A RIA combined with SPE for the determination of a dual D2-receptor and beta2-adrenoceptor agonist, AR-C68397XX, in human plasma

A radioimmunoassay has been developed for the determination of AR-C68397XX, a dual D2-receptor and beta2-adrenoceptor agonist, in human plasma. The method incorporates solid phase sample extraction and is suitable for the determination of the analyte at pg ml(-1) concentrations. The antiserum was raised in Suffolk cross sheep following primary and booster immunisations with an immunogen prepared by conjugating a carboxyphenylmethyl derivative of AR-C68397XX, to bovine serum albumin. The radioligand was prepared by the 125I-labelled iodination of a derivative of AR-C68397XX. The solid phase extraction procedure, using octadecyl sorbent, was introduced to remove matrix interferences in the plasma and to enhance method sensitivity. The calibration range is 20-500 pg ml(-1), using 0.5 ml of undiluted human plasma sample.     

Rapid formation of reduced haloperidol in guinea pigs following haloperidol administration

Haloperidol, a butyrophenone neuroleptic, is metabolically reduced in man, but not in rat and not in many other experimental animals. Here we present data that describes reductive haloperidol metabolism in guinea pigs in vivo. When haloperidol was injected intraperitoneally to guinea pigs, it was converted to reduced haloperidol so quickly that 1 hr after the injection the concentration of haloperidol was only about one fifth of that of reduced haloperidol. Dopamine metabolism was enhanced in the striatum after the administration of reduced haloperidol, but this enhancement could mostly be explained by oxidation of a small amount of reduced haloperidol back to haloperidol. The molecular mechanisms of haloperidol reduction should be further studied using guinea pigs as a model for human haloperidol metabolism.     

Interconversions of haloperidol and reduced haloperidol in guinea pig and rat liver microsomes

An alcohol metabolite of haloperidol, reduced haloperidol, is present in the tissues of haloperidol-treated patients. We have studied whether rat and guinea pig liver microsomes have the capability to reduce haloperidol and thus serve as models for human haloperidol metabolism. Interestingly, the rat microsomes did not reduce haloperidol, but possessed an NADPH-dependent, carbon monoxide-inhibited mechanism to oxidize the reduced haloperidol back to haloperidol. Guinea pig microsomes efficiently reduced haloperidol molecules in a fashion not dependent on nicotinamide cofactors and not inhibited by carbon monoxide. Both of these activities were confined to the microsomal fraction. In guinea pigs, reduction of haloperidol was observed also in kidney slices, whereas brain slices proved inactive. Reduced haloperidol was also oxidized to haloperidol to a small extent in guinea pig microsomes. These in vitro experiments confirm our findings in vivo, which showed that in rats haloperidol is not reduced, while guinea pigs have a very active mechanism for reducing haloperidol. Thus, guinea pigs constitute a model for human haloperidol metabolism, and they should be used for further characterization of the reductive drug-metabolizing system.     

Measurement of haloperidol in human breast milk by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the assay of haloperidol in human breast milk. The method involves rapid extraction of haloperidol and chromatography with a reversed-phase C18 column and a mobile phase of phosphate buffer (pH 4.0):acetonitrile (70:30, v/v). Moperone was used as the internal standard. Haloperidol and moperone were detected with ultraviolet detection at 254 nm. The sensitivity was 5 ng/mL of milk, and the standard curve was linear in the concentration range 5-250 ng/mL. The average interassay coefficient of variation for samples with drug in the concentration range 25-125 ng/mL was less than 8.8%. The absolute recovery of haloperidol by the method was greater than 94.4%. No interference with endogenous substances in human milk was observed. The method was used to determine haloperidol levels in breast milk from patients undergoing chronic haloperidol treatment.     

Biliary excretion of reduced haloperidol glucuronide

The biliary excretion of haloperidol and reduced haloperidol were investigated in the guinea pig. Bile duct cannulated guinea pigs were administered a single intraperitoneal dose of haloperidol (1 mg/kg). Bile was continually collected over a 12-h period. Aliquots of the bile samples were analyzed by HPLC for free haloperidol and reduced haloperidol. The remaining portions of the bile samples were incubated with beta glucuronidase and reanalyzed for haloperidol and reduced haloperidol. Although no significant amount of haloperidol glucuronide was detected in the bile, a new metabolite of reduced haloperidol, reduced haloperidol glucuronide, was found. The amount of reduced haloperidol excreted in the bile as the glucuronide conjugate was significantly higher than the amount of haloperidol or reduced haloperidol. These results imply that reduced haloperidol glucuronide may play a role in the disposition of haloperidol and/or its metabolite, reduced haloperidol.     

Establishment of new cloned enzyme donor immunoassays (CEDIA) for haloperidol and bromperidol

The authors have developed and verified the precision and accuracy of new automated cloned enzyme donor immunoassays (CEDIA) for haloperidol and bromperidol, and cross-validations have been performed with conventional semiautomated EIA kits (MARKIT-M) and high-performance liquid chromatographic (HPLC) methods. The CEDIA method provides a quick (about 10 minutes) assay for haloperidol or bromperidol, requiring no serum/plasma pretreatment or predilution. The CEDIA haloperidol/bromperidol assay showed little or no cross reactivity with either their metabolites or many drugs commonly coprescribed. MARKIT-M revealed considerable cross reactivity values proportional to the spiked amounts of reduced metabolites. Precision, accuracy, recovery, and linearity testing for the CEDIA assay were all sufficient for clinical use. Significant linear correlations were found between CEDIA and HPLC in measuring haloperidol (CEDIA = 1.06 x HPLC + 0.869; n = 44, rs = 0.913, P < 0.001) and bromperidol (CEDIA = 1.06 x HPLC + 0.606; n = 56, rs = 0.914, P < 0.001) concentrations. This study has, therefore, demonstrated that the CEDIA assay has a quick run time with high precision and accuracy, and this method is a useful tool for the TDM of haloperidol or bromperidol.     

Disposition of haloperidol pyridinium and reduced haloperidol pyridinium in schizophrenic patients: no relationship with clinical variables during short-term treatment

In an open clinical trial, serum concentrations of haloperidol pyridinium (C(HP+)) and reduced haloperidol pyridinium (C(RHP+)), as well as haloperidol (CH) and reduced haloperidol (C(RH)), were measured in 57 schizophrenic and schizoaffective inpatients during 6 weeks of short-term treatment. Psychopathology was monitored with the Brief Psychiatric Rating Scale (BPRS), and extrapyramidal adverse effects were assessed with the Extrapyramidal Symptom Rating Scale (EPS). Significantly linear relationships were found between haloperidol dose (D) and pyridinium metabolite serum concentrations, as well as between C(H) and the pyridinium metabolite serum concentrations. C(HP+) (range, 0.2-4.9 ng/mL) and C(RHP+) (range, 0.03-6.23 ng/mL) were low compared with C(H) and C(RH), being as mean values approximately 7% and 14% of C(H) and C(RH), respectively. Additionally, the values of C(RHP+) and the slope of the correlation of C(H) with the C(RHP+)/C(HP+) ratio were considerably lower than in a previous report of long-term treatment with haloperidol. This is explained by the shorter time of treatment of the present study. Carbamazepine comedication was found to not influence relative pyridinium metabolite serum concentrations C(HP+)/D and C(RHP+)/D. However, the aromatization ratios of haloperidol (C(HP+)/C(H)) and reduced haloperidol (C(RHP+)/C(RH)) were increased by concomitant carbamazepine. As the main result, no relationships between the pyridinium metabolite serum concentrations and clinical variables (BPRS change, EPS, dose of biperiden) were detected. For instance, the aromatization ratios C(HP+)/C(H) and C(RHP+)/C(RH) did not predict clinical improvement or extrapyramidal adverse effects. Therefore, no confirmation of the "pyridinium hypothesis," which suggests haloperidol pyridinium metabolites to be the origin of adverse effects and decreased therapeutic effect, can be derived from this study. However, the authors emphasize that pyridinium metabolites cannot be excluded as the origin of decreased therapeutic effect in long-term treatment and of adverse effects not investigated in the present study, such as tardive dyskinesia. Finally, it is concluded that the serum concentration of the parent drug remains the main variable of interest in the therapeutic drug monitoring of haloperidol during short-term treatment.     

Radioimmunoassays for a new angiotensin-converting enzyme inhibitor, zabicipril, and its active metabolite, zabiciprilat, in human plasma.

Zabicipril (Z), a new angiotensin-converting-enzyme inhibitor under development, is a prodrug that has to be deesterified in vivo to the active metabolite, zabiciprilat (ZT), to reach its antihypertensive pharmacological properties. Two radioimmunoassays have been developed for the measurement of both Z and ZT in human plasma. Anti-Z and anti-ZT antibodies were raised in rabbits against their respective lysine analogues conjugated to bovine serum albumin. [3H]Z and [3H]ZT were used as radioligands for the prodrug (Z) and the drug (ZT), respectively. Studies on anti-Z antiserum specificity showed no significant cross-reactivity (< 0.1%) with the active metabolite (ZT); similarly, Z is poorly recognized (0.5%) by the anti-ZT antiserum. Both antisera showed little cross-reaction (2%) with glucuronide metabolites. The sensitivities of the assays were 1 and 0.2 ng/mL for Z and ZT, respectively. Interassay coefficients of variation ranged from 9 to 12% for Z at 2-50 ng/mL and from 8 to 13% for ZT at 0.5-5 ng/mL. These assays were used to investigate the pharmacokinetic profile of both Z and its pharmacologically active metabolite ZT after intravenous infusion of Z in human volunteers.     

Development of a Sensitive Radioimmunoassay for Fab Fragments: Application to Fab Pharmacokinetics in Humans

Anti-sheep Fab fragment antisera were produced in rabbits using sheep digoxin-specific Fab fragments (Digidot) as immunogen. These antisera were used for the development of a radioimmunoassay (RIA) of sheep Fab fragments in human plasma and urine using ¹²⁵I-labeled Fab fragments. Interference in the assays by digoxin, human proteins, and antibodies from different species was insignificant, but cross-reactivity between anti-sheep Fab antisera and goat IgG or Fab fragments was 22 to 67%. The limit of detection was 0.1 µg/mL and the assay was linear over a 0.6–28 µg/mL range of Fab fragments. Intra- and interassay coefficients of variation were less than 6.9 and 10.5%, respectively. Accuracy of plasma and urine assays at various Fab fragment levels ranged from 96 to 106%. RIA was applied to the pharmacokinetic study of sheep digoxin-specific Fab fragments in one patient acutely intoxicated by digitoxin and treated with Digidot. The Fab elimination half-life was 12.1 hr. Steady-state volume of distribution and total-body clearance were 10.8 L and 23.4 mL/min, respectively. Unchanged Fab fragments (50 kD) and degradation products (25 kD) isolated by gel filtration chromatography of a urine sample cross-reacted with the anti-Fab antiserum.     

An ultrasensitive method for the measurement of haloperidol and reduced haloperidol in plasma by high-performance liquid chromatography with coulometric detection

A new analytical method has been developed for the simultaneous quantitation of haloperidol and reduced haloperidol in plasma. The method is based on high performance liquid chromatography (HPLC) with coulometric detection. The extraction and sample clean up procedures are simple and rapid to execute, yet yield chromatograms virtually free of interference from endogenous plasma constituents, such that the extraordinary sensitivity of the coulometric detector can be exploited fully. The detection limits for haloperidol and reduced haloperidol are 20 pg/ml plasma, and the limits of quantitation are 50 pg/ml for both drug and metabolite. Standard curves were linear down to 50 pg/ml with coefficients of variation of less than 7.0% at the limits of quantitation. The method was applied to the study of the plasma levels of haloperidol and reduced haloperidol in two healthy subjects. It was possible to monitor the plasma levels of haloperidol for at least 96 h (4 days) after the administration of a 5-mg oral dose of haloperidol. It was also possible to monitor reduced haloperidol levels over 96 h in one subject, although the metabolite was not detectable in the plasma of the other at any stage.     

Splanchnic uptake of haloperidol and release of reduced haloperidol in vivo in the guinea pig.

Splanchnic uptake of haloperidol (HAL) and release of reduced haloperidol (RHAL) were studied in vivo in guinea pigs. Anesthetized animals with implanted cannulae in the aorta, the hepatic vein and the inferior vena cava were infused intravenously with HAL at a rate of 9.6 micrograms/min/animal for 90 min. Plasma HAL and RHAL in samples taken from the arterial and hepatic venous cannulae were measured by HPLC with an electrochemical detector. Contamination of the hepatic venous samples by blood from the inferior vena cava was ruled out by the validation method of tritiated water washout [Huang MT, J Appl Physiol 71: 359-364, 1991]. HAL concentrations plateaued at 70-80 ng/mL in the aorta and 5-7 ng/mL in the hepatic vein during the final 30 min of infusion. Splanchnic extraction of HAL was 91%. Hepatic blood flow was estimated to be 1.95 +/- 0.40 (SD) mL/min/g. If assuming that splanchnic uptake of HAL took place in the liver, a rate of uptake of HAL in the liver of 79.2 +/- 18.6 (SD) ng/min/g could be calculated by the Fick principle. The uptake in the whole liver accounted for 14% of the rate of HAL infusion into the animal. Plasma RHAL in the aorta, 6.4 +/- 6.6 (SD) ng/mL at 60 min and 9.4 +/- 4.6 (SD) ng/mL at the end of HAL infusion, was about 10-12-fold less than aortic HAL. The concentrations of RHAL in the hepatic vein were not significantly different from those in the aorta, indicating that splanchnic tissues including the liver are not responsible for plasma RHAL secretion. The highly efficient uptake of HAL as well as the ketone reductases found previously in vitro in liver microsomes of guinea pigs were probably involved only in biliary excretion of HAL.     

Effects of itraconazole on the steady-state plasma concentrations of haloperidol and its reduced metabolite in schizophrenic patients: in vivo evidence of the involvement of CYP3A4 for haloperidol metabolism

The effects of itraconazole, a potent inhibitor of cytochrome P450 (CYP) 3A4, on the steady-state plasma concentrations of haloperidol and reduced haloperidol were examined in schizophrenic patients. Thirteen schizophrenic patients treated with haloperidol 12 or 24 mg/day received 200 mg/day of itraconazole for 7 days. Plasma concentrations of haloperidol and reduced haloperidol were measured by high-performance liquid chromatography together with clinical assessment by the Brief Psychiatric Rating Scale (BPRS) and the Udvalg for Kliniske Undersogelser side effect rating scale just before and during itraconazole treatment and 1 week after its discontinuation. Plasma concentrations of haloperidol and reduced haloperidol during the itraconazole treatment (16.9 +/- 11.2 and 6.1 +/- 6.6 ng/mL, respectively) were significantly (p < 0.01) higher than those observed before itraconazole treatment (13.0 +/- 7.9 and 4.9 +/- 5.1 ng/mL) or 1 week after its discontinuation (13.5 +/- 8.2 and 4.9 +/- 5.0 ng/mL). No change was found in clinical symptoms assessed by BPRS, whereas neurologic side effects were significantly (p < 0.05) increased during itraconazole coadministration. The elevated plasma concentrations of haloperidol and reduced haloperidol during itraconazole coadministration were likely due to the inhibitory effects of itraconazole on the metabolism of haloperidol and reduced haloperidol. Thus, this study may provide in vivo evidence of involvement of CYP3A4 in the metabolism of haloperidol and possibly in that of reduced haloperidol. Deterioration of neurologic side effects during itraconazole treatment may result from the increased plasma concentrations of haloperidol and reduced haloperidol during itraconazole treatment.     

Histamine H1-receptor antagonists,promethazine and homochlorcyclizine,increase the steady-state plasma concentrations of haloperidol and reduced haloperidol

The effects of histamine H1-receptor antagonists, promethazine and homochlorcyclizine, both of which are inhibitors of CYP2D6, on the steady-state plasma concentrations (Css) of haloperidol and reduced haloperidol were studied in 23 schizophrenic inpatients receiving haloperidol, 12 to 36 mg/d, for 2 to 29 weeks. Promethazine, 150 mg/d, in 11 patients and homochlorcyclizine, 60 mg/d, in the others were coadministered for at least 1 week. Blood sampling was performed before and during coadministration of promethazine or homochlorcyclizine and 1 week after the discontinuation, together with clinical assessments by Brief Psychiatric Rating Scale (BPRS) and Udvalg for kliniske undersogelser (UKU) side effect rating scale. The Css (mean +/- SD) of haloperidol and reduced haloperidol during promethazine coadministration (27.6 +/- 24.9 and 8.6 +/- 13.2 ng/mL) were significantly higher than those before the coadministration (12.7 +/- 10.8 and 5.0 +/- 6.0 ng/mL; P < 0.01) or 1 week after the discontinuation (15.6 +/- 14.8 and 5.8 +/- 7.9 ng/mL; P < 0.05). The Css of haloperidol and reduced haloperidol during homochlorcyclizine coadministration (14.9 +/- 8.1 and 6.4 +/- 5.4 ng/mL) were also significantly higher than those before the coadministration (10.9 +/- 7.2 and 3.8 +/- 3.6 ng/mL; P < 0.01) or 1 week after the discontinuation (12.9 +/- 7.4 and 4.8 +/- 4.1 ng/mL; P < 0.05). No change in BPRS or UKU score was found throughout the study. Thus, the current study suggests that coadministration of clinical doses of promethazine and homochlorcyclizine increases the Css of haloperidol and reduced haloperidol via the inhibitory effects on the CYP2D6-catalyzed metabolism of haloperidol and reduced haloperidol.     

注射用帕瑞昔布钠特殊安全性评价

目的 评价注射用帕瑞昔布钠的安全性。方法 全身主动过敏实验：豚鼠隔日ip注射用帕瑞昔布钠（供试品,20 mg/mL）、原研对照、生理盐水或人血清白蛋白3次,于末次致敏后第14、21天进行激发,观察激发后30 min内动物反应;被动皮肤过敏实验：在豚鼠背部分别sc不同稀释度的抗体血清0.1 mL进行被动致敏,24 h后iv激发,30 min后处死,测量皮肤内层的蓝斑大小;家兔血管刺激性实验：连续耳iv供试品5 d后,于给药结束及恢复期取双耳进行HE染色,观察其对注射部位血管的刺激性;体外溶血实验观察供试品3 h内有无溶血现象。结果 20 mg/mL注射用帕瑞昔布钠无豚鼠全身过敏现象及被动皮肤过敏反应,未见血管刺激性;未见溶血现象发生。结论 20 mg/mL注射用帕瑞昔布钠无明显过敏反应、刺激性、溶血现象,是安全的。     

疫苗检定用麻疹病毒抗血清的制备

目的:制备麻疹病毒抗血清,用于含麻疹病毒成分的联合减毒活疫苗的检定。方法:制备麻疹病毒Edm-3株病毒收获液,加入弗氏佐剂,对家兔和豚鼠分别经皮下多点或腹腔途径免疫3针,采血制备抗血清,经除菌过滤和补体灭活后,对抗血清进行中和效价、中和能力、细胞毒性及特异性检测。结果:用4种免疫方式制备的抗血清中和效价均高于1∶320...     

Anti-warfarin antibody preparation and its characterization for radioimmunoassay

Anti-warfarin antiserum was prepared in rabbits by immunization with a synthesized warfarin antigen, 4'-azo-warfarin human serum albumin, which possesses two enantiomorphic haptenic sites of warfarin on the molecule. The antiserum recognized both R- and S-warfarin to the same degree, 50% cross reactivities of racemic warfarin, respectively. One of the warfarin metabolites, racemic warfarin alcohol, showed 1% cross reactivity, and R- or S-warfarin alcohol have half the reactivity of the racemic alcohol. Rabbit plasma warfarin levels were determined by radioimmunoassay using this antiserum and racemic [14C]warfarin and by fluorometric assay after isolation by thin layer chromatography. After a single administration of warfarin (2 mg kg-1 orally or 500 microgram kg-1 i.v.), the plasma levels determined by both assay methods showed a good correlation (r = 0.97, P less than 0.001, Y = 1.04-0.09). The results show that the radioimmunoassay can determine total plasma warfarin without interference of plasma metabolite. The applicability and limitation of the radioimmunoassay for pharmacokinetic study are discussed.     

Development of a lateral flow dipstick for simultaneous and semi‐quantitative analysis of dihydroartemisinin and piperaquine in an artemisinin combination therapy

Dihydroartemisinin (DHA) and piperaquine (PPQ) are two drugs used in an artemisinin‐based combination therapy (ACT). The circulation of counterfeit antimalarial drugs demands the development of simple, point‐of‐care (POC) tests for monitoring drug quality. Here we aimed to design an antibody‐based lateral flow dipstick assay for simultaneous quality control of DHA and PPQ. To obtain a monoclonal antibody (mAb) for PPQ, one structural unit of the symmetric PPQ molecule was used to derive a carboxylic acid for linkage to a carrier protein as immunogen. Screening of hybridoma cells identified an mAb 4D112B2 that reacted with the PPQ‐based immunogen. A highly‐sensitive icELISA was designed based on this mAb, which showed 50% inhibition concentration of PPQ at 1.66 ng/mL and a working range of 0.35 – 7.40 ng/mL. The mAb showed 10.2, 15.9 and 30.4% cross reactivity to hydroxychloroquine sulfate, chloroquine and amodiaquine, respectively. No cross reactivity was observed to lumefantrine, mefloquine artemisinin and its derivatives. Using our previous DHA dipstick design, a lateral flow dipstick for simultaneous analysis of PPQ and DHA was developed. The indicator ranges for PPQ and DHA were 2 – 5 μg/mL and 250 – 500 ng/mL, respectively. The dipstick was used to semi‐quantitatively analyze PPQ and DHA content in commercial ACT drugs, which produced agreeable results to those determined by high‐performance liquid chromatography. This combination dipstick makes it a potential POC device for quality control of the two active ingredients in a commonly used ACT.     

Time-resolved fluoroimmunoassay for the determination of lisinopril and enalaprilat in human serum

A solid-phase immunoassay with detection based on time-resolved fluorescence (TR-FIA) has been developed for the determination of lisinopril and enalaprilat in human serum. The immunogen was prepared by coupling lisinopril to bovine serum albumin through a two-step reaction with difluorodinitrobenzene. An antiserum specific to both lisinopril and enalaprilat was used. The assay is based on the competitive immunoassay principle in which the drug competes with biotin-labeled drug for a limited quantity of primary antibody bound via sheep anti-rabbit globulin to the wells of microtitration strips. At the end of the first incubation, the unbound biotin-labeled drug is washed away. In the second step, europium-labeled streptavidin (specific to biotin) reacts with the biotin already bound to the solid-phase antibody. After a washing step, the addition of an enhancement solution dissociates the europium ions from the labeled streptavidin into solution. The fluorescence from each sample is inversely proportional to the concentration of the drug in the sample. The assay demonstrates good accuracy, reproducibility and specificity at serum concentrations down to 0.5 ng ml⁻¹. However, the useful concentration range of TR-FIA is much narrower than that obtained by double antibody radioimmunoassay (RIA).     

Radioimmunoassay method for DX-9065a,an anticoagulant agent. Development,evaluation and application to human plasma

A simple and sensitive radioimmunoassay (RIA) method was developed for determination of DX-9065a, (+)-(2S)-2[4-[[(3S)-1-acetimidoyl-3-pyrrolidinyl]oxy]phenyl]-3-[7-amidino-2-naphthyl]propanoic acid hydrochloride pentahydrate, a newly synthesized anticoagulant agent. Immunogens were prepared by condensation of a hapten with bovine serum albumin via a car☐yl group. Antisera was obtained by immunization of five rabbits with immunogen. High-titer antisera was obtained from 2 rabbits immunized with immunogen. The sensitivity of this newly developed RIA method was 100-fold greater than that of a previously used conventional HPLC method. This method was validated for determination of human plasma samples in clinical trials. The cross-reactivities of employed antisera with three steroisomers (2{ce:italic}R{/ce:italic}3{ce:italic}R{/ce:italic}-, 2{ce:italic}R{/ce:italic}3{ce:italic}S{/ce:italic}- and 2{ce:italic}S{/ce:italic}3{ce:italic}R{/ce:italic} forms) were 0.7, 20.2 and 43.9% respectively. The effect of cross-reactivity of postulated stereoisomers in clinical samples was evaluated by a parallelism study using human plasma samples obtained after oral administration of the drug to healthy Japanese volunteers. Results showed no effect on measured concentration. From these data, this method showed suitable accuracy and precision for the pharmacokinetic evaluation of DX-9065a in clinical study.The method was applied to plasma samples obtained from a healthy Japanese volunteer who had orally received 12.85 mg (10 mg as DX-9065) of the drug. The maximum plasma concentration measured was 6.2 ng ml ⁻¹ 1 h after administration.