Studies On The DDB Nanosuspensions

DDB (Bifendate, DDB) is an important synthetic intermediate of schisandrin C and is widely used in China for the treatment of chronic hepatitis. Clinical studies have shown that DDB could lower the activity of GPT (glutamic pyrumic transaminase) and GOT (glutamic oxalic transaminase) and increase the concentration of hepatic microsomes (e.g. P450) and the abilities of detoxication of liver. In addition, DDB could protect the liver from poison damage by alleviating the liver damage caused by carbon tetrachloride, anticancer drugs, antituberculotic and other chemicals. DDB is easy for patients to accept as a result of its safty, low side-effect and cost. At present, DDB is mainly employed for the treatment of chronic hepatitis with elevated activity of alanine aminotransferase and other drug-induced elevated activity of alanine aminotransferase. However, DDB is almost insoluble in water leading to a low oral bioavailability, generally only25%to30%. The efficacy of DDB preparations available is not ideal currently. New pharmaceutical teniques should be found to overcome the pharmaceutical problems due to its low solubility.This study describes the preparation of bifendate (DDB) nanosuspensions by precipitation-combined microfluidization. The preparation procedure was optimized in regard to particle size, size distribution and stability. In the precipitation process, the concentrations of polyvinylpyrrolidone K30(PVPK30) and lecithin in the anti-solvent, the concentration of DDB in the organic phase, the precipitation temperature and the stirring rate were optimized, respectively. In the microfluidization process, two important parameters, the number of cycles and the pressure were also investigated systematically. The optimized DDB-NSP was transformed into dry powders by freeze-drying. The lyophilization precedure has been optimized and the characteristics of the DDB-NSP dry powders, such as saturation solubility, dissolubility in vitro, storage stability, were evaluated. Compared with commercial DDB, an enhanced dissolution property was shown due to the increased surface area in the in vitro dissolution test. Finally, crystalline state evaluation before and after particle size reduction process were characterized by differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The pharmacokinetics and biodistribution of DDB-NSP and DDB-Sol (DDB solution) were carried out after intravenous administration using New Zealand rabbits and Kunming mice, respectively. The HPLC method was applied to study the influence of particle size on the characteristics of the tissue distribution and pharmacokinetics after intravenous administration.A series of experiments have been performed to determine the optimized formulation as follows:the ratio of DDB-to-stabilizers is4:5; Lecithin and PVPK30were chosen as stabilizers due to their good and stable performance. First,200mg DDB was weighed precisely and dispersed in5mL DMSO to form the organic phase; then, lecithin and PVPK30of the prescription amount were co-dissolved in100mL distilled water to form the aqueous phase by keeping the temperature at0-3℃by immersing the aqueous phase in an ice-water bath. Second, the organic phase was quickly injected into the pre-cooled aqueous phase at a stirring speed of1000rpm to obtain a coarse nanosuspension; next, the mixture was treated with an ultrasound for20min in ice-water bath to obtain a coarse nanosuspension. Third, the coarse nanosuspension was further processed through a microfuidizer model M-110S (MicroFuidics, American), performing2cycles at6900psig,2cycles at11650psig, and then10cycles at23300psig to obtain the final nanosuspension. The precipitation-combined microfluidization method is simple and has a relatively reproducibility. The shape of the nanoparticles under the TEM is granular crystalline. The mean particle size is200±4nm with a narrow size distribution (PI0.308±0.04) and Zeta potential is-21.92±0.96mV.In order to ameliorate either physical or chemical stability, nanosuspensions are usually transformed into solid products by freeze-drying. The prepared DDB-NSP were prefreezed at-80℃for24h using a DW-HL218(Shanghai Huayan Co. Ltd., Shanghai, China), and then freeze-dried for48h at-55℃with a pressure of15mTorr. Mannitol (5%, w/v) was added into the freshly prepared DDB-NSP as cryoprotectant. The results have shown that lyopyilization had little effects on the basic characteristics of the nanoparticles, such as particle size and Zeta potential. The DSC and XRD testified that there was no crystalline state changed in the size reduction process. Compared with the commercial DDB powder, the DDB nanocrystals noticeably increased saturation solubility of the drug from37.39μg·mL-1to72.19μg·mL-1. The in vitro dissolution studies showed that79.8%of the drug was dissolved for DDB-NSP while the dissolution of the pure DDB was less than6%after the same time period. During the120min, DDB-NSP has achieved complete dissolution while only50.4%of the raw crystals were dissolved. The results above indicated that the dissolution rate was markedly improved in the nanometer-sized system and the drug release pattern was in accord with Biexponential equation:1-Q%=-0.9747e-0.1628t+0.1863e-0.0107t, rα=0.9969, rp=0.9965. The initiatory stability research showed that the freeze-dried DDB-NSP has a shelf-life of at least3months at3-5℃or20-25℃.HPLC method was used to determine and compare the content of DDB in different tissues of mice after the tail intravenous injection of DDB-So1and DDB-NSP. All these results indicated that DDB-NSP had more significant liver-targeting characteristics compared with DDB-So1, which made DDB-NSP promising candidates for the treatment of hepatitis. Moreover, the mean residence time in plasma and liver was also prolonged. It was worth noting that the reduced mean residence time in kidney lowered side effects. In a word, nanosuspensions were helpful for DDB to improve its therapertic efficiency and obtain a long-term effect.Compared with DDB-So1, DDB-NSP exhibited a markedly different pharmacokinetic property. In contrast to rabbits treated with DDB-So1, the rabbits treated with DDB-NSP showed higher DDB concentrations at all time points, especially at the initial time points. For injection of DDB-Sol:C=2.04e-0.77t+0.1, CLz=2.83L·h-1·Kg-1, MRT=1.52h;for ORI-NSP:C=22.445e-0.355t, MRT=2.77h, CLz=0.15L·h-1·Kg-1. Compared with DDB-Sol, the pharmacokinetic behaviours of DDB-NSP displayed a longer MRT and slower clearance. The AUC values of DDB-NSP and DDB-Sol were64.281,3.741h·μg·ml-1, respectively, resulting in an approximate17.18-fold increase in AUC.Our study is the first report on the preparation of DDB nanosuspensions. The results of our study were helpful for the development of the injection of poorly soluble drugs. A tentative conclusion was made that the nanosuspension delivery system might be a simple and adequate choice for the clinical application of DDB.