头孢噻呋钠脂质体的药动学、毒性及对自由基和药酶的影响
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摘要
脂质体(liposomes)是一种具有长效缓释作用的微型囊泡,可以将药物包封于脂质双分子层内。头孢噻呋钠(ceftiofur sodium)是第三代广谱头孢菌素,在兽医临床上被广泛用于治疗家畜的呼吸道疾病和奶牛乳腺炎。头孢噻呋钠脂质体是将头孢噻呋钠包封在脂质体双分子层内所形成的一种混悬剂。本研究从以下五个方面来研究头孢噻呋钠脂质体的临床药理特点,旨在为头孢噻呋钠脂质体在兽医临床上的应用提供指导和理论依据。
1、头孢噻呋钠脂质体体外释放度的测定
通过动态透析法,用pH值为6.5的PBS缓冲液作为释放介质来测定头孢噻呋钠脂质体的体外释放度。结果显示,56.48%的头孢噻呋钠脂质体在24h内释药,而头孢噻呋钠原料药2h的累积释药率为90%,24h后累积释放率达到94.68%。提示头孢噻呋钠脂质体的释放较慢,相对于头孢噻呋钠原料药来说,具有明显的长效和缓释效果。
2、头孢噻呋钠脂质体在牛体内药物动力学试验
通过给6头黄牛静脉注射头孢噻呋钠脂质体和头孢噻呋钠原料药,采集血液并用高效液相色谱法测定一定时间内血液中的药物浓度,来考察两种药物在黄牛体内的药物代谢情况,获得头孢噻呋钠脂质体在牛体内的药物动力学参数。结果显示,单剂量分别静脉注射头孢噻呋钠脂质体和原料药后,在给药48h后仍可检测出头孢噻呋钠脂质体,说明头孢噻呋钠脂质体具有长效作用,在血液中的浓度可以维持在48h以上。头孢噻呋钠脂质体在黄牛体内的药物代谢符合二室开放模型,头孢噻呋钠脂质体在牛体内的消除半衰期(t1/2β)为头孢噻呋钠原料药的2.11倍。这表明,头孢噻呋钠经脂质体包封后具有比单独的头孢噻呋钠更长的药效,可以更有效和方便的用于治疗需要长期给药的奶牛乳腺炎等疾病。
3、头孢噻呋钠脂质体的毒性研究
按1000mg/kg、500mg/kg和200mg/kg体重给15只小鼠腹腔注射头孢噻呋钠脂质体,7d后小鼠仍然健康存活；再将56只小鼠随机分为7组,即头孢噻呋钠脂质体高、中、低剂量组(分别相当于临床剂量的20、10和5倍),头孢噻呋钠原料药高、中、低剂量组和生理盐水对照组,连续21d腹腔注射头孢噻呋钠脂质体和头孢噻呋钠后,进行血液学、血清生化学和组织病理学检查。结果显示,小鼠血常规及生化数据经t检验后显示头孢噻呋钠高剂量组对小鼠有明显的毒性,脂质体高剂量组也有一定毒性,但相比同剂量的原料药毒性有所降低。肝脏和肾脏病理切片结果显示脂质体对小鼠造成的毒性影响明显比头孢噻呋钠小。提示经脂质体包封后的头孢噻呋钠毒性明显下降,安全性提高。
4、头孢噻呋钠脂质体对小鼠自由基的影响
为测定头孢噻呋钠脂质体对小鼠肝脏自由基形成的影响,将24只昆明小鼠分为三组,连续7天腹腔注射头孢噻呋钠脂质体、头孢噻呋钠和生理盐水。完成给药后,处死小鼠并获取小鼠的肝脏组织,测定小鼠肝脏组织中SOD的活力,MDA的含量以及抑制羟自由基生成的能力。结果显示,性别差异对SOD、MDA与羟自由基活性没有明显的影响。头孢噻呋钠脂质体组的SOD活性(85.40±2.59)明显高于原料药组(75.42±3.29),而MDA含量与羟自由基活性没有明显的差异。头孢噻呋钠脂质体组的SOD活性明显高于头孢噻呋钠原料药组,这提示头孢噻呋钠脂质体比头孢噻呋钠原料药更能提高SOD的活性,从而降低由自由基引起的细胞毒性。
5、头孢噻呋钠脂质体对肝微粒体药酶的影响
为了解头孢噻呋钠脂质体对肝脏微粒体药酶的影响,将24只昆明小鼠随机分为三组,分别连续7天腹腔注射2.2mg/kg的头孢噻呋钠脂质体和头孢噻呋钠原料药,另外一组不给药作为空白对照。完成注射后处死小鼠,采集肝脏,制备肝微粒体并测定肝脏微粒体中蛋白浓度和细胞色素b5的含量,NADPH-细胞色素C还原酶、氨基比林-N-脱甲基酶、红霉素-N-脱甲基酶以及苯胺羟化酶的活性。结果显示,头孢噻呋钠脂质体对这五种药酶均表现出一定的抑制作用,但是与空白对照相比,除了对NADPH-细胞色素C还原酶的抑制作用极显著外,其余都无显著性差异。且这五种酶的活性非常低,这种极低的药酶活性很好的从根本上解释了头孢噻呋钠脂质体在小鼠体内较慢的代谢速率,从而避免药物峰浓度过高而引起的细胞毒性。
Liposome is a microscopic spherical particle can encapsulate drug in the lipid bilayer and has long-term and sustained-release effect. Ceftiofur sodium is a third generation broad spectrum cephalosporin widely used clinically to treat respiratory diseases and mastitis in livestock via intramuscular (i.m.) injection. Ceftiofur sodium liposome is the suspension formed by a lipid bilayer enclosing ceftiofur sodium. Five relative experiments were designed here to evaluate the clinical pharmacological characteristics of ceftiofur sidium liposomes, as to provide guidance and theoretical basis for the use of ceftiofur sodium liposomes in clinical veterinary.
1. In vitro release of ceftiofur sodium liposomes
In vitro release of ceftiofur sodium liposomes was deteced by dynamic dialysis method which use PBS buffer (pH6.5) as the release medium. The results showed that the fraction release of ceftiofur sodium liposomes at24h is56.48%, and ceftiofur sodium is90%at2h,94.68%after24h. Ceftiofur sodium liposome has significant long-term and sustained-release effect compared with the non-encapsulated ceftiofur sodium.
2. pharmacokinetics of ceftiofur sodium liposomes in cows
To examine the metabolism and pharmacokinetic parameters of ceftiofur sodium liposomes, six healthy, adult cows in two treatment groups were dosed intravenously with ceftiofur sodium liposomes and ceftiofur sodium, serial blood samples collected, and plasma concentrations determined by high performance liquid chromatography. The results showed that ceftiofur sodium liposome was still detectable after48h, indicating that ceftiofur sodium liposome has long-term effect. Intravenous plasma concentration profiles of liposomes best fit a two compartment model and the elimination half-life was2.11times that of ceftiofur sodium. Thus, this liposome preparation provided therapeutically effective plasma concentrations for a longer duration than with the drug alone, making it more effective and convenient for use in treating bovine mastitis that requires long duration maintenance of therapeutic plasma concentrations.
3. The toxicity of ceftiofur sodium liposomes
15mice were given ceftiofur sodium liposomes at dose of1000mg/kg,500mg/kg and200mg/kg by intraperitoneal injection, all the mice were still alive after7days of administration.56healthy SPF mice were divided into7groups randomly, were injected20,10.5times the clinical dose of ceftiofur sodium and ceftiofur sodium liposomes, one group were given physiological saline as the control. Blood routine examination, biochemical analysis and histopathological were tested after21days continuous injection. The results showed that T-tests of mice blood routine and biochemical data revealed that high dose group of ceftiofur sodium has obvious toxicity to mice. Though the low dose group of ceftiofur sodium liposomes has some toxicity to mice, the toxicity was smaller than the same dose of ceftiofur sodium. The pathology sections of liver and kidney showed that the toxicity of ceftiofur sodium liposomes to mice was significantly smaller than ceftiofur sodium. Conclusion:The liposomes which encapsulated ceftiofur sodium can decreased toxicity, increased security.
4. Effects of ceftiofur sodium liposomes on free radical formation in mice
To examine the effects of ceftiofur sodium liposomes on the free radical formation in liver of mice,24mice were assigned randomly into three groups, i.e.,1) ceftiofur sodium;2) ceftiofur sodium liposomes and3) physiological saline. Treatments were applied via intraperitoneal injections for7days. At the end of the treatment period, animals were euthanized and liver collected for analysis of superoxide dismutase (SOD) activity and malondialdehyde (MDA) contents and the ability of liver tissue to suppress hydroxyl radical formation. The results showed that gender had no significant effect on free radical formation. Ceftifur sodium liposomes-treated mice had higher activity of SOD (85.40±2.59)than ceftiofur sodium-treated(75.42±3.29) mice; however, MDA content and the ability of liver tissue to suppress hydroxyl radical formation did not reach statistical significance among groups. It was concluded that ceftiofur sodium liposomes can improve the SOD activity and reduce the cell toxicity brought by free radical compared to ceftiofur alone in mice.
5. Effects of ceftiofur sodium liposomes on drug metabolizing enzyme
To understand the effects of ceftiofur sodium liposomes on liver microsomal drug metabolizing enzyme,24Kunming mice were randomly divided into three groups, intraperitoneal injection2.2mg/kg ceftiofur sodium liposomes and ceftiofur sodium respectively for7days, another group as the control. Mice were euthanized after completion of the injection, liver was collected to prepare liver microsomes and protein concentration, content of cytochrome b5, activities of NADPH-cytochrome C reductase, aminopyrine-N-demethylase enzyme, erythromycin-N-demethylase and aniline hydroxylase were detected in this study. The results revealed that ceftiofur sodium liposomes showed inhibition effect on these five drug metabolizing enzymes, the inhibition effect of ceftiofur sodium liposomes on NADPH-cytochrome C reductase was significantly lower than control group, the rest are no significant differences. The activities of these five enzymes are very low, this low activity well explained the slower metabolic rate of ceftiofur sodium liposomes, thus avoiding the cytotoxic brought by higher drug peak concentration.
1、头孢噻呋钠脂质体体外释放度的测定
通过动态透析法,用pH值为6.5的PBS缓冲液作为释放介质来测定头孢噻呋钠脂质体的体外释放度。结果显示,56.48%的头孢噻呋钠脂质体在24h内释药,而头孢噻呋钠原料药2h的累积释药率为90%,24h后累积释放率达到94.68%。提示头孢噻呋钠脂质体的释放较慢,相对于头孢噻呋钠原料药来说,具有明显的长效和缓释效果。
2、头孢噻呋钠脂质体在牛体内药物动力学试验
通过给6头黄牛静脉注射头孢噻呋钠脂质体和头孢噻呋钠原料药,采集血液并用高效液相色谱法测定一定时间内血液中的药物浓度,来考察两种药物在黄牛体内的药物代谢情况,获得头孢噻呋钠脂质体在牛体内的药物动力学参数。结果显示,单剂量分别静脉注射头孢噻呋钠脂质体和原料药后,在给药48h后仍可检测出头孢噻呋钠脂质体,说明头孢噻呋钠脂质体具有长效作用,在血液中的浓度可以维持在48h以上。头孢噻呋钠脂质体在黄牛体内的药物代谢符合二室开放模型,头孢噻呋钠脂质体在牛体内的消除半衰期(t1/2β)为头孢噻呋钠原料药的2.11倍。这表明,头孢噻呋钠经脂质体包封后具有比单独的头孢噻呋钠更长的药效,可以更有效和方便的用于治疗需要长期给药的奶牛乳腺炎等疾病。
3、头孢噻呋钠脂质体的毒性研究
按1000mg/kg、500mg/kg和200mg/kg体重给15只小鼠腹腔注射头孢噻呋钠脂质体,7d后小鼠仍然健康存活；再将56只小鼠随机分为7组,即头孢噻呋钠脂质体高、中、低剂量组(分别相当于临床剂量的20、10和5倍),头孢噻呋钠原料药高、中、低剂量组和生理盐水对照组,连续21d腹腔注射头孢噻呋钠脂质体和头孢噻呋钠后,进行血液学、血清生化学和组织病理学检查。结果显示,小鼠血常规及生化数据经t检验后显示头孢噻呋钠高剂量组对小鼠有明显的毒性,脂质体高剂量组也有一定毒性,但相比同剂量的原料药毒性有所降低。肝脏和肾脏病理切片结果显示脂质体对小鼠造成的毒性影响明显比头孢噻呋钠小。提示经脂质体包封后的头孢噻呋钠毒性明显下降,安全性提高。
4、头孢噻呋钠脂质体对小鼠自由基的影响
为测定头孢噻呋钠脂质体对小鼠肝脏自由基形成的影响,将24只昆明小鼠分为三组,连续7天腹腔注射头孢噻呋钠脂质体、头孢噻呋钠和生理盐水。完成给药后,处死小鼠并获取小鼠的肝脏组织,测定小鼠肝脏组织中SOD的活力,MDA的含量以及抑制羟自由基生成的能力。结果显示,性别差异对SOD、MDA与羟自由基活性没有明显的影响。头孢噻呋钠脂质体组的SOD活性(85.40±2.59)明显高于原料药组(75.42±3.29),而MDA含量与羟自由基活性没有明显的差异。头孢噻呋钠脂质体组的SOD活性明显高于头孢噻呋钠原料药组,这提示头孢噻呋钠脂质体比头孢噻呋钠原料药更能提高SOD的活性,从而降低由自由基引起的细胞毒性。
5、头孢噻呋钠脂质体对肝微粒体药酶的影响
为了解头孢噻呋钠脂质体对肝脏微粒体药酶的影响,将24只昆明小鼠随机分为三组,分别连续7天腹腔注射2.2mg/kg的头孢噻呋钠脂质体和头孢噻呋钠原料药,另外一组不给药作为空白对照。完成注射后处死小鼠,采集肝脏,制备肝微粒体并测定肝脏微粒体中蛋白浓度和细胞色素b5的含量,NADPH-细胞色素C还原酶、氨基比林-N-脱甲基酶、红霉素-N-脱甲基酶以及苯胺羟化酶的活性。结果显示,头孢噻呋钠脂质体对这五种药酶均表现出一定的抑制作用,但是与空白对照相比,除了对NADPH-细胞色素C还原酶的抑制作用极显著外,其余都无显著性差异。且这五种酶的活性非常低,这种极低的药酶活性很好的从根本上解释了头孢噻呋钠脂质体在小鼠体内较慢的代谢速率,从而避免药物峰浓度过高而引起的细胞毒性。
Liposome is a microscopic spherical particle can encapsulate drug in the lipid bilayer and has long-term and sustained-release effect. Ceftiofur sodium is a third generation broad spectrum cephalosporin widely used clinically to treat respiratory diseases and mastitis in livestock via intramuscular (i.m.) injection. Ceftiofur sodium liposome is the suspension formed by a lipid bilayer enclosing ceftiofur sodium. Five relative experiments were designed here to evaluate the clinical pharmacological characteristics of ceftiofur sidium liposomes, as to provide guidance and theoretical basis for the use of ceftiofur sodium liposomes in clinical veterinary.
1. In vitro release of ceftiofur sodium liposomes
In vitro release of ceftiofur sodium liposomes was deteced by dynamic dialysis method which use PBS buffer (pH6.5) as the release medium. The results showed that the fraction release of ceftiofur sodium liposomes at24h is56.48%, and ceftiofur sodium is90%at2h,94.68%after24h. Ceftiofur sodium liposome has significant long-term and sustained-release effect compared with the non-encapsulated ceftiofur sodium.
2. pharmacokinetics of ceftiofur sodium liposomes in cows
To examine the metabolism and pharmacokinetic parameters of ceftiofur sodium liposomes, six healthy, adult cows in two treatment groups were dosed intravenously with ceftiofur sodium liposomes and ceftiofur sodium, serial blood samples collected, and plasma concentrations determined by high performance liquid chromatography. The results showed that ceftiofur sodium liposome was still detectable after48h, indicating that ceftiofur sodium liposome has long-term effect. Intravenous plasma concentration profiles of liposomes best fit a two compartment model and the elimination half-life was2.11times that of ceftiofur sodium. Thus, this liposome preparation provided therapeutically effective plasma concentrations for a longer duration than with the drug alone, making it more effective and convenient for use in treating bovine mastitis that requires long duration maintenance of therapeutic plasma concentrations.
3. The toxicity of ceftiofur sodium liposomes
15mice were given ceftiofur sodium liposomes at dose of1000mg/kg,500mg/kg and200mg/kg by intraperitoneal injection, all the mice were still alive after7days of administration.56healthy SPF mice were divided into7groups randomly, were injected20,10.5times the clinical dose of ceftiofur sodium and ceftiofur sodium liposomes, one group were given physiological saline as the control. Blood routine examination, biochemical analysis and histopathological were tested after21days continuous injection. The results showed that T-tests of mice blood routine and biochemical data revealed that high dose group of ceftiofur sodium has obvious toxicity to mice. Though the low dose group of ceftiofur sodium liposomes has some toxicity to mice, the toxicity was smaller than the same dose of ceftiofur sodium. The pathology sections of liver and kidney showed that the toxicity of ceftiofur sodium liposomes to mice was significantly smaller than ceftiofur sodium. Conclusion:The liposomes which encapsulated ceftiofur sodium can decreased toxicity, increased security.
4. Effects of ceftiofur sodium liposomes on free radical formation in mice
To examine the effects of ceftiofur sodium liposomes on the free radical formation in liver of mice,24mice were assigned randomly into three groups, i.e.,1) ceftiofur sodium;2) ceftiofur sodium liposomes and3) physiological saline. Treatments were applied via intraperitoneal injections for7days. At the end of the treatment period, animals were euthanized and liver collected for analysis of superoxide dismutase (SOD) activity and malondialdehyde (MDA) contents and the ability of liver tissue to suppress hydroxyl radical formation. The results showed that gender had no significant effect on free radical formation. Ceftifur sodium liposomes-treated mice had higher activity of SOD (85.40±2.59)than ceftiofur sodium-treated(75.42±3.29) mice; however, MDA content and the ability of liver tissue to suppress hydroxyl radical formation did not reach statistical significance among groups. It was concluded that ceftiofur sodium liposomes can improve the SOD activity and reduce the cell toxicity brought by free radical compared to ceftiofur alone in mice.
5. Effects of ceftiofur sodium liposomes on drug metabolizing enzyme
To understand the effects of ceftiofur sodium liposomes on liver microsomal drug metabolizing enzyme,24Kunming mice were randomly divided into three groups, intraperitoneal injection2.2mg/kg ceftiofur sodium liposomes and ceftiofur sodium respectively for7days, another group as the control. Mice were euthanized after completion of the injection, liver was collected to prepare liver microsomes and protein concentration, content of cytochrome b5, activities of NADPH-cytochrome C reductase, aminopyrine-N-demethylase enzyme, erythromycin-N-demethylase and aniline hydroxylase were detected in this study. The results revealed that ceftiofur sodium liposomes showed inhibition effect on these five drug metabolizing enzymes, the inhibition effect of ceftiofur sodium liposomes on NADPH-cytochrome C reductase was significantly lower than control group, the rest are no significant differences. The activities of these five enzymes are very low, this low activity well explained the slower metabolic rate of ceftiofur sodium liposomes, thus avoiding the cytotoxic brought by higher drug peak concentration.
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