections of lung tissue,tumor apoptosis and survival rate.Results:(1)The results of methodological verification for the determination of HM by HPLC showed that the method had good linear relationship within the range of 2.5-200 ?g/m L and its precision and recovery met the requirements.(2)The single-factor experiments showed that the benzalkonium bromide and HM had no significant effect on gelation temperature;but with the increase of P407 concentration,gelation temperature gradually decreased;and the gelling temperature was gradually rising with the increase of P188 concentration.The results of central composite design-response surface methodology were consistent with the results of single factor investigation,and HPMC had little influence on the gel temperature.Results of rectum retention showed that the in situ gel rectum HM did not leak after the treatment in rats,and could remain in vivo for more than 6 h.The release of HM gel in vitro was consistent with the Weibull model.The optimal formulation was finally determined as HM 1.2%,HPMC 0.93%,P188 2.13%,P4020.7%,benzalkonium bromide 0.02%.(3)The results of in vitro application showed that the HM gel had a significant inhibitory effect on the proliferation of CT26 cells;HM gel could induce thapoptosis of CT26 cells;the proportion of late apoptotic cells in HM gel group was 19.11% ±1.61%;and HM gel could inhibit the lateral migration of CT26 cells.(4)The results of in vivapplication of mice showed that tumor weights,tumor metastases in lung tissue,the apoptotiareas and survival rates of mice in HM gel group were better than those in the control group,thblank gel group and HM pure group.Conclusion:(1)The established method for the determination of HM content conformed to the experimental requirements.(2)The HM gel had good physical and chemical properties;and the process design of HM gel preparation was reasonable.(3)The results of in vitro and in vivo application showed that HM gel had a significant inhibitory effect on the growth and metastasis of colorectal cancer.The anti-tumor effect in vivo of HM gel group was better than that of HM pure group,and the toxicity of HM was reduced to a certain extent.In summary,HM gel had significant anti-tumor effect on colorectal cancer,which is expected to be a promising new drug delivery preparation for the treatment of colorectal cancer.