The Early Antitumor Drugability Research Of A Marine Fungal Source Anthracycline Compound Aspergiolide A

Anti-tumor antibiotics derived from microbial metabolites has been an outstanding representative of the anticancer drug. the Doxorcubicin and its derivatives as the representative of the anthracycline anticancer drugs in clinical practice for the treatment of solid tumors and leukemia. But these drugs cumulative cardiac toxicity and other side effects limit its efficacy. The development of reduced toxicity while retaining its antitumor activity has been the research focus of this class of compounds. The Aspergiolide A(ASP-A) is obtained from the marine mangrove roots mud fungi Aspergillus glaucus HB1-19, it has a novel structure with independent intellectual property rights and is a anthracycline lactone.In this paper, the antitumor drugability of ASP-A is evaluated preliminary, including the following aspects:1, in vitro and in vivo anti-tumor activity;2, HPLC method formethod for detection of ASP-A and pharmacokinetic inspection;3, the tissue distribution of ASP-A in mices;4, the absorption way of ASP-A in vitro;5,early safety evaluation.Part1The anti-tumor activity of ASP-A in vitro and in vivo1. MTT assay is applied to measure the proliferation inhibition of ASP-A on variety of tumor cells.The results show that the ASP-A can inhibit cell proliferate in Hela,SMMC-7721, SGC-7901and other cells,the IC50is in the2～7.07μM level, ASP-A has broad spectrum of cytotoxic activity in vitro.2. Western Blotting is applied to detect the apoptotic proteins expression in human hepatoma cell line BEL-7402when ASP-A is at different concentrations (2.5μM,5μM,10μM).BEL-7402is roled by different concentrations of ASP-A for12hours, compared with vehicle group,Caspase-9,Caspase-3,Caspase-8and PARP cleaved with varying degrees.Bax, γ-H2AX expression increased with the rise of the ASP-A doses. Those results indicated that ASP-A can cause cellular DNA broken, and induce human hepatoma cells apoptosis.3. In the mouse liver cancer model, there are six groups:a blank group, the vehicle group, the ASP-A groups(5mg/kg,15mg/kg,45mg/kg),doxorubicin group(2mg/kg),intraperitoneal injection, daily administration for consecutive8days. Experimental results showed that the ASP-A high, medium and low doses inhibition rates were66%,72%and55.4%, ASP-A can significantly inhibit the growth of H22xenograft tumors, have a good anti-tumor activity and less impact on the body weight of mice.4.In human hepatocellular carcinoma xenografts in nude mices, administration of the ASP-A concentration7mg/kg,14mg/kg,28mg/kg, intraperitoneal injection administration21days. The results showed that the inhibition rate were40.1%,48.8%and51.2%,and had no significant effect on body weight.Part2The studies of ASP-A on safety assessment, pharmacokinetics and drug distribution.1. The samples were analyzed by HPLC with internal standard.the method is selective extraction recovery, precision and quantitative linear range were in line with the specification requirements at home and abroad.2. tumor-bearing mices by intraperitoneal injection ASP-A with two-doses (15mg/kg and30mg/kg) at different time points, blood samples was analysed by HPLC,3P97software related to the pharmacokinetic parameters calculated. The results showed that the ASP-A metabolism in plasma was two-compartment model. In two concentrations,the pharmacokinetic parameters were:t1/2β(min):42.70,47.61; Tpeak(min):15.33,15.88; Cmax(mg/L):20.96,39.50; CL(L·kg-1·min-1):0.011,0.012; AUC(mg·min·L-1):1359.41,2493.94.3. Tumor-bearing mices by intraperitoneal ASP-A two-dose (15mg/kg and30mg/kg), take tissue samples at different time points to examine the distribution of drugs in mice tissues. The experimental results showed that ASP-A mainly located in the heart, kidneys and liver.4. The MTD of administration in mices was400mg/kg.5. Manual patch clamp technique detected ASP-A inhibition of the hERG channel, the rate of inhibition of the hERG current were0.9±1.6%,2.0±2.3%,3.8±4.0%and10.1±5.9%, there is no obvious inhibition.6. Caco-2monolayers were investigated in vitro uptake and transfer of ASP-A. The ASP-A was dose-dependent and time-dependent uptake in Caco-2monolayer model;Caco-2monolayer cell transfer model established in this subject was in line with relevant standards, the experimental results of ASP-A transition on the model was active transport.