14-Succinate Lysozyme Conjugate For The Renal Delivery Of Triptolide

Triptolide (TP) is one of the most important biologically active components of the Chinese herbal Tripterygium wilfordii Hook f(TWHf). A triptolide-lysozyme (TP-LZM) conjugate was synthesized to achieve renal specific delivery and to reduce the side effects of triptolide. Triptolide was coupled to lysozyme through succinic via an ester bond with an average coupling degree of 1 mol triptolide per 1 mol lysozyme. The lysozyme can specifically accumulate in the proximal tubular cells of the kidney, making it a potential carrier for targeting drugs to the kidney. The structure of triptolide succinate (TPS) was confirmed by IR,~1H-NMR, MS and UV. The concentrations of triptolide in various samples were determined by reversed-phase high-performance liquid chromatography (HPLC). In this study, the physicochemical and stability profiles of TP-LZM under various conditions were investgated the stability and releasing profiles of triptolide-lysozyme (TP-LZM) under various conditions. In vitro release trails showed triptolide-lysozyme was relatively stable in plasma (less than 30 % of free triptolide released) and could release triptolide quickly in lysosome (more than 80 % of free triptolide released) at 37℃for 24 h. In addition, the biological activities of the conjugate on normal rat kidney proximal tubular cells were also tested. The conjugate can effectively reduce NO production in the medium of HK-2 induced by lipopolysaccharide (LPS) but with much lower toxicity. These studies suggest the possibility to promote curative effect and reduce its extra-renal toxicity of triptolide by TP-LZM conjugate.We next examined the uptake and handling of TPS-LZM in human renal proximal tubular HK-2 cells and in mouse using fluoresce imaging and high-performance liquid chromatography/mass spectrometry (LC/MS) method. We found that TPS-LZM was taken up by HK-2 cells via staturable process, degraded and released free TP excreted mainly to basolateral side of the ceils. Tissue distribution and pharmacokinetic study revealed that its major accumulation site appeared to be kidney. Compared with TP at the same time, the overall targeting efficiency (TE) was significantly enhanced from 11.74 % to 95.54 % and the MRT of TPS-LZM was moderately prolonged from 3.08 h to 4.10 h. We further investigated the effects of TPS-LZM on experimental membranous glomerulonephritis (MN) induced by cationized bovine serum albumin (C-BSA) in rats. After 3 weeks of treatment, rat treated with TPS-LZM showed less severe kidney disease with significantly diminished liver toxicity. We also eatablished a rat ischemia/reperfusion model to examine the thepatutic effects of the conjugate. It can effectively reduce the apotosis of proximal tubular cells and the expression of NF-κB in the kidney. In conclusion, these results showed that renal targeting of TP can be obtained by conjugation with LZM, suggesting renal targeting delivery of TP may be useful in treating immune related renal diseases.