摘要
[目的]
去铁酮(deferiprone, DFP)是一种化学合成金属螯合剂,化学名称为1,2.二甲基-3-羟基-4-吡啶酮。去铁酮在临床常用于治疗铁负荷过多的地中海贫血患者,是目前唯一可以用于口服的金属螯合剂,对铁和铝有较强的螯合作用。低、中、高三种不同浓度的去铁酮药代动力学、组织分布与血浆蛋白结合率研究国内外均未见报道。本实验通过建立一种简单、快速、准确的测定大鼠血浆及组织中DFP的高效液相色谱.紫外检测法,用于DFP在大鼠体内的药代动力学、组织分布与血浆蛋白结合率研究,为进一步临床药代学研究和合理用药提供理论依据。
[方法]
1 DFP在大鼠体内的药代动力学实验
1.1实验动物处理
取Wistar大鼠15只,随机分为3组：低剂量组、中剂量组、高剂量组。实验前平衡3天,饥饿12h后以灌胃方式分别给DFP。在给药后不同时间点于颈静脉窦处取血0.25 ml,肝素抗凝,制成血浆,血浆经处理后取20μl进样测定。
1.2色谱条件
色谱柱：Diamonsil(R)钻石C18 (250mm×4.6mm,5μm) 色谱柱；流动相：乙腈-磷酸盐缓冲液(0.05mol·L-1磷酸氢二钠、5mmol·L-1庚烷磺酸钠和2mmol·L-1 EDTA,磷酸调pH=3.0)=8:92 (v/v);流速：1.0 ml·min-1;检测波长：278 nm；检测温度：室温；进样量：20μl。
2 DFP在大鼠体内组织分布实验
2.1实验动物处理
将20只大鼠随机分为4组(空白组；10 min组；60 min组；360 min组),每组5只。饥饿12h后,空白组给1 ml生理盐水,其余各组灌胃给DFP。各组于不同时间点断头处死,取其各组织。用生理盐水将组织样品冲洗干净,滤纸吸干,加入生理盐水制成20%的组织匀浆,离心后,上清液经处理后取20μl进样。
2.2色谱条件
色谱柱：Diamonsil(R)钻石C18 (250mm×4.6mm,5μm)色谱柱；流动相：乙腈-磷酸盐缓冲液(0.05mol·L-1磷酸氢二钠、5 mmol·L-1庚烷磺酸钠和2 mmol·L-1 EDTA,磷酸调pH=3.0)=8：92或10：90(v／v)；流速：1.0 ml·min-1;检测波长：278 nm；检测温度：室温；进样量：20μl。
3 DFP的大鼠血浆蛋白结合率实验
3.1实验动物处理
取健康雄性Wistar大鼠10只,处死,取血,肝素抗凝,制成血浆。透析内液和透析外液经处理后进样20μl。
3.2色谱条件
同“2.2色谱条件”。
3.3透析袋对药物的吸附实验
于透析袋内加入1 ml空白透析液,放入含有10 ml DFP溶液的广口瓶中,盖紧塞子,37℃生化培养箱中放置4h至其平衡,然后测定透析袋外的药物浓度,计算透析膜对药物的吸附率。
3.4平衡时间的考察和血浆蛋白结合率的测定
于透析袋中加入1 ml空白血浆,置广口瓶中悬浮于10 ml不同浓度的DFP透析液中,放置于37℃的生化培养箱中。于一定时间点,测定透析袋内、外的DFP浓度,计算DFP的血浆蛋白结合率。
4数据处理与分析
DAS2.0药代动力学智能分析软件计算药代动力学参数和房室模型；SPSS13.0版统计学分析软件进行统计描述和f检验。
[结果]
1 DFP在大鼠体内药代动力学实验结果
1.1方法学研究结果
取DFP对照品溶液经光谱扫描,本实验选用278 nm为检测波长。DFP在本实验条件的保留时间为10.8 min,在1-120 mg·L-1范围内线性良好(r=0.9999),低、中、高三种浓度绝对回收率均大于98.71%,相对回收率在99.04%～100.6%,日内变异RSD均小于0.74%,日间变异RSD均小于1.16%,于4℃存放1周和20℃存放1个月后RSD均小于1.72%,稳定性较好。
1.2药代动力学结果
大鼠单次以灌胃方式给DFP后体内的药动学过程符合二室开放模型。低、中、高三种剂量的平均吸收半衰期为18.90、16.08、12.69 min,其一级吸收速率Ka为0.05～0.17 min-1；低、中、高三种剂量的平均分布半衰期tl／2。分别为23.33、22.22、20.88 min,其表观分布容积分别为0.70、0.62、0.41 L·kg-1；低、中、高三种剂量的清除率分别为0.017、0.02、0.016 L·min-1·kg-1,平均消除半衰期t1／2β分别为53.32、50.87、46.34 min；给低、中、高三种剂量DFP后,0-∞时间内药时曲线下面积分别为2380.1、3519.4、8788.7 mg·L-1·min。统计矩法所得低、中、高三种剂量的平均滞留时间MRT(0-t)分别为109.81、95.28、88.70 min,血浆末端清除半衰期t1／2z分别为140.28、93.40、63.27 min,达到最大血药浓度的时间分别为36.00、48.00、54.00 min,最大血药浓度Cmax分别为21.60、37.13、83.25 mg·L-1,表观分布容积Vz分别为2.80、2.49、1.41 L·kg-1,清除率CLz分别为0.014、0.018、0.016 L·min-1·kg-1,得到的药时曲线下面积AUQ(0-t)值分别为2276.96、3580.54、8933.31 mg·L-1·min
2 DFP在大鼠体内组织分布实验结果
2.1方法学研究结果
DFP在小肠的色谱条件下保留时间为10.8 min,在其他组织的色谱条件下保留时间为7.8 min。各组织中的DFP在一定浓度范围内线性关系良好(r>0.9995),低、中、高三种浓度绝对回收率均大于86.0%,相对回收率在97.1%～109.6%,日内变异RSD均小于3.33%,日间变异RSD均小于4.98%,于4℃存放1周和.20-C存放1个月后RSD均小于5.49%,稳定性较好。
2.2组织分布结果
本实验给大鼠单次灌胃70mg·kg-1 DFP, 10min后在包括脑和睾丸的各组织中,均检测出有DFP分布。给药10 min和60 min后,胃中DFP的浓度为244.81mg·kg-1和58.86 mg·kg-1,远远高于同组小肠中DFP含量(P<0.001)。给药60 min和360 min后在肝脏中DFP的平均含量为359.22 mg·kg-1和56.80 mg·kg-1,显著高于其他组织(P<0.001)。给药360 min后,肾中DFP的含量为10.13 mg·kg-1,显著高于除肝脏外其他组织中DFP含量(P<0.005)。
3 DFP的大鼠血浆蛋白结合率实验结果
3.1方法学研究结果
DFP在透析内液的色谱条件下保留时间为10.8 min,在透析外液的色谱条件下保留时间为7.8 min。DFP在透析内、外液中一定浓度范围内线性关系良好(r>0.9998),低、中、高三种浓度绝对回收率均大于98.71%,相对回收率在99.04%～101.27%,日内变异RSD均小于0.74%,日间变异RSD均小于1.15%,其于37-C存放8h后RSD均小于1.94%,短期稳定性较好,于4℃存放1周和-20℃存放1个月后RSD均小于1.72%,长期稳定性较好。
3.2血浆蛋白结合率结果
低、中、高三种浓度下,透析袋对DFP的平均吸附率分别为4.53%,3.66%和3.41%。在37℃条件下,DFP经2h达到透析平衡,其血浆蛋白结合率为4.25%-5.31%,各浓度组间的血浆蛋白结合率比较差异无统计学意义(P>0.05)。
[结论]
1本实验建立的测定大鼠血浆、组织匀浆和透析液中DFP含量的高效液相色谱法具有快速、准确、灵敏,重复性好等优点,可用于DFP在大鼠体内的药代动力学研究、组织分布和血浆蛋白结合率的研究。
2灌胃给药后,DFP在大鼠体内吸收快、分布广、消除快,其药代动力学过程符合二室模型特征。
3灌胃给药后,DFP在大鼠体内分布广泛,各主要脏器组织均有分布。
4 DFP血浆蛋白结合率较低,属于低血浆蛋白结合药物,主要以游离形式存在而发挥药效。
Objective
Deferiprone(DFP), with its chemical name 1,2-dimethyl-3-hydroxy-4-pyrid-one, is a chemical synthesis of metal chelating agents. Deferiprone is usually used to treat iron overload in patients withβ-thalassemia. It has a good chelation with iron and aluminum as the first oral metal chelator. There were no reports about pharmacokinetics of low, middle and high concentrations, tissue distribution and plasma protein binding rate of DFP at home and abroad. The experiments established a simple, quick, accurate HPLC-UV method of DFP for the study of pharmacokinetics, tissue distribution and plasma protein binding rate of DFP in rats, which provided a theoretical basis for the further study on clinical pharmacokinetics and clinical rational drug use.
Methods
1 Pharmacokinetics of DFP in rats
1.1 The treatment of experimental animals
Fifteen wistar rats were randomly divided into three groups:low-dose group, middle-dose group and high-dose group. After three days'equilibrium, DFP was given to the rats through intragastric administration after 12 hours'fasting. Then about 0.25ml blood, from which plasma was collected, was collected from jugular sinus at different times with Heparin as anticoagulant.20μl sample was detected after the plasma had been treated.
1.2 Chromatographic condition
The separation was performed on a Diamonsil(R) C18 column (250 mm×4.6 mm, 5 5μm) and detected at 278nm. The mobile phase was a mixture of acetonitrile-natrium phosphate buffer (0.05 mol·L-1 Disodium hydrogen phosphate, adjust pH=3.0 with phosphoric acid, containing 5mmol·L-1 heptanesulfonic acid and 2 mmol·L-1 EDTA)=8:92 (v/v) at a flow rate of 1.0mL·min-1. Injection volume was 20μl, and detection run at room temperature.
2 Tissue distribution of DFP in rats
2.1 The treatment of experimental animals
Twenty rats were randomly divided into four groups (control group; 10 min group; 60 min group; 360 min group),and each group had 5 rats. After starvation of 12 hours, the control group was given 1 ml physiological saline, the other groups fed with DFP by intragastric administration. Each group were decapitated at different time, and organizations were taken. Samples of the tissue were rinsed by normal saline, dried by filter paper. The samples of tissue were made into 20%tissue homogenate with normal saline, then centrifuged.20μl sample was injected after the supernatant treated.
2.2 Chromatographic condition
The separation was performed on a Diamonsil(R)C18 column (250 mm×4.6 mm, 5μm) and detected at 278 nm. The mobile phase was a mixture of acetonitrile-natrium phosphate buffer (0.05 mol·L-1 Disodium hydrogen phosphate, adjust pH=3.0 with phosphoric acid, containing 5mmol-L'1 heptanesulfonic acid and 2 mmol·L-1 EDTA)=8:92 or 10:90 (v/v) at a flow rate of 1.0mL·min-1. Injection volume was 20μl, and detection run at room temperature.
3 Plasma protein binding rate of DFP in rats
Ten healthy male Wistar rats were decapitated for blood which was anticoagulated by heparin and made into plasma. Plasma protein binding rate of DFP was detected by equilibrium dialysis method, and 20μl sample was injected after dialysate treated.
3.2 Chromatographic conditions
As "2.2 chromatographic conditions".
3.3 Drug adsorption experiment of dialysis bag
lml blank dialysate was added into dialysis bag. Then the dialysis bag was suspended in a wide mouth jar containing 10ml DFP dialysate of different concentrations. The stopper was bunged tightly. Then put the bottles into biochemical incubator at 37℃. After 4 hours balancing, concentrations of the drug outside the dialysis bag were measured for calculating the rate of drug absorption of dialysis membranes.
3.4 The inspection of equilibrium time and the determination of plasma protein binding rate
lml blank plasma was added into the dialysis bag. Then the dialysis bag was suspended in a wide mouth jar containing 10ml DFP dialysate of different concentrations. Then put the bottles in biochemical incubator at 37℃. The concentrations of inside and outside of the dialysis bag were measured at a certain time for determinating the plasma protein binding rate.
4 Data processing and analysis
The compartment model and pharmacokinetic parameters were calculated by DAS 2.0 statistical analysis software. SPSS 13.0 was used for statistical description and t-test.
Results
1 Pharmacokinetics results of DFP in rats
1.1 The results of methodology study
Solution of DFP reference substance was scanned by UV-2450.278nm was selected to be the detection wavelength. The retention time of DFP was 10.8 min in the experimental conditions. It has a good correlation in the linear range from 1 to 120 mg·L-1 (r=0.9999). The absolute recovery of DFP was more than 98.7% and the relative recovery was 99.04%-100.6% at low, middle and high concentrations. The RSDs of intra-day were less than 0.74%, and those of inter-day were less than 1.16%. The RSDs were less than 1.72% after stored for 1 week at 4℃and 1 month at-20℃. The stability was good.
1.2 Pharmacokinetic results
The pharmacokinetic process of DFP in rats was two-compartment model after ig DFP. The t1/2Ka were 18.90,16.08 and 12.69min, and Ka was at the range of 0.05-0.17 min-1 at low, middle and high concentrations. The ti/2αwere 23.33,22.22 and 20.88 min, and the V1 were 0.70,0.62 and 0.41L·kg-1 at low, middle and high concentrations. The CL were 0.017,0.021 and .016L(min-kg)-1, and the t1/2βwere 53.32,0.87 and 46.34min at low, middle and high concentrations.. The AU(o-∞,) were 2380.1,3519.4 and 8788.7mg-LL1-min after given DFP of low, middle and high concentrations, respectively. The follow parameters of low, middle and high concentrations were obtained by rosenblueth method of non compartment mode. The MRT(o-t) were 109.81,95.28 and 88.70min, and the i2z were 140.28,93.40 and 63.27 min. The Tmax were 36.00,48.00 and 54.00min, and the Cmax were 21.60,37.13 and 83.25mg·L-1. The VZ were 2.80,2.49 and 1.41L·kgg1, and the CLZ were 0.014,0.018 and 0.016L-(min·kg)-1.The AUC(0-t) were 2276.96,3580.54 and 8933.3 1mg·L-1·min, respectively.
2 Tissue distribution results of DFP in rats
2.1 The results of methodology study
The retention time of DFP was 10.8min at the chromatographic conditions of small intestine, and other tissues were 7.8min The linear range of tissues were of a good correlation (r>0.9995). The absolute recoveries of DFP were more than 86.0% and the relative recoveries were 97.1%-109.6% at low, middle and high concentrations. The RSDs of intra-day were less than 3.33%, and those of inter-day were less than 4.98%. The RSDs were less than 5.49% after stored for 1 week at 4℃and 1 month at-20℃. The stabilities were good.
2.2 The results of tissue distribution
The DFP was detected in each of tissues including brain and testis 10 min after ig 70mg·kg-1DFP. The concentrations of DFP were 244.8 1mg·kg-1 and 58.86 mg·kg-1 in stomach 10min and 60min after ig DFP. They were remarkably higher than those in small intestine in the same group (＜0.001). The concentrations of DFP were 359.22mg-kg-1and 56.80mg·kg-1 in liver 60 min and 360 min after ig DFP, and they were remarkably higher than those in the other tissues (＜0.001). The concentration in kidney was 10.13mg·kg-1 360 min after ig DFP, and it was remarkably higher than those in the other tissues except liver ((P<0.005).
3 The results of plasma protein binding rate of DFP
3.1 The results of methodology study
The retention time was 10.8min at the chromatographic conditions of inside dialysate, and it was 7.8min at the chromatographic conditions outside dialysate. The linear range of dialysate were of a good correlation (r>0.9998). The absolute recoveries of DFP were more than 98.71% and the relative recoveries were 99.04%-101.27% at low, middle and high concentrations. The RSDs of intra-day were less than 0.74%, and those of inter-day were less than 1.15%. The RSDs were less than 1.94% after stored for 8 hours at 37℃, and the short-term stabilities were good. The RSDs were less than 1.72% after stored for 1 week at 4℃and 1 month at-20℃, and the long-term stabilities were good.
3.2 The results of plasma protein binding rate
The adsorption rates of dialysis bag were 4.53%,3.66% and 3.41% at low, middle and high concentrations, respectively. It needed 2 hours to get dialysis balancing at 37℃in biochemical incubator. The range of plasma protein binding rates were 4.25%-5.31%. There was no statistical significance among all concentration groups of plasma protein binding rates (P>0.05).
Comcolutions
1 The HPLC methods which were developed in this study were for the detection of DFP in rats plasma, tissue bomogenate and dialysate. They were quick, accurate, sensitive and good repeatabilities. They could be used in the studies of pharmacokinetic, tissue distribution and plasma protein binding rate of DFP in rats.
2 DFP has quick absorption, wide distribution and fast elimination in rats after ig drug. The pharmacokinetic process of DFP in rats was two-compartment model.
3 DFP was widely distributed in rats after ig drug. There were DFP in main tissues. It showed that DFP could pass through blood-brain barrier and blood-testis barrier.
4 The plasma protein binding rate of DFP is low. So DFP is a kind of low plasma protein binding rate drug, and most of the drug molecules act on body with free type.
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