Synthesis, Characterization, In Vitro And In Vivo Evaluation Of Pegylated Oridonin Conjugates

Oridonin (ORI), a diterpenoid compound isolated from Chinese medicinal herb Rabdosia rubescens, has gained great attentions due to its anti-tumor activities against a number of cancers such as esophageal cancer, primary liver cancer and colorectal carcinoma and so on. Accumulating evidences show that oridonin can hamper the progression of tumor, alleviate syndrome of cancer and mitigate tumor burden. In addition, some research results clearly demonstrate that oridonin has remarkable antileukemia efficacies on acute myeloid leukemia (AML) with low adverse effect, which might provide benefits for patients with AML and widen oridonin clinical applications. Although oridonin has been found to possess promising biological activities, its development and application as a therapeutic agent is limited by its poor aqueous solubility and rapid plasma clearance. Apparently, novel approaches should be explored to solve problems mentioned above.A prodrug strategy can increase the solubility and stability of parent drug molecules, prolong the systemic circulation time and thereby enhance the pharmacological effects. Nowadays, polymeric prodrugs with drug molecules attached to polymer backbones have been getting popular in drug delivery systems. The design of a prodrug may effectively solve the problem of poor solubility and stability of some certain drugs. The drug retention time in the blood circulation could be increased and the in-vivo distribution could be modified by using the polymeric prodrug strategies. It may also improve the bioavailability and reduce the toxicity of parent drugs. In addition, the macromolecular carriers in prodrug molecules may result in an enhanced cellular uptake of parent drugs via a endocytosis process, it may also induce a specific recognition of target cells by receptor-ligand interactions.In the area of polymeric prodrug, polyethyleneglycol (PEG) is considered to be the most useful candidate for the prodrug conjugation due to its high aqueous solubility. The utilization of PEG as a carrier to conjugate to drug molecules of interest is known as PEGylation, which has been well-established in the field of polymer-based delivery. PEG is amphiphilic and soluable in organic solvents as well as in water. The protein or cellular absorption of PEG is the lowest among any known polymers. The USFDA has approved pegylated drugs for human intravenous (i.v.), oral, and dermal applications.This study synthesized a series of conjugates of the natural antineoplastic agent oridonin with different molecular weights of mPEG-NH2. The PEG-SA-ORI conjugates were characterized by FTIR and HPLC using a size exclusion chromatographic (SEC) column. The characteristic absorption of ORI and PEG in the PEG-SA-ORI conjugates indicated the attachment of PEG into ORI. In the SEC analysis of PEG-SA-ORI conjugates and corresponding PEGs, appearance of chromatographic peaks with the ORI UV profiles in the PEG-SA-ORI conjugates also proved the successful synthesis of the PEG-SA-ORI conjugates. Drug contents of PEG-SA-ORI conjugates were calculated via an ultraviolet absorbance method, the conjugating ratio (CR) decreased from89.4to49.0while the MW of polymer rose from5kDa to40kDa. A direct observation method was used for the estimation of solubility of the PEG-SA-ORI conjugates. All the PEG conjugates exhibited higher water solubility compared to the exceedingly poor aqueous solubility of ORI. The equivalent solubility of ORI in the PEG-SA-ORI conjugates was increased4.7,14.1,34.8, and99.2fold for PEG40kDa-SA-ORI (3.51mg/mL), PEG20kDa-SA-ORI (10.6mg/mL), PEG10kDa-SA-ORI (26.1mg/mL) and PEG5kDa-SA-ORI (74.4mg/mL) conjugates, respectively.In vitro ORI release analysis from the conjugates was performed in the phosphate buffered saline solutions (PBS) at pH5.5and7.4. All the PEG-SA-ORI conjugates showed a certain degree of biphasic drug release, and the cumulative release rates at pH5.5were higher than that at pH7.4, indicating a certain degree of pH sensitivity in the drug release behaviors of the PEG-SA-ORI conjugates.The MTT assay was used to investigate the in vitro antitumor properties of PEG-SA-ORI conjugates. The MTT assay showed that PEG-SA-ORI and ORI solutions had an effect of significant inhibition of leukemia K562cells. The inhibition of K562cells by the PEG-SA-ORI conjugates increased markedly in a concentration-and time-dependent manner. Moreover, Hoechst staining further confirmed that the PEG-SA-ORI conjugates could promote the apoptosis of K562cells.The pharmacokinetic studies in rats displayed that the blood concentration-time curves of the PEG10k-SA-ORI and PEG20K-SA-ORI conjugates exhibited a markedly difference compared to the ORI solution. For injection of ORI solution:Total body clearance0.505L·h-1·Kg-1, mean residence time1.32h; for PEG10k-SA-ORI solution: Total body clearance (CL) ofO.232L·h-1·Kg-1and mean residence time (MRT) of14.50h were determined for the PEG10k-SA-ORI solution while the PEG20k-SA-ORI solution was found to have CL of0.230L·h-1·Kg-1and MRT of22.44h. Both of the above conjugates exhibited significant decreased CL and increased MRT compared to the ORI solution (CL of0.505L·h-1·Kg-1and MRT of1.32h).This work describes the synthesis, characterization and in vitro antitumor evaluation of novel PEGylated oridonin conjugates. Further nonclinical and clinical evaluation of these novel PEGylated oridonin conjugates may bring a valuable treatment option for leukemia patients.Our study results also indicate that the PEGylation technologycould be a promising method to increase solubility and stability of poorly soluble drugs.