A rapid and sensitive LC/MS/MS assay for quantitative determination of digoxin in rat plasma

Digoxin is a cardiac glycoside that is widely used for the treatment of congestive heart failure. To evaluate pharmacokinetics of digoxin in rats, a sensitive LC/MS/MS assay was developed and validated for the determination of digoxin concentration in rat plasma. For detection, a Sciex API3000 LC/MS/MS with atmospheric pressure ionization (API) mass spectrometry turbo ion spray inlet in the positive ion-multiple reaction monitoring mode was used to monitor precursor→product ions of m/z 798.6→651.6 for digoxin and m/z 577.6→433.3 for oleandrin, the internal standard (IS). The standard curve was linear (r²≥0.999) over the digoxin concentration range of 0.1–100 ng/ml in plasma for digoxin. The mean predicted concentrations of the quality control samples deviated by <5.8% from the corresponding nominal values; the intra-assay and inter-assay precision of the assay were within 8.6% relative standard deviation. At the lower limit of quantitation (LLQ) of 0.1 ng/ml, the mean deviation of predicted concentrations from the nominal value was within 3.7%. The extraction recoveries of digoxin and internal standard were 82.7±3.9 and 105.9±2.3%, respectively. The present method was successfully applied to characterization of pharmacokinetic profiles of digoxin in rats after oral administration.     

加替沙星对家兔体内地高辛血药浓度的影响

目的观察加替沙星与地高辛合用后其对地高辛血药浓度和药代动力学参数的影响,为临床安全、有效、合理使用强心苷类药物提供参考依据。方法用荧光偏振免疫法测定不同给药方法中家兔体内地高辛血药浓度,并对结果进行统计分析。结果合用加替沙星后,地高辛的血药浓度明显增高,地高辛在家兔体内的分布半衰期(T1/2)延长,表观分布容积(Vd)增大(P<0.05),而血浆清除率(CL)和曲线下面积(AUC0～∞)无显著变化(P>0.05)。结论加替沙星可显著增加地高辛的血药浓度,增加地高辛在体内的蓄积,两者合用时应及时监测地高辛血药浓度,制定个体化给药方案,确保临床疗效,减少毒性反应的发生。     

Lack of effect on brain stem and cerebral cortex Na+, K+-ATPase during heart block produced by chronic digoxin treatment.

The autonomic nervous system has been shown to play an important role in digitalis toxicity. In order to determine whether the central nervous system could be digitalis' site of action, the effect of chronic treatment with toxic doses of digitalis on brain Na+,K+-ATPase was studied in the dog. After four weeks of digoxin treatment, Na+,K+-ATPase activity of the brainstem or cerebral cortex was unaffected at the time when digitalis toxicity (heart block) was apparent. ATP-dependent (3H)-ouabain binding to these tissues was also unaffected indicating that a significant occupancy of brain Na+,K+-ATPase by digoxin did not occur during chronic drug treatment. In contrast, cardiac Na+,K+-ATPase was markedly inhibited with the concomitant binding of digoxin to the enzyme. Since Na+,K+-ATPase is the most digitalis sensitive system identified to date, it appears that digoxin does not affect neuronal function directly.     

Cardio protective effect of glucose-insulin infusion on acute digoxin toxicity in rat

We recently observed a case of digoxin and insulin self-poisoning without cardiac repercussion. We raised the hypothesis that insulin may have a cardio-protective effect in case of digoxin toxicity. We have therefore evaluated the effect of glucose-insulin infusion on mortality and ECG abnormalities during acute digoxin toxicity in rats. Before and after a hyperinsulinemia-euglycemia clamp, rats in glucose-insulin-digoxin (GID) group (n=10) received an intravenous infusion of 12ml/h or 2,5ml/h digoxin (0.25mg/ml) respectively until death occured. Animals receiving digoxin or saline solution intravenously served as control (n=10). ECG recording was performed in all animals over the entire period. Serum insulin and digoxin concentrations were measured by ELISA method after digoxin administration. When digoxin was administered after the clamp, all animals in GID group were alive, whereas 80% of animals in the digoxin group were dead (p<0.001) after 30min. The administration of Digoxin provoked rapid death of rats in the digoxin group in 15+/-12min whereas in GID group the survival period was significantly increased to 38+/-3min (p<0.001). Twenty minutes after digoxin administration, P waves disappeared for 78% of animals in digoxin group while they were present in all rats of GID group (p<0.001). Animal death occurred after a digoxin infusion volume of 7.7+/-0.6ml and 3.0+/-2.4ml in GID and digoxin group respectively (p<0.001). Five minutes after digoxin administration, potassium plasmatic level increased significantly in digoxin group as compared to GID group: 7.1+/-2mmol/l versus 4.4+/-0.4mmol/l (p<0.001). When digoxin was infused before the clamp, 40% of animals in GID group were alive after 180min and the other 60% died after 137+/-40min whereas death of rats in the digoxin group occurred within 80+/-10min (p<0.001). The death of animals was preceded by the P waves disappearing. Thirty minutes after digoxin administration, the potassium plasmatic level increased significantly in the digoxin group as compared to the GID group: 6.9+/-0.5mmol/l versus 4.9+/-0.3mmol/l (p<0.001). At the time of death, both volume of digoxin infusion and serum digoxin concentration were increased in GID group as compared to digoxin group: 5.7+/-1.6ml versus 3.3+/-0.4ml (p<0.001) and 10.7+/-8.3mg/l versus 8.5+/-4.6mg/l. CONCLUSION: Glucose-insulin infusion delayed the abnormalities in cardiac conduction and improved rat survival after acute digoxin toxicity. These results suggest a cardioprotective effect of insulin in case of acute digoxin toxicity.     

吉林生晒参慢性毒性试验的初步比较研究

目的以慢性毒性试验研究吉林人参对Wistar大鼠的毒性作用情况。方法进行慢性毒性试验,给予含有不同剂量（8.0、6.5、5.0g/kgBW）吉林人参样品的饲料喂饲大鼠180d,在试验中期和末期分别检测并比较各组大鼠体重、脏器系数、血液学指标、血生化学指标、病理变化情况。结果试验中、末期各剂量组大鼠的各种指标与对照组比较,差异无显著性（P〉0.05）。有一些血液学及血生化学指标在试验中、末期间比较,差异具有显著性（P〈0.05）。结论在180d的慢性毒性试验中,食用吉林人参未对大鼠产生明显慢性毒性作用。     

地高辛血药浓度监测在ICU的临床应用与研究

目的 总结并分析我院重症监护室患者进行地高辛血药浓度监测的情况,为临床合理用药提供参考.方法 采用荧光偏振免疫法测定地高辛血药浓度,利用有关统计学方法对所得数据进行处理,并作回顾性分析.结果 74份病历共进行TDM 104次,其中测定地高辛46例次,达有效血药浓度34例,占73.91%;未达有效血药浓度5次,占10.87%;达中毒血药浓度7例,占15.22%.结论 ICU患者基础疾病复杂,内环境紊乱,联合使用多种药物.所以使用地高辛时,剂量不能完全决定血药浓度,血药浓度也不能完全决定临床疗效,因此必须在参考TDM结果的同时,结合患者具体生理、病理及临床表现等各种情况来调整给药方案.     

采用雅培i4000快速测定血浆地高辛浓度及临床应用

目的 探讨采用雅培i4000快速检测血浆地高辛（Digoxin）浓度,以指导临床快速诊断和合理用药.方法 收集132例患者血浆在雅培i4000上检测Digoxin浓度,比较Digoxin浓度与疗效的关系;按年龄和浓度分组统计药物浓度分布规律.对0.8～2.0 μg/L治疗有效浓度之外的患者根据情况分别调整剂量并连续监测血浆浓度.结果 132例患者中,106例血药浓度在治疗剂量内的占80.30％;用药后治疗总有效率为86.36％.＜0.8μg/L组、0.8～2.0 μg/L组和＞2.0 μg/L组,其有效率分别为10.91％、95.28％和75.00％;11例浓度＜0.8 μg/L患者和4例＞2.0 μg/L的患者经调整用药剂量后,浓度回复至有效浓度内.60岁以上年龄段患者的Digoxin平均浓度高于50～ 60岁组.7例出现中毒症状的患者经调整剂量后症状消除.结论 采用雅培i4000检测血浆Digoxin浓度操作简便、快捷,结果准确可靠.     

Effect of age on levels of diazepam in plasma and brain of rats

Summary The pharmacokinetics and distribution in brain and cerebellum of diazepam after a single dose were studied in middle aged (6 months) and old (18 months) rats. Following the single intravenous bolus of 5 mg/kg diazepam was eliminated more slowly in old rats (T _1/2()=3.1h) than in middle aged rats (1.4 h). This was due to an increase in the apparent volume of distribution Vd from 11.0 l/kg (control rats) to 29.5l/kg. Concentrations of diazepam in brain and cerebellum were in the same range (0.5–1.1 ng/mg) in both groups after this dose. We conclude that the distribution of diazepam is age-dependent which might be due to an altered body composition.     

美托洛尔联合地高辛治疗缺血性心肌病合并心房颤动40例临床观察

目的：观察美托洛尔联合地高辛治疗缺血性心肌病合并心房颤动的临床效果。方法对80例缺血性心肌病合并心房颤动患者根据随机数字表法分为两组,对照组单纯使用地高辛进行治疗,观察组患者在对照组患者治疗方案基础上,加用美托洛尔,比较两组患者治疗前后超声心动图指标包括左心室舒张末内径（ LVEDD）、左室收缩末期内径（ LVESD）、每搏搏出量（ SV）和射血分数（ EF）改变情况、心率控制情况以及心房颤动的临床疗效。结果与治疗前比较,观察组LVEDD[（50．2±3．2） mm与（71．9±2．2） mm]、LVESD水平[（40．5±3．4）mm与（59．7±3．7）mm]均明显降低,SV[（69±5）mL与（39±4）mL]及EF水平[（59．7±8．7）％与（39．6±9．7）％]明显升高（t＝6．78、4．67、5．78、5．46,均P＜0．05）。观察组患者LVEDD、LVESD水平均明显低于对照组,SV以及EF水平明显高于对照组,差异有统计学意义（ t＝4．35、3．98、4．32、4.56,均P＜0．05）;经治疗,两组患者的心率水平均有下降,但观察组患者的静息期和活动期的心率水平[（71．2±8．9）次/min与（120．3±15．4）次/min]均明显低于同期对照组水平[（79．4±9．6）次/min与（140．2±13．3）次/min],差异有统计学意义（t＝3．85、4．02,均P＜0．05）。结论美托洛尔联合地高辛治疗缺血性心脏病合并心房颤动临床效果显著,改善患者心脏功能更明显,降低了心率水平,临床总有效率更高,值得临床推广。     

Kinetic analysis of saturable hepatic uptake of digoxin and its inhibition by rifampicin

Although the organic anion transporter Oatp2 plays a critical role in determining the hepatic clearance of some drugs, little quantitative information exists about its functional characteristics in relation to inhibition of sinusoidal drug uptake. We investigated the uptake kinetics of the Oatp2 substrate digoxin in the isolated perfused rat liver. In the single-pass perfused liver three consecutive digoxin doses of 15, 30 and 45 micorg were administered in the presence or absence of rifampicin (100 micorM), an inhibitor of Oatp2. Digoxin was determined in the outflow samples by HPLC and all data were analyzed by simultaneous nonlinear regression assuming a Michaelis-Menten uptake mechanism. Hepatocellular uptake of digoxin was concentration-dependent with a Michaelis constant (K(M)) of 577.8 ng/ml. Rifampicin significantly reduced uptake (K(M) increased 2.5-fold) without affecting other parameters.     

Intravenous nicotine self-administration in rats: effects of mecamylamine, hexamethonium and naloxone

The rate and pattern of lever pressing were studied in 18 rats during 24-h sessions in which responding resulted in intravenous infusions of nicotine. There were four indications of the positive reinforcing effect of nicotine: (1) a greater number of lever presses when nicotine was response-contingent compared to when saline was available; (2) a greater number of responses on the lever resulting in an infusion of nicotine than on the control lever; (3) systematic decreases in the number of contingent nicotine infusions when nicotine was delivered noncontingently; and (4) systematic changes in the frequency of lever pressing as a function of dose. Under a fixed ratio 1 (FR 1) schedule, the number of infusions first increased and then decreased as the dose of nicotine was decreased (64, 32, 16, and 8 microg/kg infusion) and nicotine intake (mg/kg every 24 h) was directly related to the infusion dose. As the FR size was increased from 1 to 6, the number of lever presses increased and the number of infusions (32 microg/kg) remained stable. At FR values greater than 6, both the number of lever presses and infusions decreased. Presession injections of mecamylamine (0.75, 1.5, and 3.0 mg/kg, s.c.) decreased the number of infusions in a dose-related manner. Presession injections of hexamethonium (1.5 and 3.0 mg/kg, s.c.) or naloxone (0.75, 1.5, and 3.0 mg/kg, s.c.) did not alter the within- or between-session patterns of nicotine self-administration. Under the conditions of the present experiment, nicotine served as an effective reinforcer and the behavior was shown to be sensitive to both FR size and infusion dose. In addition, the results suggest that nicotine self-administration involves central nicotinic receptors and that opioid receptor antagonism has no effect on nicotine's reinforcing effects in rats.     

Age-related differences in susceptibility to toxic effects of valproic acid in rats

A multi-age rat model was evaluated as a means to identify a potential age-related difference in liver injury following exposure to valproic acid (VPA), a known pediatric hepatotoxic agent. Different age groups of Sprague-Dawley (SD) rats (10-, 25-, 40-, 80-day-old) were administered VPA at doses of 160, 320, 500 or 650 mg kg(-1) (i.p.) for 4 days. Animals from all age groups developed toxicity after treatment with VPA; however, the patterns of toxicity were dissimilar within each age group. The high dose of VPA caused significant lethality in 10- and 25-day-old rats. All doses of VPA caused decrease in the platelet counts (10-, 25-day-old rats) and the rate of growth (40-day-old rats) and increases in the urine creatine concentration (high dose, 80-day-old rats). VPA induced hepatic and splenic alterations in all age groups. The most severe lesions were found mostly in 10- and 80-day-old rats. Significant changes in blood urea nitrogen, alanine aminotransferase and alkaline phosphatase were observed in 10-day-old pups after treatment with low doses of VPA. The highest VPA dose caused significant decreases in the levels of serum total protein (40- and 80-day-old rats). Principal component analysis of spectra derived from terminal urine samples of all age groups showed that each age group clusters separately. In conclusion, this study showed that the vulnerability profile of each age group was different indicating that a multi-age pediatric animal model is appropriate to assess more completely age-dependent changes in drug toxicity.     

A comparative study of the toxic and therapeutic effects of digoxin and its immobilized form

It was shown on different species of laboratory animals (frogs, mice, rats) that digoxin immobilized on the copolymer was 8-15 times less toxic that its usual preparation. In this case glycoside fixed on the copolymer preserves completely its specific cardiotonic effect, i.e., possesses a wider range of the therapeutic action.     

Intravenous butyrylcholinesterase administration and plasma and brain levels of cocaine and metabolites in rats

Butyrylcholinesterase is a major cocaine-metabolizing enzyme in humans and other primates, catalyzing hydrolysis to ecgonine methylester. Increasing butyrylcholinesterase activity may be a treatment for cocaine addiction. We evaluated the effect of 30-min pretreatment with horse-derived butyrylcholinesterase (5-15,000 U i.v.) or with the selective butyrylcholinesterase inhibitor cymserine (10 mg/kg i.v.) on the metabolism of cocaine (17 mg/kg i.p.) in anesthetized rats. Venous blood samples were collected for two hours after cocaine administration and later assayed for cocaine and metabolites by gas chromatography/mass spectroscopy. Whole brains were collected after the last blood sample and similarly assayed. Butyrylcholinesterase significantly increased plasma and brain ecgonine methylester levels and decreased cocaine plasma half-life from 26.2 min (saline) to 16.4 min (15,000 U). Butyrylcholinesterase had no significant effect on plasma or brain cocaine or benzoylecgonine levels. Cymserine had no effect on any variable. These findings suggest that butyrylcholinesterase treatment may have benefits in enhancing cocaine metabolism and in increasing levels of ecgonine methylester, which may have a protective action against cocaine.     

Excretion of digoxin and its metabolites in urine after a single oral dose in healthy subjects

The 3-day urinary excretion of digoxin, its conjugated and unconjugated hydrolytic metabolites and dihydrodigoxin, was studied in 8 healthy men after oral administration of tritiated digoxin. Analysis was performed by high pressure liquid chromatography (HPLC). The total radioactivity corresponded to 45.4±2.0 per cent (mean ± S.E.M.) of the dose. By HPLC 424 ± 2.7 per cent was recovered before and 44.0 ± 2.7 per cent after deconjugation of the samples. Digoxin and dihydrodigoxin constituted 40.3 ± 2.9 per cent; of this 0.7 ± 0.4 per cent was dihydrodigoxin. The sum of the hydrolytic metabolites was 2.1 ± 0.3 per cent before and 3.4± 0.5 per cent after deconjugation. No correlation was found between gastric pH and the production of hydrolytic metabolites. The relative amount of these metabolites was maximal (mean 13.4 per cent of the excretion) in the 4.8 h sampling period. During the first 8 h an average of 8.6 per cent of the radioactivity was not recovered by HPLC. The metabolism of digoxin as judged by urinary excretion was limited and showed great variation during the early hours after treatment. The excretion of unchanged digoxin in some individuals constituted as little as 60 per cent over the first 12 h after dosing.     

Commonly used surfactant,Tween 80, improves absorption of P-glycoprotein substrate,digoxin, in rats

Tween 80 (Polysorbate 80) is a hydrophilic nonionic surfactant commonly used as an ingredient in dosing vehicles for pre-clinical in vivo studies (e.g., pharmacokinetic studies, etc.). Tween 80 increased apical to basolateral permeability of digoxin in Caco-2 cells suggesting that Tween 80 is an in vitro inhibitor of P-gp. The overall objective of the present study was to investigate whether an inhibition of P-gp by Tween 80 can potentially influence in vivo absorption of P-gp substrates by evaluating the effect of Tween 80 on the disposition of digoxin (a model P-gp substrate with minimum metabolism) after oral administration in rats. Rats were dosed orally with digoxin (0.2 mg/kg) formulated in ethanol (40%, v/v) and saline mixture with and without Tween 80 (1 or 10%, v/v). Digoxin oral AUC increased 30 and 61% when dosed in 1% and 10% Tween 80, respectively, compared to control (P < 0.05). To further examine whether the increase in digoxin AUC after oral administration of Tween 80 is due, in part, to a systemic inhibition of digoxin excretion in addition to an inhibition of P-gp in the GI tract, a separate group of rats received digoxin intravenously (0.2 mg/kg) and Tween 80 (10% v/v) orally. No significant changes in digoxin IV AUC was noted when Tween 80 was administered orally. In conclusion, Tween 80 significantly increased digoxin AUC and Cmax after oral administration, and the increased AUC is likely to be due to an inhibition of P-gp in the gut (i.e., improved absorption). Therefore, Tween 80 is likely to improve systemic exposure of P-gp substrates after oral administration. Comparing AUC after oral administration with and without Tween 80 may be a viable strategy in evaluating whether oral absorption of P-gp substrates is potentially limited by P-gp in the gut.     

蓖麻毒素的毒性作用机制及其在肿瘤治疗中的应用

蓖麻毒素对哺乳动物细胞具有很强的毒性,属于Ⅱ型核糖体失活蛋白。其A链是活性链,可使核糖体失活,从而使蛋白质合成受阻,细胞死亡。基于蓖麻毒素的细胞毒性,人们将蓖麻毒素及其A链应用于肿瘤治疗领域,目的在于特异性杀伤肿瘤细胞,并减小对正常细胞的毒性。本文就蓖麻毒素的分子结构、毒性机制以及近年来在肿瘤治疗领域的研究进展作一综述。     

A comparison of the effects of digoxin and digitoxin on systolic time intervals and colour vision

Summary Animal studies suggest that for the same inotropism hydrophilic cardiac glycosides produce greater depression of atrioventricular (AV) conduction than lipophilic ones. This has been explained on the basis of a greater vagomimetic effect with hydrophilic agents and a greater sympathomimetic effect with lipophilic agents.In this randomized, cross-over study we investigated the effects of placebo, digoxin (relatively hydrophilic), and digitoxin (relatively lipophilic) in twelve healthy volunteers. For both drugs steady-state serum concentrations in the mid-therapeutic range were achieved.Both drugs produced the same positive inotropic effect as measured by systolic time intervals (QS₂c). There was a trend for digoxin to have a greater effect on AV conduction than digitoxin.After atropine or propranolol there was no difference between the effect of the two cardiac glycosides on AV conduction. No significant effects on colour vision were seen. We conclude that, there do not seem to be pharmacodynamic differences between digoxin and digitoxin at mid-therapeutic serum concentrations.