| Inflammatory bowel disease (IBD) is a non-specific chronic inflammation of the intestinal tract. There are two clinical forms of IBD:Crohn’s disease (CD) and ulcerative colitis (UC). IBD is the second most common inflammatory disease, which affects tens of millions of people around the world, and incidence rate is being growth every year. Molecular mechanisms underlying the pathogenesis of IBD has not yet been fully elucidated. Some reports suggested that interactions among the gut environmental interface, epithelial barrier function, genetic factors, and microorganism and host immune system are involved in the development of IBD. Presently, patients undergo some types of drug therapies including amino salicylates, glucocorticosteroids, immune modulators and antibiotics. Currently available treatments for IBD are only effective for temporary symptomatic relief while having many concomitant disadvantages. Furthermore, immunosuppressant and anti-inflammatory drugs have many undesirable side effects including diarrhea, cramps, abdominal pain accompanied by fever and high blood pressure. Besides, antibiotics administration could adversely change the environmental conditions of microflora. New drugs with good curative effect and fewer side effects are eagerly needed which have prevention or treatment of inflammatory bowel disease. There are reports show that the drugs used to treat inflammatory bowel disease, such as Sulfasalazine (SASA), Mesalazin and Azathioprine can induce hepatocellular injury, even hepatic failure. Therefore, it is important to reduce the liver injury and toxic effects of drugs in the evaluation of innovative drugs.In this paper, allyl methyl disulfide (AMDS) for treatment of inflammatory bowel disease is a bioactive components derived from garlic. Besides having beneficial effects with regards to bactericidal and antibacterial, epidemiological studies also have indicated that some of the sulfur compounds in garlic can not only reduce the risk of cancer, but also reduce cardiovascular disease. In addition, some of sulfur compounds were also shown to be effective for anti-inflammation and protecting the liver. Biological effects of garlic are attributed to its sulfur compounds. Different sulfur compounds have different pharmacological effects. Some sulfur compounds such as allicin in garlic has been developed into preparation. AMDS is a natural sulphur-containing compound present in garlic. However, there is barely a paper to report its pharmacological functions including therapeutical effect of the AMDS for IBD. The preparation of AMDS also has not been studied. The purpose of this subject is to study the pharmacological activities about preliminary therapeutical effect for IBD and to investate the solid preparation of AMDS. This thesis provides theory evidence for the clinical application of AMDS in the treatment of IBD.The main contents of this study include four sections:(Ⅰ) Evaluation of pharmacological activities of AMDS. The content of this section including the therapeuticical effect and mechanism of AMDS on IBD and protective effect of AMDS on acute liver injury. (Ⅱ) Study of the basic properties of AMDS. (Ⅲ) A preliminary study on the preparation process of solid dosage and enteric preparation of AMDS. Prepared of three kinds of solid dosage forms by different process, prepared enteric-coated pellets and investigated its quality. (Ⅳ) Study of the bio-pharmaceutics of AMDS preparation. The main metabolites were identified and the pharmacokinetic of preparation were studied.HT-29 and Caco-2 were chosen as model cell in vitro in order to study the therapeutic effect of AMDS on IBD. The inhibitory effects and the mechanism of action of AMDS on IL-8 and IP-10 induced by TNF-a in two cells were studied. First of all, SRB and LDH cytotoxicity assays were carried out to evaluate the effect of AMDS on inhibition of cell proliferation or cytotoxicity on HT-29 and Caco-2 cells. The secretions of IL-8 and IP-10 of two kinds of cells by TNF-a induced at different time points were examined by ELISA assay. The effects of AMDS on the secretion of IL-8 and IP-10 were also investigated. The effects of AMDS on IL-8 mRNA level of HT-29 cells induced by TNF-α using RT-PCR. Western blotting method to detect the effect of AMDS on the levels of NF-κB and the nuclear transfers of NF-KBp65 was studied by immunofluorescence. The results of SRB and LDH show that no effects on cell viability or proliferation were noted within the concentration range of 0～150 μM. The results also showed that the release of IL-8 and IP-10 in cells was increased after stimulation by TNF-a in a time-dependent manner. The secreting amount of IL-8 and IP-10 achieved the maximum at 24 h point. We investigated the effect of AMDS on the secretions of IL-8 and IP-10 in two cells and the results showed that the production of IL-8 and IP-10 from epithelial cell lines by TNF-a. Pretreatment of the cells with various concentrations of AMDS for 3 h prior to TNF-a challenge suppressed the secretions of the two cytokine in a concentration-dependent manner. The RT-RCR results demonstrated that TNF-a challenge caused a significant elevation of IL-8 mRNA and that AMDS inhibited IL-8 mRNA accumulation. The attenuation effect of AMDS on IL-8 mRNA accumulation was most likely not due to the general prevention of gene transcription because AMDS did not affect the mRNA levels of the housekeeping gene GAPDH. Mechanistic studies revealed that AMDS inhibited IκBα degradation and NF-κBp65 translocation into the nucleus at both the transcriptional and translational levels, suggesting that the attenuation effort of AMDS on cytokine IL-8 secretion might at least be partially related to the NF-κB signaling pathway.The effect and mechanism of AMDS on IBD in vitro were studied. The rat colitis model was established, the cytokines and antioxidant enzymes were assessed, and NF-κB signaling pathway was also explored in this section. The rat colitis model induce by trinitrobenzene sulphonic acid (TNBS) was successfully constructed. TNF-α, IL-8 and IL-10 levels in serum were measured by ELISA kits. The activities of myeloperoxidase(MPO), muperoxidase dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-PX) of colon were then assayed using commercial reagent kits. Analysis colonic lesions in histopathological aspects and observe the expressions of NF-κB by immunohisto-chemistry method. The results showed that TNBS increased the expression of TNF-α and IL-8, but suppressed IL-10 expression. Treatment with AMDS in TNBS-treated rat inhibited the expression of the TNF-α and IL-8 and increased IL-10 expression. The biochemical index of colon was measured. The results revealed that AMDS has regulation effect on MPO、SOD、MDA and GSH-PX levels. Furthermore, AMDS significantly reduced the MDA and MPO level in colon and restored the activities of antioxidant enzymes SOD, GSH-PX towards normal levels. Histological slide results of rat colon treated with TNBS showed significant injury and inflammation. However, administration of AMDS significantly reduced the release of inflammatory mediators and repaired the damage in colon, suggesting that AMDS has a protection against TNBS-induced colon damage. The immunohistochemical study indicated that AMDS can protect against the barrier dysfunction induced by TNBS in colon, and part of the mechanism might be its ability to inhibit the NF-κB activation. The immunohistochemical analysis results are consistent with the results of in vitro.The hepatoprotective effect of AMDS was investigated in this paper. The content consisted of establishment of acute liver injury model, evaluation of routine liver injury index and study of protective mechanism. The liver injury models induced by APAP in mice, to observe the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), LDH, TNF-a and IL-6 in serum pretreated with different doses of AMDS. Measure the antioxidant index in liver tissue including MDA, SOD、 GSH、GSH-PX and GSH/GSSG. The liver injury was observed by pathological examination and the immunohisto-chemistry method was used to analysis inflammation and lipid peroxidation of the liver cells. According to the experimental dose of 400mg/kg, APAP successfully induced acute liver injury. AMDS could significant restraint ALT, AST, LDH, TNF-a and IL-6 activities. The levels of MDA were decreased remarkable in liver tissue; the levels of GSH, SOD, GSH-PX and GSH/GSSG were increased. The results show that AMDS could reduce the degree of the hepatic injury. Histopathology slides and immunohistochemical results reveal that AMDS has better protective effect.In this paper, the basic properties including structural confirmation, solubility and stability of AMDS were evaluated. AMDS was characterized by different methods. An analysis method of AMDS in vitro is established. The solubility, oil/Water partition coefficient and its stability were studied. AMDS was characterized by IR,1H-NMR, 13C-NMR and GC-MS and established the spectra. Based on the UV-VIS spectrum, AMDS maximum absorption wavelength is determined and the in vitro determination method by HPLC is established, the results of methodology research show that linear relationship and specificity is good, the stability, precision and rate of recovery of this method accord with the quantitative analysis request. The results of solubility study show that AMDS in water is about 26p,g.mL-1. Liquid water partition coefficient of logP is about 2.68. The solubility of AMDS is increased with different solubilization agent. It was found that Tween-80 as solubilizer is the best. The stability test results show that the temperature has a great influence on AMDS. AMDS is relatively stable at low temperature. When AMDS dispersed in different solvents, the antioxidant could increase the stability of AMDS at room temperature.In order to expand its application, three solid dosage forms of AMDS were prepared including of inclusion complexes, liquisolid preparation and solid dispersion. The main objects of this section focused on improving stability, masking odor and reducing gastric irritation of AMDS. The experimental results showed that β-CD inclusion complex has high drug loading than that of SEB-β-CD and HP-β-CD. When P-CD used as a carrier, drug loading efficiency has exceeded 8%(w/w). Orthographical experimentation was used to search the optimum formula and the best processing condition. Through orthogonal analysis, the optimum conditions were obtained for the β-CD inclusion complex:reaction temperature 40℃, the proportion of β-CD to AMDS 6:0.7, reaction time 4 h. The β-CD inclusion complex was characterized by IR, DSC and’H-NMR spectrum. These results showed that the inclusion complex was formed. The stability test showed that the preparation of inclusion complex could improve the stability of AMDS efficiently and cover its unpleasant smell to some extent. Then the liquisolid system was studied. Solid granules were prepared by liquisolid technique. In the process of liquisolid system the PEG400 was chosen as non volatile solvent, MCC:lactose (1:1) as carrier and syloid(?) FP244 as coating material. The best preparation technological condition of liquisolid system was AMDS:PEG400=1:1, the ratio of (MCC+ lactose) with syloid(?) P244 is 2:1. The stability test of liquisolid preparation showed that the preparation method increasing the stability of AMDS efficiently. The result of stability experiment indicated that the content of AMDS slightly decreased at 30℃±2℃ within 3 months. However, experiment of long stability shows AMDS solid dispersions remains stable at low temperature. The solid dispersion was prepared by melting method, the results of the stability showed that the method improved the stability of AMDS. The dissolution results of three solid forms enteric coated capsules showed that AMDS has been completely dissolved at 3 h. Three solid forms can be used to prepare other formulations.Enteric coated pellets of AMDS were prepared including of preparation of pill and coating of pellets. AMDS enteric pellets were preparaed in order to further improve its stability and to reduce the irritation to stomach. We prepared AMDS enteric pellets in order to reduce the stomach irritation, improve the stability and increase the drug targeting on the base of AMDS solid dosage forms. The AMDS pellets were prepared with centrifugal granulation and extrusion-spheronization methods. The results showed that pellets had high drug loading and good roundness by centrifugal granulation method. Study the influence of different types of adhesive, spraying rate of hinder solution and feeding rate of powder on pellets preparation. Results showed that the yield and roundness of pellets is good when using microcrystalline cellulose (MCC) as blank pellets, the binder is HMPC E5 3%, powder supply rate is 10～15r/min, and spraying speed is 5～10 r/min. The aqueous acrylic coating material Eudragit L30D-55 was adopted to develop the AMDS enteric pellets, adding PEG6000 as shaping agent in the coating solution could improve the penetration rate of the coating film. In addition, adding talc powder as anti adhesion agent to avoid adhesion of pellets. With the yield and in vitro release rate as the evaluation index, the formulation of coating was optimized. Preparation of three batches of AMDS enteric coated pellets and investigate the release rate in vitro. The results show that the cumulative release rate in the acid were less than 7% and the cumulative release rate is over 75% in pH 6.8 buffer solution at 45min. Therefore, the preparation of AMDS enteric-coated pellets is feasible and the quality of the pellets is reliable. The stability test of AMDS pellets showed a gradual decrease of AMDS content under high temperature, indicating the pellets should be stored in cool place. The accelerated test and long run test indicated no obvious change in pellets character, content, release degree in acid and alkaline solutions.Biopharmaceutical study of AMDS preparation consists of metabolites and pharmacokinetic studies. In-vivo metabolism studies show that AMDS was metabolized quickly and no parent drug was detected. Two major metabolites were identified in rat plasma, they were AMSO and AMSO2. After repeated administration, Compared with oil dispersant of AMDS, pharmacokinetic results show that Cmax and AUC0'∞ of the rats after oral administration of enteric-coated pellets increased to 114% and 177%, respectively. MRT was extended to 134% with enteric-coated pellets. The results show metabolism time of AMSO2 is longer with pellets than other preparations.The treatment effect of AMDS in inflammatory bowel disease and the mechanism were studied by using in-vitro and in-vivo experiments for the first time in this paper. The protective effect of AMDS on acute liver injury induced by medicine was also investigated. Through experiment, we found that AMDS not only has the treatment effect of inflammatory bowel disease but also has hepatoprotective effect. In order to cover smell, reduce gastric irritation, increase drug stability, and improve drug targeting, AMDS was prepared into solid forms, and can be further prepared into enteric coated pellets. Our studies lay a theoretical foundation in clinical application. |
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