Generation and Characterization of Cytochrome P450 2J3/10 CRISPR/Cas9 Knockout Rat Model.

Cytochrome P450 2J2 (CYP2J2) enzyme attracts more attention because it not only metabolizes clinical drugs but also mediates the biotransformation of important endogenous substances and the regulation of physiologic function. Although CYP2J2 is very important, few animal models are available to study its function in vivo In particular, a CYP2J gene knockout (KO) rat model for drug metabolism and pharmacokinetics is not available. In this report, the CRISPR/Cas9 technology was used to delete rat CYP2J3/10, the orthologous genes of CYP2J2 in humans. The CYP2J3/10 KO rats were viable and fertile and showed no off-target effect. Compared with wild-type (WT) rats, the mRNA and protein expression of CYP2J3/10 in liver, small intestine, and heart of KO rats were completely absent. At the same time, CYP2J4 mRNA expression and protein expression were significantly decreased in these tissues. Further in vitro and in vivo metabolic studies of astemizole, a typical substrate of CYP2J, indicated that CYP2J was functionally inactive in KO rats. The heart function indexes of WT and KO rats were also measured and compared. The myocardial enzymes, including creatine kinase-muscle brain type (CK-MB), creatine kinase (CK), and CK-MB/CK ratio, of KO rats increased by nearly 140%, 80%, and 60%, respectively. In conclusion, this study successfully developed a new CYP2J3/10 KO rat model, which is a useful tool to study the function of CYP2J in drug metabolism and cardiovascular disease. SIGNIFICANCE STATEMENT: Human CYP2J2 is involved not only in clinical drug metabolism but also in the biotransformation of important endogenous substances. Therefore, it is very important to construct new animal models to study its function in vivo. This study successfully developed a new CYP2J knockout rat model by using CRISPR/Cas9 technology. This rat model provides a useful tool to study the role of CYP2J in drug metabolism and diseases.

A novel HPLC-MS/MS method for the simultaneous determination of astemizole and its major metabolite in dog or monkey plasma and application to pharmacokinetics.

Astemizole (AST), a second-generation antihistamine, is metabolized to desmethyl astemizole (DEA), and although it has been removed from the market for inducing QT interval prolongation, it has reemerged as a potential anticancer and antimalarial agent. This report describes a novel high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneously determining the concentrations of AST and DEA in beagle dog and cynomolgus monkey plasma with simple preparation method and short retention time. Prior to HPLC analyses, the plasma samples were extracted with simple liquid-liquid extraction method. The isocratic mobile phase was 0.025% trifluoroacetic acid (TFA dissolved in acetonitrile) and 20 mM ammonium acetate (94:6) at a flow rate of 0.25 mL/min and diphenhydramine used as internal standard. In MS/MS analyses, precursor ions of the analytes were optimized as protonated molecular ions: [M+H](+). The lower limit of quantification of astemizole was 2.5 ng/mL in both species and desmethyl astemizole were 7.5 ng/mL and 10 ng/mL in dog and monkey plasma, respectively. The accuracy, precision, and stability of the method were in accordance with FDA guidelines for the validation of bioanalytical methods. Finally this validated method was successfully applied to a pharmacokinetic study in dogs and monkeys after oral administration of 10 mg/kg AST.

Development of QTc prolongation model incorporating circadian rhythm using harmonic model.

1. QT prolongation is one of the major safety tests used in the development of a new drug. The ICH guidelines for the evaluation of QT prolongation recommend the use of the in vitro hERG assay and the in vivo telemetry test. However, QT intervals change under normal conditions due to circadian rhythm and can affect the results of the tests. In this study, we developed a PK/PD model to describe the QT interval after the administration of astemizole allowing for the normal changes by circadian rhythm. 2. The typical PK parameters of absorption rate constant (ka), volume of distribution (Vc and Vm), metabolism (km), and elimination rate constant (kel and kel-m) were 0.49 h(-1), 4950 L, 20 L, 0.0127 h(-1), 0.0095 h(-1), and 0.95 h(-1), respectively. The final PK/PD model was the biophase model with the modified harmonic model. The typical PK/PD parameters, base QTc interval (QT0), amplitude (T1, T3), period of QTc interval changing (T2, T4), and EC50 were 233 ms, 3.31, 1.5, -9.24 h, 1.85 h, and 0.81 ng/ml, respectively. 3. The PK/PD model to explain the changes of the QT interval that allows normal changes in the circadian rhythm after the administration of astemizole was developed successfully. This final model can be applied to the development of a human model.

Mapping pharmaceuticals in rat brain sections using MALDI imaging mass spectrometry.

Matrix-assisted laser desorption/ionization-tandem mass spectrometric method (MALDI-MS/MS) has proven to be a reliable tool for direct measurement of the disposition of small molecules in animal tissue sections. As example, MALDI-MS/MS imaging system was employed for visualizing the spatial distribution of astemizole and its primary metabolite in rat brain tissues. Astemizole is a second-generation antihistamine, a block peripheral H1 receptor, which was introduced to provide comparable therapeutic benefit but was withdrawn in most countries due to toxicity risks. Astemizole was observed to be heterogeneously distributed to most parts of brain tissue slices including cortex, hippocampus, hypothalamic, thalamus, and ventricle regions while its major metabolite, desmethylastemizole, was only found around ventricle sites. We have shown that astemizole alone is likely to be responsible for the central nervous system (CNS) side effects when its exposures became elevated.

Selective interaction of lansoprazole and astemizole with tau polymers: potential new clinical use in diagnosis of Alzheimer's disease.

We describe the interactions of two benzimidazole derivatives, astemizole (AST) and lansoprazole (LNS), with anomalous aggregates of tau protein (neurofibrillary tangles). Interestingly, these compounds, with important medical applications in the treatment of allergies and gastrointestinal disorders respectively, specifically bind to aggregated variants of tau protein and to paired helical filaments isolated from brains of Alzheimer's disease (AD) patients. These ligands appear to be a powerful tool to tag brain-isolated tau-aggregates and heparin-induced polymers of recombinant tau. The interactions of AST and LNS with tau aggregates were assessed by classical radioligand assays, surface plasmon resonance, and bioinformatic approaches. The affinity of AST and LNS for tau aggregates was comparatively higher than that for amyloid-beta polymers according to our data. This is relevant since senile plaques are also abundant but are not pathognomonic in AD patients. Immunochemical studies on paired helical filaments from brains of AD patients and surface plasmon resonance studies confirm these findings. The capacity of these drugs to penetrate the blood-brain barrier was evaluated: i) in vitro by parallel artificial membrane permeability assay followed by experimental Log P determinations; and ii) in vivo by pharmacokinetic studies comparing distribution profiles in blood and brain of mice using HPLC/UV. Importantly, our studies indicate that the brain/blood concentration ratios for these compounds were suitable for their use as PET radiotracers. Since neurofibrillary tangles are positively correlated with cognitive impairment, we concluded that LNS and AST have a great potential in PET neuroimaing for in vivo early detection of AD and in reducing the formation of neurofibrillary tangles.

Interaction of astemizole, an H1 receptor antagonist, with conventional antiepileptic drugs in mice.

Histamine is one of the aminergic neurotransmitters, playing an important role in the regulation of a number of physiological processes. There are several subtypes of histamine receptors-H(1), H(2), H(3) and the recently discovered H(4). H(1) receptors exist on mast cells, basophils, enterochromaffin cells and in the central nervous system, being located postsynaptically. H(1) receptor antagonists, including classical antiallergy drugs, occasionally have been expected to induce convulsions in children and epileptics. The aim of this study was to evaluate the effects of astemizole-given intraperitoneally, singly or for 7 days on the anticonvulsant activity of antiepileptic drugs (AEDs) against maximal electroshock (MES)-induced convulsions in mice. The following AEDs were administered intraperitoneally: valproate magnesium, carbamazepine, diphenylhydantoin and phenobarbital. Adverse effects were evaluated in the chimney test (motor performance) and passive avoidance task (long-term memory). Brain and plasma levels of AEDs were measured by immunofluorescence. Astemizole (a single dose and following a 7-day treatment at 2-6 mg/kg) reduced the threshold for electroconvulsions, being without effect upon this parameter at lower doses. Astemizole (1 mg/kg) did not significantly alter the protective effect of AEDs against MES (after acute and 7-day administration). Also, acute astemizole (2 mg/kg) remained ineffective in this respect. Astemizole (2 mg/kg), following chronic administration, significantly reduced the protective efficacy of phenobarbital and diphenylhydantoin, reflected by an increase in their ED(50) values (50% effective dose necessary to protect 50% of animals tested against MES) from 21.1 to 34.0 mg/kg and from 10.4 to 19.2 mg/kg, respectively. Astemizole (2 mg/kg) did not alter the protective activity of the remaining AEDs. Moreover, astemizole (2 mg/kg) did not influence the free plasma levels and brain concentration of the studied AEDs. Also, this H(1) receptor antagonist did not impair long-term memory or motor coordination when given acutely. However, 7-day treatment with astemizole (2 mg/kg) significantly decreased TD(50) (50% toxic dose required to induce motor impairment in 50% of animals) value of phenobarbital, being without effect on carbamazepine, valproate and diphenylhydantoin in this respect. Similarly, phenobarbital and diphenylhydantoin, administered alone at their ED(50)s against MES, or combined with astemizole, disturbed long-term memory in mice. The results of this study indicate that astemizole may need to be used with caution in epileptic patients.

Involvement of CYP2J2 on the intestinal first-pass metabolism of antihistamine drug, astemizole.

Orally administered astemizole is well absorbed but undergoes an extensive first-pass metabolism to O-desmethylastemizole. Desmethylastemizole is formed in the human microsomal systems of the small intestine as well as the liver, which suggests the role of cytochromes P450 (P450s) in the first-pass metabolism of astemizole. Human P450s involved in the O-demethylation of astemizole have, however, not been identified, and the involvement of twelve known drug-metabolizing P450s were denied. During the course of the P450 identification study, higher activities of the astemizole O-demethylation in the rabbit small intestine than in the liver (about 3-fold) were found. These data suggest the possible involvement of CYP2J, since P450 included in this subfamily is dominantly expressed in the small intestine of rabbits. Therefore, CYP2J2 cDNA has been isolated from the human cDNA library and expressed in COS-1 cells. A clear activity of astemizole O-demethylation was detected in recombinant CYP2J2 with K(m) = 0.65 microM and V(max) = 1129 pmol/nmol P450/min. Expression of the immunoreactive protein with CYP2J2 antibody was detected in the small intestine and liver. Expression levels of the immunoreactive protein with the CYP2J2 antibody in the small intestine were well correlated with the activities of the astemizole O-demethylation (r = 0.901, n = 5, p < 0.05). The CYP2J2 substrates, arachidonic acid and ebastine, strongly inhibited the microsomal astemizole O-demethylation in the human small intestines and recombinant CYP2J2. These results indicate the involvement of CYP2J2 in the presystemic elimination of astemizole in the human small intestine.

Second-generation antihistamines: the risk of ventricular arrhythmias.

Some second-generation antihistamines, notably terfenadine and astemizole, have been associated with prolongation of the QT interval and the development of torsades de pointes, a potentially fatal ventricular arrhythmia. This rare adverse event has been associated with greatly elevated blood levels of these agents, resulting from drug overdose, hepatic insufficiency (dysfunction), or interactions with other drugs that inhibit their metabolism. This paper reviews the data concerning the effects of selected second-generation antihistamines on cardiac conduction, particularly the QT interval, to evaluate whether ventricular arrhythmias are a class effect of these agents. Electrocardiographic studies indicate that terfenadine and astemizole, but not loratadine or cetirizine, prolong the QT interval in laboratory animals. In vitro studies demonstrate that terfenadine and astemizole block the cardiac K+ channels, leading to delayed ventricular repolarization and QT-interval prolongation; in contrast, neither loratadine nor its metabolite, desloratadine, significantly inhibits cardiac K+ channels at clinically achievable blood levels. Studies in human volunteers confirm the absence of electrocardiographic effects of azelastine, cetirizine, fexofenadine, and loratadine administered at several times the recommended dose or concomitantly with agents that inhibit their metabolism and elimination. In conclusion, the data indicate that the potential to cause ventricular arrhythmias is not a class effect of second-generation antihistamines and that loratadine, cetirizine, azelastine, and fexofenadine are not associated with torsades de pointes or other ventricular arrhythmias.

Pharmacokinetic overview of oral second-generation H1 antihistamines.

Specific H1 antihistamines have become the standard of treatment for relief of symptoms associated with seasonal allergic rhinitis. First-generation antihistamines are small lipophilic molecules that are associated with numerous adverse events largely because of their propensity to cross the blood-brain barrier and their cholinergic activity. Second-generation antihistamines, being more lipophobic, offer the advantages of a lack of CNS and cholinergic effects such as sedation and dry mouth, which are commonly seen in first-generation antihistamines. Their longer duration of action also enables a more patient-friendly dosing regimen which increases patient compliance. This paper reviews the pharmacokinetic properties of these second-generation agents and is intended to provide comparisons that help explain differences in dosing profiles and drug interactions for members of this class of drugs. With the announced withdrawal of terfenadine from the U.S. market in early 1997, 4 second-generation antihistamines are currently widely available: astemizole, loratadine, cetirizine, and fexofenadine. Terfenadine and astemizole both produce significant cardiac QT interval prolongation that may progress to a rare but fatal cardiac ventricular tachycardia known as torsades de pointes. While only terfenadine has been withdrawn due to its adverse effects profile, significant warnings were recently issued for astemizole. The pharmacokinetic profiles of loratadine and cetirizine are reflective of the advantages of these agents as non-cardiotoxic antihistamines. With respect to the newest agent fexofenadine, the major metabolite of terfenadine, published reports are minimal, but its pharmacokinetics differs from that of terfenadine.

Estado actual de antihistamínicos / Current state of antihistamine drugs

El aumento de la prevalencia mundial de las patologías respiratorias alérgicas conlleva un aumento simultáneo en la prescripción de antihistamínicos orales. Los efectos adversos que dichos medicamentos provocan en el sistema nervioso central son muy frecuentes(1), por lo que se presenta una revisión actualizada de los antihistamínicos sistémicos bajo el clásico adagio de la Medicina: "Primum non nocere" ("antes que nada no dañar"). En esta revisión se describe las generalidades de la acción de la histamina en el organismo. Los efectos terapéuticos y secundarios de los antihistamínicos de primera y segunda generación y finalmente se presenta una descripción acabada del nuevo antialérgico epinastina

Influence of itraconazole on the pharmacokinetics and electrocardiographic effects of astemizole.

AIMS: The aim of this study was to investigate the influence of chronic itraconazole treatment on the pharmacokinetics and cardiovascular effects of single dose astemizole in healthy subjects was studied. METHODS: Twelve male volunteers were taking orally 200 mg twice daily itraconazole or placebo for 14 days with a washout period of 4 weeks in between. Approximately 2 h after the morning dose of itraconazole or placebo on day 11, 10 mg astemizole was orally administered. The plasma concentrations of astemizole and desmethylastemizole were measured by radioimmunoassay up to 504 h after administration; electrocardiograms with analysis of the QTc interval were recorded up to 24 h post administration. RESULTS: Itraconazole treatment did not significantly change the peak concentration of astemizole (0.74 vs 0.81 ng ml-1) but it increased the area under the curve from 0 to 24 h (5.46 to 9.95 ng ml-1 h) and from 0 to infinity (17.4 to 48.2 ng ml-1 h), and the elimination half-life (2.1 to 3.6 days). The systemic bioavailability of desmethylastemizole was also increased. The QTc interval did not increase after astemizole administration and there was no difference in the QTc intervals between the itraconazole and placebo session. CONCLUSIONS: Chronic administration of itraconazole influences the metabolism of single dose astemizole in normal volunteers without changes of cardiac repolarization during the first 24 h after astemizole administration. However, the reduction in astemizole clearance under concomitant administration of itraconazole may result in a marked increase in astemizole plasma concentrations and QTc alterations during chronic combined intake of astemizole with itraconazole.

A study of the interaction between dirithromycin and astemizole in healthy adults.

The effect of a standard regimen of dirithromycin, a macrolide antibiotic, on the single-dose pharmacokinetics of the H (1) receptor blocker astemizole was evaluated in a sample of 18 healthy young adults (nine males and nine females). The study was conducted in a two-way cross-over fashion after the subjects had been randomly given either dirithromycin (two 250 mg tablets) or placebo (two tablets) every morning for 10 days. On the morning of the fourth dose of either dirithromycin or placebo each subject ingested a single 30-mg oral dose (three 10-mg tablets) of astemizole. The disposition kinetics of both astemizole and its major metabolite, N-desmethylastemizole, were characterized after measuring the concentrations of both analytes in the serum fraction of serial blood samples collected for 14 days after the astemizole dose. In addition, corrected QT (QT(c) ) intervals were estimated from electrocardiogram rhythm strips that were run 24 hours prior to the astemizole dose, 12 hours after the astemizole dose, and after the last treatment (dirithromycin or placebo) dose in both study periods. Pharmacokinetic parameters that were measured for both astemizole and N-desmethylastemizole during each treatment were: C(max), t(max), AUC (0-infinity), CL(oral), half-life, and volume of distribution (V). None of the parameters for N-desmethylastemizole was different when comparing data by ANOVA from the dirithromycin treatment period with that of the placebo treatment period. On the other hand, during dirithromycin treatment astemizole CL(oral) was 34% slower, volume of distribution was 24% larger, and half-life was 84% longer. Generally, all QT ( c ) intervals did not appear to be affected by dirithromycin treatment. The changes in astemizole kinetics could not be attributed to its N-demethylation since the dispositional kinetics of N-desmethylastemizole were unaffected by dirithromycin. Therefore, it is difficult to ascertain the clinical significance of the changes in astemizole kinetics. Since there were no significant differences for mean QT(c) intervals and no effect of dirithromycin treatment on N-desmethylastemizole kinetics, it is unlikely that a standard regimen of dirithromycin would place a patient taking astemizole at an increased risk of torsade de pointes or related ventricular arrhythmias.

Treating allergic rhinitis with second-generation antihistamines.

Allergic rhinitis afflicts close to 40% of the nation's population and costs more than $1.8 billion a year. The toll exacted by this disorder has been greatly alleviated by nonsedating second-generation antihistamines loratadine, terfenadine, and astemizole. The three agents effectively reduce symptoms without the sometimes intolerable adverse effects of older drugs, but they are not completely equivalent. For example, terfenadine requires twice/day dosing, whereas the others can be given once/day. Astemizole has a slow onset and extremely prolonged duration of action. Both terfenadine and astemizole may have cardiotoxic effects (e.g., torsades de pointes) when serum concentrations rise due to overdosing or drug interactions. Cetirizine, a recently approved second-generation antihistamine, has sedative and anticholinergic effects, although to a lesser degree than the first-generation antihistamines.

Cetirizine and astemizole therapy for chronic idiopathic urticaria: a double-blind, placebo-controlled, comparative trial.

BACKGROUND: Cetirizine and astemizole have been shown to be safe and effective in the treatment of patients with chronic idiopathic urticaria. Cetirizine brings about clinical benefit more rapidly. OBJECTIVE: The purpose of this study was to compare the efficacy of single daily doses of cetirizine and astemizole in relieving the symptoms of chronic idiopathic urticaria, with particular emphasis on the commencement of action. METHODS: Patients with chronic idiopathic urticaria were randomly assigned to relieve either 10 mg of cetirizine, 10 mg of astemizole, or placebo for 4 weeks in a multicenter double-blind trial. Patients rated symptom severity each night, and investigators rated symptoms weekly. RESULTS: One hundred eighty-seven patients were enrolled in the trial; 180 were included in the safety analysis and 177 were included in at least one efficacy analysis. Both cetirizine and astemizole were significantly superior to placebo in relieving symptoms of chronic idiopathic urticaria. Both patients' and investigators' ratings indicated that cetirizine acted more rapidly. Both active treatments were well tolerated, and the incidence of somnolence did not differ statistically between cetirizine (14.5%) and astemizole (10.3%). CONCLUSION: Both cetirizine and astemizole provide effective relief of the symptoms of chronic idiopathic urticaria with similar side-effect profiles. However, clinical benefit occurs significantly more rapidly with cetirizine.

Efecto de la solución oftálmica de levocabastina (antagonista de receptores H1) en los síntomas de la conjuntivitis primaveral: estudio prospectivo, aleatorio, comparativo y doble ciego / Effects of levocabastine ophthalmic solution (H1 receptor antagonist) in spring conjuntivitis symptoms: propective, comparative, aleatory, and double blind study

Se estudiaron en forma doble ciego, prospectiva y aleatoria, 40 pacientes con diagnóstico clínico de conjuntivitis primaveral y eosinofilia en la citología. Se les dividió aleatoriamente en dos grupos de 20. Un grupo recibió levocabastina (0.5-ml) y el otro solución salina. La edad promedio del grupo que recibió levocabastina fue de 9.1 años y 44 meses de evolución del padecimiento. La del grupo que recibió placebo fue de 10.1 años y 48 meses de evolución. Se administró solución oftálmica de cualquiera de los dos colirios, de acuerdo con su distribución aleatoria, una gota 2 veces al día (cada 12 horas por 7 días. Los pacientes se evaluaron diarimente a sí mismo por medio de una escala analógica visual. Se analizaron los resultados los días primero, tercero y séptimo y se sujetaron a un análisis estadístico con la prueba de U.Mann-Whitney. De los síntomas evaluados, fotofobia y lagrimeo mostraron disminución con significación estadística. Prurito, sensación de cuerpo extraño y pesantez mostraron tendencias a la mejoría con el medicamento sin tener significanción estadística. No hubo diferencias en el comportamiento de la hiperemia

The effect of activated charcoal on the absorption and elimination of astemizole.

1. The effect of activated charcoal on the absorption and elimination of astemizole and its metabolites was studied in healthy volunteers. 2. Subjects were divided into three groups containing seven subjects each. One group received 30 mg of astemizole with water only (control) and another group with 25 g of activated charcoal. The third group received multiple doses (12 g) of charcoal from 6 h onwards twice daily for 8 days. The concentrations of astemizole and its metabolites in plasma were measured by radioimmunoassay for 192 h. 3. Activated charcoal, administered immediately after astemizole ingestion, reduced the absorption of astemizole by 85% (P < 0.001). Multiple doses of activated charcoal, administered throughout the period of astemizole elimination, had no significant effect on the rate of elimination or the area under the curve from 0 to 192 h. 4. The absorption of astemizole from the gastrointestinal tract can be effectively prevented with activated charcoal. Because astemizole is rapidly absorbed, charcoal should be administered as soon as possible in acute astemizole poisoning. Multiple doses of charcoal do not seem to shorten the elimination half-life of astemizole.

Prolonged QT interval and torsade de pointes following astemizole overdose.

A 26 year-old woman was admitted to the hospital two hours after astemizole overdose. Electrocardiograph showed a prolonged QT interval. Torsade de pointes occurred 13 h after ingestion. Plasma levels of astemizole plus hydroxylated metabolites showed an apparent plasma half-life of 17 h. The possible occurrence of torsade de pointes in astemizole overdose, and the long elimination time of astemizole and hydroxylated metabolites, makes it necessary to maintain ECG monitoring until QT interval has returned to normal.

Pharmacokinetics of astemizole in children.

Astemizole is often administered to children in the treatment of rhinoconjunctivitis and urticaria with good efficacy and few side effects. Both astemizole and its major metabolite desmethylastemizole (DMA) are clinically effective without annoying side effects such as sedation. The pharmacokinetics in adults is well known. In three different studies we have investigated the pharmacokinetical properties of the drug in children. Study I (absorption): Thirty-eight children 8-16 years old (mean 12.6 years) and weighing 25-80 kg (mean 45 kg), with rhinoconjunctivitis due to birch pollinosis, were pretreated with either astemizole 5 mg daily or placebo for two weeks. Then, all children were treated with astemizole in doses increasing every week, i.e. 5, 10, 20 and 40 mg per day. There was a good correlation between the given dose per kg body weight and the plasma concentration of astemizole plus hydroxylated metabolites, indicating that astemizole is completely absorbed. Study II (time to reach steady state): A group of 21 children 7-18 years old (mean 13.9 years), plus 2 younger children, 2 and 5 years old, with allergy against birch- or grass pollen were treated with astemizole 10 mg daily for 12 weeks. Astemizole had reached steady-state plasma levels when the first sample was taken after 1 week, DMA reached steady state within 4 weeks. Study III (elimination half-life [t1/2 beta]): In 10 of the children from study II, t1/2 beta for astemizole plus DMA could be calculated (two samples) and was 10.8 days.(ABSTRACT TRUNCATED AT 250 WORDS)