5-氟尿嘧啶脂质体防治增生性玻璃体视网膜病变的实验研究
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摘要
目的 研究玻璃体内注射5-氟尿嘧啶(5-fluorouracil，5-FU)脂质体对视网膜的毒性作用，及其对动物模型的增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)的防治效果。
方法 一、家兔18只共36只眼，分3组：5-氟尿嘧啶脂质体组，浓度分别为0.8mg／0.1mL,1.6mg／0.1mL,3.2mg／0.1mL，6.0mg／0.1mL；游离5-氟尿嘧啶组，浓度为1.6mg/0.1mL；空白对照组，注射磷酸缓冲液0.1mL。每组家兔眼玻璃体内注射以上制剂后每周观察眼前后节变化，28天行视网膜电图检查，并做光镜、电镜检查，综合分析各组药物对视网膜的毒性作用。
二、家兔6只共12只眼，双眼玻璃体内注射活化巨噬细胞制作增生性玻璃体视网膜病变模型。分2组，分别注射：5-氟尿嘧啶脂质体和磷酸缓冲液0.1mL。每周观察各组玻璃体视网膜病变发展程度。28天观察各组牵引性视网膜脱离发生率。结果 一、28天结合视网膜电图及视网膜病理学检查：5-氟尿嘧啶脂质体组0.8mg，1.6mg，3.2mg剂量组未见视网膜毒性作用：1.6mg游离5-氟尿嘧啶组和6.0mg 5-氟尿嘧啶脂质体组可见视网膜电图及视网膜病理学检查异常。
三、术后3-4周，脂质体组玻璃体视网膜增生程度较对照组减轻；术后28天，脂质体组视网膜脱离发生率为16.7％，对照组为83.3％，有显著性差异。结论 脂质体包裹5-氟尿嘧啶能降低其对视网膜的毒性作用；一次性玻璃体内注射5-氟尿嘧啶脂质体能有效抑制玻璃体视网膜病变的发展，显著降低增生性玻璃体视网膜病变动物模型牵引性视网膜脱离的发生率。
Objective To examine the retinal toxicity of liposome-encapsulated 5-fluorouracil (5-FU) administered intravitreally and the inhibiting effects on the proliferative vitreoretinopathy (PVR) in an animal model.
Methods Liposome-encapsulated 5-FU was prepared in four different concentration of 0.8,1.6,3.2,and 6.0 mg,0.1mL. 18 white rabbits (36 eyes) were divided into 6 group of 3 animals each (6 eyes) . In all the 36 eyes, 24 eyes (6 eyes each) received an intravitreal injection of the four above mentioned doses,6 eyes 1.6 nig,0.1 mL of free 5-FU, 6 eyes 0.1 mL of normal saline as a normal control group. Retinal toxicity was examined by ophthalmoscopy, photopic electroretinography (ERG) ,light and transmission electron microscope on the 28th day after injection.
The rabbit proliferative vitreoretinopathy (PVR) model was induced by macrophages in both eyes. In the first group, 0.1 mL of normal saline were injected in each eye as a control group. In the second group, liposome-encapsulated 5-FU in a concentration of 1.6 mg,0.1mL were injected. All the rabbits were examined using indirect ophthalmoscopy 28 days after injection.
Results Doses of up to 3.2 mg administered intravitreally in rabbits demonstrated
no retinal toxicity by histologic or electroretinographic criteria. In an experimental animal model of PVR, intravitreal injection of homologous rabbit macrophages caused tractional retinal detachments in 83.3% of eyes after 4 weeks. The addition of 1.6 mg of liposome-encapsulated 5-FU decreased the rate of detachment to 16.7%. Conclusion Liposome-encapsulated 5-FU may markedly reduce the retinal toxicity and the rate of retinal detachment in PVR model after intravitreal injection.
方法 一、家兔18只共36只眼，分3组：5-氟尿嘧啶脂质体组，浓度分别为0.8mg／0.1mL,1.6mg／0.1mL,3.2mg／0.1mL，6.0mg／0.1mL；游离5-氟尿嘧啶组，浓度为1.6mg/0.1mL；空白对照组，注射磷酸缓冲液0.1mL。每组家兔眼玻璃体内注射以上制剂后每周观察眼前后节变化，28天行视网膜电图检查，并做光镜、电镜检查，综合分析各组药物对视网膜的毒性作用。
二、家兔6只共12只眼，双眼玻璃体内注射活化巨噬细胞制作增生性玻璃体视网膜病变模型。分2组，分别注射：5-氟尿嘧啶脂质体和磷酸缓冲液0.1mL。每周观察各组玻璃体视网膜病变发展程度。28天观察各组牵引性视网膜脱离发生率。结果 一、28天结合视网膜电图及视网膜病理学检查：5-氟尿嘧啶脂质体组0.8mg，1.6mg，3.2mg剂量组未见视网膜毒性作用：1.6mg游离5-氟尿嘧啶组和6.0mg 5-氟尿嘧啶脂质体组可见视网膜电图及视网膜病理学检查异常。
三、术后3-4周，脂质体组玻璃体视网膜增生程度较对照组减轻；术后28天，脂质体组视网膜脱离发生率为16.7％，对照组为83.3％，有显著性差异。结论 脂质体包裹5-氟尿嘧啶能降低其对视网膜的毒性作用；一次性玻璃体内注射5-氟尿嘧啶脂质体能有效抑制玻璃体视网膜病变的发展，显著降低增生性玻璃体视网膜病变动物模型牵引性视网膜脱离的发生率。
Objective To examine the retinal toxicity of liposome-encapsulated 5-fluorouracil (5-FU) administered intravitreally and the inhibiting effects on the proliferative vitreoretinopathy (PVR) in an animal model.
Methods Liposome-encapsulated 5-FU was prepared in four different concentration of 0.8,1.6,3.2,and 6.0 mg,0.1mL. 18 white rabbits (36 eyes) were divided into 6 group of 3 animals each (6 eyes) . In all the 36 eyes, 24 eyes (6 eyes each) received an intravitreal injection of the four above mentioned doses,6 eyes 1.6 nig,0.1 mL of free 5-FU, 6 eyes 0.1 mL of normal saline as a normal control group. Retinal toxicity was examined by ophthalmoscopy, photopic electroretinography (ERG) ,light and transmission electron microscope on the 28th day after injection.
The rabbit proliferative vitreoretinopathy (PVR) model was induced by macrophages in both eyes. In the first group, 0.1 mL of normal saline were injected in each eye as a control group. In the second group, liposome-encapsulated 5-FU in a concentration of 1.6 mg,0.1mL were injected. All the rabbits were examined using indirect ophthalmoscopy 28 days after injection.
Results Doses of up to 3.2 mg administered intravitreally in rabbits demonstrated
no retinal toxicity by histologic or electroretinographic criteria. In an experimental animal model of PVR, intravitreal injection of homologous rabbit macrophages caused tractional retinal detachments in 83.3% of eyes after 4 weeks. The addition of 1.6 mg of liposome-encapsulated 5-FU decreased the rate of detachment to 16.7%. Conclusion Liposome-encapsulated 5-FU may markedly reduce the retinal toxicity and the rate of retinal detachment in PVR model after intravitreal injection.
引文
1 Kirchhof B, Sorgente N. Pathogenesis of proliferative vitreoretinopathy. Dev Ophthalmol, 1989; 16:1-53
2 Weller M, Wiedemann P, Heimann K. Proliferative vitroretinopathy is it anything more than wound healing at the wrong place. Int Ophtha#nol, 1990; 14(2):105-107
3 Hui YN, Liang HC, Cai YS, et al. Corticoster and daunomycin in the prevention of experimental proliferative vitreoretinopathy induced by macrophages. Graefe's Arch Clin Exp Ophthalmol. 1993; 231 (2): 109-114
4 Blumenkranz MS, Claflin A, Hajek AS. Selection of therapeutic agents for intracular proliferative disease. Arch Ophthalmol, 1984; 102(4):598
5 曾水清，胡椿枝，吕源淑等．兔眼玻璃体内注入脂质体的观察．眼科研究1992；10(1)： 23-25
6 Stem WH, Heath TD, Lewis GP, et al. Clearance and localization of intravitreal liposomes in the aphakic vitrectomized eye. Invest Ophthalmol Vis Sci. 1987; 28(5):907-911
7 Blankenship GW. Evaluation of a single intravitreal injection of 5-fiuorouracil in vitrectomy case. Graefe's Arch Clin Exp Ophthalmol, 1989; 227(6): 565-568
8 惠延年，石一宁，张新海等．巨噬细胞诱发的实验性增生性玻璃体视网膜病变中的几种早期炎性细胞因子．中华眼科杂志．1999；35(2)：99-103
9 梁厚成，惠延年，蔡用舒．道诺霉素脂质体防治实验性增殖性玻璃体视网膜病变．中华眼底病杂志．1993；9(2)：77-79
10 Wiedemann P, Heimann K. Prolifterative Vitreoretiopathy. Pathogenesis and possibilities for treatment with cytostatic drag. Klin Monatsbl-Augenheilkd 1986; 188(6):559-564
11 Kon CH, Occleton NL, Charteris D, et al. A prospective study of matrix
metalloproteinases in proliferative vitreoretiopathy. Invest Ophthalmol Vis Sci 1998; 1524-1529
12 Wiedemann P, Weller M, Heimann L. Proliferative vitreoretiopathy: new discoveries in pathophysiology and therapy. Kiln Monatsbl-Augenheilkd,1990; 197(5):355-361
13 Blumenkranz MS, Claflin A, Hajek AS. Selection of therapeutic agents for intracular proliferative disease. Arch Ophthalmol, 1984; 102:598
14 Leaver PK. The treatment of vitreoretinal scar tissue. Eye, 1994; 8(2):210-216
15 Freilich DB,Lee PF, Freeman HM. Experimental retinal detachment. Arch Ophthalmol, 1966; 76(3):423-436
16 De Juan E,Humayun MS, Hatchell DL, et al. Histopathology of experimental periretinal neovascularization. Invest Ophthalmol Vis Sci, 1989; 30(7): 1495-1503
17 Planck SR, Andresevic J, Chen JC, et al. Expression of growth factor mRNA in rabbit PVR model systems. Curr Eye Res, 1992; 11 (11): 1031 - 1039
18 Fastenberg DM, Diddie KR, Sorgente N, et al. The role of proliferation in an experimental model of massive periretinal proliferation. Am J Ophthalmol, 1982; 93:559
19 Blumenkranz MS, Claflin A, Hajek AS, et al. Selection of therapeutic agents for intraocular proliferative disease. Cell culture evaluation. Arch Ophthaimol, 1984; 102:598
20 Rubsamen PE, Davis PA, Hernandez E, et al. Prevention of experimental proliferative vitreoretinopathy with a biodegradable intravitreal implant for the sustained release of fluorouracil.Arch Ophthalmol, 1994; 112(3):407-413
21 陈建斌．玻璃体内植入式药物缓释系统．国外医学眼科分册．2001；25(1)：57-60
22 陆彬．药物新剂型与新技术．北京．人民卫生出版社；1998：111-126
23 黄立夫．脂质体作为药物载体的可行性及其在细胞生物学上的应用．国外医学分子生物学分册．1984；6：230
24 Barza M,Stuart M,Szoka F. Effect of size and lipid composition on the pharmacokinetics of intravitreal liposomes. Invest Ophthalmol Vis Sci, 1987; 28(5):893-900
25 Stern WH,Heath TD,Lewis GP et al. Clearance and localization of intravitreal liposome in the aphakic vitrectomized eye. Invest Ophthalmol Vis Sci, 1987; 28(5):907-911
26 Liu KR, Peyman GA, Khoobehi B, et al. Intravitreal liposome-encapsulated trifluorothymidine in a rabbit model. Ophthalmology, 1987; 94(9): 1155
27 Blankenship GW. Evaluation of a single intravitreal injection of 5-fluorouracil in vitrectomy case. Graefe's Arch Clin Exp Ophthalmol, 1989; 227(6): 565-568
28 Rubsamen PE, Davis PA, Hernandez E, et al. Prevention of experimental proliferative vitreoretinopathy with a biodegradable intravitreal implant for the sustained release of fluorouracil. Arch Ophthalmol, 1994; 112(3):407-413
29 Cai J, Wei R, Ma X et al. Cytotoxic effect of antiproliferative agents on human retinal glial cells in vitro. Int Ophthalmol, 2001; 24(4):225-231
30 Gase JL, Peyman GA, Barrada A, et al. Clearance in intravitreal fluorouracil.OphthalmicSurg, 1985; 16(6): 378-381
31 Jarus G, Blumenkranz M, Hernandez E, et al. Clearance of intravitreal fluorouracil. Normal and aphakic vitrectomized eyes. Ophthalmology, 1985; 92(1): 91-96
32 Blumenkranz M, Hernandez E, Ophir A, et al. 5-fluorouracil: new applications in complicated retinal detachment for an established antimetabolite. Ophthalmology, 1984; 91(2): 122
33 Kulnig N, Binder S, Riss B. Inhibition of experimental intraocular proliferation with intravitreal 5-fluorouracil. A transmission electron microscopic study in rabbits. Ophthalmologica, 1984; 188(4);248-258
34 Assil KK, Frucht-Perry J, Ziegler E, et al. Tobramycin liposomes. Single
subconjunctival therapy of pseudomonal keratitis, Invest Ophthalmol Vis Sci, 1991; 32(11):2891-2897
35 Fishman P, Peyman GA, Hendricks R,et al. Liposome-encapsulated 3H-5FU in rabbits, Int Ophthalmol, 1989; 13(5):361-365
36 吴乐正，吴德正主编．临床视觉电生理学．北京：科学出版社，1999：17-20
37 Jay WM, Aziz MZ, Rissing JP, et al. Pharmacokinetic analysis of intravitreal caftriaxone in monkeys.Arch Ophthalmol, 1995; 103 (1): 121-123
38 梁厚成，惠延年，蔡用舒．道诺霉素脂质体玻璃体内代谢及视网膜毒性．眼科研究．1993；11(2)：87-89
39 Langer R.New methods of drug delivery. Science, 1990; 28(2):1527-1533
2 Weller M, Wiedemann P, Heimann K. Proliferative vitroretinopathy is it anything more than wound healing at the wrong place. Int Ophtha#nol, 1990; 14(2):105-107
3 Hui YN, Liang HC, Cai YS, et al. Corticoster and daunomycin in the prevention of experimental proliferative vitreoretinopathy induced by macrophages. Graefe's Arch Clin Exp Ophthalmol. 1993; 231 (2): 109-114
4 Blumenkranz MS, Claflin A, Hajek AS. Selection of therapeutic agents for intracular proliferative disease. Arch Ophthalmol, 1984; 102(4):598
5 曾水清，胡椿枝，吕源淑等．兔眼玻璃体内注入脂质体的观察．眼科研究1992；10(1)： 23-25
6 Stem WH, Heath TD, Lewis GP, et al. Clearance and localization of intravitreal liposomes in the aphakic vitrectomized eye. Invest Ophthalmol Vis Sci. 1987; 28(5):907-911
7 Blankenship GW. Evaluation of a single intravitreal injection of 5-fiuorouracil in vitrectomy case. Graefe's Arch Clin Exp Ophthalmol, 1989; 227(6): 565-568
8 惠延年，石一宁，张新海等．巨噬细胞诱发的实验性增生性玻璃体视网膜病变中的几种早期炎性细胞因子．中华眼科杂志．1999；35(2)：99-103
9 梁厚成，惠延年，蔡用舒．道诺霉素脂质体防治实验性增殖性玻璃体视网膜病变．中华眼底病杂志．1993；9(2)：77-79
10 Wiedemann P, Heimann K. Prolifterative Vitreoretiopathy. Pathogenesis and possibilities for treatment with cytostatic drag. Klin Monatsbl-Augenheilkd 1986; 188(6):559-564
11 Kon CH, Occleton NL, Charteris D, et al. A prospective study of matrix
metalloproteinases in proliferative vitreoretiopathy. Invest Ophthalmol Vis Sci 1998; 1524-1529
12 Wiedemann P, Weller M, Heimann L. Proliferative vitreoretiopathy: new discoveries in pathophysiology and therapy. Kiln Monatsbl-Augenheilkd,1990; 197(5):355-361
13 Blumenkranz MS, Claflin A, Hajek AS. Selection of therapeutic agents for intracular proliferative disease. Arch Ophthalmol, 1984; 102:598
14 Leaver PK. The treatment of vitreoretinal scar tissue. Eye, 1994; 8(2):210-216
15 Freilich DB,Lee PF, Freeman HM. Experimental retinal detachment. Arch Ophthalmol, 1966; 76(3):423-436
16 De Juan E,Humayun MS, Hatchell DL, et al. Histopathology of experimental periretinal neovascularization. Invest Ophthalmol Vis Sci, 1989; 30(7): 1495-1503
17 Planck SR, Andresevic J, Chen JC, et al. Expression of growth factor mRNA in rabbit PVR model systems. Curr Eye Res, 1992; 11 (11): 1031 - 1039
18 Fastenberg DM, Diddie KR, Sorgente N, et al. The role of proliferation in an experimental model of massive periretinal proliferation. Am J Ophthalmol, 1982; 93:559
19 Blumenkranz MS, Claflin A, Hajek AS, et al. Selection of therapeutic agents for intraocular proliferative disease. Cell culture evaluation. Arch Ophthaimol, 1984; 102:598
20 Rubsamen PE, Davis PA, Hernandez E, et al. Prevention of experimental proliferative vitreoretinopathy with a biodegradable intravitreal implant for the sustained release of fluorouracil.Arch Ophthalmol, 1994; 112(3):407-413
21 陈建斌．玻璃体内植入式药物缓释系统．国外医学眼科分册．2001；25(1)：57-60
22 陆彬．药物新剂型与新技术．北京．人民卫生出版社；1998：111-126
23 黄立夫．脂质体作为药物载体的可行性及其在细胞生物学上的应用．国外医学分子生物学分册．1984；6：230
24 Barza M,Stuart M,Szoka F. Effect of size and lipid composition on the pharmacokinetics of intravitreal liposomes. Invest Ophthalmol Vis Sci, 1987; 28(5):893-900
25 Stern WH,Heath TD,Lewis GP et al. Clearance and localization of intravitreal liposome in the aphakic vitrectomized eye. Invest Ophthalmol Vis Sci, 1987; 28(5):907-911
26 Liu KR, Peyman GA, Khoobehi B, et al. Intravitreal liposome-encapsulated trifluorothymidine in a rabbit model. Ophthalmology, 1987; 94(9): 1155
27 Blankenship GW. Evaluation of a single intravitreal injection of 5-fluorouracil in vitrectomy case. Graefe's Arch Clin Exp Ophthalmol, 1989; 227(6): 565-568
28 Rubsamen PE, Davis PA, Hernandez E, et al. Prevention of experimental proliferative vitreoretinopathy with a biodegradable intravitreal implant for the sustained release of fluorouracil. Arch Ophthalmol, 1994; 112(3):407-413
29 Cai J, Wei R, Ma X et al. Cytotoxic effect of antiproliferative agents on human retinal glial cells in vitro. Int Ophthalmol, 2001; 24(4):225-231
30 Gase JL, Peyman GA, Barrada A, et al. Clearance in intravitreal fluorouracil.OphthalmicSurg, 1985; 16(6): 378-381
31 Jarus G, Blumenkranz M, Hernandez E, et al. Clearance of intravitreal fluorouracil. Normal and aphakic vitrectomized eyes. Ophthalmology, 1985; 92(1): 91-96
32 Blumenkranz M, Hernandez E, Ophir A, et al. 5-fluorouracil: new applications in complicated retinal detachment for an established antimetabolite. Ophthalmology, 1984; 91(2): 122
33 Kulnig N, Binder S, Riss B. Inhibition of experimental intraocular proliferation with intravitreal 5-fluorouracil. A transmission electron microscopic study in rabbits. Ophthalmologica, 1984; 188(4);248-258
34 Assil KK, Frucht-Perry J, Ziegler E, et al. Tobramycin liposomes. Single
subconjunctival therapy of pseudomonal keratitis, Invest Ophthalmol Vis Sci, 1991; 32(11):2891-2897
35 Fishman P, Peyman GA, Hendricks R,et al. Liposome-encapsulated 3H-5FU in rabbits, Int Ophthalmol, 1989; 13(5):361-365
36 吴乐正，吴德正主编．临床视觉电生理学．北京：科学出版社，1999：17-20
37 Jay WM, Aziz MZ, Rissing JP, et al. Pharmacokinetic analysis of intravitreal caftriaxone in monkeys.Arch Ophthalmol, 1995; 103 (1): 121-123
38 梁厚成，惠延年，蔡用舒．道诺霉素脂质体玻璃体内代谢及视网膜毒性．眼科研究．1993；11(2)：87-89
39 Langer R.New methods of drug delivery. Science, 1990; 28(2):1527-1533