The Preparation Of Recombinant N-terminal Heparin-binding Domain Polypeptide Of Fibronectin And The Pharmacokinetics Study In Rat

Fibronectin(FN)is known to be a large multifunction glycoprotein with binding sites for many substances, including heparin, collagen, fibrinogen, fibrin and integrin cell surface receptors. It is involved in a variety of cellular processes, including tissue repair, blood clotting and cell migration or adhesion. There are many structural domains in FN molecule, and also contain two heparin-binding domains, they are N-terminal heparin-binding domain and C-terminal heparin-binding domain. Most studies suggested the synthetic peptides derived from heparin-binding domain of FN have the effects in many aspects, Such as: heparin-binding domain polypeptides of FN can enhance the biological activity of vascular endothelial growth factor(VEGF)by a singular growth factor; heparin-binding domain polypeptides of FN enhance activity in cell adhesion, inhibit hepatocarcinoma and tumor metastasis in liver; heparin-binding domain polypeptides of FN regulate apoptosis by suppression of p53 and c-myc in human hematopoietic progenitors, and it also bind lymphocyte CD44; heparin-binding domain polypeptides of FN can modulates tissue plasminogen activator to catalyze plasminogen activation. Dr Zou Qilian and Wu yong of our laboratory had constructed the recombinant yeast expressed fungus GS115 with the N-terminal heparin-binding domain polypeptid,also expressed and purified the recombinant N-terminal heparin-binding domain polypeptide (FNNHBDPP). Our previous studies indicated FNNHBDPP could be used in the treatment of sepsis and disseminated intravascular coagulation(DIC)in mice, also can inhibit the growth of melanoma and hepatocarcinoma in tumor invasion and metastasis. Therefore, we will investigat the distribution, metabolism and excretion of FNNHBDPP in rat, and get important message from this researching.In this study, we expressed FNNHBDPP with automatic fermenter, and purified HNNHBDPP successfully. We used 125I-FNNHBDPP as isotope tracer and HPLC-RAD as analytical tool to study the pharmacokinetics of FNNHBDPP in rat. Experimental result show: 1 The purity of FNNHBDPP is more than 98 percent in SDS-PAGE and HPLC; 2 Two compartment model can be used to explain the process of FNNHBDPP in the rat model, the absorption half life(T1/2α) of FNNHBDPP in rat,high dosage is 6.28min, middle dosage is 6.23min, low dosage is 5.55min. The eliminate half life(T1/2β) of FNNHBDPP in rat, high dosage is 94.37min, middle dosage is 89.49min, low dosage is 82.22min. A better linear correlation between AUC and dosage (0.993). About 74% tracer isotope was out in urine in144h, and about 6.3% tracer isotope was out in excrement in same time, about 6.5% tracer isotope was out in bile in 72h. The distribution of FNNHBDPP in mouse: the radioactivity in tissue at 5min is kidney> lung> liver> spleen> stomach> heart> bone> bowel> testis> muscle> brain; the radioactivity in tissue at 60min is: lung> kidney> liver> spleen> stomach> bone> heart> bowel> testis> muscle> brain. After 8 hours, the radioactivity of all tissue is lower than average except lung. The study demonstrated FNNHBDPP is mainly distributed in organs which have rich blood supply, such as kidney, lung, liver and spleen, and FNNHBDPP can not penetrate blood-brain barrier. We also found a short-term aggregation in lung from metabolic fragment of FNNHBDPP, which could be eliminated eventually.