| The development of water insoluble candidate compounds is a tough problem in the new drug researching. This is also the major reasons of lead to high failure probability, high risk and larger import of drugs development. Furthermore, this is also caused low clinic curative effect, increased adverse reaction and aggravated medical cost. The drug nanocrystals nanotechnology, a new pharmaceutical preparation method, which emerged in the recent decades and applied in the industries, has mini-dimension effective and quantum runnel ling effects. The most important features of insoluble compound nanocrystals are improved solubility and enhanced dissolution velocity, as well as derived the other physicochemical properties and physiological nature.Silybin, a water insoluble flavanolignan compound, which with low toxicant and high performance of protect hepatic function, is the major active constituents of Silybum marianum (L) Genrtn. Currently, the on market dosage forms of silybin are capsule and tablet. Due to the low solubility, the absolute bioavailability of Beagle dog oral administration is only about 20%. Therefore, the poor solubility of silybin may be severity impaired the clinic affectivity.The major objective of the present research is to develop silybin nanocrystals for oral and intravenous delivery by piston-crack high pressure homogenization approach, establish the silybin determine method by HPLC-DAD and LC-MS for in vitro and in vivo samples analysis, investigate silybin nanocrystals pharmaceutics properties and pharmacokinetics as well as organs distribution characters, and research the Caco-2 cell monolayer transport properties. Furthermore, the pharmacodynamics evaluation of silybin nanocrystals also will be conducted by in vitro and in vivo acute hepatic injury models.The major contents include preformulation study, technology and formulation screening of preparation silybin nanocrystals by HPH, establishment of HPLC-MS method for biological specimen analysis, pharmacokinetics study of Beagle dog oral administration and intravenous drop of silybin nanocrystals, organs distribution by mice caudal vein intravenous administration, the in vitro transport across Caco-2 cells monolayer evaluation, in vitro HL7702 cell and in vivo Beagle dog hepatic injure model for pharmacodynamics investigation.The established HPLC-DAD method for in vitro samples analysis was investigated and corresponded to technology requirement. The experiment results of preformulation study stated that the solubility of silybin increased with pH and at pH7.4 is 49.7?g·ml-1. The log of oil/water partition coefficient is 2.8, which hinted that the poor oral bioavailability of silybin was mainly due to the low solubility resulted slow dissolution velocity.There was a favorable reproducibility and stability in the HPH method for silybin nanocrystals preparation. The higher preparation pressures the smaller nanocrystals particle size. The particle size of nanocrystals of prepared under 1800 bar is one fifth of the nanocrystals of prepared under 800 bar. The more times of homogenization operations the more uniformity of the prepared nanocrystals and 15 times were selected. A L9(34) orthogonal design were conducted for silybin nanocrystals formulation screening. The different concentration of phospholipids, poloxamer 188, SDS and PVP K30 were selected for the formulation screening. And zeta electric potential was the investigate index. The experiment revealed that when the formulation contents 0.2% phospholipids,0.1% poloxamer 188,0.05% SDS and 0.05% PVP K30 the zeta electric potential was the greatest. In consideration of safety and stability, the final silybin nanocrystals formulation contents of 100 ml water,0.5 g silybin,0.2 g phospholipids and 0.1 g poloxamer 188.The protectants screening of vacuum freeze-drying was carried out between polyalcohol and high molecular compound, such as mannitol, trehalose,?-cyclodextrin, lactose, glucose and dextran. The freeze-dried nanocrystals were evaluated by appearance morphous as well as integrate format. The experiments result demonstrated that 5%(w/v) mannitol is the most suitable protectants. The freeze-dried product shows integrity round cake shape, smoothing surface, light and soft of texture. The microscope observation and evaluation revealed that the mannitol replaced the role of water of -OH and the mannitol alternated with nanocrystals, which formed order reticulate structure. The regularly structure is better for the stability of nanocrystals and prevented the contact between two nanocrystals. Therefore, the freeze-drying technology manifested that pre-freeze condition is -80?for 24 h, freeze-drying time is 48 h. The water contain of obtained products is lower than 3%.To investigate the feasibility of silybin nanocrystals for oral and intravenous administration, tow different particle size distributed nanocrystals were prepared using the following operation parameters. As stated formulation, silybin nanocrystals A (SN-A) were homogenized under 800 bar, followed freeze-drying. Silybin nanocrystals B (SN-B) were homogenized under 1800 bar, followed freeze-drying. Thereafter, facies analysis and superficial syndrome of SN-A and SN-B were conducted. Immediately after freeze-dried, the size and zeta potential (ZP) were found to be 641.8±14.7 nm (PI?0.375) and -23.1±0.6 mV for SN-A, and 127±1.9 nm (PI?0.292) and -25.5±0.7 mV for SN-B, respectively. The TEM observation revealed that with the increase of homogenization pressure, in addition to the particle size decreasing, the silybin nanocrystals appeared to be more regularly shaped. The AFM experiments demonstrated that the silybin nanocrystals with a spherical shape revealed values of 718 nm and 226 nm in their diameters, for SN-A and SN-B, respectively. Additionally, the presence of encapsulate films on the surface of the pure particles of drugs could also be observed by AFM. These results indicate that the thin films should be surfactants. Therefore, AFM analysis reinforced the presence of the stabilizer on the surface of the drug particles and this feature is in accordance with the TEM results. SEM images of the freeze-dried nanocrystals prepared under different homogenization pressure were clearly confirmed that the higher produce pressure the more uniformly nanocrystals in the morphology and the smaller particle size. Even the decreased size cannot be in proportion to the increased pressure. Hence, it was assumed that SN-B had higher uniform and smaller dimension. These results were in accordance with the study of particle size analysis and PI values. Nanocrystals were freeze-dried to obtain the dried silybin powder. TEM images indicate that the silybin powder was aggregated due to the water-removal. The DSC thermograms clearly showed that both of the two freeze-dried silybin nanocrystals displayed conspicuous melting point of silybin at the temperature with comparable physical mixture of drug and surfactants. These denoted that no any possible transformation to an amorphous state during the HPH and freeze-drying operation and this is important to long-term stability. XRPD experiment confirmed the fact that the diffraction pattern was preserved in silybin nanocrystals, which indicates that the crystalline state of silybin appeared to be unaltered following the HPH operation.In solubility experiment, nanocrystals powder provided a more than two-fold increase in solubility compared to the un-milled silybin or physical mixture. This increase is more pronounced for SN-B that has smaller particle size. Either in SN-A or in SN-B, dissolution velocity enhancement was clearly observed at both tested pH6.0 and pH7.4. Correlating with the crystalline state analyze results, the dramatically enhanced dissolution rate resulted from the increased effective surface area, and the improved solubility due to the decreased particle size, but no due to the consequence of the presence of amorphous fraction. The specific surface area is 0.435 m2·g-1 and 0.484 m·g-1, for SN-A and SN-B, respectively. The porosity and pore diameter, for SN-A is 0.00401 and 17.05±16.0 nm, for SN-B is 0.00431 ml·g-1 and 17.03±16.4 nm, respectively. The accelerated stability test manifested that the SN-A and SN-B has satisfactory stability.An LC-MS method for determination of silybin in Beagle dog plasma and mice organs was developed for the first time. The technology investigation coincided with the requirement of SFDA guidance principle of chemicals non-clinic pharmacokinetics. For Beagle oral administration (20 mg-kg-1), both nanocrystals delivery systems showed approximately two-fold or three-fold bioavailability improvements in terms rate and extent compared with the un-milled suspension. Based on comparison of the AUC0-?> values, the un-milled silybin suspensions showed significantly lower silybin exposure than the nanocrystals (SN-A and SN-B). There was no significant difference in AUC0-?, between SN-A and SN-B. The Cmax was significantly lower in the un-milled silybin suspensions than that of the nanocrystals SN-A or SN-B. The un-milled silybin suspension has the shortest MRT, the SN-A showed longer and the N-B showed the longest. The tmax was significantly shorter for the un-milled suspension than that for the nanocrystals of SN-A and SN-B. No significant statistical difference between the nanocrystals was found for this parameter. For Beagle intravenous drop administration (15 mg·kg-1), in contrast to solution, the nanocrystals formulations had prolonged t1/2 and augmented AUC. No adverse events were observed during the administration. The solution's AUC0-?was lower than that of the nanocrystals, including SN-A and SN-B. Although the solution has higher Cmax, the lower MRT and higher clearance rate as well as other parameters also lead to a distinct pharmacokinetics profile when compared to the nanocrystals plots. Therefore, these experiments demonstrated that nanocrystals formulations can be used to improve exposure of compounds with low dissolution rate and poor pharmacokinetic characteristics.To investigate the effect of nanocrystals particle size on the organ distribution, the mice vena caudalis intravenous silybin nanocrystals (20 mg-kg"1) were carried out. The evaluation index included drug targeting index, drug selectivity index, drug targeting efficiency and relative targeting efficiency, which revealed that silybin nanocrystals has liver passive targeting. In considering of silybin concentration in the organs, nanocrystals could decrease the concentration in the plasma and heart; increase the concentration in the liver. Judging from the quantity of silybin in the organs, the liver aggregation of the solution term, SN-A and SN-B is 15%-35%, 46%-63%and 31%-67%, respectively. Furthermore, in 10 h following administration SN-A, the liver aggregation stabilize at 40%-50%. This experiment revealed that the target efficiency of SN-A is better than SN-B.In the Caco-2 cell cytotoxicity experiments, when the concentration of silybin was below 125?g·ml-1, there were no significant differences between the experimental groups and the control groups. The effects of nanocrystals, SN-A and SN-B, on facilitating silybin transport across the monolayers were much higher than that of the physical mixture formulation. Additionally, the decrease in particle size could also enhance ability of silybin in SN-B system transport across the monolayers as it has a higher ability to enhance Papp. Nanocrystals presented higher Papp values compared with un-milled silybin and physical mixture. Compared with the silybin nanocrystals, the un-milled silybin presented a lower Papp value in pH 6.0 medium. Based on the Caco-2 cell monolayers permeability date, we may tentatively present a conclusion that nanocrystals could promote of poorly soluble drugs transport cross intestine epithelium.The injuring of HL7702 hepatic cell model was established by incubation with H2O2 for 24 h at concentration of 5 mM. When the silybin prophylactic administrated to cell injury model, the effective presented concentration dependent tendency. The most effectiveness were presented at 10?g·ml-1 and 50?g·ml-1, for silybin solution and SN-A, respectively. The protection efficiency of solution and SN-A was lower than the prophylactic administration experiments, when the silybin remedially administrated to cell injury model. In order to demonstrate the efficiency of SN-A on the protection of HL7702 is better than the solution formulation; the HE staining study was carried out. When the solution and SN-A administrated at 10?g·ml-1, the cells of SN-A group was presented as the endochylema turgor vitalis, the morphology normally, the nucleus and nuclear-cytoplasmic ratio closed to norm cells. Furthermore, the cells condition of solution formulation group was between model group and SN-A group, and presented some cell debris.Beagle dog acute liver injure model was constructed by subcutaneously multipoint injected of CCl4-peanut oil solution (1:1, w/w). The SN-A intravenous drop and SN-B orally administrated at dose of 15 mg·kg-1 and 20 mg·kg-1, respectively. Compound ammonium glycyrrhizinate was chosen as positive control. The model group and silybin solution control were also set. The hepatic functional parameter, such as AST, ALT, ALP, TBIL and GGT were detected. The enzymes in the liver tissue, include SOD and MDA, were determined. The pathological section were used to observation the patho-changes of liver tissue. Therefore, the safety and efficiency of orally and intravenously administrated silybin nanocrystals were evaluated. Judging from the hepatic functions detection and observation of the patho-changes, the acute liver injurer of Beagle dogs were successfully established. Compared with solution formulations, the nanocrystals, SN-A for intravenous and SN-B for oral administration, could restore the deviate parameters, including AST, ALT, ALP, TBIL and GGT, toward to normal values. Furthermore, the silybin nanocrystals also could increase the SOD activity and decrease the level of MDA. As observed in the histological section, hepatic cell balloon changes and inflammatory cell infiltrate were decreased in the SN-A and SN-B groups, compared with solution group. And large area necrosis also refrained in the nanocrystals. At the same dose, the nanocrystals could effectively protect the liver cells avoid of injure, maintenance the nucleus and membrane integrity, promote the stabilization of liver cells. Silybin nanocrystals, SN-A for intravenous and SN-B for oral delivery, could effectively prevent the CCl4 induced hepatic injury of Beagle dogs. And these demonstrated that nanocrystals could improve the oral bioavailability and target efficiency of intravenous delivery.In this study, the silybin nanocrystals for oral and intravenous administration,, could be prepared by piston-gap high pressure homogenization method. The freeze-drying, manicol used as protectant, could improve the stability of silybin nanocrystals. In the preparation, the crystals form maintained and particle size distributed uniformity. Dissolution velocity and saturation solubility is dramatically improved. The transmembrane transport ability and oral bioavailability enhanced. The nanocrystals also could implement liver passive target delivery. The nanocrystals improved the effectiveness of silybin for hepatic injury in vitro and in vivo. |
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