| Xanthan gum (XG) is a microbial extracellular heteropolysaccharide, prepared from fermenting Xanthomonas campestris. The relative molecular weight (Mr) of XG ranges from 2×106 to2×107Da. It contains repeating pentasaccharide units formed by two D-glucose, two D-mannose and one D-glucuronic acid. XG has a high viscosity even at low concentrations and a high degree of pseudoplasticity.Intra-articular (IA) injection of XG could act as a viscous lubricant during low impact movements of the joint and as an elastic shock absorber during high impact movement. Our previous study indicated that IA injection of XG could inhibit the continuous lesions on the cartilage and delayed the progression of osteoarthritis (OA).IA injection of hyaluronic acid (HA) is widely used in the treatment of OA, however, HA is easily degraded by hyaluronidase and free radicals, and then be cleared relatively quickly from the joint cavity. XG is similar to HA in viscosity and rheology, however, it seems to be more stable than HA.In an experimental model of OA induced by papain, IA injection of XG once every 2 weeks for 5 weeks and IA injection of HA once a week for 5 weeks had similar effectiveness. The therapeutic effect of XG is probably longer than that of HA. IA injection of XG is a potential treatment method for OA to avoid numerous injections. However, whether XG could be cleared from the joint cavity and its pharmacokinetics after IA injection are still unkown. In the present study,14C radiolabelled XG (14C-XG) was obtained by fermentation, and the absorption, distrbution, excretion of XG after intra-articualr injection were evaluated in animals.1 Preparation of 14C radiolabelled XG by fermentationObjective:To obtain 14C-XG with the radiochemical purity and specific activity applicable for the pharmacokinetics study in vivo. Methods:The 14C-XG was prepared through fermentation with the combination of normal glucose and 14C radiolabelled glucose (14C-glucose) as carbon source. The XG yield was controlled by controlling the total amount of carbon source. So, when the content of 14C-glucose in the fermentation medium was fixed, the specific activity of 14C-XG could be controlled. Effect of radioactivity on the XG yield and the utilization of 14C was evaluated. To obtain the 14C-XG for pharmacokinetics study, we added 1200 uCi of 14C-glucose to 15 mL of fermentation medium, and proceeded to fermentation and purification. Results:The XG yield exbibited linear relationship with the amount of carbon source in the range of 0.5%-2%. The ratio of 14C-glucose in crude XG was about 60%. Radioactivity in the fermentation medium had no effect on the XG yield and the utiliation of 14C.Finally, the total amount of carbon source for 14C-XG fermentation was 0.7%, of which 14C-glucose only consisted of 0.71%. After purification, 14C-XG for pharmacokinetics study was obtained, with a radiochemical purity of 99.5% and the specific activity of 7.27 μCi/mg. Conclusion:The 14C-XG was successfully prepared by fermentation with the combination of normal glucose and 14C-glucose as carbon source. The process was simple and showed good stability. Radiochemical purity and specific activity of the 14C-XG obtained were applicable for the pharmacokinetics study in vivo.2 Elimination of xanthan gum from the joint cavityObjective:To study the elimination kinetics of XG from the animal joint cavity. Methods:Rabbit knees were intra-articularly injected with 1%(w/v) XG labelled with green fluorescence or 14C-XG with the dosage of 0.1 mL/kg. The residual levels of the two kinds of XG in the synovial fluid were compared. Rat knees were intra-articularly injected with 14C-XG with dosages of 0.25,0.5 and 1 mg (0.5%,1% and 2%14C-XG,50 μL). Residual levels of 14C-XG in the rat joint cavities after killed at different time were measured. Non-compartmental model was used to analyze the kinetic parameters cleared from joint cavity. 14C-XG was intra-articularly injected into the rabbit knees to investigate its distribution in the joint tissues. Results:Both XG labelled with fluorescence and 14C-XG could be cleared from the joint cavity. Residual levels of fluorescence labelled XG in the synovial fluid were lower than those of 14C-XG Residual levels of 14C-XG in the synovial fluid after certain time after IA injection of were higher than that of 14C radiolabelled HA reported in the literature. After single dose of 1 mg XG IA injection, there was still 27% residual XG in the rat joint cavity on day 30 and 9.85% residual XG on day 180 after IA injection. The mean residence time (MRT) was 333 h. With a correlation coefficient of 0.9999, good liner correlation was found between the dosage and AUC. At the same time point, the residual percentage had no dfferences among each dosage group. After IA injection, small amount of XG could be distributed into the joint tissues, including the articular cartilage, synovium membrane, meniscus, fat in the joint and muscles around the joint cavity. Conclusion:XG could be cleared from the joint cavity, and its residence time was longer than that of HA. Interval time using IA injection of XG for OA therapy could be extended.3 Pharmacokinetics of xanthan gum after intra-articular injectionObjective:To investigate the pharmacokinetics of XG in rats after IA injection. Methods:Following single IA administration of 14C-XG to rats, with dosages of 0.25, 0.5 and 1 mg (0.5%,1% and 2% 14C-XG,50μL), the drug concentrations in plasma at different time were determined. Non-compartmental model was used to analyze the plasma concentration data. Intraperitoneally (IP) injected animals were set for comparison. For the animals which were intra-articulraly injected with 1 mg of 14C-XG, drug concentrations in the main tissues at different time were determined after the animals were sacrificed. The urine, bile and CO2 were collected, and their radioactivity was tested. Results:After IA injection of 14C-XG, radioactivity could be detected in the plasma, main tissues and the excretion. Good correlations were found between AUC, Cniax and the dosages, indicated that IA injection of XG exhibited dose-proportional pharmcokinetics with linear kinetic characteristic in the plasma. Pharmacokinetic parameters of IA injection and IP injection were compared. Absorption of XG after IA injection was slower than that of IP injection with a longer Tmax.The ralative bioavailability for IA injection was 47.1% of IP injection.14C-XG widely distributed to main orgains and tissues in rats, and most of it distributed in heart, liver, spleen, lung and kidney. Concentrations of 14C-XG in liver and spleen were the most highest among the organs. Total drug residue level in the liver was the highest, which accounted for 16.5% of the total dose at 15 d after IA injection. XG could be cleared from each tissue.14C-XG was mainly excreted through breathing, part through urine, and little through feces after IA injection. Cumulative amounts percentage of XG in urine at 30 d after an IA injection was 4.63%, and the excretion rate was about 0.07% of the total dose per day. Cumulative amounts percentage of XG in urine at 90 d after an IA injection was 21.4%, and the excretion rate was about 0.32% of the total dose per day. Conclusion:XG could be absorbed into the blood circulation, distributed to the main tissues, and then be cleared. However, it was a slow process for XG being eliminated from the body after IA injection.4 Preliminary study on the metabolism of XGObjective:To investigate the metabolism of XG preliminarily. Methods:The range of Mx of XG in the plasma, liver and urine was tested. Free radical and rat liver lysosomes were incubated with XG in vitro, and changes of Mr and viscoelastic behavior were measured, respectively. Results:XG could be absorbed into the blood circulation and distributed in liver in the form of macromolecules (Mr>1×105). XG with Mr<1×l14 could be excreted through urine. Free radicals could decrease the viscosity of XG, however, XG had a higher resistance to free radical degradation than HA. After incubated with rat liver lysosomes, the Mr of XG (4.2×106) was reduced to 1.1×106. Conclusion:XG could be absorbed into the blood circulation and distributed into liver in the form of macromolecule. Free radical and liver lysosomes could degrade XG. Enzymes able to degrade XG may exist in the liver lysosomes. |
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