| Isoliquiritigenin is a chalcone derivative and found in licorice and many medicinal plants. It has various biochemical activities such as Anti-tumor, anti-virus, anti-free radicals, vascular relaxation, inhibition of lipid peroxidation and other bioactive. It can significantly inhibit proliferation of HIV. ISL has widely inhibited effect on many types of carcinoma. Early works in this project found that it could significantly inhibit proliferation on hepatocellular carcinoma cell line S180and gynecological tumor H22. Therefore, ISL is a compound which has highly potential medicinal value.Solid lipid nanoparticles (SLN) are new types of submicron colloidal drug delivery systems with particle size between50-1000nm which found in1990s. It can be used for topical, oral and parenteral administration and have the advantages of targeting drug delivery, slow-release, solubilization, etc. It can be used as drug carrier of antitumor and clinical diagnosis, and so on. It is the current focus of medicinal preparation research and. development.To give full play to the antitumor activity of ISL, its SLN was developed. The advantages on this preparation of sustained release, solubilization, targeting and enhance antitumor effect were utilized. Study found that it caused blood toxicity in high dose after intravenous injection, it mainly affected blood clotting mechanism and caused vascular embolism. In order to overcome deficiency of this preparation, ISL-SLN was modified by low molecular weight heparin sodium (LMWH), and physicochemical proprieties of LMWH-ISL-SLN were studied. After accomplished preparation, we studied the difference among ISL, ISL-SLN and LMWH-ISL-SLN on tissue distribution characteristics in vivo in mouse, activity of resistance HepG2proliferation, acute toxicity of mouse and blood toxicity. Before the study of this paper, we found that absorption of ISL was a typical two compartment model pharmacokinetic characteristics after intravenous injection. But the curves of concentration-time of ISL were irregular and multiple absorption peaks manifested after oral administration. In order to find out ISL’s absorption kinetics, reveal its absorption mechanism, understand its absorption sites and study biliary excretion rule, test of intestinal absorption in vivo on SD rats and biliary excretion were done to explain absorption law of ISL after oral administration.By the study of intestinal absorption in vivo and ISL biliary excretion on SD rats, Absorption mechanism of ISL in intestine are obtained. It was passive diffusion process. Absorption rate constant, Absorption percentage, and apparent absorption coefficient in each section of intestine were different. The absorption amount on colon was significantly higher than other section intestine and stomach. Absorption in the stomach was the least. Absorption of duodenum, jejunum and ileum was more or less, but each parameter was higher than those of stomach, lower than colon. Biliary excretion of ISL was little, it illustrates that biliary excretion was not the main excretory pathways, so it was not the major factors of affecting pharmacokinetic. Ka, Papp and maximum absorption rat were basically the same, the relationship of absorption quantity and dosage was a good linear under the same section intestine. Medium pH had little effect on absorption of ISL.In this paper, the fomulations of ISL-SLN were selected by uniform design and the final prescription was ISL75mg, soybean lecithin145mg, poloxamer1%, cetyl alcohol50mg, medium chain triglycerides2mg, stearic acid5mg. The particle size, charge, encapsulation efficiency and drug loading and other basic properties were determined on this preparation prepared by above prescription and injection method. The results showed that ISL-SLN was well distributed with a mean diameter of132nm. Its zeta potential was-28.3mV and encapsulation efficiency was99.8percent. The spherical solid particles with particle size between100nm-200nm observed under electron microscope, and achieved the design requirements. In vitro release test showed that ISL-SLN’s release was slower than ISL. The stability of ISL-SLN was poor at room temperature. While mannitol as scaffolds agent and the preparations freeze dried, the stability and dispensability of ISL-SLN in water were better except the size increased. But the sizes of the particles are still within300nm.By the modification of ISL-SLN with LMWH, LMWH-ISL-SLN was got. Prescription of this preparation was:soybean lecithin100mg, cetyl alcohol46mg, medium chain triglycerides2mg, poloxamer1%, LMWH (lmg/mL)300μL. The physicochemical proprieties of LMWH-ISL-SLN were studied. The results showed that the shape of LMWH-ISL-SLN was regular and well distributed. Average size of the nanopartical was217.5nm, a zeta potential was-18.2mV and encapsulation efficiency of ISL was99.8%. The encapsulation efficiency of LMWH was39.3%. Compared with ISL-SLN, particle size increased and the zeta potential decreased. Ihe encapsulation efficiency of ISL had no significant change and the stability was the same as ISL-SLN at room temperature.After iv administration of ISL, ISL-SLN, LMWH-ISL-SLN to mice, ISL concentration in plasma and other tissues, such as heart, liver, spleen, lung, kidney and brain, were determined by HPLC. The pharmacokinetic parameters for three preparations were compared. Results showed that the pharmacokinetics processes in mouse were best fitted to two-compartment open model and Cmax of ISL Solution preparation is larger than Cmax of LMWH-ISL-SLN. The Cmax of ISL-SLN is less than two Cmax. Changes of AUC also showed the same trend. Absolute bioavailability of ISL-SLN decreases with dose increasing, while the LMWH-ISL-SLN is opposite. Apparent volume of distribution of three preparations was small that shows ISL’s distribution in mice is widely. Targeting research of three preparations indicated that distributions of ISL were mainly in liver and kidney. Ratio in liver had no significant changes with the dose increase, but percent in kidney decreases. Compared with ISL, ISL-SLN’s targeting in vivo change obviously. Ratio of heart and lung increases dramatically and percent in heart was dose-dependent. Ratio in pulmonary reduces with dose increasing. Distribution in liver, spleen and kidney were decrease relatively. Compared with ISL-SLN, LMWH-ISL-SLN’s targeting in liver and kidney ware high than ISL-SLN. Distribution of ISL-SLN and LMWH-ISL-SLN in brain and spleen were low.In the study of inhibition of the proliferation of human hepatoma cell line HepG2in vitro by ISL, ISL-SLN and LMWH-ISL-SLN, results showed that ISL have a significant inhibitory effect on human HepG2cells in a dose-dependent manner. Its median inhibitory concentration (IC50) was77.6μM. Inhibitory effect of ISL, ISL-SLN and LMWH-ISL-SLN had no significant change.Acute toxicity experimental of ISL, ISL-SLN and LMWH-ISL-SLN study found that ISL-SLN had obvious toxicity at the dose of190-340mg/Kg. It causes the death of experimental animals to varying degrees after administration of ISL-SLN In the above dose range. But the group of solution agent of ISL and the LMWH-ISL-SLN group were significantly lower in toxicity. Few experimental animals were killed. As we can see, dosage form is the main reason that causes toxicity during the injection, but the toxicity of LMWH-ISL-SLN was significantly reduced. Experimental animals have few deaths in above concentrations. Compared the changes of APTT, FIB, and TT before and after intravenous injection above drugs in mouse and found the reason by observing the dead mouse after acute toxicity test. High dose injection of SLN caused intravascular coagulation. The toxicity of LMWH-ISL-SLN was significantly reduced. |
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