Radioreceptor assay for determination of the antimuscarinic drug ipratropium bromide in man

Summary A radioreceptor assay for the determination of ipratropium bromide in human plasma has been developed, using [³H] N-methyl-scopolamine as a radioligand to label muscarinic cholinergic receptors in a membrane preparation of rat cerebral cortex. there was no interference due to the cross-reactivity of 3 metabolites of ipratropium with the parent compound (5.2, 1.5 and 0.004%, respectively). The validity of the assay was checked between 20 pg/ml and 1000 pg/ml drug.In a pilot study plasma levels following a single oral dose of 30 mg were determined to examine the applicability of the radioreceptor assay to clinical and pharmacokinetic studies, and for measurement of plasma levels after therapeutic oral doses. The peak plasma concentration in three healthy volunteers ( =322 pg/ml) occurred within 1–3 h.     

Disposition of haloperidol and reduced haloperidol plasma levels after single dose haloperidol decanoate administration

A single dose of haloperidol decanoate 100 mg was administered to 15 schizophrenic patients. Blood samples were obtained prior to injection, 1 h, 3 h, 6 h, 8 h, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, one week, two weeks, three weeks and four weeks post-injection. Haloperidol and its reduced metabolite, reduced haloperidol, plasma levels were assayed by HPLC with electrochemical detection. The pharmacokinetic parameters of haloperidol were determined. The mean time of maximal (T_max) plasma levels for haloperidol was 5·73 ± 0·80 days. The haloperidol plasma levels showed a biexponential decline with an elimination half-life of 15·78 ± 5·90 days. Reduced haloperidol was rapidly formed from the haloperidol. The T_max of reduced haloperidol was 7·00 ± 2·35 days. The mean ratio reduced haloperidol/haloperidol was 0·155 ± 0·111. Since the T_max occurs at approximately six days, a weekly loading dose of haloperidol decanoate is feasible during the transition from oral to depot therapy.     

Circadian changes in the distribution and effects of haloperidol in the rat

Striking circadian changes in behavioral sensitivity to haloperidol were found by measurements of cataleptic responses in rats trained in a controlled lighting cycle (lights on, 7:00 a.m.–7:00 p.m.). Thus, catalepsy was maximal at about 4:00 p.m. and minimal at about 4:00 a.m., virtually the opposite of the circadian rhythm of spontaneous behavioral activity in drug-free rats. At a given dose of haloperidol, catalepsy scores differed 2- to 3-fold, and the ED₅₀ shifted left nearly 10-fold from a.m. to p.m. After fixed doses of haloperidol, tissue levels of the drug, as determined by a sensitive and selective radioreceptor assay, differed by 2- to 6-fold through the 24 hr cycle and brain levels closely followed the circadian changes in behavior. These results suggest a pharmacokinetic contribution to the circadian changes in behavioral response, although additional pharmacodynamic factors are also considered.     

Application of Radioreceptor Assay of Benzodiazepines for Toxicology

Abstract: A radioreceptor assay (RRA) for determining benzodiazepines (BZ) has been developed and applied to toxicological analysis of serum samples from 21 patients with acute BZ overdosage. The method was sensitive (e.g., lorazepam 17 nM, diazepam 41 nM), and specific for pharmacologically active BZ derivatives. The reproducibility of the results was good (intra-assay variation <8%, inter-assay variation <10%). Concentrations measured by the RRA showed a good correlation with those obtained by gas-liquid chromatographic analysis of the same samples. The quantitative results represent the sum of one or several parent substances and all biologically active metabolites, in proportion to their receptor binding affinities.     

Comparison of radioreceptor assay and radioimmunoassay for atropine: Pharmacokinetic application

Summary A membrane suspension prepared from rat brain was able to bind the potent muscarinic antagonist quinuclidinyl benzilate (QNB). The K_D for binding was 0.48 nM and B_max was 1.42 pmol/mg protein. Atropine competitively inhibited the binding of tritiated QNB to muscarinic receptors. This new radioreceptor assay (RRA) for atropine has been compared with a radioimmunoassay (RIA) for atropine. The RRA measures only the active component of atropine, 1-hyscyamine and in this respect it differs from the RIA. As little atropine as 1.25 ng/ml (4.33 nmol/l) in a 25 µl serum sample could be reliably assayed by the RRA. Using both assay techniques the pharmacokinetics of atropine was studied after a single 0.02 mg/kg i.v. dose given to 8 anaesthetized patients. The half-life calculated by the RRA was 3.7±2.3 h (m ± SD) and by the RIA 4.3±1.7 h. Both the volume of distribution and the total clearance were higher according to the RRA than the RIA: 3.9±1.5 vs 1.7±0.71/kg and 15.4±10.3 vs 5.9±3.6 ml/min/kg, respectively. The AUC measured by the RRA and RIA was 29.8±18.9 and 103.9±110.7 µg·h/l, respectively. The differences in the pharmacokinetics according to the 2 methods are presumably due to preferential tissue uptake of the l-form.     

Plasma level profile of haloperidol in man following intramuscular administration

Summary Haloperidol is rapidly absorbed by normal subjects following intramuscular administration, maximum plasma level being reached within 20 min. The plasma level profile of haloperidol shows multi-compartment model kinetics with a terminal elimination half-life of 20.7±4.6 (SD) h. The prolonged elimination half-life for haloperidol suggests that a delay of approximately three to five days will occur before equilibrium plasma levels are re-established following a change in dosage regimen.     

Development of a Radioreceptor Assay for the D2-Selective Dopamine Agonist N-0437

N-0437 is a recently developed dopamine (D²) agonist, theoretically attractive in the therapy of Parkinson's disease and glaucoma. Since its high potency allows small doses of the compound in clinical use and as extensive metabolism occurs in animals, a highly sensitive assay method was required for drug-monitoring purposes. To this end we developed a radioreceptor assay (RRA), a sensitive tool for the assessment of the sample's (dopaminergic) bioactivity. The RRA is based on competition between N-0437 and its tritium-labeled analogue for binding to dopamine receptors. The assay has been optimized for the preparation of the receptor suspension and the incubation conditions. Direct application of the assay for biological samples was impossible because of matrix interferences. Therefore, a solid-phase extraction method was developed in which the combination of a polar Si column and dichloromethane as eluent resulted in an effective elimination of the interferences. Recoveries were better than 90 and 95% for plasma and urine, respectively, even at concentrations at the determination limit of the method (300 pg/ml). Relative standard deviations were less than 15%. Because RRAs are stereoselective, the method discriminates between active and inactive species.     

高效液相色谱法测定碘化N-正丁基氟哌啶醇含量

目的:用高效液相色谱法测定碘化N-正丁基氟哌啶醇 (F 2 )的含量.方法:采用Phenomenex Luna C 18 反相色谱柱( 4.6 mm×250 mm,5 μm),以乙腈-甲醇-0.025 mol·L -1 磷酸二氢钾(45∶5∶50)作为流动相,流速为 0.8 mL·min -1 ,检测波长:248 nm,柱温:35 ℃,进样体积:20 μL,按外标法以峰面积计算F 2 的含量.结果:F 2 在1～10 mg·L -1 浓度范围内,浓度与峰面积的线性关系良好,r为 0.999 2 ,RSD为 1.70% (n=5).结论:本方法简便,快速,结果准确,可靠,重现性好,可用于本品质量控制.     

Improved Radioreceptor Assay of Opiate Narcotics in Human Serum: Application to Fentanyl and Morphine Metabolism

A recently developed radioreceptor assay (RRA) (1) that employs ³H-naloxone and rat brain membrane homogenates was improved two ways. First, the brain membranes were preincubated in the presence of sodium ions, and second, manganase-II ions were added to the sample incubations. These changes enhanced the assay sensitivity and reproducibility with stored membrane preparations and reduced the effects of serum constituents (Na⁺) on ligand–receptor binding. Patient sera were assayed by radioimmunoassay (RIA) and RRA after fentanyl administration and by high-performance liquid chromatography (HPLC) and RRA after morphine administration. The results with both fentanyl assays were comparable, and no fentanyl metabolites were detectable by RRA after HPLC of serum extracts. In contrast, preliminary results with the HPLC-RRA procedure suggest the presence of an active morphine metabolite of unknown structure in sera obtained from patients on morphine therapy.     

Determination of haloperidol and reduced haloperidol in the plasma and blood of patients on depot haloperidol

We developed a sensitive HPLC assay to measure haloperidol (HA) and its metabolite, reduced haloperidol (RH), in plasma and whole blood. The conditions under which HA might be converted to RH during collection and analysis of blood were examined. Provided the blood was kept at 0° C, erythrocyte ketone reductase activity was insignificant. The solid phase extraction method did not generate RH. We studied ten patients taking 25–400 mg/month of HA decanoate and one patient for 4 weeks after the daily oral dose of 120 mg HA was ceased. In the patients on depot HA, the plasma and blood concentrations of HA were not significantly different (P>0.1). For the first time, RH was detected in plasma patients on depot drug, but only in three cases. In contrast, RH was present in the blood of eight of these patients. The accumulation of RH in red blood cells was also evident in the patient on oral HA, in whom the mean ratio of RH concentrations in whole blood to plasma was 3.6±1.1. Plasma concentrations of HA correlated highly with total neuroleptic activity measured by a radioreceptor assay. Compared to plasma, analysis of concentrations of HA and RH in blood has the advantages of greater sensitivity, of using smaller volumes of blood and of avoiding the efflux of HA and RH during separation of plasma and red cells.     

Reduced haloperidol: Effects on striatal dopamine metabolism and conversion to haloperidol in the rat

Acute injection of rats with either haloperidol or reduced haloperidol (1 mg/kg, IP) greatly increased the striatal concentrations of the acidic dopamine metabolites, indicating enhanced turnover of dopamine. The effect of reduced haloperidol was almost as great as that of haloperidol. Reduced haloperidol, however, was much less efficient (about 400 times) than haloperidol in displacing [³H]spiperone binding to striatal membranes in vitro. In agreement with the above results, reduced haloperidol was found to be oxidized to haloperidol, so that 2 h after injection of reduced haloperidol the concentrations of haloperidol and reduced haloperidol were equal in the striatum. The apparent conversion of reduced haloperidol to haloperidol was much quicker in liver than in plasma or brain, and it is suggested that the conversion primarily occurs in the liver. Before drawing any definite conclusion about the possible central activity of reduced haloperidol, further studies with other animal species are needed.     

喹硫平和氟哌啶醇治疗老年痴呆精神行为症状的对照研究

目的：观察喹硫平和氟哌啶醇治疗老年痴呆精神行为症状的疗效和安全性。方法：采用随机对照研究,喹硫平组28例,剂量(130±s 60)mg·d~(-1),25～400mg·d~(-1),氟哌啶醇组23例,剂量(4．8±1．4)mg·d~(-1),2～20mg·d~(-1),疗程12wk,治疗前后采用痴呆病理分析评定量表(BEHAVE-AD)和治疗时出现的症状量表(TESS)评定疗效和不良反应。结果：2组病人治疗后BEHAVE-AD评分显著下降(P＜0．01),2组病人之间治疗前后BEHANE-AD的减分值无显著差异(P＞0．05),氟哌啶醇组44%有锥体外系反应,明显高于喹硫平组的7%,有显著差异(P＜0．05)。结论：喹硫平和氟哌啶醇治疗老年痴呆精神行为症状疗效确切,喹硫平不良反应少。     

氟哌啶醇致不良反应的文献分析

目的探讨氟哌啶醇所致不良反应(ADR)的发生原因、规律、特点,为临床安全、合理用药提供参照。方法检索1989年1月—2017年7月中国学术期刊(网络版)、万方数字化期刊全文库、中文科技期刊全文数据库(维普)及Pub Med数据库收录的全部中医药期刊,收集报道氟哌啶醇致不良反应的文献进行统计和分析。结果共检索到94篇文献,122例病例。男性明显多于女性,年龄集中在18~40岁。用药1~7 d出现ADR的例数最多,占45.10%。氟哌啶醇引发的不良反应主要涉及神经系统、心血管系统、皮肤损害,其中神经系统损害的患者63例,占51.6%,所占比例最高。心血管系统和皮肤损害ADR占比分别为14.8%、10.7%。结论氟哌啶醇引发的不良反应较为常见,临床应重视氟哌啶醇不良反应的危害性,加强药学监护,尽量避免或减少其所致不良反应的发生,确保患者用药安全。     

Electrophysiological interactions between haloperidol and reduced haloperidol, and dopamine, norepinephrine and phencyclidine in rat brain

Haloperidol and its metabolite, reduced haloperidol, were compared as antagonists of catecholaminergic neurotransmission in central nervous system of the rat. Agonists and antagonists were applied from multibarrelled micropipettes, which were also used to record extracellularly the effects of these substances on neuronal discharge. Haloperidol antagonized dopaminergic inhibition of caudate neurons and inhibition of cerebellar Purkinje neurons induced by noradrenaline, whereas reduced haloperidol was an ineffective antagonist. Phencyclidine, which is an indirect dopaminergic agonist in the caudate, caused inhibition of the discharges of caudate neurons resembling that induced by dopamine itself. These indirect effects of phencyclidine were also antagonized by haloperidol but not by reduced haloperidol. The data suggest that the metabolite, reduced haloperidol, is not an effective neuroleptic drug in the central nervous system.     

喹硫平、氟哌啶醇对青年精神分裂症患者血清生长激素水平的影响

目的:探讨青年精神分裂症患者血清生长激素(GH)基础水平与正常青年人的差异,及喹硫平和氟哌啶醇对精神分裂症患者GH水平的影响.方法:41例青年精神分裂症患者分为喹硫平组19例和氟哌啶醇组22例,另选择20例健康志愿者作正常对照.喹硫平组平均口服剂量(580±170)mg·d-1,氟哌啶醇组平均口服剂量(26±8)mg·d-1,疗程均为8周.用酶联免疫法测定治疗前后的GH水平.结果:青年精神分裂症患者的基础GH水平与健康对照组差异无显著性(P＞0.05),治疗8周后,喹硫平组患者GH水平与治疗前无明显变化,氟哌啶醇组治疗8周后GH值为(1.42±0.87)μg·L-1,较治疗前(2.49±1.10)μg·L-1显著降低,差异有显著性(P＜0.01).结论:氟哌啶醇能使青年精神分裂症患者GH水平显著降低,喹硫平对青年精神分裂症患者GH水平无明显影响,安全性好.     

癫痫患儿丙戊酸钠血药浓度的相关分析

目的 探讨癫痫患儿丙戊酸钠血药浓度与剂量等因素的相关性.方法 126例癫痫儿童按体重口服相应剂量(10-20mg·kg-1·d-1)德巴金糖浆,3-5天后采用高效液相法测定丙戊酸钠血药浓度并采用Pearson相关及偏相关分析结果.结果 Pearson相关分析发现体重与口服剂量,体重与年龄,年龄与剂量之间存在正相关相关系数分别为0.55、0.70、0.45(P<0.001).剂量与血药浓度之间也为正相关相关系数为0.33(P<0.01).偏相关分析发现剂量、体重与丙戊酸钠血药浓度的偏相关系数分别为0.55(P<0.001)、0.30(P<0.05).结论 样本研究发现2岁以下癫痫患儿丙戊酸钠血药浓度与剂量、体重存在一定相关关系,与年龄的相关性不显著.临床用药需考虑剂量、体重对血药浓度的影响.     

Effect of quinidine on the interconversion kinetics between haloperidol and reduced haloperidol in humans: implications for the involvement of cytochrome P450IID6

Summary Haloperidol (HAL) is a potent butyrophenone antipsychotic agent which is reversibly metabolized to reduced haloperidol (RHAL). In order to determine if this reversible metabolic pathway is linked to the debrisoquine 4-hydroxylase isozyme of cytochrome P-450 (P450IID6), HAL (5 mg) or RHAL (5 mg) was orally administered to healthy male volunteers in a randomized crossover design both with and without a prior (1 h) oral dose of quinidine (250 mg bisulfate), a potent inhibitor of this isozyme. Thirteen volunteers, 11 extensive metabolizers, 2 poor metabolizers, completed all four phases of the study. Plasma samples harvested over seven days were analysed for HAL and RHAL. An expression for the apparent fractional availability of metabolite from the parent compound given (Fapp _infm ^supp ) was derived and was used to determine whether HAL or RHAL is the preferred metabolite, and whether quinidine co-administration alters Fapp for either compound.The AUC (0-t) for both HAL and RHAL were significantly greater following the administration of either compound with quinidine compared with AUC (0-t) values obtained in the absence of quinidine. The maximum plasma concentration (C_max) of the administered compound was also greater following the administration of quinidine. Quinidine had no effect on the half-lives of the administered compounds. The Fapp for HAL and RHAL were not significantly affected by the administration of quinidine, indicating that the interconversion of HAL and RHAL is not linked to P450IID6. The Fapp of RHAL after administration of HAL was significantly greater than the Fapp of HAL after RHAL administration, indicating that RHAL is the preferred metabolic form. This difference was not affected by quinidine.It is concluded that: 1) RHAL is the preferred form after administration of either compound and is not affected by quinidine, 2) the interconversion of HAL and RHAL is not affected by quinidine, indicating that this reversible metabolic process is not linked to P450IID6 and 3) there is a significant increase in the AUC (0-t) and C_max values following quinidine co-administration with either HAL or RHAL. The precise mechanism of this interaction can not be established from this study, however, the observed increases in AUC (0-t) and C_max may be explained with a simple tissue blinding displacement mechanism.     

Determination of a Novel Calcium Channel Antagonist,Mepirodipine, in Plasma by Radioreceptor Assay Using (+)-[3H]PN 200-110

Pharmaceutical Research -     

碘化N-正丁基氟哌啶醇对大鼠心肌细胞内钙离子的影响

目的:研究碘化N-正丁基氟哌啶醇(F2)对大鼠心肌细胞内钙离子([Ca2+]i)的影响。方法:采用钙荧光染料Fluo-3-AM负载心肌细胞,在激光扫描共聚焦显微镜上测定细胞内钙离子的变化。结果:在含钙(1.8mmol·L-1)台式液中,60mmol·L-1KCl使细胞内钙荧光强度由1.0增高至1.84±0.35(P<0.01,n=50),分别用0.1、1、10μmol·L-1F2预先孵育心肌细胞,F2可以浓度依赖地抑制钙荧光强度的增高,IC50为1.61μmol·L-1。0.1、1、10μmol·L-1F2使得KCl诱导增高的钙荧光强度从1.92±0.42分别降至1.88±0.39、1.31±0.36和1.09±0.05(P分别>0.05、<0.01和<0.01,n=50),IC50为1.78μmol·L-1。F2不影响静息状态下的细胞内钙的浓度。对咖啡因和三磷酸肌醇介导的细胞内钙的释放均无作用。结论:F2通过阻断心肌细胞膜电压依赖性钙通道降低心肌细胞内钙浓度,这可能是保护心肌缺血/再灌注损伤的机制。