Experimental And Clinical Research On Sustained-release 5-FU Microparticle Interstitial Chemotherapy Of Primary Hepatic Cancer

Experimental and Clinical Research on Sustained-release 5-FU Microparticle Interstitial Chemotherapy of Primary Hepatic CancerBACKGROUND & OBJECTIVE:Primay hepatic cancer (PHC) is a disease that occurs when the tumor originates in the liver and did not spread from another organ. It is now one of the most common solid tumor in the world and the third leading cause of cancer-related death. More than 500 000 new cases are currently diagnosed yearly, with an age-adjusted worldwide incidence of 10-20 per 100 000 population and its incidence is increasing worldwide because of the dissemination of hepatitis B and C virus infection. In Asia and Africa, hepatocellular cancer is the most common type of malignancy and frequently develops in patients who have liver cirrhosis. Unfortunately, PHC carries a dismal prognosis due to rapid growth rate and nonspecific clinical presentation during the early stages of the disease . The spontaneous survival time of the untreated group is only 8.4 months. Surgical resection is considered to be the curative treatment of patients with PHC. However, only 10-20% of PHC are found to be resectable at the time of diagnosis. In addition recurrence of PHC is frequent even after curative surgical treatment. Chemotherapy holds great theoretical appeal in treating this disease, and systemic chemotherapy has been shown to prolong survival in some patients with PHC . However, orthodox intravenous chemotherapy is also not widely tolerated,because it is effective in some patients but its side effect is relatively more. Thus, the development of effective chemotherapy methods is urgently needed for the control of this disease. Interstitial chemotherapy (IC) is a relatively new concept postulated in last 80's. IC is a relative targeting therapy through which therapeutic agents are locally released from a drug carrying system for an extended period of time while maintaining low systemic drug composure. The drug releasing product could be administered through implantation or injection into the needed areas such as tumor mass, tumor cavity or tumor bed (cavity) left by surgical resection, tumor surrounding tissues, and lymph nodes. The high local drug concentration is particularly useful for killing the surgical inoperable tumors, invisible tumors, isolated tumors cells, and the micro-metastases in lymph nodes.From 1990's, some researchers, including my renowned adviser prof. Wu Mengchao advanced the new concept on interstitial chemotherapy. Li W-X et al. observed the effectiveness and safety of interstitially implanted 5-FU in treating 139 patients with esophageal cancer, and found that the one year recurrence rate after surgical resection was 38.30% (18/47),23.36%(10/43) and 14.29% (7/49) , respectively, in patients treated with surgical resection alone, plus implanted 5-FU (200-400mg) and plus implanted 5-FU (400-800mg) . 800mg 5-FU was still well tolerated. Therefore, the authors believe that interstitial chemotherapy, which could provide the needed area with high drug concentration for long period of time with less systemic side effects, is expected to be a potential and promising choice for treating esophageal cancer.Based on above considerations, Anhui Wuhu Zhongren Pharmaceuticals Co., LTD has developed novel drug sustained releasing implants for treating primay hepatic cancer, including sustained release 5-fluorouracil (ZHONG REN FU AN) . This product could increase drug concentration in tumor local areas by up to thousand folds for up to 4 weeks with only trace amount of systemic exposure. Tumor growth inhibition could be increased by up to 70%-90% as compared to systemic chemotherapy.Although surgical resection is recognized as the first option for the treatment of primay hepatic cancer, recurrence after tumor resection is the main factor affecting negatively the prognosis of the patients. Residual cells are the major reasons causing recurrence leading poor prognosis. After surgery, the immune function of the hosts declines, with the variety of growth factors being simultaneously secreted, which makes residual cancer cells or precancerous lesions actively grow, therefore highly contributing to early cancer recurrence. Therefore, timely and effectively controlling the residual tumor cells appears to be very important. However, surgical trauma greatly decreases the tolerance of the body to conventional chemotherapy. Since 2003 we started implanting or injecting sustained release 5-fluorouracil into the needed areas such as tumor mass, tumor cavity or tumor bed (cavity) left by surgical resection, tumor surrounding tissues, and lymph nodes.The purpose of this study is to confirm the sustained-release 5-FU microparticles which can keep on inhibiting hepatoma carcinoma cell and inducing apoptosis by cell experimental , and to get the proof of interstitial chemotherapy of primary hepatic cancer in hepatectomy by clinical research . METHODS:1. Culture of the hepatoma carcinoma cell (HepG2) with sustained-release 5-FU and detection the growth of HepG2. by MTT(1) Culture of the hepatoma carcinoma cell (HepG2)The hepatoma carcinoma cell (HepG2) was cultured in dulbecco's modified eagle's medium(DMEM) which immersed with sustained-release 5-FU for Od, 1d, 7d, 14d, 21d and 28d.(2)Detection of the growth of HepG2 by inverted phase contrast microscope.Detection of the growth of HepG2 by inverted phase contrast microscope after 72h.(2)detection of the growth of HepG2 by MTT.Detection of the growth of HepG2 by inverted phase contrast microscope after 72h cultured in MTT.2. the cell apoptosis induced by sustained-release 5-FU(1) Hochest stainingCulture of the hepatoma carcinoma cell (HepG2) after Hochest staining and detection of the cell apoptosis .(2) flow cytometryCulture of the hepatoma carcinoma cell (HepG2 )and detection of the cell apoptosis by flow cytometry.3. evaluation of the efficacy of intraoperative sustained-release 5-FU chemotherapy300 HCC patients underwent hepatectomy from JAN 2003 to JUL 2004. All patients were randomly divided into control group and interstitial chemotherapy group according to random number table. In the control group, 150 patients were treated with hepatectomy alone, whereas in the interstitial chemotherapy group, 150 patients were treated with hepatectomy plus implanting sustained-release 5-FU microparticle on surgical margin. All patients were followed up regularly.RESULTS:1. Culture of the hepatoma carcinoma cell (HepG2) with sustained-release 5-FU anddetection the growth of HepG2 by MTT(1) The hepatoma carcinoma cell (HepG2) with sustained-release 5-FU turned into rounding, blunting, shrinking with karyopyknosis , shedding from the adherence, and suspending in the culture medium in time-dependent manner .But the control group was normal.(2) The survival rate of cells was determined by MTT assay. The survival rate of 0d, 1d, 7d, 14d, 21d and 28d was 99.5%±8.2%, 78.3%±6.2%, 43.2%±4.5%, 26.3%±3.2%, 14.7%±2.2% and 11.6%±1.5% in the interstitial chemotherapy group, respectively. So 5-FU microparticles could keep on releasing during 28days and inhibiting hepatoma carcinoma cell in time-dependent manner.2 The cell apoptosis induced by sustained-release 5-FU(1) The more time the sustained-release 5-FU released, the more cells were induced to apoptosis observated by Hochest staining. So the sustained-release 5-FU could keep on inducing hepatoma carcinoma cell to apoptosis in time-dependent manner.(2)The 0d,1d, 7d, 14d, 21d and 28d early apoptosis ratio was 0.0%, 1.19%,2.26% , 3.69%, 4.92% and 17.92% in the interstitial chemotherapy group investigated by flow cytometry, respectively. The late apoptosis cell rate was 0.10%,1.33%,3.77% , 4.95%, 6.27% and 9.35%, the total apoptosis ratio was 0.10%,2.52%,6.03% , 8.64%, 11.17% and 27.27%. The sustained-release 5-FU could keep on inducing hepatoma carcinoma cell to apoptosis in time-dependent manner.3. Evaluation of the efficacy of intraoperative sustained-release 5-FU chemotherapy The 2-, 3- and 4-, 5-year recurrent rate was 14.0%,23.3%,32.0% and 43.3% in the interstitial chemotherapy group versus 23.3%,34.7% ,47.3%and 59.3% in the control group, respectively, with a difference in the 2-, 3-year recurrent rates (0.01