左氧氟沙星透明质酸钠滴眼液眼部药动学研究
|
摘要
细菌性角膜炎是临床上常见的、致盲率较高的一类眼部感染性疾病。在20世纪60年代，细菌性角膜炎占角膜感染的首位，随着人们生活水平的提高和新型抗生素的不断问世，到20世纪80年代，细菌性角膜炎已降至第二位。但是，细菌性角膜炎仍然是威胁人们眼部健康和视力的常见疾病之一，目前主要的致病菌为表皮葡萄球菌、金黄色葡萄球菌、肺炎链球菌、绿脓杆菌、肠道杆菌等。近10年来，氟喹诺酮类药物应用于眼科临床，由于该类药物抗菌谱广，对革兰阴性杆菌包括绿脓杆菌和部分革兰阳性菌均有良好的抗菌作用，近年来已成为临床上首选的抗菌药物。其中，左氧氟沙星为该类药物中的新型药物。左氧氟沙星是氧氟沙星的左旋光学异构体，其抗菌谱更广，对包括厌氧菌在内的革兰阳性菌和革兰阴性菌均有更强的抗菌作用，抗菌活性是氧氟沙星的两倍。左氧氟沙星局部滴眼眼内通透性较高，然而普通的滴眼液由于受泪液稀释、鼻泪管排泄等因素影响眼部的生物利用度较低，作用时间也极为短暂。为了提高眼部生物利用度，延长作用时间，可以在普通左氧氟沙星滴眼液内加入黏性赋形剂，使溶液黏滞度增加，延长药物在眼部的滞留时间，提高眼部的生物利用度，从而增加治疗效果。透明质酸钠(sodium hyaluronate，SH)是一种常见的生物黏附性良好的黏性剂，在普通左氧氟沙星滴眼液内加入透明质酸钠制成缓释滴眼液，将提高左氧氟沙星在眼部的疗效。本实验利用高效液相法(HLPC)测定了0.3％左氧氟沙星透明质酸钠缓释滴眼液与同浓度普通左氧氟沙星滴眼液点眼后泪液、角膜和房水药物浓度变化，对眼部药动学和相对生物利用度进行比较研究，为临床在治疗细菌性角
郑州大学(2004届)硕士研究生毕业论文
左氧氟沙星透明质酸钠滴眼液眼部药动学研究
膜炎时选择作用更持久、渗透性更强的滴眼液提供理论依据。
1 .HpLC法测定左氧氟沙星泪液、角膜和房水中药物浓度
实验动物为健康成年白兔，体重2一3kg。色谱条件色谱柱为岛津ODS一C18
不锈钢柱(15oK46mm，5林m):流动相为柠檬酸溶液(o.osmoFI)一醋酸胺溶
液(lmo功)一乙睛(82:l:17);流速为l.Zml/min，紫外检测波长为294nm，
柱温为40oC。用滤纸片置于兔子结膜囊下弯隆处30秒采集泪液，用lml注射器
取房水，耳缘静脉推注空气处死兔子，摘除眼球采集角膜。角膜和房水样品均经
二氯甲烷提取，氮气吹干，流动相溶解后进样。该方法所测得的左氧氟沙星色谱
峰与杂质峰分离良好，无干扰，保留时间稳定。标准曲线线性关系良好，相关系
数二0.9999。角膜、房水的平均回收率分别为76.47%、76.53%。角膜、房水三
个标准浓度日内日间差RSD均小于3%。
2.两种左氧氟沙星眼内通透性和药代动力学分析
0.3%左氧氟沙星透明质酸钠缓释滴眼液(实验药)与0.3%普通左氧氟沙星
滴眼液(对照药)各分别使用白色健康兔36只，体重2一3kg，雌雄兼用，随机分
成9组，每组4只8眼。用微量取样器分别给各组家兔双眼结膜囊内滴50川0.3%
滴眼液，滴眼后分别于5、10、15、30、45、60、120、180和240分钟取样。泪
液样品经适量流动相溶解后，角膜和房水样品经提取后，采取HPLC法测量。结
果测得，泪液中两种滴眼液药物浓度由高逐渐下降，在4小时分别降至19.86林9/9
和10.02协9/9。实验组比对照组泪液中的药物浓度高，采用t检验，前30分钟时
间点(10分钟除外)差异无显著性，之后差别均具有显著性(P<0.05)。点眼后
4小时实验组比对照组泪液浓度明显提高(P<0.05)。用计算机进行拟合并计算
得动力学参数。实验组与对照组的消除半衰期tl/2分别为8.27 min和7.81min。
而实验组AUCO一40是对照组的1 .8倍。角膜中两种滴眼液药物浓度由高逐渐下降，
在4小时分别降至1.14卜眺和0.73林吮。实验组比对照组角膜中的药物浓度高，
除smin、巧min以外其余时间点差别均具有显著性(P 合符合二房室模型，实验组与对照组的分布相半衰期tl/;分别为6.23 min和
5.14min，消除相半衰期分别为146.38min和128.3lmin。而实验组 AUC。二4。是对
郑州大学(2 004届)硕士研究生毕业论文
左氧氟沙星透明质酸钠滴眼液眼部药动学研究
照组的1 .6倍。在房水中实验组与对照组的药物达峰浓度峰分别为1 .62卜岁ml和
1.00林留ml，有显著性差异(P<0.05)，达峰时间均为60 min。实验组‘的左氧氟沙
星药物浓度比对照组高(smin、10min除外)，点眼4小时后的药物浓度实验组
较对照组有明显提高(P<0.05)。用计算机进行拟合并计算得动力学参数。实验
组与对照组的消除半衰期tl/2分别为59.78 min和51.82min，而实验组的AUC你24。
是对照组的1.9倍。
实验证明，0.3%的左氧氟沙星透明质酸钠滴眼液单次滴眼后，与同浓度普通
左氧氟沙星滴眼液比较，泪液中的药物浓度下降较缓慢，角膜及房水内药物浓度
较高，说明眼内通透性提高;而且AUCo一24。是同浓度普通左氧氟沙星滴眼液单
次滴眼后的1.8、16和1.9倍，生物利用度也得以提高;与眼部常见细菌呱C90
相比，03%的左氧氟沙星透明质酸钠滴眼液较普通左氧氟沙星滴眼液有近两倍
的提高。证实了透明质酸钠作为一种溶液勤性剂提高药物豁稠度，延长药液在眼
部的滞留时间的作用
Bacterial keratitis is a kind of ocular inflamed disease which is often met clinically and even can lead to blindness. In 1960s, bacterial keratitis took the first place in all kinds of keratitis. With the improvement of living conditions and the invents of new anti-bacterial drugs, the rate of bacterial keratitis has decreased to the second place since 1980s. But this disease is still a danger for eye health and vision. The pathogens often causing bacterial keratitis are staphylococcus epidermidis, staphylococcus aureus, streptococcus pneumoniae, pseudomonas aeuroginosa and escherichia coli. Fluoroquinolones have been used in clinics for 10 years, and which have been the most often applied for more broader anti-bacterial spectrum and more poweful anti-bacterial action to G" including pseudomonas aeuroginosa and G+ in part. Levofloxacin is a new kind of fluoroquinolones which is L-isomer of ofloxacin. The anti- bacterial spectrum of levofloxacin is more broader than the former generation of fluoroquinolones,
and the anti- bacterial power is two times than that of ofloxacin. G+ including anaerobic bacterial and G" are all sensitive to levofloxacin. The ocular penetration of levofloxacin is high in documents from different writers. But the ocular bioavailability of common eye-drops topically applied is low and the residence time is short, because the concentration is decreased quikly with the tear turn-over and nasolacrimal duct excretion. In order to increase ocular bioavailability and make the
residence time longer, some viscous expient was added to the common eye-drops. With the increased drug viscosity, the ocular residence time will be longer, the ocular bioavailability will be increased. Sodium hyaluronate (SH) is a high molecular weight, naturally occurring glycosaminoglycan. Sodium hyaluronate, as an additive to ophthalmic formulations, has been claimed to increase the ocular residence time. An addition of sodium hyaluronate to 0.3% levofloxacin is expected to increase the drug treatment effect.In this experiment, we used the high-performance liquid chromatograph to assay and compare the concentration changes in tears, corneas and aqueous humors after 0.3% levofloxacin-SH and 0.3% levofloxacin topically applied to rabbits eyes, and investigated the ocular pharmacokinetics and bioavailability in order to supply data for chosing much longer residence time, much stronger penetration anti-bacterial eye-drops for bacterial keratitis.
1.The assay of levofloxacin concentration in corneas and
aqueous humors by HPLC
The healthy adult white rabbits (2-3kg) were chosen as experiment animals. The chromatograph conditions: the mobile phase was 0.05mol/l acid citric- lmol/1 ammonium acetate-acetonitrile (82:1:17). The flow-rate was 1.2ml/min. Detection wavelength was 294nm. The column temperature was 40 C. Tears were acquired by using filter papers into inferior fomix of conjuctive sac in rabbits. Aqueous humors were acquired by using the 1 mi-syringe. The rabbits were killed at different times, and the eyeballs were excavated and then corneas were acquired. All corneas and aqueous humors were extracted by dichloroethane, dried by nitrogen, dissolved by mobile phase, and injected into HPLC. The chromatograpy peak of levofloxacin was isolated well from the mixed peaks potentially which interfere with that of levofloxacin by using this method. The remain time was stable. The linearity of standard curve was proved to be good(r=0.9999). The average reproducibility of cornea and aqueous humors in this assay, expressed as the percentage of related standard deviation (% RSD), was 76.47%, 76.53% respectively. The accuracy, expressed as the percentage error in the same day or the different day, was <3% for
three standard concentrations of corneas and aqueous humors.
2.Ocular penetration and pharmacokinetics of levofloxaein in
two different preparations
36 white healthy rabbits (2-3kg) which were divided randomly into 9 groups were used for 0.3% levofloxacin-SH and 0.3% levofloxacin respectively, and each group h
郑州大学(2004届)硕士研究生毕业论文
左氧氟沙星透明质酸钠滴眼液眼部药动学研究
膜炎时选择作用更持久、渗透性更强的滴眼液提供理论依据。
1 .HpLC法测定左氧氟沙星泪液、角膜和房水中药物浓度
实验动物为健康成年白兔，体重2一3kg。色谱条件色谱柱为岛津ODS一C18
不锈钢柱(15oK46mm，5林m):流动相为柠檬酸溶液(o.osmoFI)一醋酸胺溶
液(lmo功)一乙睛(82:l:17);流速为l.Zml/min，紫外检测波长为294nm，
柱温为40oC。用滤纸片置于兔子结膜囊下弯隆处30秒采集泪液，用lml注射器
取房水，耳缘静脉推注空气处死兔子，摘除眼球采集角膜。角膜和房水样品均经
二氯甲烷提取，氮气吹干，流动相溶解后进样。该方法所测得的左氧氟沙星色谱
峰与杂质峰分离良好，无干扰，保留时间稳定。标准曲线线性关系良好，相关系
数二0.9999。角膜、房水的平均回收率分别为76.47%、76.53%。角膜、房水三
个标准浓度日内日间差RSD均小于3%。
2.两种左氧氟沙星眼内通透性和药代动力学分析
0.3%左氧氟沙星透明质酸钠缓释滴眼液(实验药)与0.3%普通左氧氟沙星
滴眼液(对照药)各分别使用白色健康兔36只，体重2一3kg，雌雄兼用，随机分
成9组，每组4只8眼。用微量取样器分别给各组家兔双眼结膜囊内滴50川0.3%
滴眼液，滴眼后分别于5、10、15、30、45、60、120、180和240分钟取样。泪
液样品经适量流动相溶解后，角膜和房水样品经提取后，采取HPLC法测量。结
果测得，泪液中两种滴眼液药物浓度由高逐渐下降，在4小时分别降至19.86林9/9
和10.02协9/9。实验组比对照组泪液中的药物浓度高，采用t检验，前30分钟时
间点(10分钟除外)差异无显著性，之后差别均具有显著性(P<0.05)。点眼后
4小时实验组比对照组泪液浓度明显提高(P<0.05)。用计算机进行拟合并计算
得动力学参数。实验组与对照组的消除半衰期tl/2分别为8.27 min和7.81min。
而实验组AUCO一40是对照组的1 .8倍。角膜中两种滴眼液药物浓度由高逐渐下降，
在4小时分别降至1.14卜眺和0.73林吮。实验组比对照组角膜中的药物浓度高，
除smin、巧min以外其余时间点差别均具有显著性(P
5.14min，消除相半衰期分别为146.38min和128.3lmin。而实验组 AUC。二4。是对
郑州大学(2 004届)硕士研究生毕业论文
左氧氟沙星透明质酸钠滴眼液眼部药动学研究
照组的1 .6倍。在房水中实验组与对照组的药物达峰浓度峰分别为1 .62卜岁ml和
1.00林留ml，有显著性差异(P<0.05)，达峰时间均为60 min。实验组‘的左氧氟沙
星药物浓度比对照组高(smin、10min除外)，点眼4小时后的药物浓度实验组
较对照组有明显提高(P<0.05)。用计算机进行拟合并计算得动力学参数。实验
组与对照组的消除半衰期tl/2分别为59.78 min和51.82min，而实验组的AUC你24。
是对照组的1.9倍。
实验证明，0.3%的左氧氟沙星透明质酸钠滴眼液单次滴眼后，与同浓度普通
左氧氟沙星滴眼液比较，泪液中的药物浓度下降较缓慢，角膜及房水内药物浓度
较高，说明眼内通透性提高;而且AUCo一24。是同浓度普通左氧氟沙星滴眼液单
次滴眼后的1.8、16和1.9倍，生物利用度也得以提高;与眼部常见细菌呱C90
相比，03%的左氧氟沙星透明质酸钠滴眼液较普通左氧氟沙星滴眼液有近两倍
的提高。证实了透明质酸钠作为一种溶液勤性剂提高药物豁稠度，延长药液在眼
部的滞留时间的作用
Bacterial keratitis is a kind of ocular inflamed disease which is often met clinically and even can lead to blindness. In 1960s, bacterial keratitis took the first place in all kinds of keratitis. With the improvement of living conditions and the invents of new anti-bacterial drugs, the rate of bacterial keratitis has decreased to the second place since 1980s. But this disease is still a danger for eye health and vision. The pathogens often causing bacterial keratitis are staphylococcus epidermidis, staphylococcus aureus, streptococcus pneumoniae, pseudomonas aeuroginosa and escherichia coli. Fluoroquinolones have been used in clinics for 10 years, and which have been the most often applied for more broader anti-bacterial spectrum and more poweful anti-bacterial action to G" including pseudomonas aeuroginosa and G+ in part. Levofloxacin is a new kind of fluoroquinolones which is L-isomer of ofloxacin. The anti- bacterial spectrum of levofloxacin is more broader than the former generation of fluoroquinolones,
and the anti- bacterial power is two times than that of ofloxacin. G+ including anaerobic bacterial and G" are all sensitive to levofloxacin. The ocular penetration of levofloxacin is high in documents from different writers. But the ocular bioavailability of common eye-drops topically applied is low and the residence time is short, because the concentration is decreased quikly with the tear turn-over and nasolacrimal duct excretion. In order to increase ocular bioavailability and make the
residence time longer, some viscous expient was added to the common eye-drops. With the increased drug viscosity, the ocular residence time will be longer, the ocular bioavailability will be increased. Sodium hyaluronate (SH) is a high molecular weight, naturally occurring glycosaminoglycan. Sodium hyaluronate, as an additive to ophthalmic formulations, has been claimed to increase the ocular residence time. An addition of sodium hyaluronate to 0.3% levofloxacin is expected to increase the drug treatment effect.In this experiment, we used the high-performance liquid chromatograph to assay and compare the concentration changes in tears, corneas and aqueous humors after 0.3% levofloxacin-SH and 0.3% levofloxacin topically applied to rabbits eyes, and investigated the ocular pharmacokinetics and bioavailability in order to supply data for chosing much longer residence time, much stronger penetration anti-bacterial eye-drops for bacterial keratitis.
1.The assay of levofloxacin concentration in corneas and
aqueous humors by HPLC
The healthy adult white rabbits (2-3kg) were chosen as experiment animals. The chromatograph conditions: the mobile phase was 0.05mol/l acid citric- lmol/1 ammonium acetate-acetonitrile (82:1:17). The flow-rate was 1.2ml/min. Detection wavelength was 294nm. The column temperature was 40 C. Tears were acquired by using filter papers into inferior fomix of conjuctive sac in rabbits. Aqueous humors were acquired by using the 1 mi-syringe. The rabbits were killed at different times, and the eyeballs were excavated and then corneas were acquired. All corneas and aqueous humors were extracted by dichloroethane, dried by nitrogen, dissolved by mobile phase, and injected into HPLC. The chromatograpy peak of levofloxacin was isolated well from the mixed peaks potentially which interfere with that of levofloxacin by using this method. The remain time was stable. The linearity of standard curve was proved to be good(r=0.9999). The average reproducibility of cornea and aqueous humors in this assay, expressed as the percentage of related standard deviation (% RSD), was 76.47%, 76.53% respectively. The accuracy, expressed as the percentage error in the same day or the different day, was <3% for
three standard concentrations of corneas and aqueous humors.
2.Ocular penetration and pharmacokinetics of levofloxaein in
two different preparations
36 white healthy rabbits (2-3kg) which were divided randomly into 9 groups were used for 0.3% levofloxacin-SH and 0.3% levofloxacin respectively, and each group h
引文
1．张文华．感染性角膜疾病．中华眼科杂志．1995；31(2)：156．
2. Smith A, Pennefather PM, Kaye SB, et al. Fluoroquinolones:place in ocular therapy. Drugs 2001;61(6):747-761.
3. Ernst ME, Ernst EJ, Klepser ME. Levofloxacin and trvafloxacin: the next generation of fluoroquinolones? Am J Health Syst Pharm 1997;54:2569-84.
4. Wimer SM, Schoonover L, Garrison ME. Levofloxacin:a therapeutic review. Clin Ther 1998;20:1049-70.
5．蔡鸿生，罗顺德，刘强，等．乳酸左氧氟沙星滴眼液在兔眼中的药动学．中国医院药学杂志，2001；21(7)：391-393．
6．陈祖基．眼科临床药理学．北京：化学工业出版社，2002．35-42．
7. Madu AA, Mayers M,Perkins R, et al. Aqueous and vitreous penetration of ciprofloxacin following different modes of systemic administration. Exp Eye Res. 1996;63(2): 129-136.
8. Liu W, Liu QF, Perkins R, et al. Pharmacokinetics of sparfloxacin in the serum and vitreous humor of rabbits: physicochemical properties that regulate petration of quinolone. Antimicrob Agents Chemother 1998;42(6): 1417-1423.
9．陈悦，张俊杰，陈祖基．左氧氟沙星滴眼液的兔眼内药代动力学．眼科研究2001；19(5)：429-431．
10. Fiscella RG, Nguyen TK, Cwik MJ, et al. Aqueous and vitreous penetration of levofloxacin after oral administration. Ophthalmology 1999; 106(12):2286-2290.
11. Garcia-Saenz MC, Arias-Puente A, Fresnadill-martubez MJ, et al. Human aqueous humor levels of oral ciprofloxacin, levofloxacin, and moxifloxacin. J Cataract Refract Surg 2001;27 (12): 1969-1974.
12. Herbert EN, Pearce IA, McGlliard J,et al.Vitrous penetration of levofloxacin in the unintlamed phakic humam eye. Br J Ophthalmol 2002; 86(4):387-389.
13. Greaves JL, Wilson CG. Treatment of disease of the eye with mucoadhesive
delivery systems. Adv Drug Deliv Rev 1993; 11(2):349-383.
14．魏刚，丁平田，崔咏艳，等．溶液黏度影响聚合物角膜滞留时间的体外评价药学学报2002；37(6)：469-472．
15. Robison JR, Mlynek GM. Bioadhesive and phase-change polymers for ocular drug delivery. Adv Drug Deliv Rev 1995; 16(1):45-50.
16. Snibson GR, Greaves JL, Soper ND, et al. Ocular surface residence times of artificial tear solutions. Cornea 1992; 11 (4):288-293.
17. Camber O, Edman P. Sodium hyaluronate as an ophthalmic vehicle:some factors governing its effect on the ocular absorption of pilocarpine. Curr Eye Res 1989;8(6): 563-567.
18. Gurny R, Ryser JE, Tabatabay C,et al. Precorneal residence time in human of sodium hyaluronate as measured by gammar scientigraphy. Graefes Arch Clin Exp Ophthalmol. 1990; 228(6): 510-512.
19. Bernatchez SF, Tabatabay C, Gurny R. sodium hyaluronate 0.25% used as a vehicle increases the bioavailability of topically administered gentamicin. Graefes Arch Clin Exp Ophthalmol 1993; 231(3):157-161.
20．黄虹，何国珍，屠晓萍，等．两种新型增粘剂在环丙沙星滴眼液中的应用．中国临床药学杂志1996；8(6)：360-363．
21．成立军，舒东，刘叶玲，等．含玻璃酸钠的毛果芸香碱滴眼液．中国生化杂志2000；21(3)：144-143．
2. Smith A, Pennefather PM, Kaye SB, et al. Fluoroquinolones:place in ocular therapy. Drugs 2001;61(6):747-761.
3. Ernst ME, Ernst EJ, Klepser ME. Levofloxacin and trvafloxacin: the next generation of fluoroquinolones? Am J Health Syst Pharm 1997;54:2569-84.
4. Wimer SM, Schoonover L, Garrison ME. Levofloxacin:a therapeutic review. Clin Ther 1998;20:1049-70.
5．蔡鸿生，罗顺德，刘强，等．乳酸左氧氟沙星滴眼液在兔眼中的药动学．中国医院药学杂志，2001；21(7)：391-393．
6．陈祖基．眼科临床药理学．北京：化学工业出版社，2002．35-42．
7. Madu AA, Mayers M,Perkins R, et al. Aqueous and vitreous penetration of ciprofloxacin following different modes of systemic administration. Exp Eye Res. 1996;63(2): 129-136.
8. Liu W, Liu QF, Perkins R, et al. Pharmacokinetics of sparfloxacin in the serum and vitreous humor of rabbits: physicochemical properties that regulate petration of quinolone. Antimicrob Agents Chemother 1998;42(6): 1417-1423.
9．陈悦，张俊杰，陈祖基．左氧氟沙星滴眼液的兔眼内药代动力学．眼科研究2001；19(5)：429-431．
10. Fiscella RG, Nguyen TK, Cwik MJ, et al. Aqueous and vitreous penetration of levofloxacin after oral administration. Ophthalmology 1999; 106(12):2286-2290.
11. Garcia-Saenz MC, Arias-Puente A, Fresnadill-martubez MJ, et al. Human aqueous humor levels of oral ciprofloxacin, levofloxacin, and moxifloxacin. J Cataract Refract Surg 2001;27 (12): 1969-1974.
12. Herbert EN, Pearce IA, McGlliard J,et al.Vitrous penetration of levofloxacin in the unintlamed phakic humam eye. Br J Ophthalmol 2002; 86(4):387-389.
13. Greaves JL, Wilson CG. Treatment of disease of the eye with mucoadhesive
delivery systems. Adv Drug Deliv Rev 1993; 11(2):349-383.
14．魏刚，丁平田，崔咏艳，等．溶液黏度影响聚合物角膜滞留时间的体外评价药学学报2002；37(6)：469-472．
15. Robison JR, Mlynek GM. Bioadhesive and phase-change polymers for ocular drug delivery. Adv Drug Deliv Rev 1995; 16(1):45-50.
16. Snibson GR, Greaves JL, Soper ND, et al. Ocular surface residence times of artificial tear solutions. Cornea 1992; 11 (4):288-293.
17. Camber O, Edman P. Sodium hyaluronate as an ophthalmic vehicle:some factors governing its effect on the ocular absorption of pilocarpine. Curr Eye Res 1989;8(6): 563-567.
18. Gurny R, Ryser JE, Tabatabay C,et al. Precorneal residence time in human of sodium hyaluronate as measured by gammar scientigraphy. Graefes Arch Clin Exp Ophthalmol. 1990; 228(6): 510-512.
19. Bernatchez SF, Tabatabay C, Gurny R. sodium hyaluronate 0.25% used as a vehicle increases the bioavailability of topically administered gentamicin. Graefes Arch Clin Exp Ophthalmol 1993; 231(3):157-161.
20．黄虹，何国珍，屠晓萍，等．两种新型增粘剂在环丙沙星滴眼液中的应用．中国临床药学杂志1996；8(6)：360-363．
21．成立军，舒东，刘叶玲，等．含玻璃酸钠的毛果芸香碱滴眼液．中国生化杂志2000；21(3)：144-143．