The Synthesis, Characterization And Activity Research Of A New Carrier Prodrug Hyaluronic Phenylbutyric Ester

Cancer treatment often harms human normal tissues due to the lack of tumor cell-specific identified ability. The research of tumor targeted drug delivery system is aim to improve the target activity and efficacy of drugs, and reduce the toxicity adverse effects. Hyaluronic acid (HA) is a naturally occurred glycosaminoglycan, it has favorable properties such as high viscoelasticity, plasticity, non-immunogenic, biocompatible and biodegradable, and now has become a hotspot of drug delivery system research. HA can not only be used as a macromolecular drug carrier, but can also specifically binds to a variety of receptors on cell surface, one of the most important receptor is CD44 which over expressed on the tumor cell surface. With these features, HA can targetedly deliver anticancer drugs to tumor tissue and tumor cells, improve the effectiveness of drugs effect on tumor cells, reduce the dosage and diminish the adverse reactions. In addition, as a macromolecular carrier, HA can take advantage of the high permeability and retention characters of tumor tissue, being gathered in the tumor tissues, thus promote the drug concentration in target tissues. Phenylbutyric acid (PBA) is an aromatic compound of fatty acid, it is a suitable model drug as can connected to HA by esterifying with hydroxyl group on disaccharide unit. PBA has a broad-spectrum inducibility of tumor cell lines, it promotes the differentiation and maturation of tumor cells towards normal cells through several regulating measures, leads to leukocyte aggregation, and changes tumor immunogenicity. PBA combining with a carrier forms a carrier prodrug that will increase its bioavailability, the carrier prodrug helps PBA to gather around the target spot, gradually be released in the body, achieves the goal of sustained release and targeted drug delivery.This paper chose HA as a drug carrier esterified with anticancer drug PBA, designed a new carrier prodrug hyaluronic-phenylbutyric ester (HA-PB). The main results for this study are as follows.1. This paper introduced for the first time a synthetic route of hyaluronic-phenylbutyrate ester, a new carrier prodrug. This experiment connected activiated PBA to HA via esterified with hydroxy group on C6 of D-glucuronic acid, and got a series of products with different esterification by changing the reaction conditions, three batches of HA-PB,140404,140426,140428, were obtained by changing the ratio of HA to PBA, with good water solubility for follow-up study.2. This paper characterized product HA-PB by IR, NMR, UV methods. The infrared spectrum results showed a new absorption peak at 1735 cm-1 was appeared for HA-PB, indicating the formation of an ester-bond; Total chemical shift assigned according to the 1H-NMR spectral data showed, all of 140404,140426,140428 batch of HA-PB introduced PB successfully which caused the chemical shift in the low field region 87.29 ppm. Compared the integral related to protons attributed to benzene ring of PB, with the integral related to protons attributed to NHCOCH3 belonging to N-acetylglucos-amine residue of HA, the degree of substitution of the three HA-PB samples were 13.2%,21.8% and 34.4% respectively.3. Established an HPLC method to analyse the related substance of HA-PB. Conducted qualitative analysis by comparing the retention time of HA-PB with several relevant substances, and conducted quantitative test by establishing standard curves for each relevant substances, thus figured out the purity of all tested HA-PB were over 99.0%. Then measured the PB content by adding NaOH to HA-PB sample for completely hydrolysis, the degrees of esterification calculated were 17.1%,21.8%, 30.3%, which basically corresponds to the result by NMR method, and had a high reliability.4. Set normal, negative control, positive control and experimental group, observed the morphological changes of mouse melanoma cells (B16) after exposed to the above mentioned samples, it was observed that the inhibiting ability was proportional to the degree of esterification of HA-PB. The inhibitory activity of HA-PB on B16 was studied by MTT assay in vitro, the results showed that half inhibitory concentration (IC50) of 140404,140426 and 140428 batch of HA-PB were 0.199,0.013 and 0.017 mmol/L (PB equivalents) respectively, all of them were lower than the IC50 value of PB:1.277 mmol/L. HA-PB was preliminary judged to have better cancer inhibition ability than the original drug PB.