肾靶向雷公藤内酯醇—溶菌酶结合物研究
摘要
肾脏疾病，尤其是免疫相关的肾脏疾病的，正成为威胁人类健康的常见疾病之一。常见的肾脏疾病包括原发性和继发性肾小球、肾小管、肾介质及肾血管等疾病，其中最常见的有急性肾炎、慢性肾炎、原发性肾病综合症及尿路感染等上述疾病不断发展，在后期可出现发展成为末期肾病(ESRD)。病人不得不依靠昂贵的血液透析或肾移植来延长生命，给患者家庭和社会带来沉重负担。目前国内临床上治疗肾脏疾病常采用激素冲击治疗，雷公藤多甙片维持治疗的治疗方案。大剂量使用激素和长期服用雷公藤多甙片所带来的毒副作用严重限制了肾脏疾病的治疗。因此，肾脏疾病临床治疗中，研究发展肾脏的靶向给药系统，以提高肾脏药物浓度和降低毒副作用，具有十分重要的意义。
1977年我国科学家在国际上首创应用中药雷公藤治疗慢性肾炎获得成功。其制剂采用雷公藤根(去皮)经粉碎、提取、精制得雷公藤多甙，压制成片剂，其成分复杂、质量不稳定和不良反应较多。本课题采用其主要有效成分雷公藤内酯醇为模型药物，选用溶菌酶为载体，研究雷公藤内酯醇的溶菌酶前体药物的合成、表征、体内外特性。为研究可以产生局部的免疫抑制和抗炎作用、毒副作用低的肾靶向新药提供科学基础。
本研究制备了雷公藤内酯醇—溶菌酶结合物(TPS-LZM)。首先合成了雷公藤内酯醇丁二酸酯，应用熔点、UV、HPLC、IR、MS、~1H-NMR等进行了鉴定，确证了雷公藤内酯醇丁二酸酯的结构。然后雷公藤内酯醇丁二酸酯与溶菌酶反应制备了目标结合物(TPS-LZM)。TPS-LZM中TP／LZM摩尔比约为1∶1，质量比约为0．25％。以3％甘露醇为冻干保护剂，将TPS-LZM结合物制成了溶液型冻干粉针剂，剂量每瓶1mg，并进行了制剂综合性能评价；本制剂有一定吸湿性，其相对临界湿度为72％，应密闭贮存。
体外理化性质研究表明：TPS-LZM：结合物在不同pH值缓冲液以及小鼠血浆中的稳定性研究结果显示其在体外环境中比较稳定。在pH=2～8溶液中，37℃下放置12 h水解产生游离TP的量小于2％；在30％大鼠血浆中，37℃条件下放置2 h，水解产生游离TP的量小于10％。在大鼠肾溶酶体溶液中能够12 h释放80％以上游离原药，表明结合物能够在到达肾脏之前稳定存在一段时间，避免在到达靶组织之前被降解；到达目的细胞器后又能够释放出活性母药。
体外吸收模型实验表明：结合物能够与近端小管细胞迅速结合，雷公藤内酯醇对溶菌酶的结合没有改变其能够被近端小管细胞摄取的特性。单层近端小管细胞吸收和释放实验发现结合物能被吸收，其活性药物的释放主要向细胞的基底膜侧。采用MTT比色法考察了结合物与原药的对近端小管上皮细胞的毒性。研究显示，结合物和原药浓度越高，对肾近段小管上皮细胞作用时间越长，则细胞毒性越大，但结合物的毒性小于原药，特别是在高药物浓度下毒性差距更大。
体内分布实验显示：与原药相比，结合物综合表现为：具有较短的血浆半衰期；具有较好的肾靶向性和滞留时间；在其它各脏器中的分布减少。药理学研究表明：结合物能够明显改善实验所致大鼠肾炎和缺血再灌注引起的损伤，其对肝脏和睾丸的毒性降低，对机体整个免疫系统的影响较小，有一定局部免疫抑制效果。
综上所述，本研究成功制备了雷公藤内酯醇—溶菌酶结合物，并对其靶向性质进行了定位、定性和定量研究，实现了本课题的设计目标。
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.
1977年我国科学家在国际上首创应用中药雷公藤治疗慢性肾炎获得成功。其制剂采用雷公藤根(去皮)经粉碎、提取、精制得雷公藤多甙，压制成片剂，其成分复杂、质量不稳定和不良反应较多。本课题采用其主要有效成分雷公藤内酯醇为模型药物，选用溶菌酶为载体，研究雷公藤内酯醇的溶菌酶前体药物的合成、表征、体内外特性。为研究可以产生局部的免疫抑制和抗炎作用、毒副作用低的肾靶向新药提供科学基础。
本研究制备了雷公藤内酯醇—溶菌酶结合物(TPS-LZM)。首先合成了雷公藤内酯醇丁二酸酯，应用熔点、UV、HPLC、IR、MS、~1H-NMR等进行了鉴定，确证了雷公藤内酯醇丁二酸酯的结构。然后雷公藤内酯醇丁二酸酯与溶菌酶反应制备了目标结合物(TPS-LZM)。TPS-LZM中TP／LZM摩尔比约为1∶1，质量比约为0．25％。以3％甘露醇为冻干保护剂，将TPS-LZM结合物制成了溶液型冻干粉针剂，剂量每瓶1mg，并进行了制剂综合性能评价；本制剂有一定吸湿性，其相对临界湿度为72％，应密闭贮存。
体外理化性质研究表明：TPS-LZM：结合物在不同pH值缓冲液以及小鼠血浆中的稳定性研究结果显示其在体外环境中比较稳定。在pH=2～8溶液中，37℃下放置12 h水解产生游离TP的量小于2％；在30％大鼠血浆中，37℃条件下放置2 h，水解产生游离TP的量小于10％。在大鼠肾溶酶体溶液中能够12 h释放80％以上游离原药，表明结合物能够在到达肾脏之前稳定存在一段时间，避免在到达靶组织之前被降解；到达目的细胞器后又能够释放出活性母药。
体外吸收模型实验表明：结合物能够与近端小管细胞迅速结合，雷公藤内酯醇对溶菌酶的结合没有改变其能够被近端小管细胞摄取的特性。单层近端小管细胞吸收和释放实验发现结合物能被吸收，其活性药物的释放主要向细胞的基底膜侧。采用MTT比色法考察了结合物与原药的对近端小管上皮细胞的毒性。研究显示，结合物和原药浓度越高，对肾近段小管上皮细胞作用时间越长，则细胞毒性越大，但结合物的毒性小于原药，特别是在高药物浓度下毒性差距更大。
体内分布实验显示：与原药相比，结合物综合表现为：具有较短的血浆半衰期；具有较好的肾靶向性和滞留时间；在其它各脏器中的分布减少。药理学研究表明：结合物能够明显改善实验所致大鼠肾炎和缺血再灌注引起的损伤，其对肝脏和睾丸的毒性降低，对机体整个免疫系统的影响较小，有一定局部免疫抑制效果。
综上所述，本研究成功制备了雷公藤内酯醇—溶菌酶结合物，并对其靶向性质进行了定位、定性和定量研究，实现了本课题的设计目标。
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.
引文
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20 Minard, F.N., Cain, J.C., Grant, D.S., Ours, C.W., Kyncl, J., Jones, P.H. and Biel, J.H., Elevation of Renal Dopamine by esters of L-γ-glutamyl dopamine, In: Proc. 172nd Meeting Am. Chem.Soc [M]. MEDI 17, 1976
21 Orlowki, M., Mizoguchi, H. and Wilk, S., N-acyl-γ-Glutamyl Derivatives of Sulfamethoxazole as Models of Kidney-Selective Prodrugs [J]. J. Pharmacol. Exp. Ther., 1979, 212:167-172
22 SU M, HE Q, ZHANG ZR, et al. Kidney-targeting characteristics of N-acetyl-L-glutamic prednisolone prodrug [J]. Acta pharmaceutica sinica(药学学报), 2003, 38 (8): 627—630
23 Drieman, J.C., Thijssen, H.H.W., Zeegers, H.H.M., Smits, J.F.M. and Struyker-Boudier, H.A.J., Renal selective N-acetyl-γ-glutamyl prodrugs: a study on the mechanism of activation of the renal vasodilator prodrug CGP 22979 [J]. Br.J.Pharmacol., 1990, 99:15-20
24 Elfarra AA, Hwang IY, Metabolism and kidney-selectivity of S-(6-purinyl)-L-cysteine analogs in rats [J]. Drug Metab Dispos 1993;21:841-845
25 Birn H, Selhub J, Christensen EI: Internalization and intracellular transport of folate binding protein in rat kidney proximal tubule [J]. Am. J. Physiol. 264 (Cell Physiol. 33): C302-C310, 1993.
26 Wang S, Luo J, Lantrip DA, Waters DJ, MathiasCJ, Green MA, Fuchs PL, Low PS: Design and synthesis of [111 In] DTPA- folate for use as a tumor-targeted radiopharmaceutical [J]. Bioconjug Chem (1997) 8(5):673-679
27 Clifford AJ, Heid MK, Muller HG, Bills ND Tissue distribution and prediction of total body folate of rats [J]. J.Nutr: 1990;120:1633-1639
28 Maack, T., Johnson, V., Kau, S.T., Figueiredo, J. and Sigulem, D., Renal filtration, transport, and metabolism of low-molecular-weight proteins: A review [J].Kidney Int. 1979,16:251-270
29 Maack, T., Park, C.H. and Camargo, M.J.F., Renal filtration, transport, and metabolism of proteins. In The Kidney, Physiology and Pathophysiology(Seldin, D.W. and Giebisch, G., eds.)[M], Raven Press, 1985, New York, pp1773-1803
30 Christensen, E.I. and Nielsen, S., Structural and functional features of protein handling in the kidney proximal tubule [J].Semin.Nephrol. 1991,11:414-439
31Haas, M, de Zeeuw, D., Zanten, A.V. and Meijer, D.K.F., Quantification of renal low-molecular-weight protein handling in the intact rat [J]. Kidney Int., 1993, 43:949-954
32 Haas, M. and de Zeeuw, D., In vivo renal low-molecular-weight protein handling in the rat [J]. J.Am.Soc.Nephrol, 1990,1:699
33 Franssen, E.J.F., Koiter, J., Kuipers, C.A.M., Briuns, A.P., Moolenaar, F., de Zeeuw, D., Kruitinga, W., Kellogg, R.M. and Meijer, D.K.F., Low molecular weight proteins for renal drug targeting: Preparation of drug-protein conjugates and drug-spacer deriivatives and their catabolism in renal cortex homogenates and lysosomal lystates [J]. J.MedChem., 1992, 35:1246-59
34 Franssen, E.J.F., Moolenaar. F. de Zeeuw, D. and Meijer, D.K.F., Low molecular weight proteins as carriers for renal drug targeting: Naproxen coupled to lysozyme via the spacer L-lactic acid, Pharmacol.Res. [J].1993,10:963-969
35 Franssen, E.J.F., van Amsterdam, R.G.M., Visser, J., Moolenaar, F., de Zeeuw, D. and Meijer, D.K.F., Low molecular weight proteins as carriers for renal drug targeting: Naproxen-lysozyme, Pharm.Res. [J]. 1991,8:1223-1230
36 Haas, M., Kluppel, A.C.A., Watna, E.S., Moolenaar, F., Meijer, D.K.F., de Jong, RE. and de Zeeuw, D., Drug targeting to the kidney: Renal delivery and degradation of a naproxen-lysozyme conjugate in vivo [J]. Kidney Int., 1997, 52:1693-1699
37 Kok, R.J., Grijpstra, F., Walthuis, R.B., Moolenaar, F., de Zeeuw, D. and Meijer, K.F., Specific delivery of captopril to the kidney with the prodrug captopril-lysozyme [J]. J.Pharmacol.Exp.Ther., 1999,288: 281-285
38 R.Folgert G.Haverdings,Marijike Haas .Renal targeting of catopril selectively enhances the intrarenal over the systematic effects of ACE inhibition in rats [J].British journal of pharmacology (2002) 136,1107-1116
39 Yamamoto,Yoko;Tsutsumi,Yasuo;Yoshioka,Yasuo; et. al. Poly(vinylpyrrolidone co-dimethyl maleic acid) as a novel renal targeting carrier[J]. Journal of Controlled Release Volume: 95, Issue: 2, March 5,2004, pp. 229-237
40 Kodaira,Hiroshi;Tsutsumi,Yasuo;Yoshioka,Ya.suo; et. al. The targeting of anionized polyvinylpyrrolidone to the renal system [J] Biomaterials Volume: 25, Issue: 18,August, 2004, pp. 4309-4315
41 Kishida,Akio A site-specific polymeric drug carrier for renal disease treatment [J].Trends in Phmacological Sciences Volume: 24, Issue: 12, December, 2003, pp.611-613
42 Mangravite,LaraM.;Bbadagnani,Ilaria;Giacomini,Kathleen M. Nucleoside transporters in the disposition and targeting of nucleoside analogs in the kidney[J].European Journal of Pharmacology Volume: 479, Issue: 1-3, October 31,2003,pp.269-281
43 Van der Wouden,Els A;Sandovici,Maria; et. al. Approaches and methods in gene therapy for kidney disease[J].Journal of Pharmacological and Toxicological Methods Volume: 50, Issue: 1, July -August, 2004, pp. 13-24
44 Noiri E, Peresleni T, Miller F, Goligorsky MS. In vivo targeting of inducible NO synthase witholigodeoxynucleotides protects rat kidney against ischemia [J]. J Clin Invest. 1996,97,2377-2383
45 Oberbauer R, Schreiner GF, Biber J, Murer H, and Meyer TW. In vivo suppression of the renal Na/Pi cotransporter by antisense oligonucleotides [J]. Proc Natl Acad Sci USA 93: 4903-4906
46 Wang ZQ, Felder RA, Carey RM. Selective inhibition of the renal dopamine subtype D1Areceptor induces antinatriuresis in conscious rats [J].Hypertension.1999; 33(pt 2) 504-510
47 Chen W, Bennett CF, Wang ME, Dragun D, Tian L, Stecker K, Clark JH, Kahan BD,Stepkowski SM. Perfusion of kidneys with unformulated "naked" intercellular adhesion molecule-1 antisense oligodeoxynucleotides prevents ischemic/reperfusion injury [J]. Transplantation 68:880-7, 1999.
48 Cheng QL, Chen XM, Li F, Lin HL, Ye YZ, Fu B.Effects of ICAM-1 antisense oligonucleotide on the tubulointerstitium in mice with unilateral ureteral obstruction[J].Kidney Int. 2000 Jan;57(1): 183-90
49 Okada H, Moriwaki K, Kalluri R, Takenaka T, Imai H, Ban S, Takahama M, Suzuki H: Osteopontin expressed by renal tubular epithelium mediates interstitial monocyte infiltration [J]. Am J Physiol 2000, 278:F110-F121
50 Han DC, Hoffman BB, Ziyadeh FN: Stimulation of TGF- type II receptor by high glucose in mouse mesangial cells and in diabetic kidney [J]. Am J Physiol 2000, 278, F628-F634
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18 Wilk, S., Mizoguchi, H. and Orlowski, M., γ-Glutamyl Dopa: A Kidney-specific Dopamine Precursor [J]. J.Pharmacol.Exp. Then, 1978, 206:227-232
19 yncl, J., Holliger, R., Ours, C.W., Minard, R.N., Jones, P.H. and Biel, J.H., Gamma-Glutamyl Dopamine, A Kidney Specific Slow Release Prodrug. In: Proc. 172nd Meeting Am. Chem.Soc[M], MEDI 19, 1976
20 Minard, F.N., Cain, J.C., Grant, D.S., Ours, C.W., Kyncl, J., Jones, P.H. and Biel, J.H., Elevation of Renal Dopamine by esters of L-γ-glutamyl dopamine, In: Proc. 172nd Meeting Am. Chem.Soc [M]. MEDI 17, 1976
21 Orlowki, M., Mizoguchi, H. and Wilk, S., N-acyl-γ-Glutamyl Derivatives of Sulfamethoxazole as Models of Kidney-Selective Prodrugs [J]. J. Pharmacol. Exp. Ther., 1979, 212:167-172
22 SU M, HE Q, ZHANG ZR, et al. Kidney-targeting characteristics of N-acetyl-L-glutamic prednisolone prodrug [J]. Acta pharmaceutica sinica(药学学报), 2003, 38 (8): 627—630
23 Drieman, J.C., Thijssen, H.H.W., Zeegers, H.H.M., Smits, J.F.M. and Struyker-Boudier, H.A.J., Renal selective N-acetyl-γ-glutamyl prodrugs: a study on the mechanism of activation of the renal vasodilator prodrug CGP 22979 [J]. Br.J.Pharmacol., 1990, 99:15-20
24 Elfarra AA, Hwang IY, Metabolism and kidney-selectivity of S-(6-purinyl)-L-cysteine analogs in rats [J]. Drug Metab Dispos 1993;21:841-845
25 Birn H, Selhub J, Christensen EI: Internalization and intracellular transport of folate binding protein in rat kidney proximal tubule [J]. Am. J. Physiol. 264 (Cell Physiol. 33): C302-C310, 1993.
26 Wang S, Luo J, Lantrip DA, Waters DJ, MathiasCJ, Green MA, Fuchs PL, Low PS: Design and synthesis of [111 In] DTPA- folate for use as a tumor-targeted radiopharmaceutical [J]. Bioconjug Chem (1997) 8(5):673-679
27 Clifford AJ, Heid MK, Muller HG, Bills ND Tissue distribution and prediction of total body folate of rats [J]. J.Nutr: 1990;120:1633-1639
28 Maack, T., Johnson, V., Kau, S.T., Figueiredo, J. and Sigulem, D., Renal filtration, transport, and metabolism of low-molecular-weight proteins: A review [J].Kidney Int. 1979,16:251-270
29 Maack, T., Park, C.H. and Camargo, M.J.F., Renal filtration, transport, and metabolism of proteins. In The Kidney, Physiology and Pathophysiology(Seldin, D.W. and Giebisch, G., eds.)[M], Raven Press, 1985, New York, pp1773-1803
30 Christensen, E.I. and Nielsen, S., Structural and functional features of protein handling in the kidney proximal tubule [J].Semin.Nephrol. 1991,11:414-439
31Haas, M, de Zeeuw, D., Zanten, A.V. and Meijer, D.K.F., Quantification of renal low-molecular-weight protein handling in the intact rat [J]. Kidney Int., 1993, 43:949-954
32 Haas, M. and de Zeeuw, D., In vivo renal low-molecular-weight protein handling in the rat [J]. J.Am.Soc.Nephrol, 1990,1:699
33 Franssen, E.J.F., Koiter, J., Kuipers, C.A.M., Briuns, A.P., Moolenaar, F., de Zeeuw, D., Kruitinga, W., Kellogg, R.M. and Meijer, D.K.F., Low molecular weight proteins for renal drug targeting: Preparation of drug-protein conjugates and drug-spacer deriivatives and their catabolism in renal cortex homogenates and lysosomal lystates [J]. J.MedChem., 1992, 35:1246-59
34 Franssen, E.J.F., Moolenaar. F. de Zeeuw, D. and Meijer, D.K.F., Low molecular weight proteins as carriers for renal drug targeting: Naproxen coupled to lysozyme via the spacer L-lactic acid, Pharmacol.Res. [J].1993,10:963-969
35 Franssen, E.J.F., van Amsterdam, R.G.M., Visser, J., Moolenaar, F., de Zeeuw, D. and Meijer, D.K.F., Low molecular weight proteins as carriers for renal drug targeting: Naproxen-lysozyme, Pharm.Res. [J]. 1991,8:1223-1230
36 Haas, M., Kluppel, A.C.A., Watna, E.S., Moolenaar, F., Meijer, D.K.F., de Jong, RE. and de Zeeuw, D., Drug targeting to the kidney: Renal delivery and degradation of a naproxen-lysozyme conjugate in vivo [J]. Kidney Int., 1997, 52:1693-1699
37 Kok, R.J., Grijpstra, F., Walthuis, R.B., Moolenaar, F., de Zeeuw, D. and Meijer, K.F., Specific delivery of captopril to the kidney with the prodrug captopril-lysozyme [J]. J.Pharmacol.Exp.Ther., 1999,288: 281-285
38 R.Folgert G.Haverdings,Marijike Haas .Renal targeting of catopril selectively enhances the intrarenal over the systematic effects of ACE inhibition in rats [J].British journal of pharmacology (2002) 136,1107-1116
39 Yamamoto,Yoko;Tsutsumi,Yasuo;Yoshioka,Yasuo; et. al. Poly(vinylpyrrolidone co-dimethyl maleic acid) as a novel renal targeting carrier[J]. Journal of Controlled Release Volume: 95, Issue: 2, March 5,2004, pp. 229-237
40 Kodaira,Hiroshi;Tsutsumi,Yasuo;Yoshioka,Ya.suo; et. al. The targeting of anionized polyvinylpyrrolidone to the renal system [J] Biomaterials Volume: 25, Issue: 18,August, 2004, pp. 4309-4315
41 Kishida,Akio A site-specific polymeric drug carrier for renal disease treatment [J].Trends in Phmacological Sciences Volume: 24, Issue: 12, December, 2003, pp.611-613
42 Mangravite,LaraM.;Bbadagnani,Ilaria;Giacomini,Kathleen M. Nucleoside transporters in the disposition and targeting of nucleoside analogs in the kidney[J].European Journal of Pharmacology Volume: 479, Issue: 1-3, October 31,2003,pp.269-281
43 Van der Wouden,Els A;Sandovici,Maria; et. al. Approaches and methods in gene therapy for kidney disease[J].Journal of Pharmacological and Toxicological Methods Volume: 50, Issue: 1, July -August, 2004, pp. 13-24
44 Noiri E, Peresleni T, Miller F, Goligorsky MS. In vivo targeting of inducible NO synthase witholigodeoxynucleotides protects rat kidney against ischemia [J]. J Clin Invest. 1996,97,2377-2383
45 Oberbauer R, Schreiner GF, Biber J, Murer H, and Meyer TW. In vivo suppression of the renal Na/Pi cotransporter by antisense oligonucleotides [J]. Proc Natl Acad Sci USA 93: 4903-4906
46 Wang ZQ, Felder RA, Carey RM. Selective inhibition of the renal dopamine subtype D1Areceptor induces antinatriuresis in conscious rats [J].Hypertension.1999; 33(pt 2) 504-510
47 Chen W, Bennett CF, Wang ME, Dragun D, Tian L, Stecker K, Clark JH, Kahan BD,Stepkowski SM. Perfusion of kidneys with unformulated "naked" intercellular adhesion molecule-1 antisense oligodeoxynucleotides prevents ischemic/reperfusion injury [J]. Transplantation 68:880-7, 1999.
48 Cheng QL, Chen XM, Li F, Lin HL, Ye YZ, Fu B.Effects of ICAM-1 antisense oligonucleotide on the tubulointerstitium in mice with unilateral ureteral obstruction[J].Kidney Int. 2000 Jan;57(1): 183-90
49 Okada H, Moriwaki K, Kalluri R, Takenaka T, Imai H, Ban S, Takahama M, Suzuki H: Osteopontin expressed by renal tubular epithelium mediates interstitial monocyte infiltration [J]. Am J Physiol 2000, 278:F110-F121
50 Han DC, Hoffman BB, Ziyadeh FN: Stimulation of TGF- type II receptor by high glucose in mouse mesangial cells and in diabetic kidney [J]. Am J Physiol 2000, 278, F628-F634